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the incidence of perilymphatic fistula has been subject to controversial discussions since they were first described by simmons . unless there is profuse gushing of fluid , there is no reliable , objective , intraoperative indicator of a perilymphatic fistula , . the search of specific protein markers , or the intravenous application of fluorescein has so far not been successful . also 2-transferrin was reported to be unique in human perilymph , but in a study of 22 patients more than half of them had known perilymph leaks 2-transferrin was falsely negative . since felgenhauer demonstrated beta - trace protein ( -tp ) to be after albumin the second most abundant protein in human cerebrospinal fluid ( csf ) -tp is completely different to 2-transferrin . with a molecular mass of 23.500 dalton -tp is identical to glutathion - independent prostaglandin d synthase , which seems to play an important role in regulating the sleep and waking state . so far , the highest concentrations of -tp were found in csf and seminal fluid . in serum , the level of -tp is below the detection limit ( < 5 mg / l ) of rocket immunoelectrophoresis . only participants with severe renal filtration barrier pathology would show a detectable concentration of -tp . with the more sensitive immunofluorometric assay , which is based on an enzymatic amplified time - resolved fluorometry , the mean levels of -tp were 0.3 mg / l in human serum and -tp was detected in homogenate of the rat cochlea and in the lateral wall of the cochlea of the mongolian gerbil . based upon our experience with -tp in the diagnosis in csf - leaks , the purpose of this study was to investigate whether -tp occurs in human inner ear fluids and if positive , in which concentrations compared to csf . to clarify the occurrence of -tp we conducted analysis of perilymphatic fluid obtained from post - mortem examinations and from inner ear surgery . in post - mortem examinations 81 perilymph , 14 perilymph samples were sampled from patients undergoing stapedotomy or labyrinthectomy at the department of otorhinolaryngology , university of cologne . the perilymph samples were taken from the lateral semicircular canal ( n=4 ) or from the oval window ( n=10 ) . also pooled csf taken by lumbar puncture from 125 patients with non - inflammatory diseases of the cns was studied . after partial mastoidectomy , removal of the posterior ear canal and the tympanic membrane including malleus and incus , the stapes was exposed and the footplate carefully removed . samples of perilymph were taken from the scala vestibuli with precision capillary bores ( wiretrol 1 - 5 l , drummond , broomall , usa - pennsylvania 19008 ) under microscopic view ( figure 1 ( fig . the volume was limited to a minimum of 4 l and a maximum of 5 l . the capillary bores were placed near the upper edge of the vestibular window , the membranous labyrinth was initially not touched . after the sampling of a second and third perilymph specimen of 5 l , the period between death and autopsy as well as the cause of death were documented . the perilymph samples were taken from the oval window or from the lateral semicircular canal using sterilized precision bore . the volume of the fluid specimen was restricted to a maximum of 5 l . because of the high specificity of -tp , small blood contamination or small dilution with local anesthetic could be ignored . in addition to the post - mortem csf samples , pooled csf taken by lumbar puncture from 125 patients with non - inflammatory diseases of the cns was studied . all specimens were investigated by a rocket immunoelectrophoresis using a monospecific , polyclonal antibody , used in rabbits . investigation took place either immediately or after storage of maximum 7 days at - 40c . the standard electroimmunoassay as described by laurrell was performed using a gel composition of 1.5% litex agarose ( litex , denmark ) , 0.02 m barbital buffer ph 8.6 , 1% antiserum , 1% polyethylenglycol 6000 ( merck , darmstadt , germany ) and an electrophoresis buffer of 0.02 m barbital buffer , ph 8.6 . electrophoresis was performed during 3 hours at 18 c at a constant voltage of 250 v. semiquantitative measurements were performed using a standard row with serial dilution of purified -tp isolated from pooled human csf ( figure 2 ( fig . after partial mastoidectomy , removal of the posterior ear canal and the tympanic membrane including malleus and incus , the stapes was exposed and the footplate carefully removed . samples of perilymph were taken from the scala vestibuli with precision capillary bores ( wiretrol 1 - 5 l , drummond , broomall , usa - pennsylvania 19008 ) under microscopic view ( figure 1 ( fig . the volume was limited to a minimum of 4 l and a maximum of 5 l . the capillary bores were placed near the upper edge of the vestibular window , the membranous labyrinth was initially not touched . after the sampling of a second and third perilymph specimen of 5 l , the utricle was punctured and endolymph was obtained . csf specimens were obtained from the third ventricle . the period between death and autopsy as well as the perilymph samples were taken from the oval window or from the lateral semicircular canal using sterilized precision bore . the volume of the fluid specimen was restricted to a maximum of 5 l . because of the high specificity of -tp , small blood contamination or small dilution with local anesthetic could be ignored . in addition to the post - mortem csf samples , pooled csf taken by lumbar puncture from 125 patients with non - inflammatory diseases of the cns was studied . all specimens were investigated by a rocket immunoelectrophoresis using a monospecific , polyclonal antibody , used in rabbits . investigation took place either immediately or after storage of maximum 7 days at - 40c . the standard electroimmunoassay as described by laurrell was performed using a gel composition of 1.5% litex agarose ( litex , denmark ) , 0.02 m barbital buffer ph 8.6 , 1% antiserum , 1% polyethylenglycol 6000 ( merck , darmstadt , germany ) and an electrophoresis buffer of 0.02 m barbital buffer , ph 8.6 . electrophoresis was performed during 3 hours at 18 c at a constant voltage of 250 v. semiquantitative measurements were performed using a standard row with serial dilution of purified -tp isolated from pooled human csf ( figure 2 ( fig . by immunoelectrophoresis the perilymph , endolymph and csf specimens from post - mortem examination precipitated clearly ( figure 2 ( fig . , there was no clear evidence for a circadian alteration ( figure 3 ( fig . to 117 hours post mortem a significant change of the -tp concentration in csf and inner ear fluids was not observed in males or females ( figure 4 a , b , c ( fig . the values ranged between 8.5 mg / l and 165 mg / l . as depicted in figure 5 a ( fig . 5 ) , the mean concentration of -tp was 49.1 17.7 mg / l in csf and 59.0 25.8 mg / l in perilymph ( scala tympani ) . in the first drawn specimen of scala vestibuli perilymph -tp was found in a concentration of 71.9 29.3 mg / l . in the second specimen from the scala vestibuli 78.7 30.4 mg / l and in the third specimen 86.4 23.1 mg / l were detected . in endolymph , samples the values were 68.0 21.7 mg / l ( first specimen ) and 80.5 10.6 mg / the first perilymph specimen from the labyrinthine vestibule showed a lower concentration compared to the second or third specimens , which originated from deeper parts of the scala vestibuli . the -tp concentration in the second endolymph specimens was also higher in comparison with the values from the first specimens . the specimens revealed a -tp - concentration of 61.5 57.8 mg / l in perilymph from the lateral semicircular canal and 51.4 48.9 mg / l in perilymph from the scala vestibuli ( figure 5 b ( fig . puncture from 125 patients with non - inflammatory diseases , a -tp - concentration of 28.4 mg / l was determined . the issue of whether perilymphatic fistula is the cause of hearing loss remains enigmatic unless a specific marker exists for reliable proof . with -tp , the occurrence and the concentration of -tp have been investigated by several authors and were re - evaluated in this study ( table 1 ( tab . 1 ) ) . -tp has become a reliable indicator for csf - leak with advantages over 2-transferrin . although it is detectable in perilymph , 2-transferrin did not provide evidence to be a reliable clinical marker for perilymph in the operative setting because of its little sensitivity with 29% positive results . this is partly due to the property of this protein as a brain - specific variant of transferrin that lacks neuraminic acid . our study now makes obvious that -tp is detectable in inner ear fluids in even higher concentration compared to csf . nevertheless , our results are the first to quantify -tp in inner ear fluids . tachibana and coworkers described prostaglandin d synthase ( pgd - s ) activity in rat inner ear homogenate . with this method it is not possible to distinct where predominantly pgd - s could be found in the inner ear . the analysis of our entire specimen revealed a high interindividual variation between 8.5 and 165 mg / l , which can not yet be explained due to the unknown physiological role of -tp . with the upcoming new nephelometryic assay it will be more feasable in the future to analyse -tp under various conditions . nevertheless , -tp proved to be a very stable protein . until 117 hours after sampling , no significant variation in the -tp concentration in the diverse samples was detected . we did not find clear evidence for a circadian alteration of the -tp - level in inner ear fluids as it could be assumed from its role in the sleep / awake rhythm and as detected in csf . but a high interindividual variation and not excludable post - mortem changes hindered a statistical proof for a circadian rhythm in our series . nevertheless , the high interindividual variation as observed can not be . by this clear confirmation for the occurrence of -tp in inner ear fluids and particularly in perilymph , assessment for clinical use is feasible without circumstantial and biasing handling , which has been described for the 2-transferrin test . this is the first published data that point out the aptitude of the -tp - test in verifying traces of perilymph in the middle ear , a valuable diagnostic tool for the existence of perilymphatic leaks as we have already shown in two cases of an vestibulocochlear disorder . nowadays , an immediate intraoperative check for -tp is possible by laser - nephelometry , which takes only 20 minutes detection time .

objective : beta - trace protein ( -tp ) has been analysed in human cerebrospinal fluid ( csf ) and other body fluids . beta - trace protein is a very sensitive and specific clinical marker and can confirm reliably the presence of csf in patients with a suspected csf leakage.design : perilymph specimens from the scala vestibuli ( n=10 ) and from the lateral semicircular canal ( n=4 ) were taken from patients undergoing stapedotomy or surgery for acoustic neuroma . during post - mortem examinations perilymph specimens from the scala vestibuli ( n=70 ) , the scala tympani ( n=11 ) , endolymph specimens ( n=21 ) and csf specimens ( n=17 ) were obtained . all specimens were analyzed by a one - dimensional immunoelectrophoresis using a polyclonal , monospecific antibody . results : specimens from live surgery showed a mean concentration of 51.5 48.9 mg / l -tp in scala vestibuli perilymph . specimens from post - mortem examinations revealed a mean concentration of 49.1 17.7 mg / l in csf , 71.9 29.3 mg / l in perilymph and 68.0 21.7 mg / l in endolymph . there was no evidence of a circadian alteration of -tp in csf or inner ear fluids.conclusions : our results demonstrated clearly that -tp is contained in human perilymph and endolymph . this is the first published data that point out the aptitude of the -tp - test in verifying traces of perilymph , a valuable diagnostic tool for the existence of perilymphatic leaks .

contact investigation around tuberculosis ( tb ) patients is considered the most effective strategy in low prevalence settings for early detection of infection and disease , and for prevention of secondary tb cases . the aims of contact investigation are primarily to find and treat secondary active tb disease to disrupt ongoing transmission . screening for latent tb infections ( ltbi ) is also performed enabling preventive treatment or follow - up screening to prevent future tb disease in contacts . in addition , source tracing investigations are performed around cases of extra - pulmonary tb ( etb ) to find undiagnosed pulmonary tb ( ptb ) cases . in the netherlands , active tb is a notifiable disease , and source and contact investigation is included in the national tb control program . contacts are differentiated according to the ' stone in the pond ' principle , a concentric circle or ring approach based on their closeness to the index , as a proxy for exposure . based on the yield of screening in the respective rings , starting with the closest ring , and the risk of transmission as estimated by the public health nurse , the investigation can be stopped or extended to include more distant contacts . factors such as the age of the index patient and contact , duration and intensity of the contact , bacteriological status of the index patient , and ethnic background of the index patient are all know to be important , but the relative importance of these factors has not been established for contact investigations in the netherlands . improved knowledge of risk factors determining the chance of contacts being infected with tb would strengthen contact investigations by improving prioritization so high - risk contacts can be targeted first . the municipal public health service ( mphs ) of rotterdam , this has resulted in a unique database that , unlike other known registries , includes information on all screened contacts , not just those who were found to have ltbi or tb . this database of index patients and their contacts offers an excellent opportunity for an in - depth evaluation of contact investigations in the control of tb . in this study , we set out to identify risk factors for tb transmission among contacts of tb patients , in order to strengthen the evidence base of the current national contact investigation policy in the netherlands . the mphs in rotterdam is responsible for tb control in the larger rotterdam - rijnmond area , with a population of 1.2 million . , data on source tracing and contact investigations have been stored systematically in a database . our study includes data on the index cases and their contacts over the period 2001 - 2006 . contact investigations are conducted according to indications of infectiousness of the index case , such as the anatomical location of tb disease , the bacteriological status , and estimated patient and/or doctor 's delay . a public health nurse interviews the index to determine which ( if any ) contacts should be investigated according to the ' stone in the pond ' principle of concentric rings , where the ring can be seen as a proxy for exposure to the index patient in the case of ptb . the first ring ( ring 1 ) contains close contacts , the second ring ( ring 2 ) contains regular contacts , while the third ring ( ring 3 ) contains community contacts . this classification is based on the frequency and duration of exposure , and physical aspects of the area where the exposure takes place ( in particular , compartment size and ventilation ) . the mphs databases on the index patients and contacts were combined and linked to create a new database of screened contacts to which the recorded characteristics of the index patient were added . for the contacts we included age , gender , date of screening , tuberculin skin test ( tst ) and/or chest x - ray ( cxr ) . for the index patients we included the type of tb disease , age , ethnic background , sensitivity of the tb strain and the diagnostic steps that were taken . a positive tst was defined as an induration of 10 mm or more to 0.1 ml of purified protein derivative , measured 48 and 72 h after tst . positive tst results were followed up by cxr to screen for abnormalities indicating pulmonary tb . contacts with a history of tb , a previous tst induration of 10 mm or more , evidence of bcg vaccination or birth before 1945 were only screened by cxr , as a tst would be unable to provide evidence of a recent infection in these contacts . in the netherlands , universal bcg vaccination for the general population has never been implemented , and is only given to travellers to endemic countries or children of parents from endemic countries . when tst was positive , but clinical and radiological signs of active tb disease were absent , the final ltbi diagnosis was made by a tb physician . the first part determined the risk factors for being diagnosed with ltbi using a tst . the second part aimed at determining the risk factors for being diagnosed with active tb . the outcome of interest in the first analysis regarding tst tested contacts was a positive tst . in the second analysis , the outcome of interest was defined as an abnormal cxr with , if possible , bacteriological confirmation of active tb . both analyses were performed with the contacts as the unit of analysis and stratified by ring . univariate logistic regression was performed to estimate the effect of the characteristics on the individual contact investigation outcomes . variables with p<0.2 in the univariate logistic model were then included in multivariate logistic regression . we used a backward - stepwise selection based on the log - likelihood ratios and excluded variables with p>0.05 . exceptionally large contact investigations were excluded from the dataset , after which the analysis was performed again to ensure these large contact subpopulations did not weigh too heavily in the regression analyses . between 2001 and 2006 , a total of 1,165 index patients with active tb were registered . of these , 731 led to investigation of at least one contact ( range 1 - 1,810 contacts ; median 6 ; iqr=3.22 ) . together , these 731 index patients had 21,540 contacts participating in source tracing or contact investigations ( figure 1 ) . their general characteristics are shown in table 1 and in more detail in the appendix ( supplementary tables 1 - 5 ) . of the contacts , 12,698 ( 59% ) were eligible ( e.g. not previously vaccinated with bcg ) and tested for latent tb infections with a tst . the database contained 20,494 unique contacts , indicating that 1,046 of the investigations ( 4.9% ) were performed in contacts who had participated in an earlier contact investigation between 2001 and 2006 . the percentage of tb infected contacts among those tested with tst was almost halved when we compared regular contacts with close contacts ( 56% ) and community contacts with regular contacts ( 50% ) . for active tb among all contacts , the decrease is even stronger ; 21% for regular contacts compared with close contacts , and 11% comparing community contacts with regular contacts . figure 1cumulative number of contacts screened over the period 2001 - 2006 in source tracing or contact investigations in the rotterdam - rijnmond area plotted against the size of the individual contact investigations . cumulative number of contacts screened over the period 2001 - 2006 in source tracing or contact investigations in the rotterdam - rijnmond area plotted against the size of the individual contact investigations . table 1characteristics of the contacts included in the source tracing and contact investigations 2001 - 2006 performed by the municipal public health service rotterdam - rijnmond , the netherlands . contacts are stratified by ring according to a concentric circle approach , as specified in the national guidelines for contact investigations in the netherlands . ring 1 contains close contacts , ring 2 contains regular contacts , while ring 3 contains community contacts.ring 1 , tst testedring 2 , tst testedring 3 , tst testedring 1 allring 2 alltotal n. contacts169762324769329610967postives*16.0%9.0%4.3%9.6% ( 1.5%*)5.7% ( 0.3%*)male gender50.1%51.1%50.1%51.9%50.7%age of contact , median ( interquartile range)25 ( 14 - 39)32 ( 21 - 43)28 ( 20 - 40)28 ( 15 - 42)34 ( 23 - 46)relationship between contact and index residential / family27.7%18.5%1.0%29.7%20.7% work / education64.4%56.8%48.8%61.6%54.6% leisure time / other4.4%21.8%29.2%5.0%21.9%unknown / censored3.5%2.9%21.0%3.7%2.7%age of index , median ( interquartile range)31 ( 23 - 47)34 ( 25 - 44)33 ( 19 - 44)33 ( 23 - 47)34 ( 25 - 47)contacts investigated by location and bacteriological status of index ptb afb+75.0%87.5%94.0%67.6%87.2% ptb afb15.5%9.7%5.9%18.8%9.4% etb9.5%2.8%0.1%13.6%3.4%contacts investigated by index cases and specific ethnic background dutch63.6%51.8%73.5%52.0%47.2% western0.8%1.1%0.1%0.8%1.2% non - western35.7%47.1%26.4%47.2%51.7%tst , tuberculin skin test ; ptb , pulmonary tubercolosis ; etb , extra - pulmonary tubercolosis.*percentage of people found positive in the diagnostic test , confirmed by the tubercolosis ( tb ) physician . percentage includes the number of latent tb infections ( ltbi ) cases in tuberculin skin test - tested persons , the percentage of tb and ltbi for the column with all contacts . for columns including all ring 1 and 2 contacts , the percentage in brackets represents active tb percentage . tst , tuberculin skin test ; ptb , pulmonary tubercolosis ; etb , extra - pulmonary tubercolosis . percentage of people found positive in the diagnostic test , confirmed by the tubercolosis ( tb ) physician . percentage includes the number of latent tb infections ( ltbi ) cases in tuberculin skin test - tested persons , the percentage of tb and ltbi for the column with all contacts . for columns including all ring 1 and 2 contacts , the percentage in brackets represents active tb percentage . of the 12,698 contacts tested with tst , 1,091 were diagnosed with ltbi while the tst follow up with cxr revealed signs of active tb in 49 . table 2 shows the multivariate regression results for the three rings ( details are to be found in the appendix , supplementary tables 1 - 3 ) . older age ( 10-year intervals ) of the contact increases the risk of having a positive tst in all rings , with odds ratios ( or ) of 1.12 , 1.17 and 1.32 for the first , second and third ring , respectively . the relationship between the index and the contacts indicates that residential or family relationships are at the highest risk of having a positive tst , while work or education related contact results in a lower chance of transmission , although the differences are not always statistically significant among the types of relationships or for every ring . the diagnosis of the index is a strong predictor for contacts with positive tst : smear positive ptb ( ptb afb+ ) results significantly more often in the diagnosis of ltbi in contacts compared to smear negative ptb ( ptb afb ) or etb in ring 1 , but the differences between ptb+ and ptb decrease in ring 2 and there is no significant difference between them in ring 3 . additionally , the ethnic background of the index patient is statistically significant in rings 1 and 3 , but not in ring 2 . as far as investigation characteristics are concerned , the infection fraction of rings closer to the index is a strong predictor for the more remote rings . for ring 2 , or is 3.19 ( 95% ci:2.4 - 4.2 ) in the multivariate analysis , with the fraction positives within an investigation as risk factor . the correlation is more pronounced in ring 3 , in which the positive fraction of ring 2 gives an or of 9.22 ( 95% ci:3.37 - 25.2 ) . table 2odds ratios and their corresponding 95% confidence intervals for all significant factors in source tracing and contact investigations among contacts of tb patients from the five multivariate logistic regression models . gender did not reach significance in any of the models and is not , therefore , shown . ring 1 contains close contacts , ring 2 contains regular contacts , ring 3 contains community contacts.tst tested contactstst and/or cxr screened contactsring 1 ltbiring 2 ltbiring 3 ltbiring 1 tbring 2 tbage of contact ( 10-year interval continuous)1.12 ( 1.03 - 1.22)1.17 ( 1.09 - 1.24)1.32 ( 0.12 - 0.55 ) leisure time / other0.57 ( 0.28 - 1.17)0.77 ( 0.60 - 0.99)0.63 ( 0.29 - 1.41 ) unknown / censored0.57 ( 0.27 - 1.19)1.59 ( 1.05 - 2.41)0.61 ( 0.08 - 4.68)age of index ( 10-year interval)0.83 ( 0.76 - 0.92)1.28 ( 1.14 - 1.44)0.78 ( 0.61 - 1.00)ethnic background dutch1.001.00 western2.08 ( 1.40 - 3.09)2.41 ( 1.29 - 4.51 ) non - western1.70 ( 1.28 - 2.27)3.44 ( 2.41 - 4.90)bacteriological status index ptb afb+1.001.001.00 ptb afb0.31 ( 0.19 - 0.50)0.98 ( 0.72 - 1.32)0.21 ( 0.09 - 0.58 ) etb0.49 ( 0.29 - 0.85)0.25 ( 0.09 - 0.68)0.09 ( 0.02 - 0.39)drug resistance susceptible1.00 resistant4.61 ( 1.90 - 11.2)bal performed yes0.080 ( 0.64 - 0.99 ) no1.00investigation size ( 100-person interval)1.05 ( 1.01 - 1.08)0.19 ( 0.06 - 0.58)quarter of starting the investigation first1.00 second2.26 ( 0.84 - 6.07 ) third4.20 ( 1.60 - 11.0 ) fourth3.92 ( 1.54 - 9.97)fraction infected in ring 1na*3.19 ( 2.40 - 4.23)na*na*3.67 ( 1.44 - 9.38)fraction infected in ring 2na*na*9.22 ( 3.37 - 25.2)na*na*tst , tuberculin skin test ; cxr , chest x - ray ; ltbi , latent tubercolosis infections ; tb , tubercolosis ; ptb , pulmonary tubercolosis ; etb , extra - pulmonary tubercolosis ; bal , benzaldehyde.*na , not applicable , as only more proximal fraction of infected contacts within a source and contact investigation was entered into the multivariate regression analysis . tst , tuberculin skin test ; cxr , chest x - ray ; ltbi , latent tubercolosis infections ; tb , tubercolosis ; ptb , pulmonary tubercolosis ; etb , extra - pulmonary tubercolosis ; bal , benzaldehyde . na , not applicable , as only more proximal fraction of infected contacts within a source and contact investigation was entered into the multivariate regression analysis . among the 21,540 contacts , 91 cases of active tb were diagnosed . of these 91 cases , 2 were found by source tracing , meaning that these 2 screen - detected ptb cases were likely the first patients in the cluster . the correlation observed for the type of relationship between index and contact in the ltbi screening analysis was also seen for contacts diagnosed with active tb ; work or education related contact has a lower relative risk of being diagnosed with tb , compared to residential , family , leisure time , or other contacts . for ring 1 , the bacteriological status of the index is a strong predictor , with an or of 0.21 for ptb and 0.09 for etb , compared to ptb+ . the infection fraction in ring 1 is also a predictor for being diagnosed with tb in ring2 , with an or of 3.67 ( 95% ci:1.4 - 9.4 ) . the age of the contacts showed an inverse relationship to active tb , compared to the association found for ltbi , as 40% of the tb cases were in children aged 14 years or under with an odds ratio of 0.70 in the multivariate model in ring 1 , with 16 cases of active tb disease in young children aged 5 years or under . it was not possible to analyze the community contacts because only 2 cases of active tb were found among the 7,277 contacts in this ring . after exclusion of the largest 5 contact investigations ( 26% of all contacts in the study ) the direction of the associations did not change ( data not shown ) . details of the analysis of the close and regular contacts are shown in the appendix ( supplementary tables 4 and 5 ) . between 2001 and 2006 , at least one contact was screened of 731 index patients , resulting in a total of 21,540 contacts . the screening of 12,698 contacts who were eligible for tst testing led to the detection of 1,091 ltbi cases . important risk factors were age of the contact , age of the index patient and the relationship to the index . infections among regular ( ring 2 ) or incidental and community ( ring 3 ) contacts were strongly associated with a higher fraction of infected close ( ring 1 ) or regular ( ring 2 ) contacts , respectively . our study is based on data from routine investigations , which has implications for the interpretation of the findings . the records of all index patients and their contacts ( if any ) were available , enabling comparison between those with positive and those with negative contact investigation outcomes . although all contacts who participated and all investigations were documented , it is difficult to determine how many contacts were missed . a fraction of contacts will be missed by the mphs , despite the extensive effort to screen all reported or possibly exposed contacts . some groups are not included in routine contact investigations , such as the homeless or illicit substance users , because there is a separate regular screening program for these subpopulations . a recent study using data from the dutch tb surveillance register showed that contacts of immigrant cases were less often investigated . despite possible incompleteness of the study population , this database provides an excellent opportunity to determine risk factors in contact investigations because the netherlands is one of few countries where contact investigations are fully documented , out of the thirteen countries known to investigate contacts of all possibly infectious index patients . firstly , close contacts ( ring 1 ) had a higher risk of infection and tb disease than regular ( ring 2 ) and community ( ring 3 ) contacts . although more refined proxies for exposure are possible , the currently used ring approach has proven to be adequate in discriminating between contact levels . it is unknown how much could be gained by using data underlying this current proxy , such as actual duration and frequency of contact , and the environmental characteristics of the place of contact . unfortunately , a more precise registration of such indicators of contact level is not practical under routine control conditions . secondly , the contagious status of the index patient , in this study recorded as bacteriological status of the index patient in combination with the infection fraction in closer contact rings , was strongly associated with the number of infections among distant contacts . it is unclear whether this effect is fully attributable to recent transmission or is because the screened contacts are part of a subpopulation with a higher risk of infection . this is one of the limitations of using the tst in contact investigations . finally , increasing age of the contacts this is understandable from the perspective that with age , the time that an individual has been at risk of coming into contact with tb ( and other cross - reacting non - tb mycobacteria ) accumulates . young children ( age < 5 years ) are more at risk for progression to active tb , which is also recognized in this study , but in our analysis this was only proven in ring 1 . these three factors have already been well established as risk factors for tb in contacts and are included in the national guidelines for source tracing and contact investigations in the netherlands . similar results have been reported in evaluations of contact investigations in comparable settings and one systematic review , while one study did not find an association between the closeness to the index and symptomatic ptb after confirming transmission through dna fingerprinting . the study was carried out before the application of the interferon- assay blood test ( igra ) in the netherlands . later studies in comparable settings show that the igra test has a higher specificity than tst and thereby reduces the number of persons who would have received prophylactic treatment in case of a false - positive tst . we do not expect that the results would change drastically if the igra test were applied instead of the tst . there is no indication that the igra test responds differently in the risk groups that we established in this study , other than detecting additional old infections in bcg vaccinated persons from endemic countries . in this respect , we would like to point out that the goal of contact investigations is primarily to prevent large clusters by stopping ongoing transmission . this study focused on a population of contacts participating in source tracing and contact investigations . we identified important risk factors for the diagnosis of tb among contacts of tb patients , based on routine practice in a low prevalence setting , and established that the findings basically match the current national guidelines . the age of the patients and their contacts , the number of infections among close contacts , the type of contact relationship , and the diagnosis of the index patient are all associated with ltbi and tb in contacts . these risk factors emphasize the importance of including personal and interpersonal factors in decision making regarding contact investigations .

contact investigations around tuberculosis patients enable early detection of infection and disease , and prevention of secondary tuberculosis cases . we aim to identify risk factors for m. tuberculosis transmission to contacts of tuberculosis patients , based on unique data from routine contact investigations by the public health service in rotterdam , the netherlands , collected between 2001 and 2006 . through logistic regression analysis , we determined the effect of various risk factors on the chance of finding a latent tuberculosis ( tb ) infection or overt tuberculosis case among contacts . a total of 1165 index patients with active tuberculosis were registered and at least one contact was investigated in 731 , resulting in 21,540 contacts overall . altogether , the contact investigations led to 91 cases of active tuberculosis . of the 12,698 contacts eligible for screening by tuberculin skin test , 1091 ( 9% ) were diagnosed with latent tuberculosis infections . risk factors were old age of the contact , old age of the index patient , and the relationship to the index . a larger fraction of infected close contacts was strongly associated with infections among more distant contacts.our findings emphasize the importance of including these personal and interpersonal risk factors in decision making in contact investigations .

more than 60 diseases and injuries resulting in approximately 2.5 million deaths per year worldwide are linked to heavy alcohol drinking ( edwards et al . , 2011 ) . the number of current available medications for the treatment of alcohol dependence is limited , and most treatments show only moderate efficacy . promising results in the treatment of alcohol dependence have been obtained with some existing drugs such as anti - epileptic drug topiramate , antispasmic baclofen , and nausea relieving ondansetrone ( sellers et al . , 1994 ; new therapeutic drug targets have also been launched during the last years including cannabinoid receptors , neuropeptide y , corticotropin - releasing factor , and ghrelin ( kraus et al . , 2005 ; thorsell et al . , 2006 ; lowery and thiele , 2010 ; maccioni et al . , 2010 ) . recent findings from our laboratory using various animal models assessing dependence - like alcohol behaviors as well as binge - like alcohol drinking suggest that histamine h3 receptor could be a potential therapeutic target in the treatment of alcohol dependence ( nuutinen et al . , 2010 , 2011a , b ) . many modulatory neurotransmitter systems converge on the same neurons in the brain . for example , the actions of acetylcholine , histamine , norepinephrine , and serotonin on human neocortical cells are similar : they all increase spiking by reducing spike - frequency adaptation ( mccormick and williamson , 1989 ) . it is thus not surprising that several transmitters or their respective receptor ligands can have similar effects on neural circuits and behavior . some of the important systems in drug abuse include the mesolimbic dopamine system and corticostriatal and corticoaccumbal glutamate systems ( koob and volkow , 2010 ; kalivas and volkow , 2011 ) . all these brain regions are also innervated by histamine - containing nerve fibers originating from the posterior hypothalamic tuberomamillary nucleus both in rodents and in humans ( panula et al . , 1989 ; following release , histamine is mainly inactivated by methylation in the brain to an inactive metabolite tele - methylhistamine , whereas in the peripheral organs oxidation by diamino - oxidase is predominant ( haas and panula , 2003 ) . concentrations of tele - methylhistamine have been shown to correlate well with histamine release in the brain . very few studies have directly analyzed the role of histamine in alcohol - related behaviors in humans , and animal studies have been carried out only recently . in postmortem analysis , histamine and tele - methylhistamine , the first metabolite of histamine , levels are clearly increased in patients who suffered from extensive liver cirrhosis following alcohol abuse ( lozeva et al . , 2003 ) . the histamine levels were elevated in both patients with high alcohol consumption and those with no alcohol use . the mechanism is thus most likely related to the highly increased levels of neutral amino acids in the blood circulation , as reviewed earlier for experimental portocaval anastomosis cases ( fogel et al . , 2002 ) . in one study on type 1 ( late onset , often females , low degree of association with violence ) and type 2 ( early onset , often males , high degree of association with violence ) alcoholics histamine levels were significantly elevated in the gray matter of type 1 alcoholics and the levels of tele - methylhistamine were elevated in type 2 alcoholics indicating that histamine turnover is altered ( alakarppa et al . , 2002 ) . in alcohol - preferring alko alcohol ( aa ) rats developed in finland in 1960s using a two - bottle choice preference selection method ( eriksson , 1968 ) several aminergic systems are abnormal . they have higher tyrosine hydroxylase activity and higher levels of dopamine and noradrenaline in striatum and limbic regions ( ahtee and eriksson , 1975 ) , higher serotonin levels in many brain areas ( ahtee and eriksson , 1973 ) , and higher histamine and tele - methylhistamine levels in many brain regions than alcohol non - preferring alko non - alcohol ( ana ) rats ( lintunen et al . , 2001 ) . higher tele - methylhistamine levels indicate also higher histamine release in aa than ana rats . this high histamine content agrees well with the higher density of histamine - immunoreactive nerve fibers in many regions of the aa rat brain , including nucleus accumbens , septal nuclei , and medial preoptic nucleus . thus , histamine from the other major storage site of brain histamine , mast cells , is not responsible for the significant difference . receptor radioligand autoradiography has shown significant differences in histamine h1 receptor binding throughout the brain of aa and ana rats . the binding densities in aa rat brain were lower , possibly as a result of high histamine release induced downregulation ( lintunen et al . , 2001 ) . receptor radioligand binding is lower in aa than ana rats in the motor cortex , nucleus accumbens , and ca1 area of the hippocampus . although similar systematic studies have not been carried out in other rat lines selected for high volunteer alcohol intake , an analysis of histamine content in the cortex of the high alcohol preference ( hap ) and low alcohol preference ( lap ) rats was not different ( kitanaka et al . , 2004 ) . however , the histamine induced phosphoinositide hydrolysis was significantly lower in hap than lap rats ( kitanaka et al . , 2004 ) . rat lines selected for high alcohol sensitivity ( alcohol non - tolerant , ant ) and low alcohol sensitivity ( alcohol tolerant , at ; rusi et al . , 1977 ) also show distinct differences in histaminergic markers in the brain : the ant rats have reduced histamine levels in many brain areas , including hypothalamus , septum , cortex , and hippocampus ( lintunen et al . , 2002 ) . the alcohol sensitive ant rats also show higher h3 receptor agonist - induced guanosine 5-o-(3-[s]thio)triphosphate binding , indicative of histamine h3 receptor activation , in motor cortex , insula , and caudate - putamen ( lintunen et al . , 2002 ) . the behavioral effects of h3 receptor ligands on these rats have not been tested , but administration of a suicide inhibitor of the histamine synthesizing enzyme histidine decarboxylase , -fluoromethylhistidine ( -fmh ) , decreases the performance of these rats in the tilting plane test ( lintunen et al . , taken together , these results suggest that there are basal differences in the histamine system markers in these rat lines . however , no mutations in genes directly related to histamine synthesis and metabolism or histamine receptors have been identified . one of the most widely used paradigms is the two - bottle choice test where animals are given free - choice access to alcohol and non - alcoholic fluid , typically water ( green and grahame , 2008 ) . alcohol consumption in this model , however , can be influenced by other factors than pharmacological action of alcohol , such as taste and calories . thus control studies with sweet , bitter and high caloric fluids are needed . using a modified prolonged 8-week version of the two - bottle choice we found that the histamine h3 receptor knockout ( h3r ko ) mice consumed alcohol at concentrations 10 and 20% significantly less than the wild type animals ( nuutinen et al . however , the food consumption in h3r ko mice was lower than in control animals leaving us with a question whether the h3r ko mice simply drink less alcohol since they need less energy ? summary of the alterations in alcohol consumption and reward followed by manipulations of the brain histaminergic system . a more newly described method for alcohol consumption is so called drinking in the dark ( did ) method where mice are given access to alcohol for a short period of time during their active time of the day ( rhodes et al . , 2005 ) . the did paradigm is a measure of binge - like alcohol drinking and usually c57bl/6j mice are used in this paradigm due to their high alcohol consumption . the method is based on the finding that rodents consume more alcohol during the first couple of hours of their active period of the day ( goldstein and kakihana , 1977 ) . in line with the two - bottle choice ( dependence - like drinking ) study we found that the h3r ko mice drank less alcohol than control mice when given a 4-h access to 20% alcohol in the did model ( nuutinen et al . interestingly , h3r ko mice also consumed less 10% sucrose than the wild type mice again pointing to the direction that the lower need of calories could be the underlying reason for the lower voluntary alcohol consumption in h3r ko mice . to study this in more detail we used h3r specific ligands in the did model and found that an h3r antagonist ciproxifan dose - dependently inhibited alcohol drinking and h3r agonist immepip increased alcohol consumption in wild type mice ( nuutinen et al . , 2011a ) . importantly , the consumption of 10% sucrose , very high in caloric content , was not modified by ciproxifan or immepip pretreatment suggesting that other reasons than a altered need for calories could underlie the change in alcohol consumption following modification of h3rs . in agreement with our mouse studies , 2011 ) found that an h3r antagonist jnj-39220675 ( 1 and 10 mg / kg ) inhibits alcohol drinking to a similar extent as naltrexone ( 5 mg / kg ) in a two - bottle choice test in selectively bred alcohol - preferring ( p ) rats . jnj-39220675 also decreased alcohol preference but not alcohol consumption after a 3-day alcohol deprivation period in p rats . these results suggest that the lack or inhibition of h3r leads to lower alcohol consumption both in mice and rats . alcohol drinking can be also studied using self - administration paradigms where an operant response , typically a lever press , is required for the access to drink alcohol . self - administration models are thought to gauge better the motivation to drink alcohol ( green and grahame , 2008 ) . operant alcohol self - administration using oral administration is more commonly used in rats than in mice since mice do not drink alcohol as readily as the rats do with the exception of c57bl/6j mouse strain . already a decade ago we found that h3r antagonists thioperamide and clobenprobit ( table 2 ) dose - dependently ( 1 , 3 , 10 mg / kg for both drugs ) decrease operant alcohol self - administration in alcohol - preferring aa rats ( lintunen et al . treatment with h3r agonist r--methylhistamine or h1r antagonist mepyramine did not affect alcohol self - administration . recently researchers at johnson & johnson tested the h3r antagonist jnj-39220675 ( 10 and 30 mg / kg ) in an operant model and found that it significantly reduced lever presses for alcohol ( galici et al . , 2011 ) . thus , data from oral self - administration studies support the role of h3r antagonism in suppressing motivation to drink alcohol . alcohol reward and reinforcement can be also studied using pavlovian conditioning ( tzschentke , 2007 ) . this approach is advisable to use in parallel with the drinking studies in order to exclude intervening variables such as anxiety ( pohorecky , 1991 ) or novelty seeking ( cloninger et al . , 1988 ) that might result in altered alcohol drinking . in our laboratory , we are routinely using an unbiased alcohol - induced conditioned place preference ( cpp ) model in mice originally developed and described by cunningham et al . ( 2006 ) . in this model mice are given an alcohol injection paired with an environmental cue ( cage floor material ) for four to eight times over a period of 23 weeks . on alternating days with the alcohol injections mice are given vehicle injections paired with another type of floor material . after the conditioning period mice are tested in a preference test where they can choose between the two cage floor materials . place preference is indexed by a significant difference in time spent on one of the conditioning cue floors between two groups that have had different cues when conditioned to alcohol . in mice lacking the histamine synthesizing enzyme histidine decarboxylase , the alcohol - evoked cpp was found to be stronger suggesting that the lack of histamine increases the reward and reinforcement by alcohol in mice ( nuutinen et al . , 2010 ; table 1 ) . next we used different h3r ligands and found that antagonists ciproxifan ( 3 mg / kg ) and jnj-10181457 ( 5 mg / kg ) totally inhibited the cpp by alcohol in wild type dba/2j mice ( nuutinen et al . , 2011b ) . importantly , h3r antagonist ciproxifan did not induce place preference or place aversion alone in 129/sv mice suggesting that h3r antagonism does not produce place aversion or preference per se ( nuutinen et al . , 2010 ) . however , studies in other mouse strains and rats and using different h3r antagonists would be important to confirm that the h3r antagonists do not have addictive or aversive properties . the h3r agonist immepip ( 30 mg / kg ; table 2 ) did not modulate alcohol - induced cpp ( nuutinen et al . , 2011b ) . we think that this was probably due to the difficulty to increase an already strong place preference . when cpp model was applied to h3r ko mice ( c57bl/6j background ) , we found a complete lack of alcohol - evoked cpp ( nuutinen et al . , 2011a ) . in agreement with the alcohol drinking studies these data suggest that inhibition or lack of h3r leads to loss of alcohol reward . stronger alcohol - induced cpp in hdc ko mice suggests that histamine could be the key neurotransmitter mediating the inhibitory effects since the lack of histamine leads to stronger cpp . the inhibitory effect of h3r antagonists may , however involve other neurotransmitter systems in addition to histamine due to the heteroreceptor function of h3rs ( schlicker et al . , 1994 ) . interestingly , brain microdialysis studies have shown that h3r antagonists thioperamide and gsk189254 do not affect histamine release in rat nucleus accumbens or striatum , the central areas in the regulation of reward and reinforcement ( giannoni et al . antagonist abt-239 did not modify dopamine release in striatum ( fox et al . , 2005 ) and jnj-39220675 treatment , which was found to inhibit alcohol drinking and self - administration in rats did not change dopamine release per se or affect alcohol - induced release of dopamine ( galici et al . . however , many of the above - mentioned drugs as well as many other h3r antagonists do increase histamine and dopamine release e.g. , in prefrontal cortex ( brioni et al . , 2011 ) , another important area of the meso - cortico - limbic reward circuit which receives dopaminergic input from the ventral tegmental area and sends glutamatergic projections to striatum and nucleus accumbens ( lasseter et al . , 2010 ) . in summary , these results demonstrate that h3r antagonists affect neurotransmitter release in a region - specific manner and it seems that the inhibitory effect of h3r antagonism on alcohol consumption and cpp is likely to result from the effects of these drugs on neurotransmitter release on brain areas other than striatum and nucleus accumbens . another intriguing mechanism for h3r antagonist - induced inhibition of alcohol reward is the possible modulation of postsynaptic dopamine receptor signaling in striatum and nucleus accumbens . accumulating evidence suggests that h3rs form functional receptor heteromers with dopamine d1 and d2 receptors ( ferrada et al . , 2008 , 2009 ; moreno et al . , 2011 ) and that specific ligands that bind to one of the receptor of the heteromer , will affect the affinity and signaling of the other . thus , it is possible that by binding to postsynaptic h3rs in striatal areas , the h3r antagonists might interfere with the enhanced dopaminergic signaling induced by alcohol . we have measured the plasma alcohol concentrations after h3r ligand administration and found no difference compared to the corresponding controls thus ruling out the possibility that h3r ligands would alter alcohol metabolism ( nuutinen et al . , 2011a ) . mechanisms leading to addictive behaviors are thought to be common for all drugs of abuse . in line with the inhibitory role of histamine in alcohol - induce place preference , a study using h1 receptor knockout mice found a stronger methamphetamine - induced cpp than in wild type controls ( takino et al . , 2009 ) and h3r antagonists thioperamide and clobenprobit reduced amphetamine self - administration in rats ( munzar et al . , 2004 ) . in contrast to our alcohol study the cpp for methamphetamine was not different in histamine h3r ko mice compared to the control animals ( okuda et al . , 2009 ) . however , the authors used a biased cpp design ( by using one conditioning cue ) and place preference was scored by comparing pre - conditioning and post - conditioning times . in our laboratory we use two distinct conditioning cues that are neutral to the mice meaning that they do not prefer either one of them initially ( unbiased design ) . this is to prevent e.g. , the anxiolytic effect of the drug used that could lead to false - positive result ( cunningham et al . , 2003 ) . further , studies have shown that mice change their preference during the conditioning period without any reinforcing drugs , so in our paradigm place preference is scored as time spent in one of the sides of the chamber during the preference test following the conditioning days ( tzschentke , 2007 ) . studies on histamine s role in morphine dependence are few but support the inhibitory role of histamine in reward and reinforcement . morphine - induced cpp was attenuated by increasing histamine levels with l - histidine and potentiated by inhibiting histamine synthesis with -fluoromethyl histidine in mice ( suzuki et al . , 1995 ; h2 receptor antagonist zolantadine potentiated morphine - induced cpp but also induced place preference alone accompanied by an increase in dopamine turnover suggesting that it has other targets , possibly dopamine transporter , in addition to h2rs . in accordance with our data with alcohol , the morphine - induced place preference was stronger in hdc ko mice ( gong et al . , 2010 ) . studies on psychostimulants ( cocaine , methamphetamine ) are not in line with the suggested inhibitory role of histamine in addictive behaviors . in contrast to other findings in hdc ko mice ( gong et al . , 2010 ; nuutinen et al . , 2010 ) , no difference in cocaine - induced cpp was found between the hdc ko and wild type mice ( brabant et al . further , a pretreatment with an h3r antagonist thioperamide enhanced cocaine s effects by inducing place preference in mice with a cocaine dose that did not induce cpp alone ( brabant et al . , 2005 ) . however , thioperamide was later found to inhibit cocaine metabolism via inhibition of liver cyp-450 enzymes ( brabant et al . , 2009 ) . thus , the observations that h3r antagonists thioperamide and clobenpropit potentiate self - administration and discrimination of methamphetamine might be due to inhibition of methamphetamine metabolism by these imidazole - based h3r antagonists ( munzar et al . , 1998 , 2004 ) . further studies with non - imidazole - based h3r compounds and with knockout / transgenic animal models are needed to reveal the role of h3r in psychostimulant - induced reward and reinforcement . numerous studies on first generation h1r antagonists support the inhibitory role of histamine in reward ( for a review see brabant et al . , 2010 ) . however , these drugs have many targets in addition to h1rs such as muscarinic receptors and more importantly , the dopamine transporter ( shishido et al . , 1991 ; oishi et al . , 1994 ) . thus , it is most probably the inhibition of dopamine uptake rather than h1r blockade that explains why h1r blockers can cause per se or increase behavioral effects typically induced by addictive substances . there is accumulating evidence from alcohol drinking and place preference studies suggesting that alcohol reward could be decreased by treatment with h3r antagonists . more studies are still needed to test the potential of h3r antagonists to prevent alcohol withdrawal symptoms or relapse . cellular and molecular mechanistic studies are also of great importance in order to more widely increase our understanding of the complex role of h3r in addictive behaviors . studies on alcohol and morphine suggest that targeting brain h3rs could be a potential means to treat drug addiction . however , due to conflicting findings with h3r antagonists in psychostimulant addiction - related behaviors , more preclinical studies are needed to really show whether h3r antagonist would be beneficial in other types of drug addictions . one of the key questions to be solved is related to the possible differential expression of h3r in different dopaminergic neurons , and analysis of direct vs. indirect effects of h3r on dopamine release . whereas it seems that histamine h3 receptor is a key element in alcohol drinking and place preference , the role of histamine in these behaviors is still unclear . the relative importance of histamine and other transmitters regulated by h3 receptor antagonism in several alcohol - related behaviors remains to be studied using specific tools .

the brain histaminergic system is one of the diffuse modulatory neurotransmitter systems which regulate neuronal activity in many brain areas . studies on both rats and mice indicate that histamine h3 receptor antagonists decrease alcohol drinking in several models , like operant alcohol administration and drinking in the dark paradigm . alcohol - induced place preference is also affected by these drugs . moreover , mice lacking h3r do not drink alcohol like their wild type littermates , and they do not show alcohol - induced place preference . although the mechanisms of these behaviors are still being investigated , we propose that h3r antagonists are promising candidates for use in human alcoholics , as these drugs are already tested for treatment of other disorders like narcolepsy and sleep disorders .

nepafenac is a nonsteroidal anti - inflammatory drug ( nsaid ) and a prodrug that is metabolized into its active form , amfenac , following ocular administration . delivering amfenac as a prodrug permits increased corneal penetration and high aqueous concentration of the compound . amfenac suppresses prostaglandin production by inhibiting cyclooxygenase ( cox ) , as do other nsaids . ciliary body was inhibited by approximately 40% within 5 min and remained unchanged thereafter , while nepafenac exhibited an inhibitory effect on prostaglandin production between 5 and 10 min after administration , with the inhibitory rate exceeding 90% between 40 and 80 min after administration . the inhibitory effect of nepafenac on extravascular protein exudation into the aqueous humor of the rabbit continued for 8 h after administration while diclofenac inhibited vascular permeation up to 4 h after administration but exhibited no significant inhibitory effect 8 h after administration . since its launch in the usa in 2005 , nepafenac 0.1% has been widely used in clinical practice in more than 70 countries worldwide . to receive marketing approval for the drug in japan , we conducted a placebo - controlled , multicenter trial to evaluate the efficacy and safety of the drug in the management of postoperative inflammation and ocular pain in japanese patients undergoing cataract surgery with phacoemulsification and aspiration ( pea ) . nepafenac ophthalmic suspension containing 1 mg of nepafenac ( fig . 1 ) per 1 ml and placebo with the same formulation as nepafenac suspension excluding nepafenac ingredient supplied in identical , opaque 5-ml bottles.fig . 1chemical structure of nepafenac chemical structure of nepafenac this was a multicenter , randomized , double - masked , placebo - controlled study conducted at 11 japanese sites ( table 1 ) from august 2006 to december 2006 . this study was conducted in accordance with the declaration of helsinki with approval from the institutional review boards . they began dosing 1 day before surgery and continued on the day of surgery and for 14 days thereafter . on the day of surgery , the test drugs are blinded to the investigators and subjects except for the appointed controller of study drugs.table 1investigators and institutionsno.institutionsinvestigators1ohtsuka eye hospitalmakoto higuchi2mitani eye clinickiichiro mitani3muramatsu eye clinictomoyuki muramatsu4nakajima eye clinictoru nakajima5kikukawa eye clinickoichiro ikeda6tokai eye clinicyoshihide nakai7kitano hospitalisao saito8nishi eye hospitalhiroki iwanishi9hirota eye clinicatsushi hirota10ohshima hospital of ophthalmologynobuyuki yabe11hayashi eye hospitalken hayashi investigators and institutions patients 20 years of age or older scheduled to undergo cataract extraction by pea and implantation of an intraocular lens ( iol ) were eligible for the study . each patient was given sufficient explanation about the purpose and details of the study and provided written consent to participate in the study . patients were excluded from enrollment if they used topical ocular or systemic steroids within 14 days of surgery or used topical ocular or systemic nsaids within 7 days of surgery , except an allowed daily dose of low - dose aspirin . patients were also excluded if they planned to have cataract surgery in their fellow , non - study eye before the 14-day postoperative study visit or had any cells , flare , or ocular pain in the operative eye at the screening examination , had planned multiple surgical procedures , exfoliation syndrome , ocular trauma , inflammatory eye disease , diabetic retinopathy which needed medical treatment , and uncontrolled diabetes mellitus , or had grade 3 or higher cataracts according to emery little classification . best - corrected visual acuity ( bcva ) and intraocular pressure ( iop ) measurement , ocular pain assessment , slit lamp examination , and laser flare cell meter ( lfcm ) measurement were performed before and 1 , 3 , 7 , and 14 days after surgery . blood pressure / pulse rate measurement was performed before and at the day of surgery and 1 , 3 , 7 , and 14 days after surgery . a laboratory examination was performed before and 14 days after surgery.table 2the examination scheduleboth eyesstudy ( operative ) eye the examination schedule study ( operative ) eye the grading criteria and scale for clinical symptom and signs are summarized in table 3 . the criteria and scale are the same as previous analysis of nepafenac [ 46 ] . patients who received the test drug , completed surgery , and had at least one follow - up visit the primary efficacy variables were the cure rate ( the percentage of subjects with an aqueous cell score + aqueous flare score = 0 ) at day 14 after surgery and the ocular pain - free rate ( the percentage of subjects with an ocular pain score of 0 ) at all postoperative visits . also , the cure rate and the ocular pain - free rate at each postoperative visit were compared between two groups . the secondary variables were aqueous cell score , aqueous flare score , cell count , flare value , and the treatment failure rate ( the percentage of subjects with either an aqueous cell score or an aqueous flare score of 3 or more , or an ocular pain score of 4 or more ) at each postoperative visit . cell count and flare value were measured with lfcm.table 3grading criteria for clinical symptom and signsgrading criteriaocular pain0 : none absence of pain1 : trace slight sensation of pain or discomfort2 : mild mild , tolerable aching of the eye3 : moderate moderate and prolonged aching sufficient to require the use of analgesics4 : moderately severe prolonged intense aching requiring the use of analgesics5 : severe prolonged sharp ocular or periocular painflareuse a 1 mm wide beam of the slit lamp aimed at the center of the pupil0 : none no visible flare when compared with the normal eye1 : mild flare visible against dark pupillary background but not visible against iris background2 : moderate flare is visible with the slit lamp beam aimed onto the iris surface as well as the dark pupillary background3 : severe very dense flare . hazy appearance of anterior segment structures when viewed with low power magnification of the slit lamp . presents as a pronounced tyndall effectcelluse narrow slit beam ( 0.5 mm in width and at least 8 mm in length ) at maximum luminance . pigment and red blood cells are to be ignored.0 : none1 : 1 to 5 cells2 : 6 to 15 cells3 : 16 to 30 cells4 : greater than 30 cells grading criteria for clinical symptom and signs all patients receiving the test drug were included in the safety analysis . safety evaluation included incidence of adverse events ( aes ) , blood pressure / pulse rate , laboratory tests , bcva , iop , slit lamp examination ( bulbar conjunctival injection , corneal edema , and chemosis ) , and fundus examination ( retina , macula lutea , choroid , and optic nerve ) . for the primary variables of cure rate at day 14 and the ocular pain - free rate at all postoperative visits , a chi - square test of independence was conducted to assess the superiority of nepafenac relative to placebo . multiplicity adjustment for the primary endpoints was made by sequential testing with fixed sequences , in which the cure rate was analyzed first followed by the ocular pain - free rate . and a chi - square test or a fisher s exact test was performed for the cure rate and the ocular pain - free rate at each study visit . the analysis of aqueous cells score , flare scores , cell counts , and flare value was conducted with a repeated measures analysis of variance at postoperative visits . a two - sample t test was used for the comparisons in aqueous cells score and flare score at baseline between two groups . the comparisons between nepafenac and placebo in treatment failures at each postoperative visit were made using nonlinear mixed model with maximum likelihood estimation from nlmixed procedure of sas software . nepafenac ophthalmic suspension containing 1 mg of nepafenac ( fig . 1 ) per 1 ml and placebo with the same formulation as nepafenac suspension excluding nepafenac ingredient supplied in identical , opaque 5-ml bottles.fig . this was a multicenter , randomized , double - masked , placebo - controlled study conducted at 11 japanese sites ( table 1 ) from august 2006 to december 2006 . this study was conducted in accordance with the declaration of helsinki with approval from the institutional review boards . they began dosing 1 day before surgery and continued on the day of surgery and for 14 days thereafter . on the day of surgery , the test drugs are blinded to the investigators and subjects except for the appointed controller of study drugs.table 1investigators and institutionsno.institutionsinvestigators1ohtsuka eye hospitalmakoto higuchi2mitani eye clinickiichiro mitani3muramatsu eye clinictomoyuki muramatsu4nakajima eye clinictoru nakajima5kikukawa eye clinickoichiro ikeda6tokai eye clinicyoshihide nakai7kitano hospitalisao saito8nishi eye hospitalhiroki iwanishi9hirota eye clinicatsushi hirota10ohshima hospital of ophthalmologynobuyuki yabe11hayashi eye hospitalken hayashi investigators and institutions patients 20 years of age or older scheduled to undergo cataract extraction by pea and implantation of an intraocular lens ( iol ) were eligible for the study . each patient was given sufficient explanation about the purpose and details of the study and provided written consent to participate in the study . patients were excluded from enrollment if they used topical ocular or systemic steroids within 14 days of surgery or used topical ocular or systemic nsaids within 7 days of surgery , except an allowed daily dose of low - dose aspirin . patients were also excluded if they planned to have cataract surgery in their fellow , non - study eye before the 14-day postoperative study visit or had any cells , flare , or ocular pain in the operative eye at the screening examination , had planned multiple surgical procedures , exfoliation syndrome , ocular trauma , inflammatory eye disease , diabetic retinopathy which needed medical treatment , and uncontrolled diabetes mellitus , or had grade 3 or higher cataracts according to emery little classification . best - corrected visual acuity ( bcva ) and intraocular pressure ( iop ) measurement , ocular pain assessment , slit lamp examination , and laser flare cell meter ( lfcm ) measurement were performed before and 1 , 3 , 7 , and 14 days after surgery . blood pressure / pulse rate measurement was performed before and at the day of surgery and 1 , 3 , 7 , and 14 days after surgery . a laboratory examination was performed before and 14 days after surgery.table 2the examination scheduleboth eyesstudy ( operative ) eye the examination schedule study ( operative ) eye the grading criteria and scale for clinical symptom and signs are summarized in table 3 . the criteria and scale are the same as previous analysis of nepafenac [ 46 ] . patients who received the test drug , completed surgery , and had at least one follow - up visit were included in the intention - to - treat ( itt ) analyses . the primary efficacy variables were the cure rate ( the percentage of subjects with an aqueous cell score + aqueous flare score = 0 ) at day 14 after surgery and the ocular pain - free rate ( the percentage of subjects with an ocular pain score of 0 ) at all postoperative visits . also , the cure rate and the ocular pain - free rate at each postoperative visit were compared between two groups . the secondary variables were aqueous cell score , aqueous flare score , cell count , flare value , and the treatment failure rate ( the percentage of subjects with either an aqueous cell score or an aqueous flare score of 3 or more , or an ocular pain score of 4 or more ) at each postoperative visit . cell count and flare value were measured with lfcm.table 3grading criteria for clinical symptom and signsgrading criteriaocular pain0 : none absence of pain1 : trace slight sensation of pain or discomfort2 : mild mild , tolerable aching of the eye3 : moderate moderate and prolonged aching sufficient to require the use of analgesics4 : moderately severe prolonged intense aching requiring the use of analgesics5 : severe prolonged sharp ocular or periocular painflareuse a 1 mm wide beam of the slit lamp aimed at the center of the pupil0 : none no visible flare when compared with the normal eye1 : mild flare visible against dark pupillary background but not visible against iris background2 : moderate flare is visible with the slit lamp beam aimed onto the iris surface as well as the dark pupillary background3 : severe very dense flare . hazy appearance of anterior segment structures when viewed with low power magnification of the slit lamp . presents as a pronounced tyndall effectcelluse narrow slit beam ( 0.5 mm in width and at least 8 mm in length ) at maximum luminance . pigment and red blood cells are to be ignored.0 : none1 : 1 to 5 cells2 : 6 to 15 cells3 : 16 to 30 cells4 : greater than 30 cells grading criteria for clinical symptom and signs safety evaluation included incidence of adverse events ( aes ) , blood pressure / pulse rate , laboratory tests , bcva , iop , slit lamp examination ( bulbar conjunctival injection , corneal edema , and chemosis ) , and fundus examination ( retina , macula lutea , choroid , and optic nerve ) . for the primary variables of cure rate at day 14 and the ocular pain - free rate at all postoperative visits , a chi - square test of independence was conducted to assess the superiority of nepafenac relative to placebo . multiplicity adjustment for the primary endpoints was made by sequential testing with fixed sequences , in which the cure rate was analyzed first followed by the ocular pain - free rate . and a chi - square test or a fisher s exact test was performed for the cure rate and the ocular pain - free rate at each study visit . the analysis of aqueous cells score , flare scores , cell counts , and flare value was conducted with a repeated measures analysis of variance at postoperative visits . a two - sample t test was used for the comparisons in aqueous cells score and flare score at baseline between two groups . the comparisons between nepafenac and placebo in treatment failures at each postoperative visit were made using nonlinear mixed model with maximum likelihood estimation from nlmixed procedure of sas software . two hundred fifteen patients , 108 treated with nepafenac and 107 treated with placebo , were enrolled in this study . two hundred fourteen remaining patients received at least one dose of test drug and were therefore included in the safety analysis . three of 214 patients discontinued from the study prior to or at the time of surgery for patient decision ( n = 1 ) , the use of nsaids ( n = 1 ) and investigator decision due to surgical difficulties . a total of 211 patients , 106 treated with nepafenac and 105 treated with placebo were included in the itt population . the demographics data for the patients included in the itt data set are summarized in table 4 , and the demographics data by institution are showed in table 5 . as indicated in table 4 , there were no statistically significant differences in gender , age , and emery s classification between treatment groups.table 4demographic data ( itt)totalnepafenac groupplacebo groupp valuen%n%n%total211100.010650.210549.8gender male9846.44946.24946.70.9489 female11353.65753.85653.3age 18 to 64 years5928.02422.63533.30.0836 65 years15272.08277.47066.7emery s classification 15023.72826.42221.00.3508 216176.37873.68379.0from chi - squaretable 5demographic data by institution ( itt)genderageemery s classificationmalefemale<656512n%n%n%n%n%n%totalnepafenac4946.25753.82422.68277.42826.47873.6placebo4946.75653.33533.37066.72221.08379.0investigator no.1nepafenac466.7233.3233.3466.7466.7233.3placebo233.3466.7350.0350.0583.3116.72nepafenac541.7758.3325.0975.0541.7758.3placebo650.0650.018.31191.7541.7758.33nepafenac533.31066.7213.31386.700.015100.0placebo640.0960.0213.31386.700.015100.04nepafenac675.0225.000.08100.0675.0225.0placebo450.0450.0225.0675.0562.5337.55nepafenac743.8956.3212.51487.500.016100.0placebo743.8956.3531.31168.800.016100.06nepafenac360.0240.0120.0480.000.05100.0placebo233.3466.7350.0350.000.06100.07nepafenac222.2777.8222.2777.8222.2777.8placebo350.0350.0233.3466.7233.3466.78nepafenac337.5562.5337.5562.5225.0675.0placebo444.4555.6444.4555.6111.1888.99nepafenac555.6444.4222.2777.8222.2777.8placebo555.6444.4555.6444.4222.2777.810nepafenac433.3866.7325.0975.0650.0650.0placebo650.0650.0541.7758.318.31191.711nepafenac583.3116.7466.7233.3116.7583.3placebo466.7233.3350.0350.0116.7583.3 demographic data ( itt ) demographic data by institution ( itt ) there was a significant difference in cure rate at day 14 between nepafenac ( 71.4% , 75/105 ) and placebo ( 28.6% , 30/105 ) ( p < 0.0001 ) , demonstrating the effectiveness of nepafenac in reducing postoperative inflammation ( table 6 ) . when comparing cure rates at each visit , nepafenac achieved significantly higher cure rates than placebo at days 7 and 14 ( p < 0.0001 for days 7 and 14 ) ( table 6 ) . the ocular pain - free rate was 96.2% ( 102/106 ) in the nepafenac group and 67.6% ( 71/105 ) in the placebo group , showing a significant difference between groups ( p < 0.0001 ) ( table 7 ) . the nepafenac group demonstrated significantly higher ocular pain - free rates than the placebo group at all postoperative visits ( p = 0.0051 for day 1 , p = 0.0003 for day 3 , p = 0.0002 for day 7 , and p = 0.0051 for day 14 ) ( table 8) . in the nepafenac group , cell scores and flare scores measured at each postoperative visit decreased as time elapsed after surgery , showing the suppressive effect of nepafenac on postoperative inflammation . in contrast , no marked decrease was observed in either the cell or flare scores in the placebo group . significant intergroup differences were observed in the cell score on day 7 and subsequent visits ( p < 0.0001 for days 7 and 14 ) and in the flare score on day 3 and subsequent visits ( p = 0.0041 for day 3 and p < 0.0001 for days 7 and 14 ) ( tables 9 and 10 ) . decreases in both cell counts and flare values were observed on day 3 and subsequent visits in the nepafenac group , whereas increases in both variables were observed on day 3 in the placebo group . significant intergroup differences were observed in cell counts and flare values on day 3 and subsequent visits ( tables 11 and 12).table 6cure rate at each study visit ( itt)treatmentall ( n)day 1day 3day 7day 14n%n%n%n%nepafenac group10521.92221.05350.57571.4placebo group10521.91615.22019.03028.6p value1.000.2822<0.0001<0.0001test = chi - square ( fisher s exact test if n < 5)one patient missing cure datatable 7percent ocular pain free ( itt)treatmenttotalpain freenn%nepafenac group10610296.2placebo group1057167.6test = chi - square , p < 0.0001table 8percentage of patients with no pain at each study visit ( itt)treatmentall ( n)day 1day 3day 7day 14n%n%n%n%nepafenac group10610397.210498.110397.210397.2placebo group1059186.78883.88581.09186.7p value0.00510.00030.00020.0051test = chi - squaretable 9aqueous cells score by treatment and visit ( itt)baselineday 1day 3day 7day 14nepafenac groupmean0.01.30.90.50.3std0.00.60.70.60.6n106105105105105min00000max04444placebo groupmean0.01.21.01.00.9std0.00.50.70.90.9n105105105105105min00000max02444p valuen / a0.69050.1638<0.0001<0.0001baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviationtable 10flare score by treatment and visit ( itt)baselineday 1day 3day 7day 14nepafenac groupmean0.00.90.50.30.1std0.00.60.60.50.3n106105105105105min00000max02222placebo groupmean0.00.90.70.80.6std0.00.50.70.80.8n105105105105105min00000max02333p valuen / a0.57930.0041<0.0001<0.0001baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviationtable 11cell count measured by lfcm by treatment and visit ( itt)day 1day 3day 7day 14nepafenac groupmean39.219.89.76.0std79.225.111.29.8n54545454min0.00.00.00.0max428.7116.452.765.3placebo groupmean40.455.332.224.1std52.866.237.331.4n53545454min0.00.00.00.0max312.2311.2177.3128.0p value0.8738<0.00010.01070.0394non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p = 0.0011 ; treatment by visit interaction p = 0.0186 ; cell count was measured by laser flare cell meter at six sitesmin minimum , max maximum , std standard deviationtable 12flare value measured by lfcm by treatment and visit ( itt)day 1day 3day 7day 14nepafenac groupmean20.620.613.411.4std24.640.912.29.1n101103103103min0.50.30.40.3max153.2396.757.857.8placebo groupmean24.347.565.652.0std34.449.877.769.9n101104104104min1.82.52.51.5max307.6333.9479.7479.7p value0.6201<0.0001<0.0001<0.0001non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviation cure rate at each study visit ( itt ) test = chi - square ( fisher s exact test if n < 5 ) one patient missing cure data percent ocular pain free ( itt ) test = chi - square , p < 0.0001 percentage of patients with no pain at each study visit ( itt ) aqueous cells score by treatment and visit ( itt ) baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation flare score by treatment and visit ( itt ) baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation cell count measured by lfcm by treatment and visit ( itt ) non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p = 0.0011 ; treatment by visit interaction p = 0.0186 ; cell count was measured by laser flare cell meter at six sites min minimum , max maximum , std standard deviation flare value measured by lfcm by treatment and visit ( itt ) non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation the incidence of treatment failures 14 days after surgery was 1.9% ( 2/106 ) in the nepafenac group and 8.6% ( 9/105 ) in the placebo group . there were more subjects who had no response to treatment in the placebo group than in the nepafenac group , although no significant difference was observed between groups ( table 13).table 13incidence of treatment failures at each study visit ( itt)treatmenttotalday 1day 3day 7day 14nn%n%n%n%nepafenac group10621.921.921.921.9placebo group10500.011.098.698.6p value0.54670.39740.20010.2236nlmixed model p = 0.6151 ; main effect of treatment ; failure defined as aqueous cells score 3 or aqueous flare score = 3 or ocular pain score 4 incidence of treatment failures at each study visit ( itt ) nlmixed model p = 0.6151 ; main effect of treatment ; failure defined as aqueous cells score 3 or aqueous flare score = 3 or ocular pain score 4 a total of 26 aes were reported in 21 subjects ( 19.6% ) in the nepafenac group and 31 aes were reported in 24 subjects ( 22.4% ) in the placebo group . two aes in the nepafenac group and six aes in the placebo group were regarded as adverse reactions ( adrs ) for which the causal relation could not be ruled out . the adrs reported in the nepafenac group were foreign body sensation in the eyes in one subject ( 0.9% ) and eye discharge in one subject ( 0.9% ) , both of which were mild and non - serious . the adrs reported in the placebo group were eye pruritus in one subject ( 0.9% ) , foreign body sensation in the eyes in one subject ( 0.9% ) subject , eye irritation in two subjects ( 1.9% ) , and increased lacrimation in two subjects ( 1.9% ) , all of which were non - serious . all of the adrs were mild in severity , except for moderate foreign body sensation in the eyes reported in one subject . in the nepafenac group , no clinically relevant findings were obtained in other examinations for safety evaluations including laboratory tests , blood pressure / pulse rate , bcva , iop , slit lamp examination , and fundus examination . two hundred fifteen patients , 108 treated with nepafenac and 107 treated with placebo , were enrolled in this study . two hundred fourteen remaining patients received at least one dose of test drug and were therefore included in the safety analysis . three of 214 patients discontinued from the study prior to or at the time of surgery for patient decision ( n = 1 ) , the use of nsaids ( n = 1 ) and investigator decision due to surgical difficulties . a total of 211 patients , 106 treated with nepafenac and 105 treated with placebo were included in the itt population . the demographics data for the patients included in the itt data set are summarized in table 4 , and the demographics data by institution are showed in table 5 . as indicated in table 4 , there were no statistically significant differences in gender , age , and emery s classification between treatment groups.table 4demographic data ( itt)totalnepafenac groupplacebo groupp valuen%n%n%total211100.010650.210549.8gender male9846.44946.24946.70.9489 female11353.65753.85653.3age 18 to 64 years5928.02422.63533.30.0836 65 years15272.08277.47066.7emery s classification 15023.72826.42221.00.3508 216176.37873.68379.0from chi - squaretable 5demographic data by institution ( itt)genderageemery s classificationmalefemale<656512n%n%n%n%n%n%totalnepafenac4946.25753.82422.68277.42826.47873.6placebo4946.75653.33533.37066.72221.08379.0investigator no.1nepafenac466.7233.3233.3466.7466.7233.3placebo233.3466.7350.0350.0583.3116.72nepafenac541.7758.3325.0975.0541.7758.3placebo650.0650.018.31191.7541.7758.33nepafenac533.31066.7213.31386.700.015100.0placebo640.0960.0213.31386.700.015100.04nepafenac675.0225.000.08100.0675.0225.0placebo450.0450.0225.0675.0562.5337.55nepafenac743.8956.3212.51487.500.016100.0placebo743.8956.3531.31168.800.016100.06nepafenac360.0240.0120.0480.000.05100.0placebo233.3466.7350.0350.000.06100.07nepafenac222.2777.8222.2777.8222.2777.8placebo350.0350.0233.3466.7233.3466.78nepafenac337.5562.5337.5562.5225.0675.0placebo444.4555.6444.4555.6111.1888.99nepafenac555.6444.4222.2777.8222.2777.8placebo555.6444.4555.6444.4222.2777.810nepafenac433.3866.7325.0975.0650.0650.0placebo650.0650.0541.7758.318.31191.711nepafenac583.3116.7466.7233.3116.7583.3placebo466.7233.3350.0350.0116.7583.3 demographic data ( itt ) demographic data by institution ( itt ) there was a significant difference in cure rate at day 14 between nepafenac ( 71.4% , 75/105 ) and placebo ( 28.6% , 30/105 ) ( p < 0.0001 ) , demonstrating the effectiveness of nepafenac in reducing postoperative inflammation ( table 6 ) . when comparing cure rates at each visit , nepafenac achieved significantly higher cure rates than placebo at days 7 and 14 ( p < 0.0001 for days 7 and 14 ) ( table 6 ) . the ocular pain - free rate was 96.2% ( 102/106 ) in the nepafenac group and 67.6% ( 71/105 ) in the placebo group , showing a significant difference between groups ( p < 0.0001 ) ( table 7 ) . the nepafenac group demonstrated significantly higher ocular pain - free rates than the placebo group at all postoperative visits ( p = 0.0051 for day 1 , p = 0.0003 for day 3 , p = 0.0002 for day 7 , and p = 0.0051 for day 14 ) ( table 8) . in the nepafenac group , cell scores and flare scores measured at each postoperative visit decreased as time elapsed after surgery , showing the suppressive effect of nepafenac on postoperative inflammation . in contrast , no marked decrease was observed in either the cell or flare scores in the placebo group . significant intergroup differences were observed in the cell score on day 7 and subsequent visits ( p < 0.0001 for days 7 and 14 ) and in the flare score on day 3 and subsequent visits ( p = 0.0041 for day 3 and p < 0.0001 for days 7 and 14 ) ( tables 9 and 10 ) . decreases in both cell counts and flare values were observed on day 3 and subsequent visits in the nepafenac group , whereas increases in both variables were observed on day 3 in the placebo group . significant intergroup differences were observed in cell counts and flare values on day 3 and subsequent visits ( tables 11 and 12).table 6cure rate at each study visit ( itt)treatmentall ( n)day 1day 3day 7day 14n%n%n%n%nepafenac group10521.92221.05350.57571.4placebo group10521.91615.22019.03028.6p value1.000.2822<0.0001<0.0001test = chi - square ( fisher s exact test if n < 5)one patient missing cure datatable 7percent ocular pain free ( itt)treatmenttotalpain freenn%nepafenac group10610296.2placebo group1057167.6test = chi - square , p < 0.0001table 8percentage of patients with no pain at each study visit ( itt)treatmentall ( n)day 1day 3day 7day 14n%n%n%n%nepafenac group10610397.210498.110397.210397.2placebo group1059186.78883.88581.09186.7p value0.00510.00030.00020.0051test = chi - squaretable 9aqueous cells score by treatment and visit ( itt)baselineday 1day 3day 7day 14nepafenac groupmean0.01.30.90.50.3std0.00.60.70.60.6n106105105105105min00000max04444placebo groupmean0.01.21.01.00.9std0.00.50.70.90.9n105105105105105min00000max02444p valuen / a0.69050.1638<0.0001<0.0001baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviationtable 10flare score by treatment and visit ( itt)baselineday 1day 3day 7day 14nepafenac groupmean0.00.90.50.30.1std0.00.60.60.50.3n106105105105105min00000max02222placebo groupmean0.00.90.70.80.6std0.00.50.70.80.8n105105105105105min00000max02333p valuen / a0.57930.0041<0.0001<0.0001baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviationtable 11cell count measured by lfcm by treatment and visit ( itt)day 1day 3day 7day 14nepafenac groupmean39.219.89.76.0std79.225.111.29.8n54545454min0.00.00.00.0max428.7116.452.765.3placebo groupmean40.455.332.224.1std52.866.237.331.4n53545454min0.00.00.00.0max312.2311.2177.3128.0p value0.8738<0.00010.01070.0394non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p = 0.0011 ; treatment by visit interaction p = 0.0186 ; cell count was measured by laser flare cell meter at six sitesmin minimum , max maximum , std standard deviationtable 12flare value measured by lfcm by treatment and visit ( itt)day 1day 3day 7day 14nepafenac groupmean20.620.613.411.4std24.640.912.29.1n101103103103min0.50.30.40.3max153.2396.757.857.8placebo groupmean24.347.565.652.0std34.449.877.769.9n101104104104min1.82.52.51.5max307.6333.9479.7479.7p value0.6201<0.0001<0.0001<0.0001non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001min minimum , max maximum , std standard deviation cure rate at each study visit ( itt ) test = chi - square ( fisher s exact test if n < 5 ) one patient missing cure data percent ocular pain free ( itt ) test = chi - square , p < 0.0001 percentage of patients with no pain at each study visit ( itt ) aqueous cells score by treatment and visit ( itt ) baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation flare score by treatment and visit ( itt ) baseline p value is from t test ; non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation cell count measured by lfcm by treatment and visit ( itt ) non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p = 0.0011 ; treatment by visit interaction p = 0.0186 ; cell count was measured by laser flare cell meter at six sites min minimum , max maximum , std standard deviation flare value measured by lfcm by treatment and visit ( itt ) non - baseline p values are lsmeans by visit ; test = repeated measure anova , main effect of treatment p < 0.0001 ; treatment by visit interaction p < 0.0001 min minimum , max maximum , std standard deviation the incidence of treatment failures 14 days after surgery was 1.9% ( 2/106 ) in the nepafenac group and 8.6% ( 9/105 ) in the placebo group . there were more subjects who had no response to treatment in the placebo group than in the nepafenac group , although no significant difference was observed between groups ( table 13).table 13incidence of treatment failures at each study visit ( itt)treatmenttotalday 1day 3day 7day 14nn%n%n%n%nepafenac group10621.921.921.921.9placebo group10500.011.098.698.6p value0.54670.39740.20010.2236nlmixed model p = 0.6151 ; main effect of treatment ; failure defined as aqueous cells score 3 or aqueous flare score = 3 or ocular pain score 4 incidence of treatment failures at each study visit ( itt ) nlmixed model p = 0.6151 ; main effect of treatment ; failure defined as aqueous cells score 3 or aqueous flare score = 3 or ocular pain score 4 a total of 26 aes were reported in 21 subjects ( 19.6% ) in the nepafenac group and 31 aes were reported in 24 subjects ( 22.4% ) in the placebo group . two aes in the nepafenac group and six aes in the placebo group were regarded as adverse reactions ( adrs ) for which the causal relation could not be ruled out . the adrs reported in the nepafenac group were foreign body sensation in the eyes in one subject ( 0.9% ) and eye discharge in one subject ( 0.9% ) , both of which were mild and non - serious . the adrs reported in the placebo group were eye pruritus in one subject ( 0.9% ) , foreign body sensation in the eyes in one subject ( 0.9% ) subject , eye irritation in two subjects ( 1.9% ) , and increased lacrimation in two subjects ( 1.9% ) , all of which were non - serious . all of the adrs were mild in severity , except for moderate foreign body sensation in the eyes reported in one subject . in the nepafenac group , no clinically relevant findings were obtained in other examinations for safety evaluations including laboratory tests , blood pressure / pulse rate , bcva , iop , slit lamp examination , and fundus examination . in japan , the ophthalmic preparations of steroids or nsaids are used for the treatment of postoperative inflammation following cataract surgery . nsaids are also used for the prevention of postoperative cystoid macular edema ( cme ) . some studies have shown that aqueous flare values in the early postoperative period are even lower in those treated with diclofenac sodium than in those treated with steroids [ 11 , 12 ] . other studies have demonstrated that nsaids are effective in preventing postoperative cme [ 1316 ] , in which an ophthalmic nsaid was administered for several months after cataract surgery . ex vivo inhibition of prostaglandin synthesis by nepafenac / amfenac was significantly greater and of longer duration than diclofenac . and the combined area under the curve of nepafenac and amfenac was higher than the bromfenac and ketorolac . in this study , nepafenac expected to prevent postoperative inflammation and ocular pain , was administered as a monotherapy to patients undergoing cataract surgery , and its efficacy and safety in the management of postoperative inflammation and ocular pain were compared with those of a placebo . the subjects were japanese patients with cataracts who were undergoing cataract surgery with pea and iol implantation . evaluations were based on cure rates 14 days after surgery and ocular pain - free rates during the first 14 days after surgery . the cure rate was 71.4% in the nepafenac group and 28.6% in the placebo group , showing a significant difference between groups . those who achieved cures in the placebo group appeared to have undergone spontaneous resolution of postoperative inflammation , as indicated by a gradual increase in cure rates over the course of treatment . in contrast , the nepafenac group demonstrated higher cure rates than those in the placebo group , with significant intergroup differences observed on days 7 and 14 postoperatively . this study revealed significant differences in cure rates between nepafenac and placebo over the course of treatment , which was consistent with the results of a study conducted in the usa using the same evaluation . the use of lfcm in the evaluation of postoperative inflammation following cataract surgery has been described in many studies [ 1820 ] . lfcm enables assessment of the degree of inflammation by quantitatively measuring such parameters as white blood cell count and protein concentration in the aqueous humor . aqueous cell counts and aqueous flare values as measured with lfcm were significantly lower in the nepafenac than in the placebo group . the mean flare value in the placebo group reached its peak on day 7 while no postoperative increase in flare values was observed in the nepafenac group . cell scores and flare scores determined by slit lamp biomicroscopy and flare values and cell counts measured with lfcm showed similar patterns of changes over time from day 3 and thereafter , demonstrating the appropriateness of the efficacy evaluation based on cell scores and flare scores determined by slit lamp biomicroscopy and the efficacy of nepafenac in the management of postoperative inflammation . since postoperative inflammation following intraocular surgery contributes to the development of cme , it is very important to reduce or resolve postoperative inflammation . in this study , evaluations were only performed up to 2 weeks after surgery , and thus the preventative effect of nepafenac on cme was not examined . however , the fact that the cure rate of nepafenac was significantly higher than placebo implies that nepafenac is likely to be effective in preventing the development of cme . the pain - free rate was 96.2% in the nepafenac group and 67.6% in the placebo group , showing a significant difference between groups . most subjects in the nepafenac group complained of no ocular pain throughout the study period while more than 30% of the subjects in the placebo group complained of ocular pain . these findings also suggest the need for the postoperative use of agents with an analgesic effect , such as nepafenac 0.1% , in clinical practice . reported aes associated with ophthalmic nsaids include diffuse superficial keratitis , corneal erosion , and corneal epithelium disorder , and more serious aes include corneal ulcer and corneal perforation . in this study , no clinically significant aes associated with nepafenac were reported , and the incidences of aes and adrs in the nepafenac group were lower than those in the placebo group , demonstrating the favorable safety profile of nepafenac . nepafenac ophthalmic suspension 0.1% is significantly more effective than the placebo in the management of postoperative inflammation and ocular pain following cataract surgery and thus is considered an essential nsaid in ophthalmic surgery .

objectivethis study aimed to examine the efficacy and safety of nepafenac ophthalmic suspension compared to placebo in the management of postoperative inflammation and ocular pain in japanese patients undergoing cataract surgery.methodsthis was a multicenter , randomized , double - masked , placebo - controlled clinical study . patients received nepafenac or placebo tid beginning 1 day before cataract surgery and continuing on the day of surgery for 14 days . one additional drop was administered on the day of surgery . the primary efficacy variables were the percentage of patients cured at postoperative day 14 visit ( cure defined as aqueous cells score + aqueous flare score = 0 ) and the percentage of patients who were pain free at all postoperative visits.resultsthe cure rate on day 14 after surgery was 71.4% ( 75/105 ) in the nepafenac group and 28.6% ( 30/105 ) in the placebo group , showing a significant difference in cure rate between groups . the nepafenac group demonstrated higher cure rates than those in the placebo group , with a significant difference in cure rate on days 7 and 14 postoperatively . the ocular pain - free rate was 96.2% ( 102/106 ) in the nepafenac group and 67.6% ( 71/105 ) in the placebo group , showing a significant difference between groups . concerning adverse events ( aes ) , 26 aes were reported in 21 subjects ( 19.6% ) in the nepafenac group and 31 aes were reported in 24 subjects ( 22.4% ) in the placebo group.conclusionnepafenac ophthalmic suspension is a nonsteroidal anti - inflammatory drug effective in the prevention of postoperative inflammation and ocular pain associated with cataract surgery .

the concept of the visual word form is one that is well - established within the psychological literature . cattel ( 1886 ) first documented whole word reading by demonstrating how briefly presented words were easier to recall than briefly presented meaningless letter strings , and letters have subsequently been shown to be better identified when presented within a word than individually ( reicher , 1969 ; wheeler , 1970 ) or within a non - word ( grainger et al . , 2003 ) . more recently , neuroimaging studies have identified an area within the left fusiform gyrus which is specialised for letter and word recognition and which may constitute the visual word form area ( vwfa ; cohen et al . , 2000 ) . given the recency of written relative to spoken language as a cultural invention , it is unlikely that a vwfa would have evolved specifically for reading . however , one suggestion is that accumulated reading experience promotes the specialisation of a pre - existing inferotemporal pathway for higher - order visual processing ( mccandliss et al . , 2003 ) . the current paper emphasises the extent of this functional specialisation by demonstrating remarkably preserved reading in the context of profoundly impaired perception of non - word stimuli . neuropsychological evidence supporting the existence of highly - specialised processes for visual word recognition has been derived from patients exhibiting letter - by - letter reading ( lbl ; also referred to as word form dyslexia or pure alexia ; e.g. , shallice and warrington , 1980 ; farah and wallace , 1991 ; binder and mohr , 1992 ; warrington and langdon , 1994 ; hanley and kay , 1996 ; cohen et al . , 2000 ) . such patients exhibit intact letter identification and relatively accurate , but slow , reading , whereby response latencies increase in a linear manner proportionate to word length . lbl reading has been suggested to reflect destruction or inaccessibility of a visual word form system , and is associated with damage to the vwfa ( warrington and shallice , 1980 ; cohen et al . , 2000 ) . the attribution of lbl reading to a specific word form deficit has been challenged on two main grounds , namely that the condition and its characteristic word length effects can be accounted for by a general visual deficit and/or a letter identification deficit . a general visual account of lbl reading suggests that reading , as a complex behaviour , can be disrupted by even the most subtle low - level visual deficits ( friedman and alexander , 1984 ; farah and wallace , 1991 ; price and devlin , 2003 ) , which propagate by a cascade process to the level of lexical and semantic representations within the visual system ( behrmann et al . a number of single case and case series studies of lbl readers have reported associated impairments on a range of perceptual tasks involving non - orthographic stimuli . for example , friedman and alexander ( 1984 ) identified an lbl patient who was impaired on tasks of letter identification , object recognition and had an elevated threshold relative to controls in detecting briefly presented pictures . furthermore , farah and wallace s ( 1991 ) patient tu performed poorly on tasks involving the perception of non - orthographic stimuli under time constraints ; these results were replicated by sekuler and behrmann ( 1996 ) . more recently , mycroft et al . ( 2009 ) found that seven lbl readers were similarly impaired for both linguistic and non - linguistic stimuli on tasks of visual search and matching , and the lbl group as a whole performed worse than the control group on a task of visual complexity . by contrast , there are documented cases of lbl readers with no discernible impairment in letter identification speed or the identification of rapidly displayed letters ( warrington and langdon , 2002 ; rosazza et al . , 2007 ) or in a range of tasks assessing visual processing , such as complex picture analysis , visual short term memory and picture recognition from unusual views ( warrington and shallice , 1980 ) . however , proponents of pre - lexical theories of lbl reading tend to dismiss such cases as reflecting insufficiently sensitive assessment of visual processing skills or the use of non - reading tasks which are not making demands comparable to those involved in reading ( behrmann et al . , 1998a , 1998b ; patterson , 2000 ) . some accounts suggest that the critical letter processing deficits may be restricted to the identification of individual letters ( e.g. , arguin and bub , 1992 , 1993 ; reuter - lorenz and brunn , 1990 ; behrmann and shallice , 1995 ) . other accounts ascribe lbl reading to a deficit in the mechanisms responsible for rapid , parallel processing of letters , leading to the less efficient serial encoding of the component letters of a word ( patterson and kay , 1982 ; behrmann et al . , 2001 ; cohen et al . , 2003 ) one such possible mechanism is the inability to use the optimal spatial frequency band for letter and word recognition , with letter confusability effects emerging at lower spatial frequencies ( fiset et al . , 2006 ) . it should also be noted that some authors have argued that deficits in letter processing are common to all lbl readers , while speculating that such deficits may be due to a more basic visual impairment ( behrmann et al . , 1998a , 1998b ) . one observation regarding both the general visual account of lbl reading is that the evidence base is largely associative in nature ; that is , most studies claim that the co - occurrence of the characteristics of lbl reading ( i.e. , accurate but slow reading , with prominent word length effects ) and a particular deficit ( e.g. , impaired perception of non - lexical stimuli ) confers support for their chosen position . in addition , proponents of the general visual impairment account have claimed support for their position from control brain - damaged patients who show the complementary association of no perceptual deficit and no impairment of reading ( e.g. , patient ol ; mycroft et al . , 2009 ) . by contrast , in the current study it is argued that such evidence does not prove a causal link between general visual deficits and lbl reading behaviour . this is achieved by presenting evidence from two patients who exhibit profound visual dysfunction in the presence of accurate and rapid word reading . rather than demonstrating a selective impairment to the visual word form system in the absence of general visual dysfunction , these patients reading abilities are remarkably preserved despite grave and diffuse impairments to their visual system . the two patients reported in this study have a diagnosis of posterior cortical atrophy ( pca ) , a neurodegenerative condition involving progressive visual impairment in contrast to relatively spared memory functions . the most frequent underlying pathology is alzheimer s disease ( ad ) , with pca patients showing a greater distribution of senile plaques and neurofibrillary tangles in posterior regions of the parietal cortex , the occipital cortex and temporo - occipital junction relative to more anterior cortical areas ( rogelet et al . characteristic symptoms of pca include early visual processing deficits , and disorders of higher - order visuoperceptual and visuospatial processing ( benson et al . , 1988 ; mendez et al . reading difficulties are often a prominent feature of pca , occurring in about 80% of patients ( mendez et al . , 2002 ) and studies on reading ability in pca have identified a range of deficits , including neglect dyslexia ( mendez and cherrier , 1998 ) , attentional dyslexia ( saffran and coslett , 1996 ) , lbl reading ( catricala et al . , 2011 ) and spatial alexia ( crutch and warrington , 2007 ) . the main aim of this study was to evaluate the hypothesis that general visual dysfunction necessarily leads to lbl reading . the general visual account predicts that basic visual impairments should be associated with slow , inefficient reading , with prominent word length effects characterised by considerable increases in reading latency with each additional constituent letter . contrary to these predications , we report two pca patients who demonstrate highly accurate and rapid reading with equivocal or absent word length effects despite profound visual dysfunction . this preservation of reading skills was observed despite significantly impaired performance on non - lexical chequerboard perception and rapid serial visual letter presentation tasks , failure on which has previously been linked to lbl reading by proponents of the general visual accounts . the reported distinction between intact reading and impoverished visual function raises questions as to whether the evidence cited for general visual accounts of lbl reading truly reflects causation , or merely the association of deficits elicited by damage to contiguous brain regions . the study participants were two individuals who met current criteria for a diagnosis of pca owing to probable ad ( mendez et al . , 2002 ; tang - wai et al . , this diagnosis was made based on clinical and neuroimaging data , together with the fulfilment of behavioural criteria employed routinely at the dementia research centre . these criteria require an individual to demonstrate episodic memory function above the 5th percentile and at least two out of four scores below the 5th percentile on tests of posterior function , which include the number location and object decision tests from the visual object and space perception battery ( vosp : warrington and james , 1991 ) and graded difficulty tests of arithmetic and spelling ( jackson and warrington , 1986 ; baxter and warrington , 1994 ) . written informed consent was obtained using procedures approved by the national hospital for neurology and neurosurgery . the patients were selected for the current study following the observation of visuoperceptual and visuospatial impairment but preserved performance on a screening test for reading ( see table 1 ) . fol is a 58 year - old right - handed retired administrator for the national health service ( nhs ) who was referred to the specialist cognitive disorders clinic at the national hospital of neurology and neurosurgery in 2010 with a 4-year history of progressive visual impairment . , with early symptoms of visual dysfunction including difficulty in locating objects in front of her and problems reading clocks . fol fulfilled the pca behavioural criteria ( failing tests of arithmetic and spatial and object perception ) but her spelling was well preserved . 1 ) showed predominantly biparietal atrophy somewhat more marked on the right with relative preservation of the hippocampi , medial temporal lobe structures and no significant vascular burden . cla is an 86 year - old right - handed retired classics teacher who was first seen at the national hospital in january 2011 as part of a clinical assessment . presenting symptoms included being unable to judge depth and movement and failing to see objects in front of her . cla fulfilled the pca criteria , failing tests of spatial and object perception , but spelling and arithmetic were well preserved and she demonstrated strong performance on a test of verbal memory . 1 ) revealed bilateral atrophy of both posterior cerebral hemispheres , more prominent on the right with anterior extension into bilateral peri - sylvian cortices and the inferior and medial right temporal lobe but relative sparing of the left inferior temporal lobe ; additional mild frontal lobe atrophy was evident bilaterally , and there was a mild to moderate degree of small vessel ischaemic damage . the controls were split into two groups appropriate for each patient , matched as closely as possible for age , gender and years of education [ fol controls ( n = 4 ) : mean age 58.4 yrs ( range 5660 ) , all female , mean education : 16 yrs ; cla controls ( n = 5 ) : mean 83.5 yrs ( range 8184 ) , all female , mean education : 14.8 yrs ] . in addition to the behavioural screening tests , cla and fol completed a battery of background neuropsychological tests . their scores on each task and an estimate of their performance relative to appropriate normative data sets are shown in table 1 . on the mini mental state examination ( mmse ) , fol performed below the normal range . she performed well on tests of concrete synonyms , cognitive estimates and naming , and her praxic skills were only mildly impaired to verbal command . she made no errors on a screening test for reading and one error on a non - word reading task . her concrete synonym comprehension performance was within normal limits but she was impaired on tests of cognitive estimates and naming . cla had some difficulties on a test of praxic skills , specifically in pantomiming using a toothbrush and hammer . cla made no errors on a screening test for reading and three errors on a non - word reading task . patients fol and cla completed a battery of standardised tests examining early visual , visuoperceptual and visuospatial processing : early visual processing(i)visual acuity test from the cortical visual screening test ( corvist ; james et al . , 2001 ) : task required discrimination of squares , circles and triangles at decreasing stimulus sizes corresponding to snellen form acuity levels.(ii)shape detection test from the vosp(warrington and james , 1991 ) : figure - ground discrimination task involving random black pattern stimuli ( n = 20 ) , half with a degraded patients were requested to state whether an x was present.(iii)shape discrimination : the stimuli ( n = 60 ) for this boundary detection task , adapted from efron ( 1968 ) , were a square ( 50 50 mm ) or an oblong matched for total flux . there were 3 levels of difficulty : oblong edge ratio 1:1.63 ( level i ) , 1:1.37 ( level ii ) , and 1:1.20 ( level iii ) . the task was to discriminate whether each shape presented was a square or an oblong.(iv)hue discrimination ( from the corvist ) : the stimuli ( n = 4 ) comprised 9 colour patches , 8 of the same hue but varying luminance and one target colour patch of a different hue . visual acuity test from the cortical visual screening test ( corvist ; james et al . , 2001 ) : task required discrimination of squares , circles and triangles at decreasing stimulus sizes corresponding to snellen form acuity levels . shape detection test from the vosp(warrington and james , 1991 ) : figure - ground discrimination task involving random black pattern stimuli ( n = 20 ) , half with a degraded shape discrimination : the stimuli ( n = 60 ) for this boundary detection task , adapted from efron ( 1968 ) , were a square ( 50 50 mm ) or an oblong matched for total flux . there were 3 levels of difficulty : oblong edge ratio 1:1.63 ( level i ) , 1:1.37 ( level ii ) , and 1:1.20 ( level iii ) . the task was to discriminate whether each shape presented was a square or an oblong . hue discrimination ( from the corvist ) : the stimuli ( n = 4 ) comprised 9 colour patches , 8 of the same hue but varying luminance and one target colour patch of a different hue . visuoperceptual processing(i)object decision ( from the vosp ) : stimuli ( n = 20 ) comprise 4 silhouette images , one of a real object ( target ) plus 3 non - object distractors.(ii)fragmented letters ( from the vosp ) : participants were asked to identify visually degraded letters ( n = 20).(iii)unusual and usual views ( warrington and james , 1988 ) : participants are asked to identify with photographs of real objects ( n = 20 ) pictured from an items not identified from the non - canonical perspective are subsequently re - presented photographed from a more usual , canonical perspective . object decision ( from the vosp ) : stimuli ( n = 20 ) comprise 4 silhouette images , one of a real object ( target ) plus 3 non - object distractors . fragmented letters ( from the vosp ) : participants were asked to identify visually degraded letters ( n = 20 ) . unusual and usual views ( warrington and james , 1988 ) : participants are asked to identify with photographs of real objects ( n = 20 ) pictured from an items not identified from the non - canonical perspective are subsequently re - presented photographed from a more usual , canonical perspective . visuospatial processing(i)number location ( from the vosp ) : stimuli ( n = 10 ) consist of two squares , the upper square filled with arabic numerals in different positions , and the lower square with a single black dot . participants are requested to identify the arabic numeral whose spatial position corresponds to that of the target dot.(ii)dot counting ( from the vosp ) : stimuli ( n = 10 ) are arrays of 59 black dots on white background.(iii)a cancellation ( willison and warrington , 1992 ) : participants are requested to mark as quickly as possible with a pencil the location of 19 targets ( letter as ) presented among distractors ( letters b e ) in a grid on an a4 sheet . number location ( from the vosp ) : stimuli ( n = 10 ) consist of two squares , the upper square filled with arabic numerals in different positions , and the lower square with a single black dot . participants are requested to identify the arabic numeral whose spatial position corresponds to that of the target dot . dot counting ( from the vosp ) : stimuli ( n = 10 ) are arrays of 59 black dots on white background . a cancellation ( willison and warrington , 1992 ) : participants are requested to mark as quickly as possible with a pencil the location of 19 targets ( letter as ) presented among distractors ( letters b e ) in a grid on an a4 sheet . visuoperceptual / visuospatial processing(i)chequerboard experiment : a set of 24 chequerboard patterns was designed based on an experiment originally developed by ichikawa ( 1985 ) and employed in previous investigations of pure alexia ( mycroft et al . , 2009 ) . chequerboards were composed of either 3 3 or 4 4 grids with the height / width of individual grid squares being kept constant ( subtending .5 of visual angle at a viewing distance of 50 cm ) . each chequerboard comprised a pattern of white and black squares , constructed so as to avoid obvious patterns and many squares of the same colour being adjacent to one another ( see table 4 ) . each chequerboard pattern was paired once with itself and once with another pattern that differed by a single square . this produced a total of 48 pairs , with each pair consisting of chequerboards being presented one above the other at the centre of the screen . each pair of chequerboards was preceded by a fixation point presented for 1000 msec . participants were asked to decide whether the chequerboards in each pair were the same or different as quickly and accurately as possible by verbal response . the pairs remained on screen until a response was given and there was a 1000 msec inter - trial interval . each block contained an equal number of 3 3 and 4 4 chequerboards . chequerboard experiment : a set of 24 chequerboard patterns was designed based on an experiment originally developed by ichikawa ( 1985 ) and employed in previous investigations of pure alexia ( mycroft et al . , 2009 ) . chequerboards were composed of either 3 3 or 4 4 grids with the height / width of individual grid squares being kept constant ( subtending .5 of visual angle at a viewing distance of 50 cm ) . each chequerboard comprised a pattern of white and black squares , constructed so as to avoid obvious patterns and many squares of the same colour being adjacent to one another ( see table 4 ) . each chequerboard pattern was paired once with itself and once with another pattern that differed by a single square . this produced a total of 48 pairs , with each pair consisting of chequerboards being presented one above the other at the centre of the screen . each pair of chequerboards was preceded by a fixation point presented for 1000 msec . participants were asked to decide whether the chequerboards in each pair were the same or different as quickly and accurately as possible by verbal response . the pairs remained on screen until a response was given and there was a 1000 msec inter - trial interval . each block contained an equal number of 3 3 and 4 4 chequerboards . in order to gather a sizeable body of reading responses , all participants were requested to read aloud 3 corpora yielding a total of 250 words . each corpus was as follows : 1.brown and ure words ( brown and ure , 1969 ) : 72 words taken from the brown and ure ( 1969 ) corpus , which was composed of a subset of words at three levels of length ( 4 , 6 and 8 letters ) matched on two levels of frequency and two levels of concreteness.2.schonell reading list ( schonell and goodacre , 1971 ) : 100 words of decreasing frequency , ranging in length from 3 to 14 letters.3.coltheart regular / irregular words ( coltheart et al . , 1979 ) : 39 pairs of regular and irregular words ranging from 3 to 8 letters long , matched for word frequency ( kucera and francis , 1967 ) , concreteness , part of speech and number of letters , syllables and morphemes . brown and ure words ( brown and ure , 1969 ) : 72 words taken from the brown and ure ( 1969 ) corpus , which was composed of a subset of words at three levels of length ( 4 , 6 and 8 letters ) matched on two levels of frequency and two levels of concreteness . schonell reading list ( schonell and goodacre , 1971 ) : 100 words of decreasing frequency , ranging in length from 3 to 14 letters . coltheart regular / irregular words ( coltheart et al . , 1979 ) : 39 pairs of regular and irregular words ranging from 3 to 8 letters long , matched for word frequency ( kucera and francis , 1967 ) , concreteness , part of speech and number of letters , syllables and morphemes . all words were presented in arial unicode ms for an unlimited duration within a rectangular fixation box at the centre of the screen ; letter height corresponded to a visual angle of 1.2 from a viewing distance of 50 cm . a series of letter processing tasks were administered , with all stimuli presented within a central fixation box to ameliorate the effects of visual disorientation:1.letter naming all participants were requested to read the letters of the alphabet , excluding i , j , o , q , w and x , in upper case . letter height corresponded to a visual angle of 1.2 from a viewing distance of 50 cm.2.rapid serial visual presentation ( rsvp ) letter / number identification letter strings of six letters each were presented serially in the same central spatial position , without an interval between successive letters , as described by previous studies in lbl reading ( warrington and langdon , 2002 ; behrmann and shallice , 1995 ) . there were three exposure durations of 150 , 200 and 250 msec / letter ; all participants were tested in nine blocks of 10 strings , with three blocks at each of the three durations arranged in a latin square design . before the presentation of each letter string , a target letter was named ; participants were asked to decide whether the target letter was present in each string . the target item occurred randomly in positions two to five in each string , with the target item being present in half of all trials . in a subsequent experiment , the number of trials was halved , resulting in nine blocks of 5 strings.3.flanked letter identification all participants were requested to read aloud upper - case letters in 120 trials under the following flanking conditions : a.letters ( n = 24 ; e.g. , znh ) : alphabetic items excluded the letters i , j , o , q , w and x , and occurred with equal frequency within each condition ( target , left flanker , right flanker).b.shapes ( n = 24 ; e.g. , n ) : shape flankers consisted of triangles presented at different orientations . the line thickness of targets and distracters was matched.c.numbers ( n = 24 ; e.g. , 6n5 ) : number flankers consisted of two single digit number flankers chosen from a range between 2 and 9 . letter naming all participants were requested to read the letters of the alphabet , excluding i , j , o , q , w and x , in upper case . letter height corresponded to a visual angle of 1.2 from a viewing distance of 50 cm . letter strings of six letters each were presented serially in the same central spatial position , without an interval between successive letters , as described by previous studies in lbl reading ( warrington and langdon , 2002 ; behrmann and shallice , 1995 ) . there were three exposure durations of 150 , 200 and 250 msec / letter ; all participants were tested in nine blocks of 10 strings , with three blocks at each of the three durations arranged in a latin square design . before the presentation of each letter string , a target letter was named ; participants were asked to decide whether the target letter was present in each string . the target item occurred randomly in positions two to five in each string , with the target item being present in half of all trials . in a subsequent experiment , flanked letter identification all participants were requested to read aloud upper - case letters in 120 trials under the following flanking conditions : a.letters ( n = 24 ; e.g. , znh ) : alphabetic items excluded the letters i , j , o , q , w and x , and occurred with equal frequency within each condition ( target , left flanker , right flanker).b.shapes ( n = 24 ; e.g. , n ) : shape flankers consisted of triangles presented at different orientations . the line thickness of targets and distracters was matched.c.numbers ( n = 24 ; e.g. , 6n5 ) : number flankers consisted of two single digit number flankers chosen from a range between 2 and 9 . letters ( n = 24 ; e.g. , znh ) : alphabetic items excluded the letters i , j , o , q , w and x , and occurred with equal frequency within each condition ( target , left flanker , right flanker ) . shapes ( n = 24 ; e.g. , n ) : shape flankers consisted of triangles presented at different orientations . the line thickness of targets and distracters was matched . numbers ( n = 24 ; e.g. , 6n5 ) : number flankers consisted of two single digit number flankers chosen from a range between 2 and 9 . in each flanking condition , the distance between the target letter and flankers was .875 mm in the condensed condition and 8.75 mm in the spaced condition , with the height of stimuli corresponding to a visual angle of 1.0. the same combination of flankers was used for each target letter under both spatial conditions . the stimuli were presented in blocks of 6 items with the same spacing between the target letter and flankers , with blocks being administered in an abba design . responses were recorded using an olympus ds-40 digital voice recorder ; reading latencies were manually determined from the temporal distance between the onset of audio waveforms corresponding to each stimulus onset and the participant s spoken response using the digital audio editor audacity ( http://audacity.sourceforge.net ) . latency data for erroneous responses and responses where participants had become overtly distracted from the task were removed from the analysis . analyses of the brown and ure ( 1969 ) and schonell ( schonell and goodacre , 1971 ) corpora were conducted using multiple linear regression , as neither fol nor cla made enough errors to allow the use of a logistic regression model . the regression model was used to relate response latencies to the effects of frequency and length . overall regression analysis was conducted using a linear mixed model , which was fitted to reaction times with random subject and item effects and fixed effects of length , diagnosis , their interaction and frequency . comparisons between both patients and their matched control groups were conducted using a modified t - test developed by crawford and garthwaite ( 2002 ) specifically to identify abnormality of test scores in single case studies . comparisons between differences in a patient s scores on two tasks and differences between the control groups performance on the same two tasks were conducted the revised standardized difference test ( rsdt ) developed by crawford and garthwaite ( 2005 ) . the results of patients fol and cla on each early visual , visuoperceptual and visuospatial processing task are shown in table 1 , together with the corresponding normative data . fol failed every single early visual , visuoperceptual and visuospatial task administered except for visual acuity . on the chequerboard experiment , fol exhibited significantly poorer performance than controls ( t = 32.7 , p < .001 ) on 3 3 and 4 4 chequerboards ( 15/24 and 14/24 , respectively ) and disproportionately identified chequerboards as being the same ( 96% ) rather than different ( 25% ) ( d prime score = 1.057 ) . cla was also impaired on all tests of early visual processing except for only mild weakness on a test of visual acuity . she was also impaired on all visuoperceptual tasks and all but one visuospatial task ( dot counting ) . on the chequerboard experiment , cla exhibited significantly poorer performance than controls ( t = 27.7 , p < .001 ) on 3 3 and 4 4 chequerboards ( 16/24 and 15/24 , respectively ) and was more likely to identify chequerboards as being the same ( 71% ) rather than different ( 58.5% ) ( d prime score = .759 ) . the total ( and percentage ) correct responses and mean ( and standard deviation ( sd ) ) reading latency data for word reading performance by fol , cla and their relevant control samples are shown in table 2.1.brown and ure words there was no significant difference between fol s response latencies and those of the control group . regression analysis found a significant effect of length ( t = 2.2 , p < .05 ) , but not of frequency ( t = .89 , p > .3 ) or concreteness ( t = 1.54 , p > .1 ) on fol s response latencies . when examining control responses at the group level , neither frequency nor length was significantly related to response latencies , although length was related to response latencies in one individual control.neither cla nor her control group made any error responses . regression analysis found no significant effects of length , frequency or concreteness on the response latencies of cla or her controls.2.schonell reading list fol made three error responses ; two of these were regularisation errors ( colonel , homonym ) , with the remaining error being a visually - based neologism ( ineradicable inerascible ) . fol showed a trend towards being less accurate and having longer latencies relative to controls ; however , neither of these effects reached formal levels of significance . regression analysis found a significant effect of length but not of frequency on response latencies for fol ( t = 4.01 , p < .001 ) and at the group level for her matched controls ( t = 4.18 , p < .001).cla again made no error responses ; the control group made a total of five errors between 3 participants . regression analysis found a significant effect of length but not of frequency on response latencies for both cla ( t = 2.11 , p < .05 ) and , at the group level , her matched controls ( t = 5.4 , p < .001).3.coltheart regular / irregular words fol made only one visual error response reading irregular words ( gauge gauche ) . the control group made no errors ; consequently it was not possible to use a modified t - test for error analysis . there was no significant difference between fol and her control group in the size of regularity effect ( rsdt : t = .4 , p > .4).neither cla nor the control group made any errors . cla s response latencies were significantly longer than those of controls for both regular and irregular words . the rsdt identified cla as being significantly slower for irregular than regular words relative to her control group ( t = 5.1 , p < .005 ) . there was no significant difference between fol s response latencies and those of the control group . regression analysis found a significant effect of length ( t = 2.2 , p < .05 ) , but not of frequency ( t = .89 , p > .3 ) or concreteness ( t = 1.54 , p > .1 ) on fol s response latencies . when examining control responses at the group level , neither frequency nor length was significantly related to response latencies , although length was related to response latencies in one individual control . regression analysis found no significant effects of length , frequency or concreteness on the response latencies of cla or her controls . schonell reading list fol made three error responses ; two of these were regularisation errors ( colonel , homonym ) , with the remaining error being a visually - based neologism ( ineradicable inerascible ) . fol showed a trend towards being less accurate and having longer latencies relative to controls ; however , neither of these effects reached formal levels of significance . regression analysis found a significant effect of length but not of frequency on response latencies for fol ( t = 4.01 , p < .001 ) and at the group level for her matched controls ( t = 4.18 , p < cla again made no error responses ; the control group made a total of five errors between 3 participants . regression analysis found a significant effect of length but not of frequency on response latencies for both cla ( t = 2.11 , p < .05 ) and , at the group level , her matched controls ( t = 5.4 , p < .001 ) . coltheart regular / irregular words fol made only one visual error response reading irregular words ( gauge gauche ) . the control group made no errors ; consequently it was not possible to use a modified t - test for error analysis . there was no significant difference between fol and her control group in the size of regularity effect ( rsdt : t = .4 , p > .4 ) . cla s response latencies were significantly longer than those of controls for both regular and irregular words . the rsdt identified cla as being significantly slower for irregular than regular words relative to her control group ( t = 5.1 , p < .005 ) . overall reaction time and word length analysis reading latencies for words of up to 12 letters , summing across the 3 reading corpora , are shown in fig . when examining the response latencies of fol and her control group , there was a main effect of length ( z = 2.5 , p < .05 ) but not diagnosis ( p > .3 ) . there was a significant interaction between diagnosis and length ( z = 2.3 , p < .05 ) . however , there was significant variation in the size of word length effect within the control group ; this was demonstrated by fitting the same model to the control data , plus a second model extended to allow length effects to vary by control participant . comparison of the two models by a likelihood ratio test identified a highly significant difference in length effects between controls ( p < .0001 ) . when examining reading latencies of cla and her control group , there was a main effect of length on reading latencies ( z = 3.1 , p < .005 ) , but only a trend towards a main effect of diagnosis ( z = 1.9 , p = .06 ) . there was no interaction between diagnosis and length ( p > .2 ) . the total ( and percentage ) correct responses and mean ( and sd ) latency data for letter processing performance by fol , cla and their relevant control samples are shown in table 3.1.letter naming neither fol nor her control group made any error responses . there was no significant difference between fol s reading latencies and those of her control group . however , cla was significantly slower than her control group.2.rapid letter / number identification : letters overall letter identification was significantly lower for fol than her controls ; this overall effect reflected significantly lower performance when stimuli were presented for 150 msec but not 200 or 250 msec . cla also made significantly more errors overall , and specifically when stimulus duration was 150 msec or 250 msec but not 200 msec . this difference was significant for numbers being displayed for 150 msec , but ceiling effects in the other temporal conditions prevented analysis using a modified t - test . there was no significant difference between cla and her controls for stimuli at any of the tested exposure durations.3.flanked letter identification neither fol nor her control group made any errors on the flanked letter identification tasks . flanker spacing had a significant effect on response latency in only one flanker condition , where target letters were read slower with spaced than condensed number flankers ( z = 2.2 , p < .05 ) . there was a trend towards there being an interaction between flanker condition and spatial condition ( t = 1.9 , p = .08 ) . flanker spacing had a significant effect upon response latency in one flanker condition , where target letters were read slower with condensed than in spaced letter flankers ( z = 2.0 , p < .05 ) . there was also one main effect of flanker type , with cla s responses in the letter flanker condition significantly slower than in the number flanker condition ( z = 2.5 , p < .05 ) . overall , there was a significant interaction between the group spacing condition , with target letters being read more slowly with condensed rather than spaced flankers relative to controls ( t = 7.5 , p < there was no significant difference between fol s reading latencies and those of her control group . rapid letter / number identification : letters overall letter identification was significantly lower for fol than her controls ; this overall effect reflected significantly lower performance when stimuli were presented for 150 msec but not 200 or 250 msec . cla also made significantly more errors overall , and specifically when stimulus duration was 150 msec or 250 msec but not 200 msec . this difference was significant for numbers being displayed for 150 msec , but ceiling effects in the other temporal conditions prevented analysis using a modified t - test . there was no significant difference between cla and her controls for stimuli at any of the tested exposure durations . flanked letter identification see fig . 3 for fol and cla s reading latencies . neither fol nor her control group made any errors on the flanked letter identification tasks . flanker spacing had a significant effect on response latency in only one flanker condition , where target letters were read slower with spaced than condensed number flankers ( z = 2.2 , p < .05 ) . there was a trend towards there being an interaction between flanker condition and spatial condition ( t = 1.9 , p = .08 ) . flanker spacing had a significant effect upon response latency in one flanker condition , where target letters were read slower with condensed than in spaced letter flankers ( z = 2.0 , p < .05 ) . there was also one main effect of flanker type , with cla s responses in the letter flanker condition significantly slower than in the number flanker condition ( z = 2.5 , p < .05 ) . overall , there was a significant interaction between the group spacing condition , with target letters being read more slowly with condensed rather than spaced flankers relative to controls ( t = 7.5 , p < the current paper describes two pca patients , fol and cla , who demonstrate preserved reading ability in spite of profoundly impaired visual function . both patients were impaired on neuropsychological tests of early visual , visuoperceptual and visuospatial processing . despite these grave visual impairments , both patients were able to read aloud words with perfect to near - perfect accuracy . reading performance was also rapid , with fol s latencies not significantly different to controls on any of the 3 tests of reading , and cla significantly slower on 2/3 sets but showing only a trend to slower reading overall once frequency was taken into account . in addition , word length effects were equivocal or absent , with fol showing a modestly increased length effect relative to controls ( amongst whom effects of length upon reading latency were also evident ) and cla showing no increase in word length effect . in further contrast to their gravely impaired visual processing , at the single letter level there was only minimal evidence of impaired processing , with patient cla showing slow ( but accurate ) single letter identification under normal viewing conditions . considering each patient s performance in more detail , fol s results seem to indicate her reading ability is almost entirely spared . in each reading corpus , fol did not differ from her control group in either accuracy or reading latency . regression analyses conducted on all 250 reading responses ( summing across tasks a1 , a2 and a3 ) did reveal a diagnosis ( fol vs controls ) length ( number of letters ) interaction . however , the same analyses found effects of length on reading latencies within matched controls , and length has been shown previously to influence reading speed in normal readers ( oregan and jacobs , 1992 ; spieler and balota , 1997 ) . more importantly , the absolute increase in mean reading latency for each additional letter as estimated from the regression model was 36 msec / letter , a small increase which is comparable to that of controls ( control mean : 13 msec / letter ; control 4 : 32 msec / letter ) and an order of magnitude different to the increases of 907000 msec per additional letter reported in previous descriptions of lbl reading ( e.g. , fiset et al . , 2005 ; mccarthy and warrington , 1990 ; mycroft et al . , 2009 ; see fig . it should also be noted that the trend towards a difference between fol and the control group s reading latencies for the schonell reading test may reflect the particularly low frequency of various words in this corpus ( somnambulist , the reading accuracy of patient cla was also excellent , with not a single error recorded on any of the reading corpora . for example , her faultless performance on the demanding schonell reading test conveys an estimated intelligence quotient ( iq ) of at least 118 ( nelson and mckenna , 1975 ) . her reading latencies did not differ from controls on the brown and ure words ( a1 ) , but reading speed did fall below that of controls on the coltheart and schonell tests ( a2 and a3 ) , with a significant regularity effect ( irregular words slower than regular words ) on the coltheart set . despite this , the overall difference in latencies across all 250 words failed to reach formal levels of significance . there was also no significant difference between cla and her controls in the effect of increasing word length . the main aim of the current paper was to evaluate the claim that general visual dysfunction can account for the acquired peripheral dyslexic syndrome known as lbl reading . general visual function accounts propose that even minor low - level perceptual deficits propagate to or limit activation of lexical representations , ultimately resulting in impaired reading behaviour . one specific prediction of such accounts is that pronounced word length effects are an inevitable consequence of deficits in general pre - lexical processing ( e.g. , farah and wallace , 1991 ; behrmann et al . the data presented in the current study fail to support this prediction . apart from demonstrating accurate and , particularly in the case of fol , rapid word reading , word length effects were equivocal ( fol ) or absent ( cla ) . this was despite the inclusion of very long words ( up to 14 letters ) which should maximise any chance of eliciting abnormal word length effects . this failure to detect the dramatic word length effects routinely observed in lbl readers can not be attributed to preserved visual function , as both patients exhibited dramatic impairments on a wide variety of perceptual tasks . these included a chequerboard task previously used to support the claim that lbl readers have a perceptual impairment that extends beyond alphanumeric stimuli ( mycroft et al . however , in asserting that such general visual accounts of lbl reading are incompatible with the data presented here for fol and cla , we would wish to state unambiguously that we are not denying that some forms of visual impairment may have an inevitable cost for reading function . rather we would argue against ( i ) the pejorative and under - specified use of terms such as general visual impairment , and ( ii ) the assumption that any form of visual impairment can cause reading impairment . we have previously proposed that visual crowding ( the excessive integration of visual features , sometimes referred to as lateral masking ) may be one of several specific visual deficits which can cause a particular form of dyslexia ( crutch and warrington , 2007 , 2009 ) . indeed , we predicted that any patient demonstrating visual crowding on flanked letter identification tasks would also show some form of visual dyslexia . in line with this prediction , neither fol nor cla ( whose reading is largely preserved ) showed crowding ; cla did show slowed target letter identification particularly with condensed rather than spaced flankers ( task b4 ) , but unlike visual crowding , this flanking effect was only present for flankers of the same category ( letter flankers but not number or shape flankers ) . given the degenerative nature of the pca syndrome , we would predict that fol and cla s reading skills will eventually become affected ; the task going forward will be to identify any components of visual dysfunction that play a causative role in this predicted deterioration . the other aim of the paper was to evaluate the hypothesis that impaired letter processing plays a causal role in lbl reading . such accounts posit that whole reading requires fast parallel letter identification , and that deficits in letter processing inevitably give rise to reading dysfunction and word length effects ( e.g. , bub et al . , 1989 ; howard , 1991 ; behrmann and shallice , 1995 ; hanley and kay , 1996 ; price and devlin , 2003 ) . while both fol and cla were significantly less accurate than controls at identifying rapidly serially presented single letters , it is likely that this performance reflects a combination of their basic visual deficits rather than a specific problem of letter processing , particularly as fol also demonstrated poorer accuracy on an equivalent task looking at rapidly presented numbers . the absence of strong evidence of a deficit in single letter processing suggests that intact parallel letter identification may account for their preserved reading in both patients . to adequately counter the general visual processing difficulties position it needs to be shown that any visual processing difficulty of the patients shown on some other perceptual task plausibly arises from impairment to a processing system necessary for word reading and not some potentially unrelated visual process . however on these grounds one can make the extremely strong statement that none of the component visual processes required for normal performance on any of the 10 visual tasks evaluated in this study ( which examine different levels of the visual system and involve different levels of task difficulty : figure - ground discrimination , shape discrimination , hue discrimination , number location , dot counting , object decision , fragmented letters , canonical and non - canonical view perception , grid experiment ) , are necessary for intact reading because our patients failed every single task . furthermore , the impaired processes highlighted by these tasks also do not fall into the poorly - defined category of general visual dysfunction which advocates of the general visual account claim cause lbl reading . however , at the much more relative level , the crashing visual deficits highlighted in our patients are an order of magnitude greater than the often subtle deficits claimed for patients cited in support of the general visual account . having documented grave visual impairments , it remains to be established what mechanisms support reading in fol and cla . the accurate and rapid reading shown by both patients suggests preservation of word form representations or parallel letter processing mechanisms . however , we note that the mri scans of fol and cla ( fig . 1 ) both indicate relative preservation of the left fusiform gyrus , commonly cited as the locus of the vwfa ( cohen et al . , 2000 ) and an area in which lesions often result in lbl reading ( binder and mohr , 1992 ; leff et al . , 2001 ; cohen et al . this area perhaps provides an anatomical substrate for preserved reading ability in these patients , with one possibility being that strong reading performance is supported by preservation of certain inputs to the vwfa that bypass other impaired aspects of early visual processing . support for this notion centres on evidence that the vwfa has connections to the primary visual cortex ( rockland and van hoesen , 1994 ; tanaka , 1997 ; haynes et al . , 2005 ) whose relative integrity in fol and cla may be indicated by their continued strong or adequate performance on tests of visual acuity . however this suggestion involves the visual word form system maintaining its efficacy , even in the presence of widespread dysfunction at lower levels of the visual system . irrespective of whether the observed reading is attributable to preservation of the word form and/or aspects of parallel letter processing , the performance of these two pca patients represents an impressive demonstration of the resilience and efficiency of the reading system in the face of profound visual dysfunction .

despite substantial neuroscientific evidence for a region of visual cortex dedicated to the processing of written words , many studies continue to reject explanations of letter - by - letter ( lbl ) reading in terms of impaired word form representations or parallel letter processing in favour of more general deficits of visual function . in the current paper , we demonstrate that whilst lbl reading is often associated with general visual deficits , these deficits are not necessarily sufficient to cause reading impairment and have led to accounts of lbl reading which are based largely on evidence of association rather than causation . we describe two patients with posterior cortical atrophy ( pca ) who exhibit remarkably preserved whole word and letter reading despite profound visual dysfunction . relative to controls , both patients demonstrated impaired performance on tests of early visual , visuoperceptual and visuospatial processing ; visual acuity was the only skill preserved in both individuals . by contrast , both patients were able to read aloud words with perfect to near - perfect accuracy . reading performance was also rapid with no overall significant difference in response latencies relative to age- and education - matched controls . furthermore , the patients violated a key prediction of general visual accounts of lbl reading that pre - lexical impairments should result in prominent word length effects ; in the two reported patients , evidence for abnormal word length effects was equivocal or absent , and certainly an order of magnitude different to that reported for lbl readers . we argue that general visual accounts can not explain the pattern of reading data reported , and attribute the preserved reading performance to preserved direct access to intact word form representations and/or parallel letter processing mechanisms . the current data emphasise the need for much clearer evidence of causality when attempting to draw connections between specific aspects of visual processing and different types of acquired peripheral dyslexia .

radiological changes of the hips occur in about 30% of as patients often resulting in functional impairment . in cases with end - stage hip disease , might evolve from early inflammatory infiltrations to lymphoid neogenesis and eventual fibrosis or fibrosclerosis . in 1982 , revell and mayston observed considerable lymphoid cell infiltration , lymphoid follicle formation , igg- , iga- , and igm - containing plasma cells , and a varying extent of fibrosis in joints from as patients . however , the pathogenesis of hip involvement in as is still unclear and few have focused on it in current reports . ectopic lymphoid tissue , also known as tertiary lymphoid tissue , has been observed recently in chronically inflamed tissue [ 6 , 7 ] where ectopic germinal centers ( gcs ) have been identified and are characterized by follicular dendritic cell ( fdc ) networks . current limited studies reported ectopic lymphoid tissue to be related to the increased severity of disease [ 7 , 8 ] and the production of autoantibodies . ectopic lymphoid tissue with gcs and its pathophysiology remain to be investigated extensively in inflammatory diseases , especially in those with lymphoid follicle formation in inflamed tissues . on the other hand , the presence of igg4 in some chronic inflammatory sclerosing diseases has been confirmed . the designation isrds refer to a collection of various conditions and clinically respond well to steroid therapy , reportedly . prominent pathological features include massive infiltrations of igg4-producing plasma cells , lymphoid follicle formation , and fibrosclerosis in single or multiple organs . the igg4-producing plasma cells can be detected in other autoimmune inflammatory diseases as well . as is currently considered an inflammatory joint disease ; however , lymphoid follicles , igg- , iga- , and igm - containing plasma cells have been found in inflamed peripheral joints . it is unclear whether inflamed joints in as have pathological changes similar to findings in autoimmune inflammatory diseases . this study aims to investigate the synovial tissue of as patients ' hips with end - stage disease , using immunohistochemistry to detect the possible presence of ectopic lymphoid tissue with gcs and igg4-positive plasma cells . seven as patients undergoing total hip replacement surgery in 2010 - 2011 for severely damaged hip joints were selected for study inclusion . all patients met the modified new york criteria for ankylosing spondylitis and had no evidence of autoimmune rheumatic disease nor igg4-related diseases . clinical data including gender , age , disease duration , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) level , and radiographic data were collected . after surgery , synovial tissue was obtained from joints by longitudinally cutting the synovium along the surface of the femoral head and fixed in 40% formaldehyde for 24 hours . prior to continued preparation and evaluation , 3 m sample sections were deparaffinized with xylene and then rehydrated with 100% , 95% , and 80% ethanol . he staining . staining with hematoxylin and eosin synovial tissue evaluation was made by an experienced observer at x200 magnification against 5 consecutive fields . 3 m sections of sample were deparaffinized with xylene and then rehydrated with 100% , 95% , and 80% ethanol . slides were treated with citrate sodium at 95c for 10 min and then incubated with 3% h2o2 . primary antibodies were applied to the slides overnight at 4c , followed by incubation with horseradish peroxidase - labeled secondary antibodies for 1 h at room temperature with repeated washes in between . primary antibodies included rabbit anti - human cd3 polyclonal antibody , mouse anti - human cd20 monoclonal antibody , mouse anti - human cd21 monoclonal antibody , and mouse anti - human cd38 monoclonal antibody . primary antibodies included rabbit anti - human iga polyclonal antibody ( dako ) , rabbit anti - human igm polyclonal antibody ( dako ) , rabbit anti - human igg polyclonal antibody ( dako ) , mouse anti - human igg4 monoclonal antibody ( invitrogen ) , rabbit anti - human c1q polyclonal antibody ( dako ) , rabbit anti - human c3d polyclonal antibody ( abcam ) , and rabbit anti - human c4d polyclonal antibody ( biomedica ) . horseradish peroxidase - labelled secondary antibodies were purchased from lsbio ( maixing company , china ) . for plasma cells expressing igg and igg4 assessment , five random fields were checked and photographed for each slide , whereby positive cells were counted and averaged . the mean age and disease duration of patients were 37.1 9.0 years ( range : 2452 years ) and 13.7 12.4 years ( range : 240 years ) , respectively . radiographic evaluation of hips showed narrowed joint spaces ranging from 0 to 7 mm , including 3 of 7 patients with complete disappearance of the joint space ( figure 1 ) . obvious fibrosis was present in all 7 cases , as was a varying infiltration of inflammatory cells . synovial lining ( intimal cell ) hyperplasia was observed in 5 cases but was nearly unobservable in 2 cases with complete fibrosis . . massive presence of inflammatory cells as well as moderate to severe synovial tissue fibrosis was observed in 5 patients ; two had lymphoid neogenesis ( figure 2(a ) ) ; two patients showed complete fibrosis with few inflammatory cells infiltrating . using anti - cd molecule antibodies , lymphoid - like structures were observed in two cases , consisting of a typical cd21 + fdcs network ( figure 2(b ) ) , numerous cd20 + cells ( figure 2(c ) ) and cd3 + t - cells ( figure 2(d ) ) . cd38 + plasma cells were found primarily nearby lymphoid - like structures and in the surrounding small vessels ( figure 3(a ) ) . the evaluation of immunoglobulin - producing plasma cells revealed that igg - positive plasma cells ( figure 3(b ) ) were more predominant compared with igm- and iga - positive plasma cells . igg4-producing plasma cells were observed only in patients with ectopic lymphoid tissues ( figures 3(c ) and 3(d ) ) . one patient had igg4-positive cells averaging 99/hpf ( figure 3(c ) ) , with a percentage of igg4/igg of 69.1% . another case had fewer igg4-expressing plasma cells with only 5.9% in igg - positive cells ( figure 3(d ) ) . c1q , c3d , and c4d positive cells were barely observed except a few in endothelial cells . a previous study of the synovia of 14 patients who underwent synovectomy or hip replacement surgery showed that lymphocyte aggregation was present in all patients ' synovia and lymphocyte follicle formation occurred in over half the samples . in comparison , our patients undergoing total hip replacement presented with more extensive fibrosis and less lymphocyte follicle formation . in our study , both ectopic lymphoid tissue with germinal centers and igg4-producing plasma cells ( figures 3(c ) and 3(d ) ) were identified among 2 cases . ectopic germinal centers in these cases demonstrate a structurally distinct feature of cd21 + fdc networks ( figure 2(b ) ) as described by humby et al . . in several reports , either ectopic lymphoid tissue or ectopic germinal centers have been related to disease severity in several autoimmune diseases [ 15 , 16 ] . the ectopic lymphoid structures with fdc networks have also been reported to support the ongoing production of class - switched autoantibodies . in the coincidental presence of ectopic gcs and igg4-producing plasma cells in this study might suggest a link between them . regarding the igg4-producing plasma cells , their proportions to igg+ cells were 69.1% and 5.9% for the two cases . these observations are pathologically in accordance with the feature of irsds mentioned by both kamisawa and okamoto and cheuk and chan [ 10 , 11 ] and hint at the clinical rationale of corticosteroid therapy . in irsds [ 1012 ] , the pathological role of igg4-expressing plasmacytes is not clear at present , but the frequent coincidence with fibrosis might suggest an association in pathological processes . above all , findings of ectopic lymphoid tissue with germinal centers and igg4-producing plasma cells might account for the pathophysiology of severe hip joint damage in as patients . considering that igg4 positivity was observed in only 2 of the 7 as patients ' synovia , future studies should be planned to carefully examine the entire joint synovia of a larger sample to determine if these findings are incidental or of pathomechanistic significance . our current study is the first to identify ectopic lymphoid tissue with fdcs networks and igg4-positive plasma cells in the inflamed synovial tissue of as patients with end - stage hip joint disease . this discovery may be a clue to better understand the mechanisms of the structural destruction of hip joints in as , particularly among the hla - b27 + patient population .

introduction . ectopic lymphoid neogenesis and the presence of igg4-positive plasmacytes have been confirmed in chronic inflammatory sclerosing diseases . this study aims to investigate hip synovial tissues of ankylosing spondylitis ( as ) patients for igg4-positive plasma cells and ectopic lymphoid tissues with germinal centers ( gcs ) . methods . synovial samples were collected from 7 as patients who received total hip replacement and were evaluated using immunohistochemistry for the presence of cd20 + b - cells , cd3 + t - cells , cd21 + follicular dendritic cells ( fdc ) , and cd38 + plasma cells . furthermore , immunoglobulin g ( igg and igg4 ) , iga , igm , and complement components c3d and c4d in synovia were evaluated . both synovial cd21 + fdcs and igg4-producing plasmacytes were analyzed . results . all seven patients had severe fibrosis . massive infiltrations of lymphocytes were found in 5 out of 7 patients ' synovia . ectopic lymphoid tissues with cd21 + fdc networks and igg4-positive plasma cells were observed coincidentally in two patients ' synovia . conclusion . the pathophysiological mechanism of as patients ' hip damage might be related to the coincidental presence of ectopic lymphoid tissue with fdcs network and igg4-positive plasma cells identified here for the first time in as patients ' inflamed synovial tissue .

twenty - four enterovirus sequences , mainly from hev - a group were retrieved from the national center for biotechnology information website . phylogenetic analysis , performed with the mega4 program ( 12 ) , indicated that ca16 strains shzh00 - 1 , shzh05 - 1 , and gz08 did not cluster with ca16-g10 ( figure 1 , panel a ) , although they indeed belonged to hev - a and clustered with ev71a ( brcr ) , ev71b ( ev71/9/97/sha89 ) , and ev71c ( s10862-sar-98 ) ( table ) . thus , the so - called ca16 full - length sequences ( shzh00 - 1 , shzh05 - 1 , and gz08 ) from china are distinct from ca16-g10 full - length sequences . classification of coxsackievirus 16a ( ca16 ) sequences from the people s republic of china into human enterovirus ( hev ) groups . a ) phylogenetic analysis performed by using all hev reference samples from china classified as hev - a but not as ca16 . mega4 software ( 12 ) was used as the analytic program and kimura 2-parameter as the model . b ) bootscanning analysis of shzh00 - 1 . for all hev - a sequences , together with sequences from 2 outgroups , shzh00 - 1 showed possible recombination with ca4 and enterovirus 71a . the vertical red lines with numbers show the possible recombination break points as determined by genetic algorithm recombination detection ( 13 ) . the sequences used corresponded to positions 27,407 in shzh00 - 1 . bootscanning was performed with a window size of 500 nt and step of 20 nt . because of gaps in alignment , break points 211 , 660 , 3,756 , 4,608 , and 6,072 correspond to positions 207 , 647 , 3,555 , 4,406 , and 5,854 in shzh00 - 1 , respectively . * ca , coxsackievirus a ; ev , enterovirus . because the shzh00 - 1 , shzh05 - 1 , and gz08 sequences clustered within the hev - a group and were determined to be ca16 on the basis of the vp1 region ( data not shown ) , we examined these 3 sequences for evidence of recombination . it has 7,410 nt in its genome , including the 5 untranslated region ( utr ) ( 1745 ) , structural protein ( 7463331 ) , and nonstructural proteins p2 ( 33325065 ) and p3 ( 50667327 ) , with the rest of its genome as 3 utr ( 73287410 ) . bootscanning with a sliding window of 500 nt , overlapping by 20 nt , was performed with the simplot program ( version 3.5.1 ) ( 14 ) to investigate the possibility of recombination within the shzh00 - 1 sequence . the results indicated that the 5 utr of the shzh00 - 1 sequence had relatively high similarity to ca4 ( figure 1 , panel b ) . the p1 ( vp4 , vp2 , vp3 , and vp1 ) region was more similar to that of ca16-g10 . however , part of the p2/p3 region of the shzh00 - 1 sequence ( 2c , 3a , 3b , and 3c ) had relatively high similarity to ev71a but not to ca16-g10 ( figure 1 , panel b ) . similar results were obtained for the shzh05 - 1 and gz08 sequences ( data not shown ) . thus , shzh00 - 1 , shzh05 - 1 , and gz08 are recombinant type a hevs that contain ca4 , ca16 , and ev71 . to examine the circulation status of recombinant ca16 in china , we characterized 24 additional ca16-related sequences from hfmd patients from central ( hangzhou , zhejiang province ) and northeastern ( changchun , jilin province ) china ( table ) . the break point around position 3555 interested us most because it not only roughly separated the p1 region from p2/p3 but also divided the open reading frame ( 7467327 ) into ca16-like and non ca16-like fragments . thus , we selected this region spanning the recombination break point between the ca16 sequence and the downstream sequence . bootscanning of these new sequences showed that the sequences from changchun and hangzhou have a recombination pattern similar to that of shzh001 ( figure 2 , panel a ; data not shown ) . however , when shzh00 - 1 was used as the reference sequence , bootscanning of these changchun and hangzhou sequences showed a high similarity to shzh00 - 1 through the entire sequenced region ( data not shown ) . phylogenetic analysis indicated that 23 of the changchun and hangzhou sequences formed a strong cluster with shzh00 - 1 , shzh05 - 1 , and gz08 ( figure 2 , panel b ) . notably , 1 sequence clustered with the ca4 reference sequence , indicating that ca4-related viruses are also circulating among hfmd patients in china . these data suggest that shzh00 - 1 and these changchun and hangzhou sequences are likely derived from a common ancestor . changchun104 was shown for bootscanning analysis with human enterovirus a ( hev - a ) sequences and shzh00 - 1 as references . b ) hangzhou212 , hangzhou023 , and all changchun sequences clustered with shzh00 - 1 , shzh05 - 1 , and gz08 , with a very high bootstrap value ( 100% ) . indicates hangzhou sequences ; indicates changchun sequences , and indicates shzh00 - 1 , shzh05 - 1 , and gz08 . c ) map of the people s republic of china indicating the provinces where shzh00 - 1like ca16 sequences were characterized . as many as 40% of cases of hfmd in china have been attributed to ca16 infection on the basis of partial viral genome determination ( 11 ) . in the current study , we demonstrated that circulating ca16 viruses in china are actually complex recombinant viruses involving multiple type a hevs , including ca4 , ca16 , and ev71 ( figure 1 ) . the 5 utr region ( 207647 bp ) of these viruses had the highest similarity to ca4 . the nonstructural protein domains ( p2 and p3 ) had a 1.5-kb fragment ( 4,4065,854 bp ) that was most similar to ev71a . several regions of shzh00 - 1 remain unclassified , including part of 2a , 2b , part of 2c , and 3d . over the past 30 years , numerous large outbreaks of ca16-associated hfmd , along with outbreaks caused by ev71 , have been reported in china . however , full molecular characterization of circulating ca16 viruses in china had not been conducted . the current study provides evidence that circulating recombinant forms of the ca16-related viruses are prevalent among hfmd patients throughout china ( figure 2 , panel c ) . the origin , including the place and date , of the current recombinant ca16 viruses is not clear . the exact parental viral strains involved in the generation of ca16 recombinant viruses are also unknown . some of the parental virus strains of china ca16 could have become extinct or are yet to be discovered . notably , ca4 was detected in 1 of our samples from zhejiang province ( central china ) and has also been reported in gansu province ( northwestern china ) ( 15 ) . the identification of ca16-related hfmd , based mainly on vp1 sequences , has been widely reported in many parts of china , including guangdong , fujian , jiangsu , and inner mongolia provinces , and beijing and shanghai . unfortunately , the sequences of other parts of these viral genomes , especially the regions spanning the recombination site , have not been determined . further detailed characterization of hev sequences from hfmd patients is still needed , but the information obtained thus far has implications for addressing the future emergence of new pathogenic hevs and for vaccine development to manage the increasing prevalence of hfmd .

to determine the relationship of coxsackievirus a16 ( ca16 ) to prototype ca16-g10 , we conducted a phylogenetic analysis of circulating ca16 strains in china . complex recombinant forms of ca16-related viruses involving multiple human enteroviruses , subgroup a ( ca4 , ca16 , and enterovirus 71 ) , are prevalent among patients with hand , foot , and mouth disease .

aqueous dispersions of micrometer - sized droplets of thermotropic liquid crystals ( lcs ) provide versatile platforms for the design of stimuli - responsive soft matter systems . the responsiveness of these droplet - based lc systems to various types of stimuli results from the fine scale of energetics that controls the equilibrium ordering of lcs . specifically , past studies have established that the ordering of lcs within micrometer - sized droplets reflects contributions to the free energy that arise from orientation - dependent anchoring of the lc at the surfaces of the droplets , elastic strain of the lc associated with the accommodation of surface anchoring , and the formation of topological defects . recent studies , however , have also led to observations involving lc droplets in aqueous dispersions that reveal our understanding of factors controlling the ordering of lc droplets to be incomplete . in particular , existing theories of lc droplets can not account for ( i ) changes in the internal configurations of the lc droplets that occur with changes in droplet size , ( ii ) the effects of simple salts on the ordering of the lcs , or ( iii ) the partitioning of amphiphiles to the defects of lc droplets . in this paper , we seek to advance our understanding and ability to design aqueous dispersions of stimuli - responsive lc droplets by exploring the properties of lc droplets encapsulated ( or caged ) inside thin and porous polymeric membranes . our results reveal that the interactions of the lc droplets with the interfaces of the polymeric cages give rise to surfactant - triggered shape changes and resulting internal ordering transitions that are not seen in free lc droplets . of particular relevance to this article are recent studies that have investigated the ordering of spherical lc droplets encapsulated in capsules composed of polyelectrolyte multilayers ( pems ) . in those studies , the spherical polymeric capsules were formed using noncovalent interactions ( e.g. , electrostatic interactions or hydrogen bonding ) , and the lcs were observed to fill the interiors of the capsules completely . the study reported in this article moves to investigate the ordering of lcs within multilayer capsules prepared by covalent reactions of branched polyethylenimine ( pei ) and poly(2-vinyl-4,4-dimethylazlactone ) ( pvdma ) . in contrast to capsules formed from pems , multilayer capsules formed using pei and pvdma have been found to fill only partially with lc , resulting in the formation of micrometer - sized lc droplets that are trapped or caged in aqueous solutions contained within the polymer capsule . these systems are a particularly interesting class of responsive soft matter , as the capsules can be designed to bind to the surfaces of mammalian cells , with the caged lc droplets undergoing changes in optical appearance upon exposure to cytotoxic analytes ( thus providing the basis for sensors of local concentrations of toxic analytes near cells , etc . ) . the work reported here sought to provide insight into some of the observations reported above and explore further the properties of caged lc droplets in the presence of aqueous solutions of surfactants . first , we report on the origins of the partial filling of pei / pvdma capsules by the nematic lc e7 . in this context , we describe the design of new amphiphilic polymeric capsules that were prepared to present a hydrophobic inner surface to the lc droplet ( to promote filling ) and a charged ( cationic ) outer surface to promote the dispersal of the capsules in water . we establish that the partial filling of the capsules by e7 is also observed with amphiphilic capsules and that it arises largely from the swelling of the capsules upon transfer into water from the bulk lc phase after filling of the capsules with lc . second , because the capsules are partially filled by lc ( the remainder of the internal volume of the capsule is an aqueous solution ) , we explore changes in the shapes of the caged lc droplets that are driven by surfactant - induced changes in the wetting of the lc on the inner surfaces of the capsules . as detailed below , we observed the addition of surfactants to aqueous solutions of caged lc droplets to promote wetting and ordering transitions of a complexity not observed in free lc droplets , which may permit new designs of stimuli - responsive lc - droplet - based systems . the fabrication of hollow polymer microcapsules was performed according to a previously published procedure for the layer - by - layer ( lbl ) formation of covalently assembled polymeric capsules . on the basis of a past report , we used monodisperse silica microspheres with a diameter of 4.99 0.22 m as a template for the polymer microcapsules to produce lc droplets of a size that is sensitive to amphiphilic adsorbates . solutions of pei and pvdma ( or pvdmafl labeled with 6-aminofluorescein to facilitate fluorescence - based imaging ) ( figure 1a ) were prepared in acetone ( 20 mm with respect to the molecular weight of the polymer repeat unit ) . sio2 microparticles ( 100 l in plastic microcentrifuge tubes ) were rinsed with 1 ml of acetone and centrifuged prior to the addition of polymer . in brief ( see si and past reports for details ) , an initial layer of pei was adsorbed onto the silica microparticles through electrostatic interactions , and subsequent layers of pvdma ( or pvdmafl ) and pei were sequentially added , layer - by - layer , to build up a covalently cross - linked multilayer film . between the addition of each polymer layer , particles were rinsed three times by centrifugation ( 2 min at 1500 rpm ) and resuspension in acetone . films fabricated using this method have been shown previously to contain residual azlactone groups , providing a reactive handle for postfabrication functionalization with other chemical functionalities . we selected two different structural motifs , shown in figure 1b ; further discussion is included below . after the fabrication of two pei / pvdma bilayers , the coated microparticles were rinsed with tetrahydrofuran ( thf ) and functionalized by adding 1 ml of decylamine ( da , figure 1b ) in thf ( 20 mm ) for 1 h to install the hydrophobic functionality . these da - functionalized coated microparticles were then rinsed with thf three times and dispersed in acetone prior to resuming the fabrication of an additional 2.5 bilayers of pei and pvdma . finally , to functionalize these outer 2.5 bilayers with protonatable hydrophilic functional groups , the microparticles were washed with thf three times and then dispersed in 1 ml of 3-(dimethylamino)propylamine ( dm ) in thf ( 20 mm ) for 1 h. polymer - coated silica microparticles were rinsed with thf three times by centrifugation and then suspended in a small volume of water ( 100 l ) in preparation for aqueous etching . the silica core was removed from the polymer membrane by treatment with a commercially available buffered oxide etching ( boe , 10:1 ) solution containing hydrofluoric acid ( hf ) ; see materials section in si . hf solutions and vapors are extremely poisonous and corrosive and may cause extreme burns that are not immediately painful . handle with extreme caution in a chemical fume hood , and use appropriate protective equipment ( gloves , face / eye protection , laboratory coat , etc . ) , and neutralize waste appropriately . ) the samples were incubated with 800 l of boe at room temperature for 510 min . the resulting hollow capsules were centrifuged ( 4500 rpm for 5 min ) and rinsed five times in 1 ml of water . after the silica template was removed to obtain hollow polymeric microcapsules , the microcapsules were filled with lc following a previously reported protocol . in brief , the capsules were rinsed with ethanol twice , centrifuged to remove as much of the supernatant ethanol as possible , and suspended in a mixture of 5% ethanol and 95% e7 . this mixture of ethanol and e7 forms an isotropic phase that can infiltrate the polymeric capsules . the ethanol was slowly evaporated from the sample by leaving the tube containing the sample uncapped on a shaker plate for 24 h , resulting in a nematic phase containing the capsules . the capsules were then extracted into an aqueous phase by removing excess e7 by centrifugation , contacting the sample with water , and shaking gently . this procedure resulted in the spontaneous transfer of capsules containing lc droplets to the aqueous phase . dispersions of either bare or coated microparticles , hollow capsules , and lc - containing capsules were placed on a coverslip and optically imaged in bright - field polarized light ( crossed polarizers ) using fluorescence modes with an olympus ix-71 inverted microscope ( center valley , pa ) with a 100 oil - immersion objective ( with or without an additional 1.6 optical zoom ) and equipped with a 100 w mercury lamp . for fluorescence - based imaging of capsules fabricated using pvdmafl , an olympus u - mnb2 fluorescence filter cube was used with a 470490 nm excitation filter and a 520 nm emission filter . a monochrome hamamatsu 1394 orca - er ccd camera ( bridgewater , nj ) was controlled with simplepci software ( compix , inc . images were later analyzed using imagej software ( nih , bethesda , md ) . for each sample , the bulk solution was imaged using polarized light microscopy to determine the ordering of lc droplets in capsules that were freely diffusing . however , since the diffusion quickly moved the capsules out of the plane of focus or the field of view , capsules could be imaged in all three imaging modes ( bright field , crossed polarizers , and fluorescence ) only after sedimentation onto the coverslip surface . in our experiments , we did not observe any orienting effect on the lc resulting from contact with the coverslip , as reported in a past study . ( a ) chemical structure of branched pei and pvdma used for the covalent layer - by - layer assembly of multilayer films . chemical functional groups : decylamine ( denoted r1 or da ) was used to functionalize the first two bilayers , and dimethylaminopropylamine ( denoted r2 or dm ) was used to functionalize the outer 2.5 bilayers ( see text ) . in contrast to prior studies described in the introduction , the capsules used in this current investigation were prepared to have walls with hydrophobic functionality facing the interior of the capsule and hydrophilic functionality on the outer surface . this was accomplished by functionalizing the interior with decylamine ( da ) and the exterior with dimethylaminopropylamine ( dm ) such that the hydrophobic interior contacted the lc and the hydrophilic exterior surface contacted the bulk aqueous phase . ( see figure 1 and the experimental section for details . ) in the remainder of this article , we refer to these capsules as having an amphiphilic structure and by using the acronym dadm to identify the da and dm motifs decorating the interior and exterior walls of the capsules . in the interest of brevity , the confirmation of the successful synthesis of these dadm capsules is detailed in the supporting information ( si ) . specifically , we present evidence of the incorporation of the da and dm into the capsule walls ( figure s1 ) and show scanning electron micrographs of polymer - coated microparticles and hollow capsules ( figure s2 ) . measurements made using the scanning electron micrographs reveal the size of the dried , polymer - coated microparticles to be 4.9 0.2 m . after treatment with lc using methods that lead to the complete filling of pem - based capsules in past studies , we observed the dadm - functionalized pei / pvdma capsules used here to be only partially filled with lc ( see figure 2f , middle row ) . specifically , we measured the average diameters of the dadm capsules and the lc droplets to be 6.7 0.3 and 4.9 0.5 m , respectively ; that is , the lc filled approximately 40% of the internal volume of the capsules . to provide insight into the reasons underlying partial filling , we imaged the film - coated microparticle precursors and the hollow capsules in each step of the synthesis ( figure 2 ) . the images in the middle row of figure 2 are bright - field micrographs , and images in the bottom row are fluorescence micrographs of fluorescein - labeled capsules that clearly define the sizes and shapes of the capsules . first , the dadm - coated silica microparticles in water were measured to have a diameter of 5.3 0.3 m ( figure 2b ) , a value that is indistinguishable from the diameter measured for the bare silica microparticles in water ( 5.2 0.3 m as in figure 2a ) . second , after etching of the silica templates , we measured the diameter of the resulting hollow capsules to vary significantly depending upon the solvent in which they were suspended ( figure 2c e ) . specifically , we measured capsule diameters to be 6.7 0.3 m in water , 5.6 0.3 m in 90% acetone/10% water , and 4.7 0.2 m in nematic e7 . third , upon extraction of lc - containing dadm capsules into an aqueous phase , the capsule diameter was again measured to be 6.7 0.3 m ( figure 2f ) , a value that is indistinguishable from that of empty capsules in water . overall , these observations reveal that the volume of the lc encapsulated within the polymeric shell is determined by the size of the capsule when it is present in the nematic lc ( i.e. , during filling ; compare panels e and f of figure 2 ) . when lc - filled capsules are extracted into an aqueous phase , the capsules swell , thus resulting in capsules that are only partially filled . we emphasize that the volume of the lc droplets caged within the polymer capsules when dispersed in water is roughly equal to the interior volume of the capsules when they are dispersed in nematic lc during filling . the top row shows a cartoon of the sample composition , the middle row consists of bright - field images , and the bottom row contains the corresponding fluorescence microscopy images of ( a ) bare silica microparticles in water , ( b ) dadm - coated silica microparticles in water , ( c ) empty dadm capsules in water ( after hf treatment and rinsing ) , ( d ) empty dadm capsules in a solution of 90% acetone and 10% water , ( e ) dadm capsules in nematic e7 , and ( f ) dadm capsules partially filled with e7 in water . the average capsule size is noted below the fluorescence microscopy image , and the lc droplet size is also denoted below ( f ) . bright - field and fluorescence microscopy images are not shown for all states , e.g. , where the sample was not in focus or not fluorescent . ( a , b ) micrographs of the same group of capsules imaged with different locations of the focal plane such that in ( a ) the circular capsule on the right of the image was in focus and in ( b ) the crescent - shaped capsule in the center of the group was in focus . ( c ) micrograph of a separate region showing a group of capsules with more spherical than nonspherical capsules . finally , we comment that a fraction of capsules observed in nematic e7 ( approximately one in six capsules prior to extraction into water ) possessed nonspherical crescent shapes , consistent with a buckling instability in the capsule wall ( figure 3 ) . for these nonspherical capsules , we measured the long axis ( i.e. , the largest diameter that could be measured ) to be 4.5 0.3 m , corresponding to a diameter similar to that of the spherical capsules ( 4.9 0.5 m ) , and the short axis ( perpendicular to the long axis ) was measured to be 2.3 0.6 m . this small subpopulation impacted the underfilling of the capsules with lc and contributed to heterogeneity in the size and shape of the capsules and droplets . we note that the majority of capsules that we observed to be nonspherical in the e7 phase returned to a spherical shape when transferred to the aqueous phase ; a very small percentage of capsules ( approximately 2% ) remained nonspherical in shape . figure 4a c shows representative caged lc droplets imaged in bright - field and polarized light modes as well as schematic illustrations of the ordering of the lc in the droplets . a number of features of the droplets imaged between crossed polarizers led us to conclude that the orientational ordering of the lc within these droplets corresponds to a so - called bipolar configuration . in this configuration , aqueous interface with two point defects at diametrically opposite poles , as depicted schematically in the right column of figure 4 . in figure 4a , the key identifying features are the two dark spots on the left and right sides of the droplet , bright lobes above and below these spots , and a relatively dark center of the droplet , indicating that the lc in the center is aligned with either the polarizer or analyzer . the narrow dark spots correspond to point defects on the droplet surface known as boojums that define the poles and , thus , the symmetry axis of the bipolar nematic droplet . ( the angle that this symmetry axis makes with the polarizer is indicated on the polarized light images . ) typically , boojum defects can be seen in bright - field images as dark regions because they possess a local refractive index environment that is distinct from that of the rest of the lc droplet and thus scatter light . however , optical distortions resulting from the polymer capsule prevented us from identifying the boojums in many of our bright - field images . bright - field and polarized light micrographs of dadm capsules in water ( left and center columns , crossed polarizers indicated by white arrows ) . corresponding schematic illustrations of the lc director profiles are shown in the right column . the angle that the axis of symmetry of the droplet makes with the polarizer is 0 in ( a ) , 14 in ( b ) , and 43 in ( c ) . ( d ) two droplets with asymmetric shapes also exhibit signature features of bipolar ordering with arrows indicating the locations of boojum defects . figure 4b shows a droplet that has its symmetry axis , defined again by dark spots that correspond to the boojums , at an angle between the polarizer and analyzer . the rotation of the symmetry axis away from the polarizer / analyzer results in a birefringent texture within the droplet that is brighter than that shown in figure 4a . this droplet also has curved dark bands within the bright central region in a baseball - like pattern , a characteristic that is frequently used to identify bipolar droplets . figure 4c shows a bipolar lc droplet within a capsule aligned with its axis of symmetry at an angle of 43 from the polarizer and analyzer . in this image , we observed only one bright region next to each boojum , as the other lobe is more closely aligned with a polarizer . in summary , the observations reported above are consistent with a bipolar configuration of roughly spherical lc droplets within the capsules . while the majority of the caged lc droplets appeared to be spherical , we also observed a subpopulation of nonspherical , asymmetric droplets ( approximately one in four droplets was asymmetric ; see discussion below for a further analysis of shape ) . figure 4d shows an example of two capsules that contain nonspherical lc droplets . in these images , it appears that the lc has partially wet the inner surface of the capsules , causing the droplets to distort to asymmetric lemonlike shapes . droplets that assumed this asymmetric shape , however , still exhibited birefringent textures that closely resembled those of the spherical droplets . we conclude that the lc ordering in these cases is a distorted bipolar configuration ( figure 4d ) , with the two boojums located close to the contact line defined by the lc polymer interface . here , we also note that the shape of the polymeric capsules did not appear to be perturbed by partial wetting of the inner surface of the capsule by the lc . previous studies have shown that addition of surfactants to aqueous dispersions of lc droplets can trigger a bipolar - to - radial ordering transition within the droplets . inspired by these results , we investigated the response of lc droplets confined within dadm capsules to model anionic and cationic surfactants ( sodium dodecyl sulfate ( sds ) and dodecyl trimethylammonium bromide ( dtab ) ) . the first section below describes lc wetting transitions within amphiphilic capsules induced by sds and dtab , and the second section describes changes in the internal ordering that occur in response to each of the two surfactants . we note that the highest concentrations of surfactant used in the experiments described below were less than the critical micelle concentrations to avoid the solubilization of the lc . bright - field ( top row ) , polarized light ( middle row , crossed polarizers ) , and fluorescence ( bottom row ) micrographs of representative regions showing lc ordering and wetting in partially filled capsules as a function of increasing sds concentration ( indicated below each column ) . labels above each column indicate the internal ordering of the lc at each concentration . figure 5 shows the appearance of lc droplets within dadm capsules upon exposure to increasing concentrations of sds . an inspection of figure 5 reveals that the addition of sds caused a continuous decrease in the contact angle ( ) of the lc on the inner surfaces of the capsules ( see figure 6 for the definition of and values of measured from bright - field micrographs ) , resulting in a change in the shape of the lc droplet . figures 5 and 6 , when combined , first reveal that low concentrations of sds ( 0.050.25 mm ) caused the lc droplets to adopt a convex lens shape ( 130 10 ) . at moderate sds concentrations ( 0.251 mm ) , we observed hemispherically shaped lc droplets ( 89 5 ) , with approximately planar ( low curvature or a large radius of curvature ( rc ) , see discussion below ) lc aqueous interfaces inside the capsules . we note that the shape ( and internal ordering ) of the lc droplets was not homogeneous within a sample ; for example , at 0.25 mm sds , we observed both lens - shaped droplets and nearly hemispherical droplets . ( see also figure s3 for information regarding the distribution of for samples in water or at different surfactant concentrations . ) lastly , at higher sds concentrations ( 0.510 mm ) , we observed a large population of droplets that adopted a concave lc aqueous interface within the capsules ( < 85 ; measurements on these samples were difficult because the angle was occluded by the lc when the interface was concave ; see also a representative video included in the si ) . these observations of a continuous change in the contact angle of the lc droplet on the inner wall of the polymeric capsule suggest that the interaction of sds with the capsule walls triggered this wetting transition . plot of contact angles of caged lc droplets on the interiors of polymeric microcapsules as a function of sds concentration ( number of droplets measured n > 30 for each data point ) . in contrast to the above - described influence of sds on the wetting of lc on capsule walls , we did not observe a substantial change in the shape or wetting of lc droplets in the presence of dtab ( see figure 7 ; the average increased from 157 11 in water to 162 10 in 10 mm dtab ; see also the si for histograms of ) . in contrast to our results using sds , this result suggests that dtab permeates but does not adsorb to the wall of the capsule to an extent that it leads to measurable changes in the contact angle , likely because of the positive charge of the dimethylamine functional groups decorating the outer walls of the dadm capsules . bright - field ( top row ) , polarized light ( middle row , crossed polarizers ) , and fluorescence ( bottom row ) micrographs of representative regions showing lc ordering and wetting within partially filled capsules as a function of increasing dtab concentration ( indicated below each column ) . labels above each column indicate the internal ordering of the lc at each concentration . concurrent with the surfactant - induced wetting transitions on the inner surfaces of the capsules , we observed the ordering of the lc within the droplets to change with the addition of sds and dtab ( middle row of images in figures 5 and 7 ) . as discussed above , the encapsulated lc droplets exhibited bipolar ordering in water ( i.e. , no surfactant , see figure 4 ) . when dtab was added to an aqueous dispersion of encapsulated lc droplets , we observed very little change in the shapes of the droplets ( figure 7 ) , and the sequence of internal configurational states that we observed was similar to that of free lc droplets . the states observed include ( i ) saturn - ring disclination line - containing states ( at 2.5 mm dtab ) , ( ii ) preradial configurations with a single point defect on the surface ( from 2.5 to 5 mm ) , and ( iii ) radial configurations ( from 5 to 10 mm ) . we note , in particular , that the appearance of the radial configuration is consistent with very weak interactions between the lc droplets and the inner surfaces of the capsules . past studies have noted that the contact of lc droplets with surfaces often pins the configurational state of lc droplets at high surfactant concentration in the preradial state . next , we characterized the ordering states of the lc within the lens- and hemispherical - shaped lc droplets ( 85127 ) that formed in the presence of sds . figure 8a d shows selected lc droplets in the presence of 0.25 mm sds ( 90 ) . we interpret the optical appearance of these droplets to indicate that they possess ordering consistent with an axial or saturn - ring configuration in which the axis of symmetry of the lc ordering follows the axis of symmetry of the droplet shape . we note that this optical texture is observed for lens - shaped droplets in the presence of 0.05 mm sds ( figure 5 ) , and it is also observed in samples at higher concentrations of sds ( up to 1.0 mm ) . as seen from a side view ( figure 8c , d ) , this director profile exhibits a disclination line near the planar interface such that the profile resembles that of a saturn - ring configuration . ( see figure s4 for images of bare e7 droplets with saturn - ring and radial configurations . ) schematic director profiles ( left column ) with corresponding bright - field ( middle column ) and polarized light ( right column , crossed polarizers indicated by white arrows ) micrographs of ( a ) top , ( b ) tilted , and ( c , d ) side views ( at different angles with respect to the polarizer / analyzer ) of hemispherical lc droplets contained within dadm capsules in 0.25 mm sds . ( e , f ) side views at different angles of hemispherical lc droplets in 1 mm sds . ( g , h ) schematics show possible director configurations for inhomogeneous boundary conditions . additionally , the top view ( figure 8a ) of the hemispherical droplets has four white lobes surrounding a dark cross , consistent with the axial symmetry indicated in figure 8 . as the sds concentration was increased , the lc droplets transitioned to an internal configuration that resembled a deformed radial droplet ( figure 8e , f , 1 mm sds , see also the video in the si ) with a point defect clearly visible in the bright - field and polarized light micrographs . we note that another possible director configuration that is consistent with the micrographs is shown in figure 8 g , as further discussed below . the key results of the study reported in this article relate to the response of caged lc droplets to surfactants . in contrast to free lc droplets , where sds and dtab trigger qualitatively similar configurational transitions and cause a minimal change in shape , we observed dadm - caged lc droplets to undergo wetting transitions in the presence of sds but not dtab . the change in droplet shape that accompanied the wetting transition resulted in differential effects of the two surfactants on the internal configurations of the lc within the droplets . we emphasize that this situation also differs from past reports that examined the anchoring of lcs confined to capillaries ( diameter 10200 nm ) , microgrooves , silica pores , polymer - dispersed cavities , and so forth because the internal volumes of those structures were completely filled with lc . ( thus , changes in shape were not observed ; see ref ( 34 ) for one exception . ) below , we discuss in more detail the coupled wetting transitions and ordering transitions described in this article . to provide insight into the wetting transitions , we calculated the shapes of the caged droplets by solving the laplace equation under the assumption that gravity and the elasticity of the lc have a negligible influence on shape . the neglect of gravity is justified by an evaluation of the bond number , calculated as bo = gl/ , where is the difference in densities of the fluid phases , g is the acceleration due to gravity , l is the characteristic length ( for which we use the droplet radius ) , and is the interfacial tension . for our experimental system as noted above , we also neglect the effect of the elasticity of the lc on the shape of the lc droplets since the ratio of the elastic energy to the surface energy , given as k/v ( where k is either the splay or bend elastic constant and k 10 n ) , is 10 for our experimental system . aqueous interface will have constant curvature within the caged lc droplets , as predicted by the laplace equation under the above - stated conditions . figure 9a shows the shapes of lc droplets on ( i ) planar surfaces and ( ii ) the curved interior surfaces of the microcapsules , both calculated as a function of [ and by assuming that the capsules are rigid ( see experimental observations above ) and of constant size ] . for these calculations , we used a volume of lc that corresponded to 40% of the interior of the capsule volume ( also consistent with the experiments reported in this article ) . to enable comparisons of the lc droplet behavior on a flat substrate and in a spherical cavity , figure 9b shows a plot of the interfacial area between the lc and polymer ( i.e. , the wall of the capsule or the surface of the flat substrate ) and between the lc and aqueous phase , both as a function of . finally , figure 9c shows the radii of curvature of the lc aqueous interfaces , plotted as a function of for the two geometries . from figure 9a first , an inspection of figure 9b reveals that , within a spherical cavity defined by the polymer capsule , there exists a crossover at which the interfacial area between the lc and polymer capsule exceeds the interfacial area of the lc aqueous interface . for the lc volume and capsule size used in our calculations , this occurs at 130. in contrast , on a planar surface , the interfacial area between the droplet and supporting substrate is always less than the aqueous interface . we also note that , on a planar substrate , the areas of the two interfaces of the lc droplets approach each other only in the limit of 0. in this limit , a thin film of infinite area is predicted . in contrast , however , in a spherical cavity , a thin film of finite thickness ( 0.5 m thick , as shown in figure 9a ) is formed on the interior surface of the capsule wall in the limit of 0. from this result , we conclude that the caging of droplets within capsules provides , in general , the basis of a versatile method for control of the relative interfacial areas of liquids that is not possible on flat surfaces . ( a ) calculated shapes for lc droplets of constant volume ( using the average experimental volume ) with varying contact angle for two geometries : ( i ) on a flat surface and ( ii ) within a spherical cavity equal in size to the dadm capsules in water . black lines represent the flat surface or spherical cavity , and blue lines represent the lc aqueous interface . axes are square , with the grid on the x axis marking 10 m ( each droplet is plotted with centers 10 m apart ) , and contact angles are used to label the x axis . ( b ) graph of interfacial areas ; interfaces are indicated within the legend . ( c ) graph of the radius of curvature ( rc ) of the lc aqueous interface for each geometry . ( d ) shapes plotted for a constant contact angle of 90 for varying volumes of lc , denoted on the plot relative to v , the average volume measured experimentally . the second set of observations that we make from figure 9 relates to the radius of curvature ( rc ) of the lc interface . specifically , an examination of figure 9c reveals that the caging of lc droplets within capsules leads to radii of curvature of the lc interfaces that are strikingly different from those on planar substrates . in particular , as the contact angle of the lc with the capsule wall approaches 84 , we calculate rc of the lc aqueous interface to diverge to positive or negative infinity . ( see also experimental observations in figure 5 . ) in contrast , the rc of lc on a planar interface diverges only in the limit of 0. this fundamental difference in the behavior of rc is significant , as rc dictates the laplace pressure difference , = pin pout = 2/rc , across the interface of the droplet . thus , rc also affects the chemical potential of the lc inside a droplet , which is given by i(rc ) = i( ) + 2vi / rc , where i( ) is the chemical potential of species i with a flat interface and vi is the molar volume of species i. in particular , we note that for all contact angles ( figure 9c ) , the rc of the lc aqueous interface for lc contained within a spherical cavity is either negative ( at < 84 ) or larger ( 84 < < 180 ) than that of the lc on a flat surface . this observation , when combined with the influence of the laplace pressure on the chemical potential of the lc , leads us to conclude that the chemical potential of caged lc is lower than that of an lc droplet on a flat surface . if two such droplets were present in a system , then we would predict that ostwald ripening would cause the growth of the encapsulated droplet at the expense of decreasing the size ( and rc ) of the droplet of the same volume on a flat surface . the effects of changes in droplet volume for caged lc droplets and lc droplets supported on flat surfaces are shown in figure 9d ( for constant = 90 ) . for caged droplets , an increase in volume leads to an increase in rc before turning negative . significantly , the chemical potential of the lc in the caged droplet decreases more rapidly with increasing volume as compared to droplets on flat surfaces . this observation leads us to predict that capsules containing droplets with rc < 0 will be stable with respect to both uncaged droplets as well as bulk lc . a second key result reported in this article is that the wetting transitions described above are coupled to changes in the internal ordering and topological defects of the lc . in order to discuss this coupling , two key concepts need to be introduced , namely , the topological charge of defects and the euler characteristic of closed surfaces . the topological charge ( n ) of a defect is related to the number of times the lc director rotates through all possible angles encountered on a surface that encompasses the defect . for example , in the case of a bipolar droplet , a topological charge of n = + 1/2 is assigned to each of the boojum defects by placing a hemispherical surface around the boojum and evaluating the director orientations . for the case of a radial droplet with a central point defect , the defect can be surrounded by a spherical surface that encounters all possible angles of the director , and it therefore has a topological charge of n = + 1 . for droplets in transition states that connect the bipolar and radial configurations of spherical lc droplets and possess uniform boundary conditions ( the tilt angle with respect to the surface normal is the same everywhere on the droplet surface ) , gauss s theorem requires the conservation of topological charge . for example , the transition state that comprises a saturn - ring disclination line located slightly inside the surface of the droplet must have a topological charge of + 1 ( it can be encompassed by a spherical surface that encounters all orientations of the lc director ) . another commonly encountered transition state that lies between the bipolar and radial configurations is the preradial configuration with a topological charge of n = + 1 . the above - described discussion addresses the well - studied case of configurational transitions in spherical droplets whereas the experiments reported in this article address the more complicated situation where changes in the shapes of the lc droplets accompany internal configurational transitions . to address this situation the euler characteristic of a sphere , and any surface that can smoothly be transformed into a sphere ( e.g. , a football and a bowl are both topologically equivalent to a sphere ) , is two . this result is useful because , for uniform boundary conditions , the sum of the topological charges ( n ) must be half of the euler characteristic of a closed surface . this leads us to conclude that the euler characteristic of the caged lc droplet shapes observed in our experiments is unaltered as compared to that of spherical droplets because all shapes observed for the caged lc droplets can be smoothly transformed into a sphere . thus , the wetting transitions reported in this article do not change the conservation of topological charge discussed in the context of past studies of free ( spherical ) droplets . specifically , for lc droplets exposed to sds ( figures 5 and 8) , the wetting transition results in multiple shapes ( lenses , hemispheres , and concave hemispheres ) , all of which are smooth deformations of a spherical surface . thus , if the boundary conditions are uniform at all surfaces of the lc , then the internal configurations of the lc in the presence of sds will be topologically equivalent to bipolar , axial / saturn - ring , and radial configurations ( schematics in figures 4 and 8a f ) . whether the anchoring of the lc at all surfaces of the caged droplets is uniform , however , remains to be established . it is possible that the caged lc droplets possess inhomogeneous anchoring , for example , due to differential adsorption of sds between the lc aqueous interface or the lc polymer interface ( figure 8g , h ) . in this scenario , due to inhomogeneous anchoring , the relationship of the topological charge and the euler characteristic is changed from that discussed above . the topological charges , however , will still satisfy gauss s theorem and will be determined by the specific profile of the director at the surfaces of the lc droplets . in figure 8g , h , we present two possible director configurations in the caged lc droplets that are consistent with the nonuniform anchoring of the lc at its surfaces . in each case polymer interface , providing a net topological charge of + 1/2 ( see above ) . as noted in the results section , the lc anchoring shown schematically in figure 8 g is consistent with the experimental observations shown in the micrographs in figure 8e , f . however , we do not observe birefringent textures that are consistent with the configuration in figure 8h . we end by noting that in the limit of 0 the complete wetting of lc on the inner surface of the capsule would lead to the formation of a shell of lc . this transition , although not yet observed in our experiments , leads to the creation of a new surface and thus the shell possesses an euler characteristic of + 4 ( and thus a topological charge of + 2 ) . for additional discussion of this geometry , we refer the reader to studies of lc shells created from double emulsions . in summary , the key advances reported in this article are two - fold . first , we provide insight into the physical processes that underlie the formation of caged lc droplets contained within cross - linked polymer multilayer capsules . we demonstrate that dadm capsules swell reversibly and that the extent of swelling correlates with the dielectric constant of the solvent . this result suggests principles by which the extent of filling of the capsules can be systematically varied in future studies . we emphasize that the partial filling of the capsules reported here underlies our observations of the lc wetting transitions . the second key advance involves surfactant - induced changes in both the wetting and ordering of caged lc droplets . whereas the effects of dtab and sds on the internal ordering of free lc droplets are similar , we reveal that the two surfactants trigger strikingly different wetting and configurational transitions when the lc droplets are caged within the polymeric capsules . specifically , the addition of anionic sds resulted in a progression of complex droplet shapes as the contact angle of the lc on the inner surfaces of the capsules decreased ; in contrast , no significant change in droplet shape was observed upon addition of cationic surfactant dtab . although additional measurements are needed to quantify the orientation of the lc at the surfaces of the complex shapes formed by the caged lc in the presence of the sds , under the assumption of uniform anchoring the various internal states of the droplets are topologically equivalent to axial and radial configurations ( i.e. , consistent with the conservation of topological charge ) . although topologically equivalent to free droplets , the contributions that surface anchoring , lc elasticity , and topological defects make to the free energy of caged lc droplets will differ from those of free droplets . as a result , caged lc droplets offer the promise of new and versatile sources for the design of lc - droplet - based responsive soft matter that can not be realized in dispersions of free droplets . we also note that the physics of caged lc droplets is conceptually similar to that of liquid - crystalline dna toroids confined in viral capsids . as such , caged lc droplets may offer the basis of a model system for understanding some aspects of dna packing in viruses .

we report a study of the wetting and ordering of thermotropic liquid crystal ( lc ) droplets that are trapped ( or caged ) within micrometer - sized cationic polymeric microcapsules dispersed in aqueous solutions of surfactants . when they were initially dispersed in water , we observed caged , nearly spherical droplets of e7 , a nematic lc mixture , to occupy 40% of the interior volume of the polymeric capsules [ diameter of 6.7 0.3 m , formed via covalent layer - by - layer assembly of branched polyethylenimine and poly(2-vinyl-4,4-dimethylazlactone ) ] and to contact the interior surface of the capsule wall at an angle of 157 11. the internal ordering of lc within the droplets corresponded to the so - called bipolar configuration ( distorted by contact with the capsule walls ) . while the effects of dodecyltrimethylammonium bromide ( dtab ) and sodium dodecyl sulfate ( sds ) on the internal ordering of free lc droplets are similar , we observed the two surfactants to trigger strikingly different wetting and configurational transitions when lc droplets were caged within polymeric capsules . specifically , upon addition of sds to the aqueous phase , we observed the contact angles ( ) of caged lc on the interior surface of the capsule to decrease , resulting in a progression of complex droplet shapes , including lenses ( 130 10 ) , hemispheres ( 89 5 ) , and concave hemispheres ( < 85 ) . the wetting transitions induced by sds also resulted in changes in the internal ordering of the lc to yield states topologically equivalent to axial and radial configurations . although topologically equivalent to free droplets , the contributions that surface anchoring , lc elasticity , and topological defects make to the free energy of caged lc droplets differ from those of free droplets . overall , these results and others reported herein lead us to conclude that caged lc droplets offer a platform for new designs of lc - droplet - based responsive soft matter that can not be realized in dispersions of free droplets .

w18o49 was prepared by using a solvothermal reaction , as described in refs . 14 , 17 . in a typical procedure , wcl6 ( 200 mg ) was dissolved in 1butanol ( 30 ml ) with sonication for 3 min . the obtained blue solution was transferred into a 50 ml teflonlined autoclave and kept at 200 c for 24 h , and then cooled to room temperature naturally . the product was washed with water and ethanol four times by using centrifugation . the product w18o49 samples with tunable oxidation states were prepared by addition of nano3 or nabh4 as the redox agent in a solvothermal system . w18o49 ( 3 mg ) was dispersed in deionized water ( 10 ml ) by ultrasonic treatment . then , dispersed w18o49 was measured by using a light absorption spectrometer to obtain the absorption spectra . the dispersed w18o49 ( 2 ml ) was sealed in a quartz cell and irradiated by using a 808 nm laser at a power density of 3.5 w cm to observe the temperature increase in every 30 s. when the system reached a stable temperature ( temperature change less than 0.2 c min ) , the laser was turned off . the above laser irradiation process was repeated for five cycles to evaluate the photothermal stability of rw18o49 . empyrean xray powder diffraction with cu k radiation over a range of 1070 ( 2 ) with 0.02 per step . xps ( thermo escalab 250 ) was performed by using monochromatic al k radiation ( 1486.6 ev ) . the epr spectra were obtained on a jesfa 200 epr spectrometer with a microfrequency of 9.45 ghz . uv / vis solid reflectance spectra were obtained by using a uv / vis solid spectrometer ( perkinelmer lambda 950 ) . w18o49 was prepared by using a solvothermal reaction , as described in refs . 14 , 17 . in a typical procedure , wcl6 ( 200 mg ) was dissolved in 1butanol ( 30 ml ) with sonication for 3 min . the obtained blue solution was transferred into a 50 ml teflonlined autoclave and kept at 200 c for 24 h , and then cooled to room temperature naturally . the product was washed with water and ethanol four times by using centrifugation . the product w18o49 samples with tunable oxidation states were prepared by addition of nano3 or nabh4 as the redox agent in a solvothermal system . w18o49 ( 3 mg ) was dispersed in deionized water ( 10 ml ) by ultrasonic treatment . then , dispersed w18o49 was measured by using a light absorption spectrometer to obtain the absorption spectra . the dispersed w18o49 ( 2 ml ) was sealed in a quartz cell and irradiated by using a 808 nm laser at a power density of 3.5 w cm to observe the temperature increase in every 30 s. when the system reached a stable temperature ( temperature change less than 0.2 c min ) , the laser was turned off . the above laser irradiation process was repeated for five cycles to evaluate the photothermal stability of rw18o49 . empyrean xray powder diffraction with cu k radiation over a range of 1070 ( 2 ) with 0.02 per step . xps ( thermo escalab 250 ) was performed by using monochromatic al k radiation ( 1486.6 ev ) . the epr spectra were obtained on a jesfa 200 epr spectrometer with a microfrequency of 9.45 ghz . uv / vis solid reflectance spectra were obtained by using a uv / vis solid spectrometer ( perkinelmer lambda 950 ) . 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 reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

abstractw18o49 with a tunable oxidation state was prepared by addition of nano3 or nabh4 as a redox agent in the solvothermal system . the addition of redox agents has no influence on the crystallization of w18o49 . the obtained w18o49 structures keep their morphology as a bundle of nanowires with a regular hexagonal on the crosssection . w18o49 exhibits strong valencedependent absorption features in the nearir region . reduced w18o49 with more w5 + has a higher concentration of oxygen vacancies , which enhances the localized surface plasmon resonance effect . reduced w18o49 exhibits a high photothermal conversion efficiency of 59.6 % and has good photothermal stability .

street vended food ( svf ) includes food and beverages that are prepared and sold outdoors or in public spaces by street merchants for consumption at the location or later without further preparation . poor hygiene and sanitation practices are one of the major bottlenecks in street food vending . salad can be defined as a food made primarily of mixture of raw vegetables and/or fruits . the possibility of foodborne diseases is more when salad vegetables are consumed without any heat treatment , sometimes without washing and peeling . water used for rinsing the vegetables and sprinkling to keep them fresh is also a source of contamination . fresh vegetables and fruits become contaminated with microorganisms during production , harvest , packing , and distribution . ready - to - eat fruit and vegetables requiring minimal or no further processing prior to consumption have been implicated as vehicles for transmission of infectious microorganisms of foodborne outbreaks related to gastrointestinal illness associated with fruit and vegetables . in this context the present study was undertaken to analyse the microbiological quality of salads served along with street foods of hyderabad city . the study was carried out in hyderabad which is the capital of andhra pradesh , india . the twin cities of hyderabad and secunderabad come under ambit of a single municipal unit , the greater hyderabad municipal corporation . for administrative purpose random sampling procedure was adopted to select four zones ( alwal : east zone , yousufguda : west zone , old city : south zone , and secunderabad : north zone ) . the sample required for the study personal interview of the street vendor was taken and flow chart was prepared from which critical control points of contamination were examined . data on salad preparation , handling , and storage practices was collected using structured questionnaire that had both observational and responsive questions . equipment used for the preparation of salad , source of water for both utensil cleaning and vegetables , utensil cleaning methods , and hand washing were considered . salad preparation , handling practices , storage of leftover salad , storage length , and salad storage practices were observed and noted . information on status of the premises , storage conditions for salad before cooking , cutting and chopping place , status of the serving plate , peeling and cleaning of vegetables , cleanliness of the cloth and clothing used , provision for waste disposal , presence of rodent droppings in the outlet , and exposure to insects was gathered . haccp was carried outin order to understand the critical points for the contamination ; information on source of purchasing of salad vegetables , their transportation , storage , and further processing before consumption was obtained through personal interviews of the street food vendors ( n = 53 ) . based on this information the process flow diagram was constructed . haccp is a system which identifies , evaluates , and controls hazards which are significant for food safety . the hazard analysis includes observing salad preparation and practices to identify the sources and modes of contamination . samples of hand washings , knife swab , and chopping board swab were collected from six street food vendors ( n = 6 ) and examined for presence of food pathogens . the polythene zip lock bags with salad samples were kept in an ice box maintained at 610 degree centigrade and processed within 24 hrs . twenty - five grams of each salad sample was weighed and transferred to 225 ml of sterile buffered peptone water . identification and enumeration of foodborne pathogens ( s. aureus , salmonella spp . , and yersinia enterocolitica ) were performed as described by standard methods of bacteriological analytical manual . after thoroughly mixing the food sample ( 25 g ) in buffered peptone water ( 225 ml ) by 1 : 10 dilution , the diluents were inoculated onto their respective selective media such as msa ( mannitol salt agar ) for staphylococcus aureus with small round pale colored colonies , xld ( xylose lysine deoxycholate agar ) for salmonella spp . with red colored colonies with black centre , and ysa ( yersinia selective agar ) for yersinia spp.with transparent colonies with pink centre . morphological tests such as grams staining and motility tests ( coagulase test for s. aureus ) were conducted followed by biochemical tests using hi assorted biochemical test kit ( a combination of 12 tests for identification of gram - negative rods . kits contain sterile media for citrate utilization , lysine utilization and ornithine utilization , urease detection phenylalanine deamination ( tda ) , nitrate reduction , h2s production test , and 5 different carbohydrates for utilization test : glucose , adonitol , lactose , arabinose , and sorbitol ) followed by latex agglutination test kit supplied by himedia . the analysis was carried out by descriptive analysis ( mean , minimum , and maximum ) for each category of the group . difference between the groups was tested by nonparametric kruskal - wallis anova considering the heterogeneity of variance . individual pair difference was tested by mann - whitney u test ( spss-14.8 windows version was used ) . microbiological analysis of salads indicated that both onion and carrot were contaminated with three pathogens . salmonella was present in 58% in carrots and 45% of onion samples . compared to onions , carrots harboured higher percentage of contamination ( table 1 ) . among the carrot samples procured from four zones of hyderabad , old city , that is , south zone , the mean concentration ranges of staphylococcus , salmonella , and yersinia in carrot and onion are shown ( table 2 ) . seventy - six percent of onion samples from east zone had staphylococcus which is high when compared to other zones . in old city , that is , south zone , 76% of onion samples had salmonella which is high when compared to other zones ( figure 2 ) . process flow diagram of onion and carrot indicated that , after peeling the outer skin of the onions , they were directly cut into small pieces and served without washing them . it was observed that the peeling which is very important step in processing of carrots was not done ; they were directly grated and served ( figure 1 ) . haccp study on contamination of salmonella in salads indicated that food handlers were responsible for salmonella contamination ( table 4 ) . most of the vendors got vegetables from whole sale market and 56% of the vendors stored them in their house and 44% of them stored it in the stall itself . about 98% of the vendors did not wash the vegetables before processing and 56% of the vendors did not peel carrots before cutting . ninety - two percent of the vendors used knife to cut the vegetables out of which 77% of the knives were wet and dirty and 88% of the vendors did not wash knife frequently . about 90% of the vendors ' cleaning cloths were untidy and none of them wore gloves before serving salads . there were about 60% of the vendors whose nails were uncut . about 64% of the stalls are adjacent to road ( table 3 ) . the study demonstrated that salads served along with popularly sold street foods are contaminated with one or many pathogens . a recent foodborne disease outbreak during 2011 in europe and germany was mainly due to contamination of salads such as cucumber , tomatoes , and lettuces by harmful strain of e. coli o104:h4 . a study conducted in dehradun , india , has shown that different types of salads such as carrot , radish , tomato , coriander leaves , turnip , onion , cucumber , and beetroot were contaminated with salmonella ( 32.28% ) , e. coli ( 33.14% ) , s. aureus ( 50.57% ) , and p. aeruginosa ( 60.85% ) . in this present study carrot and onion were chosen because they are the most commonly served salads in street foods of hyderabad . about 58% of carrots and 45% of onions contained salmonella and 68% of carrots and 24% of onions had yersinia . type of pathogens reported in salads , specifically with carrot , was e. coli o157:h7 , salmonella spp . , and l. monocytogenes and for onions was e. coli and salmonella typhimurium . the presence of microbes in salads can be linked to a number of factors such as improper handling and processing , use of contaminated water during washing and dilution , cross - contamination from rotten vegetables , or the use of dirty processing utensils like knife and trays [ 11 , 12 ] . the present study indicated that the bacterial load of carrots was high when compared to onions because carrots were not peeled . peeling as expected reduces the bacterial load ; a study done by ankitha et al . indicated that a reduction of about 40% of bacterial load was observed in peeled carrot . the contamination by staphylococcus aureus may be due to its carriage in nasal passages of food handlers or infected workers . the contamination of salmonella spp . in vegetables was due to washing of vegetables with contaminated water and handling of vegetables by infected workers , vendors , and consumers in the market place which helps spread pathogenic microorganisms . haccp study revealed that raw vegetables ( carrot and onion ) themselves carried pathogens and since they were not washed they continued to be present at the time of consumption . previous studies have demonstrated that salmonella cross - contamination occurs frequently through the use of contaminated vegetables that are improperly cleaned and undisinfected [ 1416 ] . most of the vendors were not using hand gloves while preparing and serving street food . similarly escherichia coli was detected in hand washings of high income and low income mothers in india at levels of 7.0 4.2 log 10 cfu / ml and 9.0 5.7 log 10 cfu / ml , respectively . a significant difference ( p < 0.01 ) was observed in load of salmonella spp . in wet , dirty chopping board when compared to the load of salmonella in clean and dry chopping board . similarly the load of yersinia spp . was high in wet , dirty chopping board when compared to the load of yersinia spp . was observed in the load of staphylococcus spp . in cleaning cloth which was dirty used by the vendor when compared to the load of staphylococcus in neat cloth used by the vendor . the pathogenic bacteria population was significantly higher in south circle because demographically this circle belongs to the old part of the city wherein the density of the population is high and most of the population was inhabited by lower income group which may be one of the factors contributing to poor hygienic practices . the present study revealed that the potential hazard of street vended salad vegetables may be due to handling practices of vendors and poor hygienic conditions in which they are sold . a simple thorough washing of vegetables with safe running water before further processing reduces the risk of microbiological hazards . there is a need to provide basic training on food hygiene to food vendors to ensure food safety and also create consumer awareness of consuming improperly processed salads as one of the possible sources of microbial hazards .

a study has been done to analyse the microbiological quality of salads served along with street foods of hyderabad . a total of 163 salad samples , 53 of carrot and 110 of onion samples , were collected from four different zones of hyderabad . about 74% and 56% had staphylococcus aureus in carrots and onions , respectively . fifty - eight percent of carrots and forty - five percent of onions samples contained salmonella , 68% of carrots and 24% of onions had yersinia . haccp study was carried out with 6 street food vendors to identify the source of salmonella contamination in salads . food handlers were found to be responsible for salmonella contamination in salads . the present study revealed the potential hazards of street vended salad vegetables , considering the handling practice usually carried out by vendors . ninety - eight percent of the vendors did not wash the vegetables before processing and serving while about 56.6% of the vendors did not peel the vegetables . majority of street vendors ' nails were uncut . a significant difference ( p < 0.01 ) was observed in yersinia spp . and salmonella spp . in wet - dirty chopping board when compared to clean - dry chopping board . a significant difference ( p < 0.05 ) of staphylococcus spp . was observed when the status of cleaning cloth was neat / untidy .

the study population included all persons in toronto for whom active tb had been reported to toronto public health from january 1 , 1998 , through december 31 , 2007 . data were extracted from the reportable disease information system and the integrated public health information system for all case - patients with a risk setting of shelter / rooming house or a risk factor of homeless . health case management files were reviewed to ensure accuracy of database entries ; additional data were abstracted when necessary . cases were included in the analysis for persons with active tb who met the following eligibility criteria in the year before diagnosis : 1 ) any shelter stay , 2 ) any rooming house stay , 3 ) no fixed address , or 4 ) use of services for homeless persons > 1 per week . cases were excluded for persons with active tb who 1 ) were foreign - born and received a diagnosis of active tb within 1 month of arrival in canada , 2 ) received a diagnosis of active tb while in a shelter designed exclusively for resettlement of newly arrived refugees , 3 ) were not residents of toronto when they received a diagnosis of active tb , or 4 ) had incomplete records . we collected data on patient demographics , clinical features of tb disease , medical management and health outcomes of patients , and molecular fingerprinting of the tb bacterium . case types were classified according to the public health agency of canada definition of new and re - treatment tb cases ( 14 ) . susceptibility testing for mycobacterium tuberculosis was performed at the central public health laboratory of the ontario agency for health protection and promotion . all isolates from new tb cases were tested for susceptibility to first - line drugs ( isoniazid , rifampin , pyrazinamide , and ethambutol ) according to recommended standard protocols , by using the commercial broth system , bactec mgit 960 ( becton , dickinson and company , sparks , md , usa ) . isolates resistant to rifampin or any 2 first - line drugs were also tested for susceptibility to second - line drugs ( 15 ) . restriction fragment - length polymorphism was performed for strain genotyping by using established methods ( 16 ) . genotypes were analyzed by using bionumerics 5.0 ( applied maths , saint - martens latem , belgium ) . hiv test results were recorded when available ; information about use of antiretroviral therapy for hiv / tb co - infected patients was not available . comparisons were made between canada - born and foreign - born case - patients and between 5-year periods ( 19982002 and 20032007 ) using the 2-sided fisher exact test or test , as appropriate . kaplan - meier plots were generated to determine time to death from all causes during the 12 months after tb diagnosis . the log - rank test was used to compare survival curves between the 2 groups . all analyses were performed by using sas version 9.1.3 ( sas institute , cary , nc , usa ) . from january 1 , 1998 , through december 31 , 2007 , a total of 3,685 active tb cases were reported to toronto public health ; among these , 102 ( 2.8% ) met the study inclusion criteria . incomplete records for 11 patients resulted in a final sample size of 91 ( figure 1 ) . most patients were absolutely homeless ( i.e. , living on the street or in a shelter ) ; 86 ( 95% ) patients reported staying in a shelter , having no fixed address , and/or using services for homeless persons > 1 per week . five ( 5% ) patients did not fall into any of these categories but had lived in a rooming house during the past year . birthplace was available for 88 patients ; nearly one third ( n = 28 ; 32% ) were born outside canada ( table 1 ) . the proportion of foreign - born patients increased over time from 24% ( n = 10 ) in 19982002 to 39% ( n = 18 ) in 20032007 ( table 2 ) . among the canada - born homeless persons with tb , 13 ( 22% ) were aboriginal . the number of reported cases of active tb over the study period by place of birth is shown in figure 2 . approximately equal numbers of cases were reported during the 2 periods : 44 ( 48% ) during 19982002 and 47 ( 52% ) during 20032007 . inclusion exclusion criteria for study of active tuberculosis ( tb ) in homeless persons , toronto , ontario , canada , 19982007 . * 1 of 22 countries that account for 80% of all new of tuberculosis cases annually ( high - burden countries ) . iqr , interquartile range ; na , not applicable ; , no statistical test performed ; amtd , amplified mycobacterium tuberculosis direct test . probability of a significant difference between canada - born and foreign - born persons for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . probability of a significant difference between the 2 periods for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . public health agency of canada definition : documented evidence or adequate history of previously active tuberculosis ( tb ) that was declared cured or treatment completed by current standards , and at least 6 mo have passed since the last day of previous treatment , and a diagnosis with a subsequent episode of tb that meets the active tb case definition . includes shelter screening , routine screening at other centers , and other detection methods . number of reported cases of active tuberculosis in homeless persons , toronto , ontario , canada , 19982007 . demographic information , clinical characteristics , and concurrent medical conditions for patients are presented in tables 2 and 3 . homeless persons with tb were often highly contagious at the time of diagnosis , as demonstrated by the large proportion of patients who had cavitating pulmonary disease and sputum smears with numerous acid - fast bacilli . the median duration of symptoms for persons with pulmonary disease before diagnosis was 2.5 months ( interquartile range 0.63.1 months ) . pulmonary disease was found in 67 ( 74% ) patients , among whom 29 ( 46% ) showed numerous acid - fast bacilli in sputum smear . the proportion of pulmonary tb patients with cavitary disease increased over time from 14% ( n = 5 ) in 19982002 to 32% ( n = 11 ) in 20032007 . * birthplace information available for 88 persons. , no statistical test performed ; copd , chronic obstructive pulmonary disease . probability of a significant difference between canada - born and foreign - born persons for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . probability of a significant difference between the 2 periods for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . includes cirrhosis , viral hepatitis b or c. in terms of treatment information and outcomes , 75% of homeless persons with tb started treatment within 4 days of diagnosis ( median 1 day ; interquartile range 04 days ) ( table 4 ) . most patients received closely monitored treatment within hospitals or as outpatients under directly observed therapy ( dot ) ( median treatment duration 2.0 and 6.2 months , respectively ) ; few received self - administered therapy for any substantial period of time . only 1 patient required a court order for treatment in a tb sanitarium . * birthplace information available for 88 persons . probability of a significant difference between canada - born and foreign - born persons for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . probability of a significant difference between the 2 periods for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . # 1 patient continues treatment at the time of this report . * * one foreign - born case - patient from 20032007 period died 14 mo after onset of treatment . after 12 mo , causes of death other than tb become more relevant ; therefore , deaths occurring after 12 mo are not included in this estimate . almost 1 of 5 ( n = 17 ; 19% ) patients died ( from any cause ) within 12 months of diagnosis ( table 4 ) ; most ( n = 12 ; 86% ) patients who died were born in canada ; 4 were hiv positive , 1 was hiv negative , and the remaining 12 had unknown hiv status . among patients who survived , most ( n = 70 ; 96% ) homeless persons with tb successfully completed treatment and only 3 were lost to follow - up or refused further care . probability of survival during the 12 months after diagnosis was lower for canada - born versus foreign - born homeless persons ( p = 0.06 ; figure 3 ) . no changes in survival probabilities were seen between the 2 periods , 19982002 and 20032007 ( data not shown ) . probability of death from all causes during the 12-month period after tuberculosis diagnosis among canada - born and foreign - born homeless persons with active tuberculosis , toronto , ontario , canada , 19982007 . of the 4 strains known to circulate among toronto s homeless population ( a , b , c , or d ) , isolates from 90% of canada - born patients belonged to one of these strains , and isolates from > 50% of foreign - born patients belonged to none of them . the proportion of isolates not belonging to these 4 strains increased over time , from 14% ( n = 4 ) during 19982002 to 32% ( n = 12 ) during 20032007 . almost all ( n = 84 ; 98% ) isolates were susceptible to first - line tb medications . only 2 isolates demonstrated evidence of antimicrobial drug resistance : 1 , from a canada - born patient , was resistant to ethambutol only , and 1 , from a foreign - born patient , was resistant to isoniazid only . probability of a significant difference between canada - born and foreign - born persons for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . probability of a significant difference between the 2 periods for each variable ; calculated by using the 2-sided fisher exact test or test , as appropriate . represents 1 canada - born person whose isolate was resistant to ethambutol only and 1 foreign - born case whose isolate was resistant to isoniazid only . homeless persons in our cohort received nearly all health care services for tb in hospital or under careful observation in the outpatient environment . all outpatients received dot , were accompanied by public health staff to all clinic visits , and received intensive social assistance . despite these efforts , all - cause mortality rates for our cohort were extremely high . among canada - born homeless persons with tb in our study , 20% died within 1 year of their diagnosis ; in comparison , among all persons with tb in toronto during 19992002 , only 7.4% died ( 17 ) . all - cause mortality rates for homeless populations in general are disproportionately high ; rates among men who use shelters in toronto are 28 higher than rates for the general population ( 18 ) . homeless persons often have more concurrent medical conditions ( e.g. , hiv , liver disease ) , mental health conditions ( e.g. , schizophrenia ) , and/or dependence on substances , any of which may raise their risk for primary or reactivated tb , complicate delivery of health services , and negatively affect treatment outcomes . our findings reflect the increased rates of illness and death among homeless persons and suggest that urgent measures are needed to improve tb treatment outcomes for this vulnerable population . recent research suggests that homeless immigrants in toronto are in general healthier and possess fewer concurrent illnesses than canada - born homeless persons , which may explain the lower prevalence of concurrent illnesses among foreign - born tb patients and the differences in mortality rates according to place of birth ( 19 ) . homeless tb patients represent 3% of all tb patients in toronto , of which a growing proportion are foreign - born , likely reflecting the changing demographics in the city overall and in the homeless population itself . our findings suggest that canada - born patients with tb were more likely to be infected with strains known to circulate within shelters and other social networks in toronto . in contrast , active tb in foreign - born patients was more likely to result from reactivation of latent infection with strains acquired overseas ( 20,21 ) . to date , drug resistance among homeless tb patients is rare ; laboratory evidence of drug resistance was demonstrated for only 2% of homeless tb patients compared with 14% of all culture - positive tb patients in toronto ( 1 ) . because being born outside canada is a known risk factor for drug - resistant tb , the rise in foreign - born homeless tb patients and the corresponding increase in heterogeneity of strain genotypes are concerning and may pose serious and growing threats to the homeless shelter system ( 20,2225 ) . the outbreaks of multidrug resistant tb in new york city during the 1980s and early 1990s highlight the potential dangers of introducing drug - resistant infections into the shelter system and call for increased prevention and control efforts ( 26 ) . despite the increase in foreign - born homeless persons with tb over time , most homeless tb patients in our sample were canada - born ( 68% ) , a substantial proportion of whom were of aboriginal origin ( 22% ) . by comparison , in 2008 , only 6% of persons with active tb in toronto were canada - born ( 1 ) . although tb in canada is primarily a disease of foreign - born persons ( 14 ) , our results suggest that tb transmission persists among canada - born inner city homeless populations . these findings also underscore the need to address tb transmission within the homeless shelter system . furthermore , the disproportionately high prevalence of canada - born aboriginal persons in our sample suggests that further efforts are needed to address the high incidence of tb in this population . homeless tb patients tend to seek care when disease is advanced and highly contagious , defined by abnormal chest radiographic findings ( cavitation ) and numerous acid - fast bacilli in sputum smear . although many homeless patients had pulmonary disease , the number was proportional to the prevalence of pulmonary disease among all tb patients in toronto ( 1 ) . in patients with pulmonary tb , an increasing prevalence of cavitary disease on chest radiographs was observed over time , despite increasing intervention and active case - finding initiatives during the more recent 5-year period of this study . however , the increase in cavitary disease could be related to delays in seeking health care , as indicated by the increase in median time from symptom onset to diagnosis over the 2 periods of the study . homeless tb patients often have difficulty accessing the health care system and may prioritize subsistence needs such as food and shelter over health services , especially those perceived as discretionary ( 27 ) . these factors , as well as cultural and language barriers among foreign - born patients ( 28,29 ) , may contribute to delays in seeking health care , which lead to advanced disease and hospitalization ( 27,30,31 ) . for our sample , hospitalization rates were high ; > 80% of patients were hospitalized . this is noteworthy in canada , where most tb patients are treated as outpatients , even at the time of diagnosis ( 32 ) . the inability to isolate infectious homeless patients in outpatient settings such as shelters largely explains the high rate of hospitalization for patients in our sample . adherence to treatment is often challenging for patients who are homeless or living in transient , substandard housing and who may have concurrent substance use or mental health problems . consequently , dot is usually implemented for homeless persons with tb in canada ( 32 ) . in the province of ontario , all patients with active tb are eligible to receive either inpatient or outpatient tb treatment , regardless of their insurance coverage . most patients in our sample received their entire treatment closely monitored within hospitals , with outpatient dot , or both . a few patients self - administered treatment for short periods . despite the common perception that homeless tb patients are noncompliant with treatment ( 3335 ) , 80% of patients in our sample completed treatment , which is equivalent to the treatment completion rate for all tb patients in toronto receiving dot ( 1 ) . intensive case management by public health and clinic staff as well as small incentives and enablers ( e.g. , food vouchers or cash ) helped ensure high completion rates for this population . for most homeless tb patients who did not complete treatment , the reason was that they died ; only a few were lost to follow - up or refused further care . the strength of this study is that it provides a comprehensive description of all cases of tb among homeless persons in a large , ethnically diverse city in canada over a 10-year period . only patients who were residents of the city of toronto at the time of diagnosis were included in the analysis ; consequently , homeless persons with active tb who may have been exposed in toronto shelters or rooming houses and later moved elsewhere were not detected . furthermore , homeless persons with tb were not included in the analysis if they had a history of shelter use > 1 year before diagnosis with active tb . as a result , some patients who acquired tb infection while homeless but who subsequently acquired housing may have been missed . furthermore , our retrospective study used public health surveillance data ; consequently , our analyses are subject to limitations in how the data were originally collected . we excluded 14 patients with active tb because their records were incomplete ; hence , we were unable to determine whether they differed demographically from included patients . this limitation could have influenced the results of our analyses that were stratified by birthplace . molecular fingerprinting data were unavailable for 20 isolates , which may have influenced the genotyping trends we observed over time . because we were unable to definitively determine the number of patients who died directly or indirectly as a result of tb , the mortality rates represent death from all causes in the year after tb diagnosis . although our study was conducted in a single metropolitan urban center , our findings and recommendations may be relevant to other large cities where levels of immigration and poverty are high . prior research among homeless persons in new york city shows decreasing trends in rates of active tb during 19922006 and demonstrates that public health prevention and control efforts ( e.g. , latent tb infection screening ) in this population can be effective ( 36 ) . in our study , several homeless patients were originally identified as tuberculin skin test positive contacts before active tb developed , but they were unwilling to start treatment for latent tb infection , were deemed poor candidates for treatment because of serious underlying medical or mental health conditions , or could not complete a course of treatment because of adverse drug reactions . lack of treatment for latent tb occurred despite substantial incentives and enablers for persons to initiate and continue therapy ( e.g. , cash for attending clinic visits , free passes for taxis or public transit , use of dot for latent tb infection ) . hence , for this cohort the opportunity to mitigate the risk for development of active tb through the treatment of latent tb infection was limited by the above challenges . primary prevention efforts should focus on shelter - based control measures , which have proven effective at reducing person - to - person spread of drug - resistant tb in other urban centers ( 37 ) . improved ventilation systems at shelters will help reduce the spread of tb during an outbreak ( 38,39 ) . smaller shelter sizes and strategies to reduce mobility ( e.g. , eliminating length of stay restrictions at shelters ) may also help limit the extent of transmission . additionally , expansion of sustainable housing programs for homeless and marginally housed populations will help reduce the number of persons needing to use shelters , subsequently decreasing the likelihood of tb exposure at these congregate settings . control of tb in homeless populations within canada will require further progress in primary prevention ( e.g. , improved ventilation and other infection control measures in shelters ) , secondary prevention ( e.g. , earlier detection and treatment of tb infection or disease through greater access to primary care ) , and tertiary prevention ( e.g. , treatment of active tb by health care providers with experience treating tb in homeless persons ) ( 17 ) . furthermore , canada s interconnectedness with the global community , and consequent interdependence with global tb , necessitates continued vigilance to confront the emerging threat of drug - resistant tb in the world ( 40 ) .

while tuberculosis ( tb ) in canadian cities is increasingly affecting foreign - born persons , homeless persons remain at high risk . to assess trends in tb , we studied all homeless persons in toronto who had a diagnosis of active tb during 19982007 . we compared canada - born and foreign - born homeless persons and assessed changes over time . we identified 91 homeless persons with active tb ; they typically had highly contagious , advanced disease , and 19% died within 12 months of diagnosis . the proportion of homeless persons who were foreign - born increased from 24% in 19982002 to 39% in 20032007 . among foreign - born homeless persons with tb , 56% of infections were caused by strains not known to circulate among homeless persons in toronto . only 2% of infections were resistant to first - line tb medications . the rise in foreign - born homeless persons with tb strains likely acquired overseas suggests that the risk for drug - resistant strains entering the homeless shelter system may be escalating .

fractures of the clavicle are one of the most common injuries to the bone in childhood , but posttraumatic nonunion of pediatric clavicle fractures are extremely rare , with only isolated reports in literature . we report a case of a posttraumatic painful nonunion of a clavicle fracture in a 13-year - old boy that caused symptomatic compression of the external jugular vein ( ejv ) and the formation of an arteriovenous fistula . the fracture was treated successfully with open reduction and internal fixation with a contoured recon plate 6 months following the injury . the patient made a full recovery 6 months following surgery and was asymptomatic with full range of shoulder movement . fracture union was confirmed by computed tomography ( ct ) scanning and no residual fistula was found . fractures of the clavicle are one of the most common bony injuries of childhood , accounting for 15% of the fractures in this population . the majority of these occur in the midshaft region and approximately half are displaced fractures . nonunion of the clavicle is a recognized complication in the adult population , with reported rates of between 1% overall and 15% in displaced fractures . it is an extremely rare complication in children , with seven published cases in the english literature . we describe a case of a posttraumatic clavicle nonunion in a 13-year - old boy which caused significant dilatation of the external jugular vein ( ejv ) as a result of a traumatic arteriovenous fistula . a 13-year - old boy was referred to clinic with a prominent dilatation on the right side of his neck that had been gradually enlarging since he sustained a displaced fracture of the middle third of his right clavicle 6 months previously . the patient was uninhibited in daily activities , but complained of ongoing pain at the fracture site and was concerned about his cosmetic appearance . his past medical history consisted of a congenital atrial septal defect with partial anomalous pulmonary venous drainage , which was surgically corrected at the age of 18 months . on examination , there was a nonpulsatile dilation in the right anterior triangle of his neck overlying the clavicle which was tender on palpation . there was no other visible deformity and he had full range of motion of the ipsilateral shoulder . ( b ) computed tomography ( ct ) three - dimensional ( 3d ) reconstruction demonstrating fracture union . ( c ) ct venogram showing enlarged right external jugular vein with no residual fistula present an ultrasound and computed tomography ( ct ) scan were arranged , which revealed a hypertrophic angulated nonunion of the clavicle fracture . the surrounding callus formation was shown to be compressing the ejv , impeding its drainage , and causing the striking distension measuring 2 cm in diameter . following multidisciplinary discussion , a decision was made to operatively reduce and fix the clavicle nonunion . however , an arteriovenous fistula was also discovered in a vascular cavity in the callus between a branch of the subclavian artery and the ejv . the fistula was excised and adjacent veins ligated and closed by vascular surgeon before the clavicle fracture was reduced and fixed with a six - hole contoured plate without the use of bone graft . follow - up , the ejv was less prominent and there was no residual pain around the shoulder . at 6-months follow - up , the patient was asymptomatic with full range of movement of the ipsilateral shoulder . radiographs and ct scans showed union of the fracture site with good alignment of the clavicle [ figure 1b ] . a ct venogram revealed an enlarged right ejv with a stenosed lower end and tortuous collaterals to the right internal jugular vein and subclavian vein , but no residual arteriovenous fistula [ figure 1c ] . complications associated with clavicle fractures include malunion , nonunion , thoracic outlet syndrome , vascular injury , and brachial plexus injury . fracture displacement is associated with an increased risk of nonunion in adults , although this does not seem to be a risk factor in the pediatric population . vascular complications as a result of clavicle fractures are rare , but are recognized as either an immediate complication due to transection of the vessel by the displaced fracture or as a late complication , secondary to compression from abundant callus formation . we are not aware of any other reported case in english literature that describes the formation of a traumatic arteriovenous fistula and symptomatic compression of the ejv following clavicle fracture . there are no known associations between congenital cardiac anomalies and the later development of arteriovenous fistulas . complications following pediatric clavicle fractures are uncommon and healing usually occurs within 4 - 6 weeks . a study by calder and colleagues recommended that children with isolated fractures of the clavicle with no complications at initial follow - up may be safely discharged from further follow - up . posttraumatic nonunions of clavicle fractures in children are extremely rare , with only isolated reports in the literature [ table 1 ] . all cases presented with hypertrophic nonunion between 6 and 72 months following injury and all but one case were treated operatively . compression plate fixation was the most common procedure , and half of the procedures also involved bone grafting of the fracture site . union was only achieved through surgery , although the functional outcome was satisfactory in all cases . pediatric posttraumatic clavicle nonunions in literature this is the first reported case of nonunion of a clavicle fracture causing symptomatic ejv compression and a traumatic arteriovenous fistula formation . this required operative fixation of the fracture and ligation of the fistula to achieve a satisfactory functional outcome for the patient . although current literature advises against routine follow - up for isolated pediatric fractures , the authors believe that follow - up for displaced pediatric clavicle fractures is good practice . open reduction and internal fixation reliably restores clavicle length and angulation , with low complication rates and good radiological and clinical outcomes for pediatric clavicle nonunions .

context : fractures of the clavicle are one of the most common injuries to the bone in childhood , but posttraumatic nonunion of pediatric clavicle fractures are extremely rare , with only isolated reports in literature.case report : we report a case of a posttraumatic painful nonunion of a clavicle fracture in a 13-year - old boy that caused symptomatic compression of the external jugular vein ( ejv ) and the formation of an arteriovenous fistula . the fracture was treated successfully with open reduction and internal fixation with a contoured recon plate 6 months following the injury . the fistula was treated by ligation and closure.conclusion:the patient made a full recovery 6 months following surgery and was asymptomatic with full range of shoulder movement . fracture union was confirmed by computed tomography ( ct ) scanning and no residual fistula was found .

alex krill and co - authors in 1969 , were the first to use multifocal choroiditis ( mfc ) to describe a non - infectious , idiopathic ocular disorder resembling the presumed ocular histoplasmosis syndrome nozik and dorsch in 1973 described a similar chorioretinopathy associated with anterior uveitis in two patients with fundus lesions similar to pohs , but with the presence of vitreous and anterior chamber inflammation , but no evidence of infectious disease . dreyer and gass in 1984 were the first to report mfcpu in a larger series of 28 cases confirming it as a distinct clinical entity from pohs . in the same year , watzke et al described ten myopic women with mfc but with smaller chorioretinal lesions and called these cases punctate inner choroidopathy these authors believed that pic is a distinctive entity in which small yellow - gray dots in the posterior pole with no signs of ocular inflammation develop into atrophic chorioretinal scars and become progressively more pigmented with time . joondeph and tessler in 1990 were the first to use the terms mfc and pic as inflammatory disorders distinct from pohs , which predominantly affect young myopic females . they described mfc as a chronic bilateral disease that was variably associated with vitritis , and often with anterior chamber inflammation which may also show progressive subretinal fibrosis and macular choroidal neovascularization . since then , there has been no evidence to support mfc and pic as separate entities : their clinical course and treatments are similar , and both conditions are idiopathic . causes of mfc lesions can be classified into infectious ( pohs , tuberculosis , brucellosis , coccidiomycosis , candidiasis and other fungal septicemias , syphilis , west nile virus , etc . ) , noninfectious [ sarcoidosis and other granulomatous diseases ( e.g. , blau syndrome ) ] , and idiopathic [ without uveitis , or with uveitis ( mfcpu ) ] . idiopathic mfc generally describes a relatively uncommon , chronic , inflammatory chorioretinopathy that predominantly affects young ( median age of 30 years ) healthy myopic white women with no known associated systemic disease or other recognized ocular syndromes , and a high risk of secondary cnv , circumscribed areas of chorioretinal atrophy and subretinal pigmented fibrotic scars . although idiopathic mfc may represent an ocular manifestation of an autoimmune disease in genetically susceptible patients , the precise etiopathogenesis still remains unclear . clinically , patients may complain of a temporal scotoma , metamorphopsia , floaters , photopsias , photophobia , and decreased vision . affected eyes typically show multiple punched - out chorioretinal lesions ranging from 50 to 350 m in size , both the posterior pole and periphery , often with clustering of lesions nasal to the disc , with minimal or no anterior uveitis or vitritis and no signs of progressive or diffuse subretinal fibrosis . other findings include peripapillary atrophy , scarring , and curvilinear chorioretinal streaks ( schlaegel lines ) in the far periphery [ figure 1 ] . patients have negative or nonreactive results on relevant diagnostic modalities ; such as histoplasmin skin reaction , syphilis ( non - treponemal and treponemal ) serology , tests for tuberculosis ( tuberculin skin test or interferon- release assays and chest radiographs ) , tests for sarcoidosis ( serum angiotensin - converting enzyme and chest radiograph or computed tomography scan ) and neoplastic / non - neoplastic anti - retinal antibodies . color fundus photograph of a 21-year - old man , presenting with blurred vision and floaters in both eyes over several months . note multiple , diffuse and discrete nummular outer retinal / choroidal white spots and equatorial linear / curvilinear streak lesions ( schlaegel lines ) in the left eye . primary vitreoretinal lymphoma ( also known as primary central nervous system lymphoma with ocular involvement ) , causes subretinal pigment epithelium ( sub - rpe ) nodular elevations about 100 m in diameter which demostrate autofluorescence and fluorescein angiographic findings similar to those seen in mfc . usually idiopathic mfc can be distinguished from masqueraders based on clinical manifestations , fundus imaging , and clinical course . peripheral mfc is a non - infectious type of the disease and strongly associated with sarcoidosis , characterized by a rather poor visual outcome , macular edema and relatively high prevalence of complications . mfcpu is a variant of idiopathic mfc that has been described in association with anterior uveitis and/or vitritis , multiple punched - out atrophic chorioretinal lesions of variable size ( typically 5001000 m in size ) in the posterior pole and midperiphery of the retina , older age and more visual impairment at presentation . this type of the disease is more frequently associated with a non - remitting clinical course and higher frequency of structural complications such as cataract , cystoid macular edema ( cme ) , and epiretinal membrane ( erm ) related to intraocular inflammation . mfcpu has a poor visual prognosis for both eyes , and normally requires higher levels of maintenance immunosuppression . haplotyping of elderly patients with mfcpu has shown significantly reduced frequency of hla b-7 , hla- dr1 , and hla- dr15 . some authors have used the term pic to designate the non - inflammatory aspect of the disease , but , most authors believe that idiopathic mfc and pic represent the same underlying disease entity and both are associated with identical interleukin-10 and tumor necrosis factor ( tnf ) haplotypes . idiopathic mfc generally describes a relatively uncommon , chronic , inflammatory chorioretinopathy that predominantly affects young ( median age of 30 years ) healthy myopic white women with no known associated systemic disease or other recognized ocular syndromes , and a high risk of secondary cnv , circumscribed areas of chorioretinal atrophy and subretinal pigmented fibrotic scars . although idiopathic mfc may represent an ocular manifestation of an autoimmune disease in genetically susceptible patients , the precise etiopathogenesis still remains unclear . clinically , patients may complain of a temporal scotoma , metamorphopsia , floaters , photopsias , photophobia , and decreased vision . affected eyes typically show multiple punched - out chorioretinal lesions ranging from 50 to 350 m in size , both the posterior pole and periphery , often with clustering of lesions nasal to the disc , with minimal or no anterior uveitis or vitritis and no signs of progressive or diffuse subretinal fibrosis . other findings include peripapillary atrophy , scarring , and curvilinear chorioretinal streaks ( schlaegel lines ) in the far periphery [ figure 1 ] . patients have negative or nonreactive results on relevant diagnostic modalities ; such as histoplasmin skin reaction , syphilis ( non - treponemal and treponemal ) serology , tests for tuberculosis ( tuberculin skin test or interferon- release assays and chest radiographs ) , tests for sarcoidosis ( serum angiotensin - converting enzyme and chest radiograph or computed tomography scan ) and neoplastic / non - neoplastic anti - retinal antibodies . color fundus photograph of a 21-year - old man , presenting with blurred vision and floaters in both eyes over several months . note multiple , diffuse and discrete nummular outer retinal / choroidal white spots and equatorial linear / curvilinear streak lesions ( schlaegel lines ) in the left eye . primary vitreoretinal lymphoma ( also known as primary central nervous system lymphoma with ocular involvement ) , causes subretinal pigment epithelium ( sub - rpe ) nodular elevations about 100 m in diameter which demostrate autofluorescence and fluorescein angiographic findings similar to those seen in mfc . usually idiopathic mfc can be distinguished from masqueraders based on clinical manifestations , fundus imaging , and clinical course . peripheral mfc is a non - infectious type of the disease and strongly associated with sarcoidosis , characterized by a rather poor visual outcome , macular edema and relatively high prevalence of complications . mfcpu is a variant of idiopathic mfc that has been described in association with anterior uveitis and/or vitritis , multiple punched - out atrophic chorioretinal lesions of variable size ( typically 5001000 m in size ) in the posterior pole and midperiphery of the retina , older age and more visual impairment at presentation . this type of the disease is more frequently associated with a non - remitting clinical course and higher frequency of structural complications such as cataract , cystoid macular edema ( cme ) , and epiretinal membrane ( erm ) related to intraocular inflammation . mfcpu has a poor visual prognosis for both eyes , and normally requires higher levels of maintenance immunosuppression . haplotyping of elderly patients with mfcpu has shown significantly reduced frequency of hla b-7 , hla- dr1 , and hla- dr15 . some authors have used the term pic to designate the non - inflammatory aspect of the disease , but , most authors believe that idiopathic mfc and pic represent the same underlying disease entity and both are associated with identical interleukin-10 and tumor necrosis factor ( tnf ) haplotypes . idiopathic mfc is a chronic progressive bilateral disease and one of the most common causes of visual loss in this entity is related to development of cnv . the rate of increase in the size and number of lesions and development of new or recurrent cme and/or cnv is also high despite the relative absence of anterior uveitis or vitritis . this observation reflects the need for long term follow - up and lends further support to the proposition that inflammation in idiopathic mfc can be confined to the outer retina / rpe . despite these and other complications , visual acuity can be maintained over many years and the visual prognosis is good in most cases . according to spaide et al the principal sites involved appear to be the sub - rpe and outer retinal spaces . acute lesions are often associated with more widespread involvement of the ellipsoid zone ( ez ) extending beyond the zones of rpe involvement , as discontinuity or elevation due to deposition of material , which may be homogenous or heterogenous . active lesions show a consistent appearance of rpe elevation due to deposition of homogenous material of medium reflectivity on sd - oct examination . the rpe elevations are usually conical although larger ones are broader in the lateral dimension but similar in height . some of the solid rpe detachments appear to rupture through the rpe resulting in an outpouring of infiltrates into the outer retina . with separation or attenuation of the rpe line , there is increased light penetration into the choroid and deeper structures . edi - oct imaging of the choroid beneath the active lesions show a small increase in choroidal thickness as compared with the surrounding choroid in some eyes . other than these rather subtle findings , there do not appear to be any other choroidal perturbations . by autofluorescent however , if there is dehiscence of the rpe , a corresponding spot of absent autofluorescence is observed . the infiltrative changes in the outer retina extend both above and laterally from the apex of the ruptured rpe elevation . in some patients as the inflammatory episodes continue to recur , additional heterogeneous zonal , multizonal or diffuse outer retinal atrophy may appear similar to the development of chorioretinal scars , and some may even progress to end - stage patterns of chorioretinal atrophy ( a new variant ) . this variant of idiopathic mfc with atrophy , involving the posterior pole and peripheral fundus , may demonstrate chorioretinal lesions and a spectrum of zonal , multizonal or diffuse outer retinal , rpe , and/or chorioretinal atrophy . there may be foveal sparing until late into the disease course in the involved eyes . this phenomenon is distinct from focal lesions seen in eyes with typical mfc . within the chorioretinal atrophic zones , spectral domain optical coherence tomography ( sd - oct ) and fundus autofluorescence ( faf ) inflammation may initially affect only the outer parts of photoreceptors and cause an associated visual field scotoma . as bouts of inflammation recur , continued progression to multizonal or diffuse disease may occur . eventually , involvement of the rpe and choroid leads to end - stage disease , which causes severe chorioretinal atrophy . foveal sparing is common until late in the disease course in these patients , and the majority of patients maintain good visual acuity . munk et al , however , reported eight patients with idiopathic mfc with atrophy associated with adjacent photoreceptor attenuation , an acute / subacute onset of decreased visual acuity and scotomata with vision loss as severe as counting fingers / hand motion . visual acuity fully recovered in only three of the eight cases after treatment with corticosteroids and/or systemic mycophenolate mofetil ( mmf ) but no other agents such as cyclosporine , methotrexate , azathioprine , infliximab , or tacrolimus were used . this variant of idiopathic mfc with atrophy , unlike classic acute zonal occult outer retinopathy , does not demonstrate the pathognomonic orange to - gray demarcating line on ophthalmoscopic examination and the typical trizonal degeneration , visualized on faf , indocyanine green angiography ( icg ) , and oct images . interestingly , genetic factors including inflammatory complement factor h have a strong association with mfc and age - related macular degeneration . having an underlying genetic susceptibility may predispose a single patient to more than one inflammatory disease . the occurrence of mfc in siblings also raises the possibility of a familial association in the pathogenesis of the disease . currently , when there is no evidence of cnv , there is no universally accepted standard treatment for the majority of patients with idiopathic mfc as the visual prognosis is excellent . the only exception to this would be those patients with inflammatory lesions very close to fovea in whom medical treatment should be considered . systemic corticosteroid therapy is typically the first - line treatment , and in conjunction with immunomodulatory therapy , has shown to reduce the amount of inflammatory infiltration of the subretinal space and outer retina , and reduce damage seen on multimodal imaging . some authors believe that single - agent immunosuppression is able to achieve treatment success in the majority of patients , but dose escalation of mycophenolate mofetil ( mmf ) is typical , and a second immunosuppressive agent is needed in about one - fifth of the patients . from the authors point of view , both anti - inflammatory and anti - vegf therapy ( in the presence of cnv ) are necessary in the acute stage to control the inflammation and cnv . however , when neovascularization has subsided there is no standard method of management based on clinical trials for long - term immunosuppression therapy of the quiescent stage in young female patients of child - bearing age .

idiopathic multifocal choroiditis ( mfc ) and/or punctate inner choroidopathy ( pic ) describe a chronic progressive bilateral inflammatory chorioretinopathy that predominantly affect healthy myopic white women with no known associated systemic or ocular diseases . the principal sites of involvement are the retinal pigment epithelium ( rpe ) and outer retinal spaces ; the choroid is not affected during the active phase of the disease . idiopathic mfc with atrophy is a recently described variant . although there is no generally accepted standard treatment , anti - inflammatory and anti - vegf ( vascular endothelial growth factor ) agents are necessary in the acute stage to control the inflammation and choroidal neovascularization ( cnv ) .

as an essential element , manganese ( mn ) is required for normal function of the central nervous system ( cns ) and other tissues . as with all other metals , manganese toxicity can occur with excessive exposure . a variety of clinical effects are associated with manganese toxicity , including manganism , a parkinsonian movement disorder that primarily affects dopaminergic and -aminobutyric acid- ( gaba- ) containing mid - brain structures that control motor functions . for example , workers exposed chronically to manganese can develop changes in visual reaction time , hand steadiness , and eye - hand coordination . these neurotoxic syndromes develop when either manganese intake is excessive ( e.g. , following high - dose oral , inhalation , or parenteral manganese exposure ) or when hepatobiliary clearance of this metal is impaired . this observation suggests that the dose of manganese delivered to target regions within the cns is the primary determinant for manganese neurotoxicity . environmental protection agency 's ( usepa ) list of hazardous air pollutants includes manganese compounds . the usepa and health agencies in other countries have raised concerns that chronic inhalation of low levels of manganese in ambient air may pose a risk to public health due to the possible accumulation of manganese in target tissues . these concerns prompted the usepa to call for a series of pharmacokinetic studies , as well as the development of physiologically based pharmacokinetic ( pbpk ) models for manganese as part of the testing requirements for the organometallic fuel additive methylcyclopentadienyl manganese tricarbonyl ( mmt , a registered trademark of afton chemical corporation ) . part i of this two part series discussed the development of the usepa 's alternative tier ii testing program for mmt that collected critical pharmacokinetic data for manganese in rodents and nonhuman primates . all test reports and correspondence related to the alternative tier 2 testing for mmt can be found in the federal docket management system ( fdms ) at http://www.regulations.gov/ identified by docket number epa - hq - oar-2004 - 0074 . one objective of the mmt alternative tier 2 program was to generate data to support the development of pbpk models for manganese [ 5 , 6 ] . key pharmacokinetic data needed to support pbpk model development and a paradigm for a tissue - dose - based health risk assessment for manganese were initially described by andersen and coworkers in 1999 and helped guide future studies . numerous animal experiments have subsequently addressed many of the data gaps raised by andersen and coworkers ( reviewed in [ 5 , 6 , 8 ] ) . the development of the pbpk models proceeded in a step - wise , iterative fashion with increasing model complexity being added at each step . table 1 provides an overview of the initial first generation models developed for this research program . the earliest dosimetry models were adapted from pharmacokinetic models developed for zinc , copper , and other essential metals that focused on dietary intake and deficiency . features of these models that were deemed important for manganese include features of these models that were deemed important for manganese include the ability to describe homeostatic control of an essential element under normal and deficient dietary conditions , and the use of compartmental and linear exchange rates to distribute the essential element into tissues and cellular compartments . the earliest manganese models were used to quantitatively test assumptions regarding the movement of manganese from the rodent gastrointestinal tract ( git ) and liver and to ascertain the degree to which systemic and orally derived manganese are handled similarly in the liver . the resulting pharmacokinetic models accurately described the decreased gastrointestinal ( gi ) manganese uptake and increased hepatobiliary elimination that is seen with rising levels of manganese in the diet . these models evaluated the kinetic behaviors of manganese in the brain , liver , and respiratory tract during and after manganese inhalation [ 12 , 13 ] . ultimately , manganese kinetics were best described using a model that included dose - dependent saturable tissue binding as well as free and bound manganese . in this context free manganese was rapidly cleared following exposure , thereby returning tissue manganese concentrations to their original basal levels . this rise of free brain manganese concentration was described with diffusion rate constants ( kin and kout ) . peak tissue manganese concentrations were constrained by the tissue maximal binding capacity ( bmax ) . importantly , dose dependencies predicted by the nong model were consistent with the total manganese tissue levels measured in rats following manganese inhalation . the model also replicated the rapid increases in tissue manganese concentrations seen at the highest inhaled manganese concentrations , as well as the rapid return to baseline after exposure ceased . the model developed by nong and coworkers for the adult rat incorporated these and other features and was used as the basis for all subsequent second generation animal pbpk models ( table 2 ) . starting in 2009 , the focus of the modeling effort began to shift to the development of more complete pbpk models for animals ( table 2 ) . these models retained many of the features found in the nong model , including dose - dependent saturable tissue capacities and asymmetrical diffusional flux of manganese into various tissue compartments . the second generation models also used airway deposition models based on particulate aerodynamics to describe manganese delivery to the respiratory tract . descriptions of the upper airways were broadened to include descriptions of the nasal cavity and olfactory epithelium using data published by schroeter et al . . regarding the cns , separate compartments for the olfactory bulb , striatum , pituitary gland , and cerebellum were developed . specific influx and efflux diffusion rate constants ( kin , kout ) and binding constants ( bmax , ka , kd ) for different brain regions were used to account for the differential increases in regional brain manganese concentrations seen under various experimental conditions . these modifications led to the publication of the revised adult rat model depicted in figure 1 . additional models were subsequently developed to describe lactational and gestational transfer of manganese in rats . in all cases , model output was compared to inhalation data obtained under this test program and that from the available literature . in 2009 , nong and coworkers also described the development of a pbpk model for nonhuman primates from the revised adult rat model . the monkey pbpk model was viewed as a critical step in the evolution of appropriate human models ( figure 2 ) . one goal of the modeling effort was to retain as many features present in the rat model as possible . body weight , tissue volumes , olfactory and respiratory tissues surface areas , ventilation rates , blood flows , and certain other model parameters were adjusted to describe monkey physiology while others ( biliary clearance and brain diffusional fluxes ) were allometrically scaled based on body weight . tissue - specific binding capacities were scaled to their respective tissue volumes while tissue - binding rate constants ( ka and kd ) were nearly constant from rat to monkeys . dietary uptake and basal biliary excretion rates were also adjusted to fit measured background tissue manganese concentrations . the final steps in the modeling program were to develop pbpk models for humans ( table 3 ) . the starting point for this effort was the monkey pbpk model developed by nong et al . with appropriate changes in physiological descriptions , allometric scaling of biliary clearance and brain diffusional fluxes to body weight , and small changes in tissue binding rate constants ( ka and kd ) . a significant change in the model involved the use of a more physiological description of the git to address an apparent delay in gi absorption evident in tracer mn studies in primates and the differential enterocyte turnover rates across lifestages . schroeter and coworkers included a series of gut compartments ( e.g. , gi lumen and epithelium ) to better describe the absorption of ingested manganese and storage of this metal . the epithelial linings of the small and large intestine have a high cellular turnover and contain rapidly proliferating cells ( enterocytes ) which replace those that are shed into the lumen . enterocytes are an important site for metal uptake and ultimately excretion through the sloughing of these cells . in our model , manganese transfer from the upper git epithelium to the lower git resulted from sloughing of enterocytes from the epithelial layer . the manganese found in shed enterocytes was ultimately excreted into feces without entering the systemic circulation . this allowed for the differential rates of enterocyte sloughing found in different life stages to be accounted for [ 19 , 20 ] . the fraction of manganese absorbed by the git ( fdietup ) and the biliary excretion rate constant ( kbilec ) were calibrated based on steady - state tissue concentrations and mn tracer studies . these changes in model structure were sufficient to capture the observed dose - dependent changes in manganese absorption by the git and biliary excretion by the hepatobiliary system . schroeter and coworkers used a step - wise approach to model development by first developing a revised monkey pbpk model , followed by an adult human model , which was validated by the available human mn tracer data . the final step in the modeling efforts culminated in the development of a model that described gestational and lactational transfer of manganese in humans . several homeostatic mechanisms have evolved to tightly regulate these tissue manganese concentrations within a normal range of values . for most tissues , normal manganese concentrations in humans range from 0.15 to 4 g mn / g of wet tissue . as noted earlier , manganese neurotoxicity occurs when manganese intake exceeds elimination , resulting in manganese accumulation in brain regions including the globus pallidus , which is particularly sensitive to manganese accumulation during overexposure . although manganese neurotoxicity is sensitive to exposure dose , it is relatively insensitive to route of exposure , as similar neurological responses have been linked to prolonged high - dose manganese inhalation , drinking water ingestion , long - term total parenteral nutrition ( tpn ) , or impaired manganese clearance because of hepatobiliary dysfunction . because of the ubiquitous nature of manganese and the role of dietary manganese in establishing steady - state tissue concentrations , risk assessments of inhaled manganese should consider the essentiality of manganese from diet to establish the tissue concentrations that will be altered with increasing levels of inhaled or ingested manganese . therefore , to understand the risk to humans from excessive manganese exposure , it is important to determine the exposure conditions that result in manganese concentrations in the brain that are increased significantly compared with brain manganese concentrations arising from normal dietary intake . pharmacokinetic models can be used to help establish safe exposure levels by predicting exposure conditions that lead to toxicologically significant increases in tissue manganese . one of the first attempts at applying pbpk models in scenarios relevant to human health risk assessment was performed by schroeter and colleagues . these investigators used their pbpk model to predict brain manganese concentrations in monkeys and people following subchronic manganese inhalation ( figure 3 ) . the predicted globus pallidus manganese concentrations for monkeys ( figure 3(a ) ) compared favorably with those observed by dorman et al . in monkeys subchronically exposed to manganese sulfate ( mnso4 ) , giving added confidence that the pbpk models were designed and parameterized appropriately . the human simulations performed by schroeter mimicked an 8 hr / day 5 day / week occupational exposure . the larger magnitude changes predicted in monkeys compared with humans at higher inhalation exposure concentrations may be due to saturation of manganese binding sites in the monkey at the higher manganese concentrations in the diet . human diets are typically low in manganese content compared to diets in laboratory animal chows , which are often supplemented to much higher ( ~100 ppm ) levels . at the lowest human exposure concentration used in our simulations ( 0.001 mg mn / m ) , the model predicted no appreciable increase ( < 1% change from basal concentrations ) in human globus pallidus manganese concentrations above the background levels associated with normal dietary exposure ( figure 3(b ) ) . at an exposure concentration of 0.01 mg mn / m , slight increases ( 5% ) in globus pallidus manganese concentration above background levels were predicted during the inhalation exposure period . more significant ( > 30% ) increases in globus pallidus manganese concentrations were predicted at the higher exposure concentrations ( > 0.1 mg mn / m ) . these data are consistent with derivations of benchmark concentrations for subclinical neurological effects from occupational studies at concentrations of 0.2 mg mn / m and indicate that significant increases in tissue manganese concentration above normal background variability are required for subclinical effects to be manifested . in light of our success in describing the rat and monkey tissue data and concordance with human responses and specific exposures , we conducted additional simulations using the available pbpk manganese models identified in tables 2 and 3 to address other exposure scenarios of concern to toxicologists and risk assessors . our goal was to predict tissue concentrations in individuals with altered physiology due to developmental life stage ( scenario 1 ) or disease ( scenario 2 ) . a second goal was to use the pbpk models to predict brain manganese concentrations with prolonged inhalation exposure and variable dietary manganese intake ( scenario 3 ) . the results of these simulations could help guide risk assessors in the application of intra- or interspecies uncertainty factors ( ufs ) as they establish exposure guidelines for the general public ( e.g. , an inhalation reference concentration or rfc ) or workers ( e.g. , threshold limit value or tlv ) . in most risk assessments , ufs are applied to lower the acceptable air concentration to protect potentially susceptible subpopulations or account for species differences in response . for example , the current us epa manganese rfc derivation incorporates a composite uf of 1000 that included ufs of 10 to protect sensitive individuals , 10 for use of a loael , and 10 for database limitations , such as less than chronic periods of exposure , inadequate information regarding developmental and reproductive toxicity , and uncertainty about the toxicity of various forms of manganese . the alternative pbpk model - based approach we present in this manuscript results in the development of pharmacokinetic chemical - specific adjustment factors ( csafs ) ( or data - derived extrapolation factors ( ddefs ) ) that could be used in lieu of default ufs used in most risk assessments [ 27 , 28 ] . a pharmacokinetic csaf is a ratio in humans or animals of a measurable metric for internal exposure to the active compound such as area under the curve ( auc ) ( auc is a surrogate for the daily and/or cumulative manganese dose received by an individual ) , cmax , or clearance between a baseline and potentially susceptible subpopulation . while serving the same purpose as ufs , these extrapolation factors are based on data directly pertinent to the chemical of interest , rather than having their basis on default assumptions about inter- and intraspecies variability . this approach leads to a higher confidence in the calculated adjustment factor and contributes to consistency in regulatory processes and decisions . unless otherwise noted , all simulations in these scenarios provide results for total tissue manganese concentration . consideration of potentially susceptible subpopulations based on lifestage and pregnancy statusage - related changes in physiology can influence the pharmacokinetics of xenobiotics , and some experimental data suggest that that the aged nervous system may be at increased risk following exposure to manganese . for example , manganese - induced depletion of striatal glutathione is more severe in aged ( 20 months old ) rats than in young ( 3-month - old ) rats following repeated ( 7 day ) high - dose ( 15100 mg mn / kg / day ) oral exposure to manganese chloride . occupational and environmental exposure studies indicate that increased age may be a risk factor for manganese - induced neurobehavioral deficits . to explore this question more quantitatively , we used the rodent pbpk model , as data were available in the literature regarding the degree to which pulmonary function declines with age.model simulations for aged rats ( figure 4 ) used a 25% decrease in minute volume consistent with reported reduction in pulmonary function in aged rats [ 22 , 23 ] . aged rats had lower target brain tissue manganese concentrations than middle - aged animals at the same exposures . this difference is likely due to the decreased breathing rates and pulmonary capacity of aged animals . since the manganese tissue concentration in the potentially susceptible subpopulation was less than in adult males , a pharmacokinetic csaf for the aged life stage should be < 1 . extending this simulation from rats to humans will require some changes to the physiological parameters used in the current models . for example , changes in liver volume and hepatic blood flow that significantly impact xenobiotic metabolism and clearance occur in geriatric human patients . likewise , aged humans have altered biliary , pulmonary , and gastrointestinal function that may alter manganese pharmacokinetics . these complexities require a human model simulation of this scenario that lies outside of the focus of the present manuscript . however , we have provided the template for generating a pharmacokinetic csaf for aged individuals . this preliminary analysis indicates that large adjustment factors may not be necessary to extrapolate from adult males , which typically are the subject of the occupational epidemiological studies that determine the point of departure in manganese risk assessments , to aged life stages.evaluation of other life stages is also important since they are also covered in the extrapolations from occupational studies by ufs . epidemiological studies in children have reported an association between elevated dietary manganese exposure and neurobehavioral and neurocognitive deficits . concerns for potential vulnerability to manganese neurotoxicity during fetal and neonatal development have been raised by regulatory agencies . pbpk models developed for this program can be used to quantitatively estimate the role that higher intestinal absorption of ingested manganese and a lower basal biliary excretion rate seen in children ( see for review ) affect dose - to - target - tissue during manganese inhalation . to this end , yoon and coworkers used their human pbpk model to estimate average expected daily aucs for the globus pallidus among different life stages ( nonpregnant , pregnant , and lactating women , fetuses , nursing infants , and 3-year - old children ) for three different exposure conditions , namely , 0 , 0.001 , and 0.01 mg mn / m for 24 hr / day 7 day / week ( see , figure 7 ) . exposure durations in the different simulations varied with life stage ( ~9 month pregnancy for women and their fetuses ; 4 months for nursing infants and their mother , and 3 years after birth for children ) . the average daily auc to the globus pallidus was calculated at the end of each simulation after running the model for appropriate durations for the selected life stages . pregnancy and lactation did not greatly affect the simulated internal dose of mn in the brain . the average daily aucs were greater in men than in women at high - dose environmental exposure of mn , that is , exposures where brain mn concentration started to rise from the basal level . the change in the average daily globus pallidus auc resulting from high - dose manganese inhalation was greater in nursing infants compared to other life stages due to the lower ( marginally deficient ) basal levels of tissue manganese at this age . for example , the estimated average auc in the highly exposed infants was approximately 11.5 g mnhr / g / day compared to approximately 7.9 g mnhr / g / day in unexposed infants , while the auc in the adult males increased from 9.3 g mnhr / g / day to 11.5 g mnhr / g / day . this apparent increase in sensitivity toward increasing tissue concentration is due to the lower basal tissue levels in neonates and likely represents the enhanced demand for manganese during this life stage [ 20 , 38 ] . both in adults and children after weaning , the relative contribution of inhalation ( 0.01 mg / m ) was smaller than their normal dietary intake at these inhalation exposures . because no life - stage achieved a higher auc than adult males , the pk simulations support a pharmacokinetic csaf of 1 for fetal , neonatal , pregnant , and nonpregnant female life - stages . age - related changes in physiology can influence the pharmacokinetics of xenobiotics , and some experimental data suggest that that the aged nervous system may be at increased risk following exposure to manganese . for example , manganese - induced depletion of striatal glutathione is more severe in aged ( 20 months old ) rats than in young ( 3-month - old ) rats following repeated ( 7 day ) high - dose ( 15100 mg mn occupational and environmental exposure studies indicate that increased age may be a risk factor for manganese - induced neurobehavioral deficits . to explore this question more quantitatively , we used the rodent pbpk model , as data were available in the literature regarding the degree to which pulmonary function declines with age . model simulations for aged rats ( figure 4 ) used a 25% decrease in minute volume consistent with reported reduction in pulmonary function in aged rats [ 22 , 23 ] . aged rats had lower target brain tissue manganese concentrations than middle - aged animals at the same exposures . this difference is likely due to the decreased breathing rates and pulmonary capacity of aged animals . since the manganese tissue concentration in the potentially susceptible subpopulation was less than in adult males , a pharmacokinetic csaf for the aged life stage should be < 1 . extending this simulation from rats to humans for example , changes in liver volume and hepatic blood flow that significantly impact xenobiotic metabolism and clearance occur in geriatric human patients . likewise , aged humans have altered biliary , pulmonary , and gastrointestinal function that may alter manganese pharmacokinetics . these complexities require a human model simulation of this scenario that lies outside of the focus of the present manuscript . however , we have provided the template for generating a pharmacokinetic csaf for aged individuals . this preliminary analysis indicates that large adjustment factors may not be necessary to extrapolate from adult males , which typically are the subject of the occupational epidemiological studies that determine the point of departure in manganese risk assessments , to aged life stages . evaluation of other life stages is also important since they are also covered in the extrapolations from occupational studies by ufs . epidemiological studies in children have reported an association between elevated dietary manganese exposure and neurobehavioral and neurocognitive deficits . concerns for potential vulnerability to manganese neurotoxicity during fetal and neonatal development have been raised by regulatory agencies . pbpk models developed for this program can be used to quantitatively estimate the role that higher intestinal absorption of ingested manganese and a lower basal biliary excretion rate seen in children ( see for review ) affect dose - to - target - tissue during manganese inhalation . to this end , yoon and coworkers used their human pbpk model to estimate average expected daily aucs for the globus pallidus among different life stages ( nonpregnant , pregnant , and lactating women , fetuses , nursing infants , and 3-year - old children ) for three different exposure conditions , namely , 0 , 0.001 , and 0.01 mg mn / m for 24 hr / day 7 day / week ( see , figure 7 ) . exposure durations in the different simulations varied with life stage ( ~9 month pregnancy for women and their fetuses ; 4 months for nursing infants and their mother , and 3 years after birth for children ) . the average daily auc to the globus pallidus was calculated at the end of each simulation after running the model for appropriate durations for the selected life stages . pregnancy and lactation did not greatly affect the simulated internal dose of mn in the brain . the average daily aucs were greater in men than in women at high - dose environmental exposure of mn , that is , exposures where brain mn concentration started to rise from the basal level . the change in the average daily globus pallidus auc resulting from high - dose manganese inhalation was greater in nursing infants compared to other life stages due to the lower ( marginally deficient ) basal levels of tissue manganese at this age . for example , the estimated average auc in the highly exposed infants was approximately 11.5 g mnhr / g / day compared to approximately 7.9 g mnhr / g / day in unexposed infants , while the auc in the adult males increased from 9.3 g mnhr / g / day to 11.5 g mnhr / g / day . this apparent increase in sensitivity toward increasing tissue concentration is due to the lower basal tissue levels in neonates and likely represents the enhanced demand for manganese during this life stage [ 20 , 38 ] . both in adults and children after weaning , the relative contribution of inhalation ( 0.01 mg / m ) was smaller than their normal dietary intake at these inhalation exposures . because no life - stage achieved a higher auc than adult males , the pk simulations support a pharmacokinetic csaf of 1 for fetal , neonatal , pregnant , and nonpregnant female life - stages . consideration of individuals with moderate - to - severe hepatobiliary dysfunctionindividuals with severe hepatobiliary disease often develop elevated brain manganese concentrations when compared with normal individuals . for example , rose and coworkers reported brain manganese concentrations in autopsy samples taken from patients with liver cirrhosis . when compared to age - matched controls , cirrhotic patients had significantly elevated brain manganese concentrations . these increases were most marked in the globus pallidus , where patients with severe cirrhosis had mean ( sem ) pallidal manganese concentrations of 4.04 1.54 g / g versus 1.41 0.91 g / g in controls . rose and coworkers also examined whether rat models using an end - to - side portacaval anastomosis or an intracholedochal injection of formalin and concomitant ligation of the bile duct to create an experimental model of biliary cirrhosis could mimic these findings . as expected , rats with biliary cirrhosis or a portacaval - shunt had increased pallidal ( increased by 27% to 57% ) and caudate / putamen ( increased by 57 to 67% ) manganese concentrations when compared with sham operated or normal control groups . it is important to note that the humans and animals studied by rose had significant clinical liver disease . for example , the cirrhotic patients studied by rose et al . died of an extreme form of hepatic encephalopathy ( i.e. , hepatic coma).individuals with liver or biliary cirrhosis can also develop significant changes in hepatic blood flow that may affect manganese pharmacokinetics . annet and coworkers reported that patients with chronic liver disease of various grades ( i.e. , child - pugh classes a , b , and c ) had mean ( sd ) apparent liver perfusion rates of 36.29 17.96 ml / min/100 ml liver volume versus 65.22 24.73 ml / min/100 ml liver volume in patients without liver cirrhosis ( as measured using magnetic resonance imaging ) . an approximately 50% reduction in total hepatic blood flow has been seen in dogs with side - to - side or end - to - side portacaval anastomoses . other investigators have reported significant ( 50% ) reductions in hepatic blood flow in rats with common bile duct ligation . these studies indicate that there should be a 50% reduction in hepatic blood flow in the pbpk model parameters for consistency with the observations of experimentally induced hepatobiliary dysfunction.impaired secretion of bile acids , bilirubin , and other organic anions , consistent with reduced biliary function , is also observed in liver disease . for example , rats with mild hepatic stenosis have an approximate 15% reduction in basal bile flow when compared with control animals . one animal model in which biliary function has been quantitatively examined is liver dysfunction induced by the subchronic to chronic administration of tpn , an intravenous diet given in severe cases of gi disorders . reported that rabbits receiving tpn will develop qualitatively similar decreases in bile flow ( reduced by 60% ) , bile acid secretion ( 52% ) , and sulfobromophthalein ( bsp ) excretion ( 38% ) when compared with control animals . thus , the available data support using a 50% reduction in bile flow in the pbpk model simulation.in this study , we used the schroeter et al . model to simulate globus pallidus manganese concentrations in humans following a one year inhalation mnso4 exposure to either 0.00005 ( the usepa rfc ) or 0.2 mg ( the current acgih tlv ) mn / m for 8 hr / d , 5 d / wk . model simulations for people with hepatobiliary impairment had a 50% decrease in liver blood flow and a concomitant 50% decrease in biliary excretion ( kbilec ) consistent with changes reported in people and/or animals with liver disease as discussed above . the simulations show that , regardless of inhalation concentration , the model predicted higher pallidal brain manganese concentrations due to hepatic dysfunction alone ( figure 5 ) , which was expected based on available data . inhalation at the rfc had no significant effect on pallidal concentrations , regardless of hepatobiliary function ( figure 5(a ) ) . inhalation concentrations at the tlv produced an increase in end - of - exposure pallidal manganese concentrations that were in addition to the increase from hepatobiliary disease ( approx . 0.85 g / g versus 0.68 g / g in controls , figure 5(b ) ) . a csaf of ~1.25 ( 0.85/0.68 ) is supported by the pbpk modeling for this extremely sensitive subgroup . since this csaf value was determined at occupational exposure levels , and no changes were observed at the rfc , this disease - related csaf is likely conservative for environmental exposures which do not cause tissue accumulation . individuals with severe hepatobiliary disease often develop elevated brain manganese concentrations when compared with normal individuals . for example , rose and coworkers reported brain manganese concentrations in autopsy samples taken from patients with liver cirrhosis . when compared to age - matched controls , cirrhotic patients had significantly elevated brain manganese concentrations . these increases were most marked in the globus pallidus , where patients with severe cirrhosis had mean ( sem ) pallidal manganese concentrations of 4.04 1.54 g / g versus 1.41 0.91 g / g in controls . rose and coworkers also examined whether rat models using an end - to - side portacaval anastomosis or an intracholedochal injection of formalin and concomitant ligation of the bile duct to create an experimental model of biliary cirrhosis could mimic these findings . as expected , rats with biliary cirrhosis or a portacaval - shunt had increased pallidal ( increased by 27% to 57% ) and caudate / putamen ( increased by 57 to 67% ) manganese concentrations when compared with sham operated or normal control groups . it is important to note that the humans and animals studied by rose had significant clinical liver disease . for example , the cirrhotic patients studied by rose et al . died of an extreme form of hepatic encephalopathy ( i.e. , hepatic coma ) . individuals with liver or biliary cirrhosis can also develop significant changes in hepatic blood flow that may affect manganese pharmacokinetics . annet and coworkers reported that patients with chronic liver disease of various grades ( i.e. , child - pugh classes a , b , and c ) had mean ( sd ) apparent liver perfusion rates of 36.29 17.96 ml / min/100 ml liver volume versus 65.22 24.73 ml / min/100 ml liver volume in patients without liver cirrhosis ( as measured using magnetic resonance imaging ) . an approximately 50% reduction in total hepatic blood flow has been seen in dogs with side - to - side or end - to - side portacaval anastomoses . other investigators have reported significant ( 50% ) reductions in hepatic blood flow in rats with common bile duct ligation . these studies indicate that there should be a 50% reduction in hepatic blood flow in the pbpk model parameters for consistency with the observations of experimentally induced hepatobiliary dysfunction . impaired secretion of bile acids , bilirubin , and other organic anions , consistent with reduced biliary function , is also observed in liver disease . for example , rats with mild hepatic stenosis have an approximate 15% reduction in basal bile flow when compared with control animals . one animal model in which biliary function has been quantitatively examined is liver dysfunction induced by the subchronic to chronic administration of tpn , an intravenous diet given in severe cases of gi disorders . . reported that rabbits receiving tpn will develop qualitatively similar decreases in bile flow ( reduced by 60% ) , bile acid secretion ( 52% ) , and sulfobromophthalein ( bsp ) excretion ( 38% ) when compared with control animals . thus , the available data support using a 50% reduction in bile flow in the pbpk model simulation . in this study , we used the schroeter et al . model to simulate globus pallidus manganese concentrations in humans following a one year inhalation mnso4 exposure to either 0.00005 ( the usepa rfc ) or 0.2 mg ( the current acgih tlv ) mn / m for 8 hr / d , 5 d / wk . model simulations for people with hepatobiliary impairment had a 50% decrease in liver blood flow and a concomitant 50% decrease in biliary excretion ( kbilec ) consistent with changes reported in people and/or animals with liver disease as discussed above . the simulations show that , regardless of inhalation concentration , the model predicted higher pallidal brain manganese concentrations due to hepatic dysfunction alone ( figure 5 ) , which was expected based on available data . inhalation at the rfc had no significant effect on pallidal concentrations , regardless of hepatobiliary function ( figure 5(a ) ) . inhalation concentrations at the tlv produced an increase in end - of - exposure pallidal manganese concentrations that were in addition to the increase from hepatobiliary disease ( approx . 0.85 g / g versus 0.68 g / g in controls , figure 5(b ) ) . a csaf of ~1.25 ( 0.85/0.68 ) is supported by the pbpk modeling for this extremely sensitive subgroup . since this csaf value was determined at occupational exposure levels , and no changes were observed at the rfc , this disease - related csaf is likely conservative for environmental exposures which do not cause tissue accumulation . consideration of dietary mn variability and chronic manganese inhalationthe strengths of using pbpk models in risk assessment include the ability to use the models to examine both dietary and inhaled intakes and support extrapolations from high to low doses , across routes , for different animal species , and for durations of exposure longer than those used in the studies that the models were based on . to date , the most complete pharmacokinetic datasets for inhaled manganese available for pbpk model development and validation were developed for rats and monkeys using exposure durations of up to 90 exposure days ( typically 6 hr / day , 5 days / week , reviewed in ) . the manganese pbpk models described in this manuscript can be used to extrapolate beyond these exposure conditions . this scenario demonstrates this ability by predicting globus pallidus manganese concentrations in people following a continuous ( 24 hr / day ) chronic ( 1 year ) manganese exposure ( figure 6).the monkey pbpk model developed by nong et al . scaled to humans was used to simulate globus pallidus manganese concentrations in people while varying the dietary intake . normal variation of manganese concentration in globus pallidus due to the fluctuation in daily dietary exposure was simulated in an adult human population of 10,000 using monte carlo techniques . these simulations varied the daily dietary intake of manganese using published data ( mean [ sd ] : 2.43 1.8 mg / day ; range 0.076.2 mg / day ) . published distribution values ( mean ) were used for body weight ( 70 kg ) , tissue volumes ( as % body weight ) for blood ( 8% ) , bone ( 12% ) , brain ( 2% ) , liver ( 3% ) , lung ( 1% ) , and the remainder of the body ( 0.74% ) [ 49 , 50 ] . distribution values ( mean ) for tissue blood flow ( as % cardiac output ) for bone ( 4% ) , brain ( 11% ) , liver ( 23% ) , and nose ( 1% ) were also used [ 49 , 50 ] . the coefficient of variation used for body weight , tissue volume , and blood flow parameters was 0.30 . mean values for cardiac output and pulmonary ventilation were set at 13 l / hr / kg and 20 l / hr / kg , respectively . all parameter distributions were truncated by two standard deviations and statistical correlations of parameters were not included in our analysis . air manganese concentrations ranged from current usepa rfc ( 0.00005 mg mn / m ) to 0.5 mg mn / m , a concentration that represents potential occupational exposure levels , although with continuous exposure in this case.a second question that we wanted to explore is the effect of exposure duration on the rate at which globus pallidus manganese concentrations change following manganese inhalation . here , we compared the end - of - exposure tissue concentrations of a subchronic ( 90-day ) versus 2-year exposure duration with the nonhuman primate model ( figure 7 ) . these simulations are in accord with observations reported by dorman and coworkers who reported that rhesus monkeys exposed ( 5 d / week ) for 15 , 33 , or 65 exposure days to mnso4 at 1.5 mg mn / m developed mean ( sem ) globus pallidus manganese concentrations of 1.92 0.40 , 2.41 0.29 , and 2.94 0.23 g mn / g tissue wet weight , respectively , all of which were significantly different ( p < 0.05 ) than background tissue levels of 0.48 0.04 g mn / g tissue wet weight . extending the simulation to 2 years produced a very slight leftward shift of the exposure - accumulation curve , but it did not change the threshold for tissue accumulation . for example , at 0.2 mg mnso4/m ( the current acgih tlv ) , a pharmacokinetic csaf for subchronic to chronic duration is 1.06 . once exposure passes the threshold for tissue accumulation , a pk csaf for duration of exposure is maximally 1.1.results of these simulations show several important findings . brain manganese concentrations are controlled over a wide range of low - to - moderate exposure conditions at and above typical environmental exposures . due to homeostatic controls , changes in globus pallidal manganese concentrations from exposures that exceed the current rfc even by several orders of magnitude ( 0.05 mg mn / m ) are small when compared to those seen as a result of normal variation in the dietary intake of manganese as demonstrated by the monte carlo analysis of dietary variation ( figure 6 ) . however , once these homeostatic mechanism(s ) are overwhelmed , pallidal manganese concentrations rise rapidly . the threshold for this response appears to occur at approximately 0.0010.01 mg mn / m ( figure 7 ) . the strengths of using pbpk models in risk assessment include the ability to use the models to examine both dietary and inhaled intakes and support extrapolations from high to low doses , across routes , for different animal species , and for durations of exposure longer than those used in the studies that the models were based on . to date , the most complete pharmacokinetic datasets for inhaled manganese available for pbpk model development and validation were developed for rats and monkeys using exposure durations of up to 90 exposure days ( typically 6 hr / day , 5 days / week , reviewed in ) . the manganese pbpk models described in this manuscript can be used to extrapolate beyond these exposure conditions . this scenario demonstrates this ability by predicting globus pallidus manganese concentrations in people following a continuous ( 24 hr / day ) chronic ( 1 year ) manganese exposure ( figure 6 ) . the monkey pbpk model developed by nong et al . scaled to humans was used to simulate globus pallidus manganese concentrations in people while varying the dietary intake . normal variation of manganese concentration in globus pallidus due to the fluctuation in daily dietary exposure was simulated in an adult human population of 10,000 using monte carlo techniques . these simulations varied the daily dietary intake of manganese using published data ( mean [ sd ] : 2.43 1.8 mg / day ; range 0.076.2 mg / day ) . published distribution values ( mean ) were used for body weight ( 70 kg ) , tissue volumes ( as % body weight ) for blood ( 8% ) , bone ( 12% ) , brain ( 2% ) , liver ( 3% ) , lung ( 1% ) , and the remainder of the body ( 0.74% ) [ 49 , 50 ] . distribution values ( mean ) for tissue blood flow ( as % cardiac output ) for bone ( 4% ) , brain ( 11% ) , liver ( 23% ) , and nose ( 1% ) were also used [ 49 , 50 ] . the coefficient of variation used for body weight , tissue volume , and blood flow parameters was 0.30 . mean values for cardiac output and pulmonary ventilation were set at 13 l / hr / kg and 20 l / hr / kg , respectively . all parameter distributions were truncated by two standard deviations and statistical correlations of parameters were not included in our analysis . air manganese concentrations ranged from current usepa rfc ( 0.00005 mg mn / m ) to 0.5 mg mn / m , a concentration that represents potential occupational exposure levels , although with continuous exposure in this case . a second question that we wanted to explore is the effect of exposure duration on the rate at which globus pallidus manganese concentrations change following manganese inhalation . here , we compared the end - of - exposure tissue concentrations of a subchronic ( 90-day ) versus 2-year exposure duration with the nonhuman primate model ( figure 7 ) . these simulations are in accord with observations reported by dorman and coworkers who reported that rhesus monkeys exposed ( 5 d / week ) for 15 , 33 , or 65 exposure days to mnso4 at 1.5 mg mn / m developed mean ( sem ) globus pallidus manganese concentrations of 1.92 0.40 , 2.41 0.29 , and 2.94 0.23 g mn / g tissue wet weight , respectively , all of which were significantly different ( p < 0.05 ) than background tissue levels of 0.48 0.04 g mn / g tissue wet weight . extending the simulation to 2 years produced a very slight leftward shift of the exposure - accumulation curve , but it did not change the threshold for tissue accumulation . for example , at 0.2 mg mnso4/m ( the current acgih tlv ) , a pharmacokinetic csaf for subchronic to chronic duration is 1.06 . once exposure passes the threshold for tissue accumulation , a pk csaf for duration of exposure is maximally 1.1 . brain manganese concentrations are controlled over a wide range of low - to - moderate exposure conditions at and above typical environmental exposures . due to homeostatic controls , changes in globus pallidal manganese concentrations from exposures that exceed the current rfc even by several orders of magnitude ( 0.05 mg mn / m ) are small when compared to those seen as a result of normal variation in the dietary intake of manganese as demonstrated by the monte carlo analysis of dietary variation ( figure 6 ) . however , once these homeostatic mechanism(s ) are overwhelmed , pallidal manganese concentrations rise rapidly . the threshold for this response appears to occur at approximately 0.0010.01 mg mn / m ( figure 7 ) . the scenarios that we have explored here can easily be broadened to address other concerns raised in relation to the human health risk assessment of manganese . another scenario that may prove useful to risk assessors is a consideration of the effect of altered iron homeostasis on manganese pharmacokinetics , since iron deficiency and iron - deficient anemia exist worldwide . inadequate tissue iron status resulting from dietary iron deficiency or anemia can lead to altered brain manganese deposition in animals [ 5254 ] . it is unknown whether interactions between iron and manganese for divalent metal transporter 1 ( dmt1 ) and other shared cellular membrane metal transporters account for this effect [ 55 , 56 ] . once these pathways and interactions are more fully elucidated , especially with quantitative measurements , these features can be incorporated into the existing pbpk models . although the pbpk models were originally created to support the risk assessment of combustion products of the fuel additive mmt ( see ) , they have much broader application to toxicologists and risk assessors . pbpk models can consider the impact of particle size and solubility on manganese dosimetry , especially as it relates to nanomaterials . for example , inhaled nanomaterials are deposited extensively in the nasal cavity ; in addition , a large percentage ( ~75% ) of the nanomaterials that reach the alveolar region remain at that site , with less than 5% of inhaled nanoparticles translocating out of the lungs ; charged nanoparticles are more likely to travel to the brain via axonal transport within the olfactory nerve than are neutral nanoparticles ; and nanoparticle size also influences organ distribution and renal excretion [ 61 , 62 ] . these models were parameterized and validated using experimental pharmacokinetic data collected for different nanomaterials ( e.g. , iridium , silver , or technetium - labeled carbon nanoparticles ) using data obtained from rats or humans . some work examined translocation of manganese oxide ( mno2 ) nanoparticles from the nasal cavity to the brain ; however , these investigators relied on the use of nasal instillation rather than inhalation . elder and coworkers exposed rats to mno2 nanoparticles with individual aerodynamic diameters of 38 nm ( note these particles form 30 nm agglomerates in the exposure system ) for 6 hr / day , 5 days / wk , for a total of 12 inhalation exposure days . in this study , the olfactory bulb showed the greatest changes in proinflammatory gene expression when compared to the midbrain , striatum , and other brain regions . this finding supports the conclusion that inhaled manganese nanoparticles , like larger particles , can undergo olfactory transport from the nasal cavity to the olfactory bulb . however , pbpk modeling in rodents and mri analysis in primates have demonstrated that the olfactory pathway does not appear to significantly impact manganese delivery to tissues outside of the olfactory pathway [ 67 , 68 ] . while research on manganese nanoparticles is still limited , there is some evidence that soluble manganese may be more bioavailable and cause more effects relative to equivalent amounts of nanoparticle manganese . whereas most of the deposited nanomaterial appears to stay in the lung , furthermore , manganese nanoparticles appear to be less toxic than an equivalent dose of soluble mn . daily intratracheal instillation of soluble mncl2 in rats for 36 weeks led to increased brain manganese levels , a reduction in body weight gain , and a decrease of open field motility when compared to controls , whereas the equivalent dose of mno2 nanoparticles had no significant effects . also , mno2 nanoparticles were less toxic to pc-12 cells in vitro by the mtt assay than an equivalent dose ( in ug / ml ) of soluble mn acetate . thus , the current pbpk modeling , based on soluble manganese ( mnso4 ) , may represent a worst - case scenario relative to nano - manganese after accounting for differences in pulmonary deposition . this expectation will be further clarified as more data become available comparing the pharmacokinetics and pharmacodynamics of nano- and soluble manganese . another potential application of the manganese pbpk models for risk assessment is the evaluation of the literature on the neurological outcomes of manganese exposure in primates , potentially identifying tissue concentrations that lead to adverse effects . the models could then be used to select the most appropriate dose metric for establishing a point of departure in future risk assessments . a previous attempt to evaluate dose response for the effects of mn in experimental animals relied on estimated cumulative intake of mn as the only measure for comparison across studies with different doses , durations , and exposure routes . alternative toxicologically relevant dose metrics , including estimated peak concentration , average concentration , and cumulative dose ( i.e. , auc ) during the mn exposure period could be estimated using a pbpk model known to accurately account for dose dependencies of mn distribution in the monkey for combined inhalation and dietary exposures . a large nonhuman primate response literature exists for analysis , including exposures by inhalation , oral , intraperitoneal , and subcutaneous dose routes , and spanned durations up to 2 years . this type of analysis using pbpk models is currently underway and will make it possible to provide a consistent description of the dose response relationship for the effects of mn independent of exposure route . finally , pbpk models may be used in an alternative dosimetric - based risk assessment strategy for essential elements considering dietary intake , natural tissue background levels , and dietary and population variability . an upper safe exposure value could be based on an air concentration that changes brain tissue levels by no more than some fraction of the normal variability within a healthy population [ 7 , 72 ] . the relationship between exposure levels and target - tissue levels would be determined by the use of pbpk models , which would account for the existence of the dose - dependent transition ( i.e. , threshold level ) for accumulation . this methodology is inherently conservative with respect to neurological outcome , as the air guideline would be set to prevent only tissue accumulation . potentially sensitive subpopulations as described in the scenarios here can be quantitatively taken into account with pbpk modeling instead of the application of ufs as described in the scenarios here . another key advantage of a pharmacokinetic approach for risk assessment is that it is not reliant upon existing occupational studies , which have limitations with respect to exposure assessment , evaluation of adverse effects , and establishing causation ( reviewed in ) , to establish a point of departure . similar pbpk model - based dosimetry approaches should also be considered for risk assessments with other essential metals , such as copper and zinc . however , the development of a comprehensive pbpk model for any essential element depends on the availability of a sufficiently diverse and robust data set to enable model construction and validation . these data now exist for mn , due in large part to the alternative tier 2 testing program for mmt . the development of pbpk models facilitates more rigorous quantitative analyses of the available pharmacokinetic data and allows the comparison and consideration of dose to target tissue in risk assessment decisions . while pbpk modeling for many exogenous compounds has become routine , there are significantly more challenges in understanding the full set of biological factors that control uptake , distribution , and clearance of manganese and other essential nutrients that exert toxicity at high doses . an overarching goal of our modeling efforts was to evaluate situations that may lead to increased brain accumulation due to altered manganese regulation in healthy and potentially susceptible human populations . these subpopulations , identified as part of the alternative tier 2 testing program for mmt , include adult males , females , the aged , fetuses , neonates , and pregnant women , as well as those with high or low dietary manganese intake . pharmacokinetic csafs were calculated to extrapolate from adult males , which are the typical subjects evaluated in occupational manganese studies , to these other life stages . these values were all 1 , indicating that no pharmacokinetic adjustment is needed to account for these populations . regarding duration of exposure , a csaf of 1 to 1.1 is calculated depending on the inhalation concentration once exposure levels increase above the threshold for tissue accumulation ( i.e. , 0.0010.01 ug / m ) . in addition , while diseased individuals are not typically included in the extrapolations done in typical health risk assessments of environmental exposure , we have now extended the simulations to include those with moderate to severe hepatobiliary insufficiency to represent a population at a higher risk of manganese effects . the simulations suggest that an impaired individual may have elevated brain manganese concentrations regardless of manganese inhalation levels , and typical environmental levels do not increase this burden . at higher exposure levels , a csaf of 1.25 is derived for the extremely sensitive subgroup . in total , the pharmacokinetic csafs developed here are less than the pharmacokinetic portion of the typical uf of 10 for human variability incorporated in current risk assessments of ambient manganese exposure . future efforts can refine the scenarios presented here in humans , examine the effect of iron homeostasis , and evaluate the effects of particle size and solubility , including manganese nanoparticles . a second outcome of these efforts is the increased confidence in the quantitative predictions of elevated manganese levels that might serve as a basis for a dosimetry - based risk assessment . an underlying assumption to this risk assessment approach is that elevated brain manganese concentration is a prerequisite for the development of manganese neurotoxicity . from a risk assessor 's perspective , an upper safe exposure value could be based on an air concentration that changes brain tissue levels by no more than some fraction of the normal variability within a population [ 7 , 72 ] . while some data gaps still exist regarding the biology of manganese transport and storage , the models described in this manuscript capture the main dose - dependent characteristics of manganese disposition . using a monte carlo analysis to simulate population variability on target tissue manganese levels , modeling simulations indicate that air manganese concentrations of 0.0010.01 mg / m are required to begin to influence natural background tissue concentrations in adult males , which are the most sensitive subgroup regarding manganese tissue accumulation currently examined . with the forthcoming evaluation of monkey studies of mn toxicity using the model to assess dosimetry and with applications to human datasets , the current pbpk model should be an important component of future tissue - dose - based approaches for mn risk assessment . in summary , data collection efforts associated with the alternative tier 2 testing program for mmt over the past 10 to 15 years on tissue mn after inhalation exposures at different dietary levels and associated pbpk modeling have greatly improved understanding of the integration of multiple processes that collectively control mn concentrations in various tissues . these efforts produced a multidose route , multispecies pbpk model that recapitulates dose - dependent brain accumulation on excessive exposures . the key biological characteristics required in fitting the model to the rat and monkey tissue time course data were finite capacities for tissue binding , slow dissociation of bound mn , dose - dependent elimination from liver and dose - dependent uptake from the diet . as expected with control process for an essential metal with high - exposure toxicity , the physiological processes preserve body stores of mn at low intakes accelerate excretion and reduce oral absorption at higher intakes . these dose - dependent processes are well known in a general manner , but not in terms of every biochemical detail . biological determinants for tissue binding , membrane transport , mn retention in enterocyte and sloughing of these cells into the intestinal lumen are under investigation . one area ripe for inclusion in the next - generation pbpk model is increased knowledge of metal transporters [ 7375 ] . more detailed dose - dependencies for these transporters would elaborate the asymmetric , dose - dependent uptake processes into brain in the present model . however , the relative rate constants can not vary significantly from the current ones since the simulations with the current model show very good correspondence with all available tissue time - course data . thus , despite some gaps in understanding the underlying biology , the pbpk modeling with mn shows clearly how similar processes work to control basal mn levels to a remarkably common concentration across species and how they accomplish control of tissue mn in the face of widely different dietary intake . controls for mn after inhalation include enhanced elimination but lack the ability to restrict absorption through the lung epithelium afforded by the gut epithelium for oral ingestion of the metal . clearly , the current human pbpk model stands poised to assist in further integration of emerging knowledge into a more quantitative and biologically complete description of regulation of mn in the body . future work , potentially including uncertainty and sensitivity analyses , will examine the inherent limitations of modeling and scaling to help determine the precise level of confidence that can be ascribed to the current predictions of human globus pallidus manganese concentrations . however , in its present form , the human pbpk model for mn already provides a solid foundation for improving risk assessment for this essential metal that also causes neurotoxicity in humans with higher dose exposures .

recently , a variety of physiologically based pharmacokinetic ( pbpk ) models have been developed for the essential element manganese . this paper reviews the development of pbpk models ( e.g. , adult , pregnant , lactating , and neonatal rats , nonhuman primates , and adult , pregnant , lactating , and neonatal humans ) and relevant risk assessment applications . each pbpk model incorporates critical features including dose - dependent saturable tissue capacities and asymmetrical diffusional flux of manganese into brain and other tissues . varied influx and efflux diffusion rate and binding constants for different brain regions account for the differential increases in regional brain manganese concentrations observed experimentally . we also present novel pbpk simulations to predict manganese tissue concentrations in fetal , neonatal , pregnant , or aged individuals , as well as individuals with liver disease or chronic manganese inhalation . the results of these simulations could help guide risk assessors in the application of uncertainty factors as they establish exposure guidelines for the general public or workers .

the ability of mesenchymal stem cells ( msc ) to differentiate along multiple pathways , together with their relatively easy isolation from bm and their extensive capacity for in vitro expansion , makes msc potentially the most attractive population among stem cells for tissue engineering and cell therapy applicable on a variety of congenital and acquired diseases [ 13 ] . in addition to their role in regenerative therapies , msc have been consistently shown to possess immunomodulatory properties , which may play a role in the maintenance of peripheral tolerance , the induction of transplantation - related tolerance and control autoimmunity [ 4 , 5 ] . there are preliminary data supporting their clinical efficacy in controlling steroid resistant graft versus host disease ( gvhd ) and improving engraftment of donor cells in the allogeneic stem cell transplant setting . however , the beneficial role of msc in gvhd prophylaxis is being questioned by small - scale randomized clinical trials [ 6 , 7 ] . further large - scale randomized studies are needed to clarify the benefits and hazards of msc administration for the prevention and therapy of gvhd . in all clinical protocols , msc have been isolated from bone marrow mononuclear cells ( bm - mnc ) and amplified in culture media supplemented with foetal calf serum [ 8 , 9 ] , or human platelet lysate . such protocols , however , result in a heterogeneous initial population of adherent bm cells , of which a significant proportion represents adherent monocytic cells . even when msc have been harvested after three or more culture passages ( > 4weeks culture time ) , the proportion of macrophages identified represented 144% of stromal cells [ 11 , 12 ] . recent studies have shown that positive selection using several surface markers including stromal ( stro)-1 , cd105 , cd271 and stage specific embryonic antigen ( ssea)-4 makes it possible to obtain a homogenous msc population without contaminating cultures with haematopoietic derived cells [ 1316 ] . however , no such an enrichment method has yet been used in clinical scale ex vivo expansion of msc . here we describe the isolation and ex vivo expansion of msc from bm - cd105 cells in culture media enriched with autologous human serum for clinical application . bm was harvested from the posterior iliac crest of 10 normal donors for a related stem cell transplant , aged 1030 years old , after informed consent , according to the ethical committee of the aghia sophia children s hospital . initially , we performed immunomagnetic isolation of bm - cd105 cells using milteny microbeads according to the manufacturer s instructions ( miltenyi biotech , bergisch gladbach , germany ) . after the isolation , bm - cd105 cells were suspended in dmem ( stem cell technologies , vancouver , bc , canada ) enriched with 10% autologous serum , and placed into a 75 cm flask ( corning life sciences , corning , ny , usa ) at a concentration of 2 10 cells / cm . fibroblastoid cells first became evident 2 to 4 days after inoculating culture flasks with cd105 cells ( fig . at this time - point cells were detached with 0.25% trypsin - ethylenediaminetetraacetic acid ( gibco brl , grand island , ny , usa ) for 5 min . at 37c counted and subsequently re - plated at 4 10 cells / cm in 175 cm flasks until confluency . cells derived from primary cultures of cd105 cells were defined as passage 0 ( p0 ) and each cycle of reseeding of msc after trypsinization was considered to be one additional passage . after a total time of 3 weeks , the cultures were discontinued . surface marker expression of msc derived from both primary ( p0 ) and p1 cultures were analysed by flow cytometry . cells were stained with monoclonal antibodies against human leukocyte antigen ( hla)-dr , cd14 , cd29 , cd34 , cd45 , cd31 , cd73 , cd105 , cd44 , cd73 , and cd90 . in order to investigate the multipotentiality of msc , msc derived both from p0 and p1 were directed towards the osteogenic , chondrogenic and adipogenic lineages . for osteogenic differentiation , cells were cultured in complete osteogenic differentiation medium ( stem cell technologies ) . after a culture period ranged from 3 to 5 weeks , monolayer cultures were analysed morphologically for the formation of large osteogenic nodules . for adipogenic differentiation , cells were cultured for 2 weeks in complete adipogenic differentiation medium ( stem cell technologies ) . chondrogenic differentiation was performed in high - density pellets containing 2.5 10 cells per pellet , cultured for 21 days in stempro chondrogenesis differentiation medium ( invitrogen , carlsbad , ca , usa ) . total rna was isolated from differentiated cells using rneasy mini kit ( qiagen gmbh , hilden , germany ) . one microgram of each rna sample was reverse transcribed with superscript iii ( invitrogen ) with use of oligo(dt ) . the resulting cdna was then used as a template for pcr amplification with platinum taq ( invitrogen ) ; the following genes have been inquired : alkaline phosphatase ( alp , f : tcagaagctcaacaccaacg ; r : gtcagggacctgggcatt ) for osteogenesis , lipoprotein lipase ( lpl , f : gagatttctctgtatggcacc ; r : ctgcaaatgagacactttctc ) for adipogenesis and collagen 2a1 ( col2a1 , f : ggaaactttgctgcccagatg ; r : tcaccaggttcaccaggattgc ) for chondrogenesis . to estimate the immune inhibitory function of msc , we used ki-67-antigen staining in mixed lymphocyte cultures ( mlc ) in the presence of msc as third party . mlc were carried out with human peripheral blood mononuclear cells ( hpbmc ) from two hla - drb mismatched individuals used as effectors and stimulators . effectors and stimulators were seeded in triplicate at the concentration of 1 10 cells/500 l / well in 24-well round - bottom plates ( nunc , roskilde , denmark ) in the absence ( control ) or presence of decreasing number of third - party mscs ( ratio msc / pbmc = 1 , 0.5 and 0.1 ) . after an incubation period of 96 hrs , intracellular expression of ki-67 was determined by immunohistochemistry . an array - based comparative genomic hybridization ( array - cgh ) analysis was conducted in order to investigate the genomic stabilility of msc . genomic dna was extracted from four msc samples derived at the end of expansion culture using the qiamp dna mini kit ( qiagen gmbh , hilden , germany ) . agilent human genome cgh 4 44k microarrays with an average spatial resolution of 12 kb were used in the study ( agilent technologies , santa clara , ca , usa ) . genomic dna from mscs and pooled reference dna of the same gender ( promega corporation , madison , wi , usa ) were digested with alui and rsai ( promega corporation ) and labelled using agilent genomic dna labelling kit according to manufacturer s instructions . mscs and reference dna were labelled with cy3 and cy5 , respectively , and were co - hybridized to arrays for 40 hrs at 65c in a rotating oven ( agilent technologies ) at 20 rpm . the arrays were then washed with agilent wash buffer and scanned by using an agilent microarray scanner . data were extracted using feature extraction 9.1 software ( agilent technologies ) and analysed using cgh analytics 3.4 software ( agilent technologies ) . genomic copy number changes were identified with the assistance of the aberration detection method 1 algorithm with a threshold of 6 . centralization and fuzzy zero corrections were applied to remove putative variant intervals with small average log2 ratios . for the location of genes in the deleted / duplicated genomic segments the ucsc ( http://genome.ucsc.edu/ ) and the database of genomic variants ( http://projects.tcag.ca/variation/ ) ( human genome build 18 ) were used . ( a ) morphology of immunomagnetically isolated bm - cd105 cells after they were cytocentrifuged and counterstained with may - grnwald solution . ( b ) three colour flow cytometric analysis of sorted cells demonstrated two distinct cd105 cell populations : cd105glycophorin - acd45 and cd105glycophorin - acd45 . ( c d ) fibroblastoid morphology of cells derived from cd105 cells after 3 ( c ) and 10 days ( d ) in culture before first passage ( primary cells ) . from bm samples of 1025 ml ( 16 ml average ) we were able to immunomagnetically isolate 5.4 10 0.9 10 cells . 1b ) : cd105clycophorincd45 ( erythroid cells ) and cd105clycophorincd45 cells ( mesenchymal cells ) comprising 78.6 16.2% and 18.6 4.8% of the total isolated cd105 cells , respectively , whereas the proportion of the latter in the initial bm mononuclear cell population was 0.16% 0.12% . light microscopic examination of cytospins prepared with freshly sorted cd105 cells disclosed , apart from typical erythroblasts , median size agranular cells with heterochromatic nuclei and numerous projections of the cell membrane resembling reticular cells ( fig . primary culture yielded 8.6 10 1.2 10 msc within a median time of 10 days . after replating at 4 10 cells / cm , p0 msc were further cultured for an additional period of 11 days , and resulted to a total production of 2.5 10 0.7 10 msc ( fig . 2 ) . flow cytometry revealed that p0 msc did not express certain haematopoietic and endothelial markers such as cd45 , cd31 , cd14 , cd34 and cd62l . on the other hand , cultured cells were found positive for cd105 , cd90 , cd73 , cd44 , cd29 and class i hla ( fig . growth profile of cd105 cells sorted on day 0 and plated at 2 10 cells / cm culture surface . on day 10 , adherent cells were detached with thrypsin and replated at 4 10 cells / cm in culture for an additional period of 11 days . boxes represent the interquartile range ; lines inside boxes represent the median value ; whiskers represent the lowest and highest observations , respectively . flow cytometric plots showing the immunophenotype of cultured cells at the end of primary culture on day 10 . gray histograms show specific antibody staining profile and open histograms show isotype control igg staining profile . msc from both primary and passage 1 ( p0 and p1 ) cultures treated with osteogenic medium underwent a change in their morphology from spindle shaped to cuboidal , and formed large nodules after 18 days of induction . chondrogenic , adipogenic and osteogenic differentiation were confirmed by col2a1 , lpl and alp expression , respectively ( fig . chondrogenic , adipocytic and osteogenic differentiation was demonstrated by the expression of specific genes col2a1 , lpl and alp , respectively . we next assessed the ability of msc to inhibit the alloreaction observed in mlc . as expected , mlc without msc led to an increase in proliferation of effector lymphocytes quantified by the percentage of ki-67 cells . both p0 and p1 msc , when added to mlc at msc / effector ratio of 1:1 , efficiently reduced the percentage of ki-67 cells by approximately 85% . with decreasing msc / effector ratios the proportion of proliferating effector cells was increased . however , even at low msc to effector ratio of 0.1 there was an inhibition of proliferating cells of about 2530% ( fig . 5 ) . hpbmc from healthy donors were co - cultured in one - way mlc with irradiated allogeneic pbmc in the absence ( control ) or presence of decreasing number of unrelated msc . at 96 hrs of culture , cells were stained for intracellular ki-67 antigen . bars represent the percentage of ki-67 cells in three independent experiments at 3 msc to effector ratios of 1 , 0.5 and 0.1 . we performed a whole - genome microarray - based cgh in order to look for chromosomal rearrangements with multiple loci involvement and for genome - wide aberrations in our msc samples from normal donors . cgh - plotter software showed balanced profiles for all four msc samples after culture expansion ( fig . we detected several small ( average size of 15 kb to 1.5 mb ) autosomal copy number variations , both deletions and duplications , which are also observed in normal individuals . the x - axis represents the 22 autosomes , the x and y chromosomes , and the y - axis shows the log2 of the fluorescence intensity ratio of all spots of the chromosome . every coloured vertical peak represents a commonly found copy number variations ; duplications are represented above the baseline with log2 ratio > 0.25 and deletions below the baseline with log2 ratio < 0.25 . our aim was to establish a culture system where sufficient numbers of pure msc could be generated within reasonable time for emergency type therapeutic strategies in the context of allogeneic stem cell transplant , like treatment of steroid - resistant gvhd and graft failure / rejection . it is important to note that msc contaminated with a low percentage of cd45 cells and msc of late passage do not exhibit in vivo significant anti - gvhd effect , as recently suggested , indicating that early passage and significant immunodepletion are required for msc - based cell therapy of gvhd . therefore , we had to develop an in vitro approach which would strictly fulfil two requirements : ( 1 ) the generation of a 2 10/per kg adult patient weight , that is the upper cell dose used for an adult patient , within a maximum of 3 weeks , capable to suppress the alloreaction in vitro and ( 2 ) the absence of any contamination of msc with haematopoietic or / and endothelial cells . based on our previous experience with chimerism studies of msc derived from immunoselected cd105 bm cells and the reduced seeding density approach established by prockop s group , we combined these methods aiming to obtain adequate numbers of msc with immunosuppressive properties within 3 weeks . starting with cd105 cells isolated from small volume bm samples , we achieved msc propagation which corresponded to a cell dose of > 2 10/kg for an adult patient within 3 weeks . more importantly , even msc from primary culture could be given to patients safely , because they were completely devoid of haematopoietic cells . we also investigated the maintenance of genomic stability using a whole - genome microarray based cgh at the end of the expansion procedure . although we analysed msc after a high in vitro proliferation rate , they did not show genomic alterations . by comparing our results with published data from clinical studies , similar numbers of msc were obtained after culturing bm - mnc isolated from > 40 ml bm samples for 46 weeks [ 23 , 24 ] . according to our own experience , when cultures started from bm - mnc population , a larger volume of bm is needed ( > 30 ml ) and the median time of expansion is much longer , almost 35 days to obtain adequate numbers for msc infusion . two recent , independent studies , however , described the successful production of a large msc number ( > 2 10 ) within fewer than 4 weeks derived from small bm aspiration samples [ 25 , 26 ] . both research groups were based on the fact that reduced msc seeding density results in marked increase of msc proliferation . nevertheless , the fact that the cultures were initiated with unmanipulated bm or bm - mnc and msc for clinical use were derived from primary culture or p1 , respectively , makes the contamination with haematopoietic cells a matter of concern . at the time of writing this report , poloni et al were able to produce 1 10 msc using bm - cd271 cells immunoselected from 5 ml bm in 30 days . however , they did not mention whether the produced cells possessed in vitro immunomodulatory abilities . in our hands , expansion cultures of cd271 cells using foetal bovine serum ( fbs ) or human serum did not produce msc solely , but also a percentage of neurosphere - like structures indicating a spontaneous differentiation to neural cell lineage . it is of major importance to use a rapid method to produce adequate numbers of uncontaminated msc with proven immunosuppressive potential . especially , in the clinical setting of resistant gvhd or graft failure , alternative treatments are required urgently , timing is crucial and shortening the culture period even by a few days can prove beneficial . the use of purified cd105 bm cells makes the method more expensive , but in terms of cost - effectiveness , the benefit from the time saved can prove valuable . we have shown that our method of deriving msc from enriched cd105-bm cells fulfils the criteria for a clinically applicable cell therapy protocol .

abstractcurrent clinical protocols used for isolation and purification of mesenchymal stem cells ( msc ) are based on long - term cultures starting with bone marrow ( bm ) mononuclear cells . using a commercially available immunoselection kit for enrichment of msc , we investigated whether culture of enriched bm - cd105 + cells could provide an adequate number of pure msc in a short time for clinical use in the context of graft versus host disease and graft failure / rejection . we isolated a mean of 5.4 105 0.9 105 cd105 + cells from 10 small volume ( 1025 ml ) bm samples achieving an enrichment > 100-fold in msc . seeding 2 103 immunoselected cells / cm2 we were able to produce 2.5 108 0.7 108 msc from cultures with autologous serum enriched medium within 3 weeks . neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product . expanded cells fulfilled both phenotypic and functional current criteria for msc ; they were cd29 + , cd90 + , cd73 + , cd105 + , cd45 ; they suppressed allogeneic t - cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential . moreover , comparative genomic hybridization analysis revealed chromosomal stability of the cultured msc . our data indicate that adequate numbers of pure msc suitable for clinical applications can be generated within a short time using enriched bm - cd105 + cells .

the liver is the most common site of hematogenous spread of primary colorectal carcinoma ( crc ) [ 1 , 2 ] and is affected in approximately 1025% of patients having surgery . surgical resection is the most effective and potential curative therapy for metastatic crc to the liver . the treatment strategies and outcomes for these patients have undergone many evolutionary changes [ 46 ] . minimally invasive laparoscopic surgery improves postoperative recovery , diminishes postoperative pain , reduces wound infections , shortens hospitalization , propagates rapid return to full activity , and yields superior cosmetic results , without compromising oncological outcome [ 7 , 8 ] . currently , laparoscopic resection of primary crc is performed in more than 40% of all patients in the netherlands according to the dutch surgical colorectal audit . however , the use of laparoscopy in liver surgery is still limited in the netherlands . there are several treatment options for crc patients presenting with synchronous liver metastases ( slms ) depending on primary tumor location ( rectum or colon ) and extent of hepatic disease . performing a simultaneous or staged resection of the primary tumor and liver metastasis is one of the items being discussed . recently , a systematic review showed that combined resection resulted in shorter hospitalization and fewer complications in comparison with staged resection although a tendency was seen towards a higher postoperative mortality after simultaneous resection . in contrast to the extensive literature on staged laparoscopic colorectal and laparoscopic liver surgery , there are only a few reports on combined laparoscopic colorectal and liver resection . the aim of this study is therefore to evaluate our initial experiences of simultaneous laparoscopic resection of primary crc and slm . five patients with primary crc and a clinical diagnosis of slm underwent combined laparoscopic colorectal and liver surgery between march 2011 and january 2012 in the academic medical center , amsterdam . treatment decisions in patients with crc and slm were based on location and complexity of resection of the primary tumor , extent of liver resection , feasibility of a laparoscopic approach , and physical condition of the patient . the institutional protocol of the academic medical center indicates that simultaneous resection is preferred in patients who require minor liver resection ( <3 segments ) independent of primary tumor location . neoadjuvant treatment preceding simultaneous resection is given for rectal primaries , consisting of short - course radiotherapy ( 5 5 gy ) followed by 3 to 6 courses of systemic chemotherapy ( capecitabine and oxaliplatin ) . if major hepatectomy is indicated , a liver first approach is preferred with neoadjuvant treatment consisting of 3 to 6 courses of systemic chemotherapy which is preceded by short - course radiotherapy in case of a rectal primary . systemic chemotherapy is completed in the adjuvant setting to a total number of 8 courses in patients with adequate clinical condition . all patients are discussed by the multidisciplinary team , and treatment is tailored to the individual patient with well - documented arguments for not following the institutional protocol ( i.e. , simultaneous right hemihepatectomy with right hemicolectomy in a fit young patient ) . laparoscopic colorectal surgery was performed using a medial to lateral approach with intracorporeal dissection and vascular control . for right hemicolectomy a pfannenstiel incision was used for specimen extraction in left - sided resections followed by an intracorporeal anastomosis using the double - stapling technique with circular stapler . complete laparoscopic liver resection was performed with the surgeon in between the patient 's legs . for metastasectomy in segments 7 and 8 , a hand - assisted laparoscopic approach was performed with the surgeon standing at the left side of the patient . total laparoscopic liver resection was started with insertion of a 10 mm trocar at the umbilicus followed by insufflation . for tumorectomy , two 5 mm trocars were positioned in the right and left upper abdomen . for left lateral sectionectomy , two 10/12 trocars were placed in each upper quadrant and an additional 5 mm trocar left subcostally . for tumorectomy in segments 7 and 8 , a hand port was placed via a vertical umbilical incision followed by a 10/12 mm trocar in the right lower quadrant and a 5 mm trocar in the midline above the hand port . in one patient , parenchymal transection was performed by using an ultrasonic dissection device with additional haemostasis using bipolar diathermy . the left segmental ( 2/3 ) portal pedicle and left hepatic vein were transected using a laparoscopic 60 mm stapler in case of left lateral sectionectomy . the liver specimen was put in a plastic bag in case of total laparoscopic resection and extracted via the umbilical or pfannenstiel incision . four men and one woman with a pathological diagnosis of crc and clinical or pathological diagnosis of slm were included . the average body mass index was 29.0 ( range 23.930.1 ) kg / m . one patient ( number 5 ) underwent laparoscopic resection of right - sided renal cell carcinoma two months earlier . during laparoscopy , a liver lesion was found and subsequent pet scanning and endoscopy revealed an additional sigmoid tumor . preoperative treatment of rectal cancer consisted of short - course radiotherapy ( 5 fractions of 5 gy ) followed by three to four courses of systemic chemotherapy ( oxaliplatin and capecitabine ) . the following procedures were performed : total laparoscopic right hemicolectomy with tumorectomy of segment 2 ( extraction via umbilical incision ) , total laparoscopic sigmoid resection with tumorectomy of segment 4/5 including the gallbladder and tumorectomy of segment 3 ( extraction via pfannenstiel ) , assisted laparoscopic low anterior resection and diverting ileostomy with total laparoscopic left lateral sectionectomy ( extraction via pfannenstiel ) , and two laparoscopic intersphincteric abdominoperineal excision with hand - assisted laparoscopic tumorectomy of segment 7 and segment 8 , respectively ( extraction via the umbilical hand port ) . the incision created for hand port and/or extraction of the specimen varied between 5 and 10 cm . median operation time was 303 minutes ( range 151384 ) with an estimated total blood loss of 700 ml ( range 200850 ) . intraoperative complications consisted of a small perforation of the right hepatic vein during liver mobilisation in one patient , which was sutured using an additional 5 mm trocar . one patient had surgery - related complications with perineal wound infection and delayed gastric emptying ( patient number 3 ) . other complications consisted of postoperative myocardial infarction necessitating reanimation and angioplasty ( patient number 1 ) , and pneumonia with delirium managed by intravenous antibiotics and haloperidol ( patient number 2 ) . an r0 resection of the primary tumor and liver lesions was achieved in all patients . definitive pathology of the liver in the patient with renal cell carcinoma and sigmoid carcinoma showed an adenocarcinoma originating from the upper gastrointestinal tract . at present , the origin of this third primary tumor is unknown and this patient receives palliative chemotherapy because of progressive liver disease . in two of the three patients who already had induction chemotherapy , systemic treatment the patient with myocardial infarction did not receive any ( neo)adjuvant systemic chemotherapy because of his poor physical condition . the advantages of performing laparoscopic colorectal or hepatic resections by experienced surgeons have led to a prominent increase for these operative approaches in recent years . separately for primary crc and crc liver metastases , laparoscopic resection has been shown to result in enhanced recovery and reduced morbidity with similar oncological outcome [ 7 , 8 ] . this suggests that a laparoscopic combined approach will also benefit patients who are candidates for simultaneous resection . open resection of both primary tumor and synchronous liver metastases often requires an extensive incision , especially if the location of the liver metastasis is opposite to the primary tumor location ( i.e. , right liver lobe and rectum ) . using a laparoscopic approach , exposure can be improved due to a magnified visualization from different angles , even in the narrow pelvis or not easily accessible places of the upper abdomen . the feasibility of a simultaneous laparoscopic approach has been demonstrated by our initial experience in five patients , and the results presented confirm findings from the limited literature on this topic ( table 4 ) . patients with a solitary , peripherally located metastasis in segments 26 are the most ideal candidates for simultaneous laparoscopic resection . two additional trocars can provide adequate access to the liver besides the standard trocar placement for the colorectal procedure . two patients in this series were diagnosed with a peripheral lesion in the posterior segments 7 and 8 , requiring full mobilization of the right liver . both liver mobilization and parenchymal transection could be accomplished in these patients using an umbilical hand port . others also described the use of a hand port placed in an upper midline incision for liver mobilization [ 12 , 17 ] . if major hepatectomy is indicated , the midline incision can subsequently be used for vascular control and parenchymal transection . but even total laparoscopic major hepatectomy in combination with colorectal resection has been shown to be feasible in three patients ( table 4 ) [ 23 , 25 ] . this allows for a small pfannenstiel incision to extract the specimens , which has been proven to result in the lowest incidence of incisional hernia . there are no randomized controlled trials comparing simultaneous and staged resections and the existing comparative studies are obviously difficult to interpret due to selection bias . theoretical arguments against simultaneous resection are the combination of a clean and contaminated procedure , and the impaired protein synthesis of the liver increasing the risk of infection and compromising anastomotic healing . however , according to a systematic review based on 14 comparative studies , combined resection was associated with lower morbidity . this led the authors to conclude that simultaneous resection can be undertaken in selected patients by surgeons specialized in both fields of colorectal and hepatobiliary surgeries . patient selection and expertise are essential for this complex type of surgery , and the multidisciplinary team should decide on optimal timing within multimodality treatment schedules . life expectancy of patients with liver metastasis from colorectal origin has been increasing as a result of improvements in liver surgery and systemic chemotherapy . repeat surgery for recurrent liver metastasis has been shown to have similar outcome compared with the first liver resection [ 2729 ] . an initial laparoscopic approach reduces adhesion formation and facilitates repeat resection , which has also been shown to be beneficiary in patients who need subsequent liver transplantation in case of patients with hcc . given the improved oncological outcome , quality - of - life issues related to abdominal wall integrity and cosmesis become more important , underlining the potential benefits of laparoscopy . in conclusion , our initial experience in combination with the previously published data indicate that simultaneous laparoscopic resection of primary crc and synchronous liver metastases is feasible and advisable in selected patients , provided that appropriate expertise is available .

introduction . simultaneous resection of primary colorectal carcinoma ( crc ) and synchronous liver metastases ( slms ) is subject of debate with respect to morbidity in comparison to staged resection . the aim of this study was to evaluate our initial experience with this approach . methods . five patients with primary crc and a clinical diagnosis of slm underwent combined laparoscopic colorectal and liver surgery . patient and tumor characteristics , operative variables , and postoperative outcomes were evaluated retrospectively . results . the primary tumor was located in the colon in two patients and in the rectum in three patients . the slm was solitary in four patients and multiple in the remaining patient . surgical approach was total laparoscopic ( 2 patients ) or hand - assisted laparoscopic ( 3 patients ) . the midline umbilical or transverse suprapubic incision created for the hand port and/or extraction of the specimen varied between 5 and 10 cm . median operation time was 303 ( range 151384 ) minutes with a total blood loss of 700 ( range 200850 ) ml . postoperative hospital stay was 5 , 5 , 9 , 14 , and 30 days . an r0 resection was achieved in all patients . conclusions . from this initial single - center experience , simultaneous laparoscopic colorectal and liver resection appears to be feasible in selected patients with crc and slm , with satisfying short - term results .

this cohort study included 44 consecutive patients with the following inclusion criteria : ( 1 ) age 1 - 16 years , ( 2 ) comitant esotropia 12 prism diopters ( ) , ( 3 ) hyperopia + 2.0 diopters ( d ; spherical equivalent ) , ( 4 ) patients cooperative for reliable measurement of ocular deviation , ( 5 ) minimum 3-month follow - up , and ( 6 ) 100% compliance to spectacle wear . exclusion criteria were ( 1 ) associated ocular comorbidity , namely , coloboma , nystagmus , albinism , or cataract , ( 2 ) poor fixation , and ( 3 ) systemic abnormalities , namely , birth asphyxia , cerebral palsy , or down 's syndrome . deviation was measured with the prism cover test using the age appropriate accommodative target for near ( 40 cm ) and distance ( 6 m ) . the measurements were taken at ( 1 ) presentation , without optical correction and without cycloplegia ; ( 2 ) after 3 days , under complete cycloplegia , without optical correction ; and ( 3 ) after 3 months of spectacle wear with full refractive correction and without cycloplegia . in patients with convergence excess ( ce ) , the near deviation was measured with + 3.0 d addition . in children < 3 years ( n = 7 ) , the corneal reflex test by krimsky 's method was utilized to measure the distance deviation . cycloplegia was achieved with 1% atropine eye ointment applied twice a day for 3 days and once on the day of examination ( fourth day ) . a child was diagnosed with refractive accommodative esotropia ( rae ) if the deviation was corrected to < 10 with spectacles [ fig . 1 ] ; partial accommodative esotropia ( pae ) if there was a significant reduction of the deviation ( 10 ) with spectacles , yet there was a residual esotropia of 10 [ fig . 2 ] ; and nonaccommodative esotropia ( nae ) if spectacle correction did not have a significant effect on the deviation [ fig . 3 ] . ce was diagnosed when the near deviation exceeded the distance deviation by 8 [ figs . serial face photographs of a child with rae showing ( a ) left esodeviation without refractive correction , ( b ) orthotropia with full spectacle correction , and ( c ) orthotropia under cycloplegia serial face photographs of a child with pae showing ( a ) right esodeviation without refractive correction , ( b ) residual esodeviation with spectacle correction , and ( c ) residual esodeviation under cycloplegia serial face photographs of a child with nae showing ( a ) right esodeviation without refractive correction , ( b ) no change in esodeviation with spectacle correction or ( c ) under cycloplegia , and ( d ) orthotropia after surgery serial face photographs of a child with refractive rae with ce showing ( a ) right esodeviation without refractive correction , ( b ) reduced esodeviation with spectacle correction , ( c ) orthotropia with spectacle correction and + 3.0 d addition , and ( d ) orthotropia under cycloplegia serial face photographs of a child with pae with ce showing ( a ) left esodeviation without refractive correction , ( b ) residual esodeviation with spectacle correction , ( c ) residual esodeviation with spectacle correction and + 3.0 d addition , and ( d ) residual esodeviation under cycloplegia serial face photographs of a child with nae with ce showing ( a ) orthotropia for the distance ( plano lens ) , ( b ) left esodeviation for the near ( plano lens ) , ( c ) orthotropia with + 3.0 d addition , and ( d ) left esodeviation under cycloplegia the ocular deviation under cycloplegia was compared with the ocular deviation after spectacle correction ( + 3.0 d addition for the near in patients with ce ) . pearson 's correlation coefficient ( r ) and intraclass correlation coefficient ( icc ) were calculated to assess the correlation . the sample size was calculated using the formula n = ( z1-/2 z1-)sd / d . at 5% significance and 90% power of the study , to detect the difference of 4 , with a standard deviation of difference 8 forty - four children aged 5.2 2.4 years ( sd , range 1 - 9 ) of whom 25 were males were included . all patients with rae and rae with ce had orthotropia under cycloplegia [ table 2 ] . in pae , the deviation measured under cycloplegia was equal to that measured with full refractive correction . in pae with ce , the deviation measured under cycloplegia was equal to that measured with full refractive correction with + 3.0 d addition . in the patients with nae , distribution and the angle of deviation of esotropic patients in various groups age , refractive error , and ocular deviation in different groups of esotropia a child with nae with ce ( nonaccommodative ce ) with emmetropia ( not included in the study ) had orthotropia with the bifocals but esotropia for near under cycloplegia [ fig . r for the ocular deviation under cycloplegia and full refractive correction ( with + 3 d addition in the patients with ce ) was 1.0 . hence the ocular deviation under cycloplegia was useful to predict the effect of spectacle correction on ocular deviation . in a previous study , in patients with rae , hyperopic lasik produced orthotropia when there was postoperative emmetropia and in patients with pae , residual esotropia remained despite a postoperative emmetropia . we treated a 3-year - old child with rae and amblyopia , whose esotropia disappeared after bilateral cataract surgery with intraocular lens implantation , despite a postoperative hyperopia of + 1.75 d in both eyes . it seems , in patients with accommodative esotropia , the central will to accommodate disappears with the full refractive correction of hyperopia / addition lenses if there is ce , after cataract extraction and under complete cycloplegia . complete cycloplegia for a prolonged duration is an absolute prerequisite for reliable measurements . with incomplete cycloplegia or shorter duration of cycloplegia , an accommodative effort a clinician should routinely perform dynamic retinoscopy and give adequate time for the cycloplegia to persist . in one patient with nae with ce ( hypoaccommodative convergence excess ) , probably the nonaccommodative / hypoaccommodative ce in these patients makes it impossible to block the convergence drive for near fixation even under cycloplegia after two days . we examined two of the pae patients nearly 3 years prior to their recruitment in this study . at that time they demonstrated orthotropia under cycloplegia during the rae stage and residual esotropia during the pae stage . another patient ( not included in the study ) had rae that rapidly decompensated to nae ; she had orthotropia under atropine during the rae stage and large esodeviation under atropine during the nae stage [ fig . another child with + 6.0 d in both eyes and intermittent exotropia developed consecutive rae ; after squint surgery ( not included in the study ) he demonstrated an orthotropia with full refractive correction and under complete cycloplegia [ fig . the measurement of deviation under cycloplegia could be helpful to differentiate the accommodative component from the nonaccommodative component in patients with esotropia and hyperopia . serial face photographs of a child with decompensated rae showing ( a ) right esodeviation without refractive correction , ( b ) orthotropia with full spectacle correction , and ( c ) orthotropia under cycloplegia . after decompensation , ( d ) right esodeviation without refractive correction , ( b ) right esodeviation with full spectacle correction , and ( c ) right esodeviation under cycloplegia serial face photographs of a child with consecutive rae following a surgery for intermittent exotropia : ( a ) left eye exotropia , ( b ) right eye esotropia following the surgery ( c ) , orthotropia with full spectacle correction , and ( d ) orthotropia under cycloplegia further studies are necessary to know whether or not the residual esotropia under cycloplegia will be controlled with fusional divergence once the deviation is reduced with spectacle correction and the effect of other cycloplegic agents .

this cohort study included children with esotropia and hypermetropia of + 2.0 diopters ( d ) . the deviation was measured at presentation , under atropine cycloplegia and 3 months after full refractive correction . of 44 children with a mean age of 5.2 2.4 years , 25 were males . eighteen ( 41% ) had fully refractive accommodative esotropia ( rae ) , 10 ( 23% ) had partial accommodative esotropia ( pae ) , and 5 ( 11% ) had nonaccommodative esotropia ( nae ) . eleven ( 25% ) had convergence excess ( ce ) . under cycloplegia , all with rae and rae with ce had orthotropia . there was no significant change in the deviation in the patients with nae . the deviation under cycloplegia and that with full refractive correction in pae and pae with ce ( with + 3.0 d addition ) were not different . the intraclass correlation coefficient for deviation under cycloplegia and after full refractive correction ( + 3.0 d addition for ce ) was 0.89 . it was concluded that ocular deviation under cycloplegia can help to predict the accommodative component in esotropia with hypermetropia .

dental anomalies can be classified into different groups such as anomalies of volume , anomalies of number , anomalies of form , anomalies of position and anomalies by union.1 fusion and gemination are developmental anomalies which occur due to the union of one or more adjacent teeth during development.2 fusion and gemination are terms frequently used to describe the clinical presentation of double teeth.2 knezevic et al2 examined 3517 plaster models in order to find the prevalence of double teeth ( fusion and gemination ) among the individuals tested based on gender , distribution in the maxilla or mandible , and whether the anomaly occurred bilaterally or unilaterally . the results of their investigation on a prevalence of double teeth appeared as 0.2% ; 57.2% of them fused and 42.9% geminated . it is generally accepted that fusion results from the conjoining of two teeth buds , while gemination originates when one tooth bud attempts to split into two.3 some authors included geminated teeth among cases of fusion in which union occurred to a supernumerary element . however , it is not known if fusion and gemination are part of the same developmental disturbance , or if they may be considered as pathological events leading to the formation of supernumerary elements.4,5 fused teeth are usually found in the mandible , while cases of geminated teeth are more frequently seen in the maxilla.6 this paper reports a rare case of a fused mandibular premolar with supernumerary tooth and its treatment with combined orthodontic and endodontic treatment . the patient was a 15 year old girl seeking orthodontic treatment for the correction of maxillary and mandibular crowding . extra - oral examination revealed a symmetrically proportionate face and a convex profile ( figure 1 ) . intra - oral examination indicated that the patient had an angle class i malocclusion with an anterior tete a tete bite , and a midline deviation of 3 mm to the right at the mandibular dental arch . the left mandibular dental arch presented fused mandibular premolar with supernumerary tooth ( figure 2 ) . the panoramic radiographic examination disclosed that all the secondary teeth were present but there seemed to be an extra tooth between mandibular left canine and first premolar ( figure 4 ) . the periapical radiography showed a fused tooth with two separate pulp chambers and two separate root canals connected in the apical third area ( figure 5 ) . the goals of the treatment were as follows : maintain angle class i relationshipmaintain the overjet and overbitecorrect the midlinealign the teeth in both arches and achieve good intercuspation . maintain angle class i relationship maintain the overjet and overbite align the teeth in both arches and achieve good intercuspation . after initial leveling , the fused teeth required non - surgical endodontic treatment for stripping , which was performed below cemento - enamel junction to correct midline deviation and preserved class i canine relationship . when a supernumerary tooth is joined with a permanent tooth , it is clinically hard to distinguish between fusion and gemination . due to the difficulties about the discrimination of fusion and gemination in the permanent dentition , mader7 suggested that all permanent teeth joined by dentine should be regarded as fusion . according to bashkar8 gemination occurs when a germ is divided in two but not in total , forming one root and two crowns . on the other hand , in fusion law et al9 declares that gemination is a similar dental malformation but consists of one pulp canal and one root , whereas in fusion the teeth are combined by dentine and have independent pulp canals . the clinical and radiographic examination reveals two pulp chambers where the crown and the roots are combined through the dentine and cement level . moreover , the pulp chambers of these teeth are also joined through the apex level . it is considered in terms of aesthetic and functional values that the treatment of the fused teeth requires a complicated multidisciplinary approach . as described in the previous studies , a possible treatment method would be extraction of the tooth after the completion of the canal therapy of the fused tooth , removal of the unwanted part and reimplantation into its original site.10,11 however , depending on the information obtained from the previous studies , ankylosis might be expected on the root surface of the reimplanted tooth . this arises due to the lack of periodontal membrane on the root surface.12 taking this information into account , in order to provide proper aesthetics and function , an alternative treatment approach was planned to avoid the ankylosis which may develop after reimplantation of the tooth , where the excess part was removed following the root canal treatment . after performing the canal therapy on the fused tooth , selective stripping was done on the unwanted supernumerary tooth to improve the crowding and the deviated midline . after the endodontic treatment of the fused teeth , the stripping of the supernumerary tooth was performed to establish a class i canine relationship and to correct the midline deviation . at the end of the treatment , the crowding was resolved as well as positive overjet and overbites were achieved ( figures 6 , 7 and 8) . after successful endodontic treatment , there was no need for surgical approach . we believe that under normal circumstances , stripping could be performed below cemento - enamel junction . as a result , significantly improved facial appearance with a satisfactory smile , vastly improved occlusion , and increased self - esteem can be achieved .

the objective of this report is to describe combined orthodontic and endodontic treatment of a fused mandibular premolar with supernumerary tooth . the patient was a 15 year old girl seeking orthodontic treatment for the correction of maxillary and mandibular crowding . cephalometric examination revealed skeletally class i relationship . the panoramic radiograph showed a fused tooth with two separate pulp chambers and two separate root canals connecting in apical third . after the endodontic treatment of the fused teeth , the stripping of the supernumerary tooth was performed to establish a class i canine relationship and to correct midline deviation . at the end of the treatment , the crowding was resolved and positive overjet and overbite was achieved .

early childhood caries is again on the rise in australian children despite considerable public health initiatives , including fluoridation of drinking water and use of fluoridated toothpaste . dental caries continues to affect large numbers of children with nearly 50% of australian 6-year - olds having a history of decay in their primary teeth and 10% having at least 8 affected teeth . for children under the age of 15 years , dental procedures are the most common reason for undergoing a general anaesthetic in australia . dental caries is not only affecting the most vulnerable people in our community , leading to significant human costs of pain , discomfort , and issues of self - esteem , but management of dental caries is also associated with considerable financial cost to individuals and governments . a greater understanding of the behaviour of cariogenic bacteria in the oral environment , together with improved knowledge of the nature of the interplay between a person 's genetic makeup and their exposure to environmental factors , should lead to better methods for assessing caries risk and , in turn , establishing more effective prevention strategies . caries is recognised as a multifactorial disease as a result of the findings of many studies that have investigated the ecology of dental plaque , including the different types of microflora that may be present , the levels of various oral bacterial species , and also the patterns of microbial transmission observed within families [ 35 ] . until recently , relatively little was known about the role of genetic factors in dental caries initiation in humans . our focus in this paper is to throw new light onto how genetic and environmental factors influence observed variation on the timing of colonization of streptococcus mutans ( s. mutans ) in the oral cavities of a large sample of monozygotic ( mz ) twins . s. mutans is the most well - documented species of the microbiological genus mutans streptococci ( ms ) . ms are generally considered to be some of the major pathogens associated with the process of dental caries , and s. mutans is frequently isolated from carious lesions . ms exist as part of the oral biofilm 's ecosystem , and they are characteristically anaerobic , acidogenic , aciduric , and carbohydrate metabolizers . the oral microenvironment consists of many different bacteria , and it is the balance of these bacteria that determines both health and disease of the oral tissues . several models have been proposed to explain the commencement and progression of dental caries . the extended caries ecological hypothesis explains the caries process , comprising a stable stage , an acidogenic stage , and an aciduric stage . there is a shift in bacteria within the oral biofilm from non - ms and actinomyces at the stable stage , to ms and lactobacilli in the aciduric stage , although it is possible to reverse this process [ 5 , 7 ] . proposed a window of infectivity for the initial colonization of ms coinciding with the emergence of the primary teeth . it was found that the average age of colonization was around 26 months of age , about the time when most of the primary teeth had emerged into the oral cavity . studies have shown that earlier colonization of ms can lead to an earlier onset of dental caries in children under five years of age [ 1012 ] . our research group has been conducting dental research involving australian twins and their families for over 25 years . by using twins we can clarify how genetic and environmental influences affect the timing of dental development and also the timing of colonization of ms within the oral cavity . for example , we have shown already that there is a very strong genetic contribution to the timing of emergence of the primary teeth [ 13 , 14 ] . in this paper we will focus on the differences rather than the similarities between mz co - twins , who share a common genetic makeup and often a common environmental background . this should allow us to gain greater insight into unique environmental effects operating on the twins as individuals as well as epigenetic influences . the advantage of mz twins for these types of studies is that they are matched perfectly for age and sex , and share the same genes . given the lack of information on genetic and environmental contributions to variation in ms colonization , the aim of this study is to clarify whether there is a definite pattern of association between the timing of emergence of the first primary tooth and the timing of colonization with s. mutans in pairs of mz twins . we hypothesize that colonization will occur after emergence of the first primary tooth , and also that mz co - twins should show a similar sequence of first tooth emergence and colonization , reflecting underlying shared genetic influences . the cohort used in this study is from a larger longitudinal study of twins focusing on primary tooth emergence and oral health . the study sample consists of 151 mz twin pairs who were recruited into our study between 0 and 1 year of age and are now aged between 2 and 8 . the co - twins have all been raised together , are all of european ancestry , and are all in good health . twins enrolled in this study were recruited through the australian twin registry , australian multiple births association , newspaper birth announcements , hospitals and prenatal classes . zygosity of the mz twins has been confirmed by dna analysis of 10 highly polymorphic genetic loci ( d3s1358 , vwa , fga , amel , d8s1179 , d21s11 , d18s51 , d5s818 , d13s317 , d7s820 ) covering 10 chromosomes from buccal swabs . the study sample includes 67 pairs of mz males and 84 pairs of mz females . ethical approval has been obtained by the university of adelaide human research ethics committee ( h-78 - 2003 ) . tooth emergence for the twins was determined by parental reports using specially designed recording charts . parents were given detailed instructions and were advised to note the date when the tooth first broke through the gingival surface and how to palpate for the tooth . the accuracy of parental reports has been confirmed by clinical examination of randomly selected twins aged 3 months to 2 years . birth weight and gestational age were obtained from the parents via a questionnaire administered before age one , which captures significant developmental time points . this questionnaire consists of questions relating to the conditions surrounding the pregnancy , birth and early months of life of the twins . the parents were asked questions about problems that may have occurred during pregnancy , type of delivery , placenta type , twins ' birth weights and lengths , and parental lifestyle habits . specifically engineered collection kits were mailed to parents quarterly , commencing at 3 months of age , to collect saliva and plaque samples of oral bacteria from the twins . each kit contained two swabs per person for collection of one morning and one evening sample on a single day . parents were instructed to wipe over the oral cavity , including the gums and tongue , and also teeth when present , using a sterile cotton swab for approximately 10 seconds . they then placed the swab tip into a sterile ependorf tube containing a semisolid transport medium to ensure the survival of the oral bacteria during transportation . the ependorf tubes were then sealed tightly and posted to the laboratory in adelaide . upon arrival , each sample was plated out on selective media ( tys20ba ) then incubated for 48 hours at 37c in an atmosphere of 95% nitrogen and 5% carbon dioxide . after incubation , plates were scored visually under a dissecting microscope for presence or absence of s. mutans based on colony morphology . a subsample of colonies identified as positive through visual scoring was confirmed as s. mutans by analysis of carbohydrate fermentation patterns . twins were tested every three months until three contiguous positive scores for both twins were obtained . at least three collection kits were administered to the families covering a period of no less than 9 months . the date at which the first of the three positive scores was identified for each twin was used as their colonization date . descriptive statistics ( interval scale variables means , standard deviations ; dichotomous variables frequencies , relative frequencies ) were calculated using one randomly selected twin per pair for all variables . where variable means are presented in the text , they are accompanied by the sample standard deviation . intra- and interobserver errors for colonization scoring were very low ( cohen 's kappa ~ 0.9 ) . sexes were compared using variance ratio ( f ) tests and student 's t - tests . the relationships between timing of both first tooth emergence and colonization and between twin pairs for interval scale data were examined using pearson 's correlation coefficient . twin pairs considered premature ( < 37 weeks gestation ) comprised 62% of the sample ( males 63% , females 61% ) . males ( 2.5 0.6 kg ) were heavier , on average , than their female twin counterparts ( 2.3 0.6 kg ) . optimal birth weight of twins is 2.5 kg or greater , with those individuals less than this classified as either low ( 1.52.5 kg ) or very low ( 1.5 kg and less ) birth weight . in our study 46% of males and 62% of females the first tooth to emerge was generally a lower central incisor , with no evidence of directional asymmetry in emergence times . the first tooth erupted significantly earlier in males ( 7.8 1.6 months ) than females ( 8.8 2.0 months ) . table 1 lists the proportion of concordant pairs for emergence of the first tooth , illustrating the trend as a progressively more liberal interpretation of concordance was applied . allowing for a discrepancy of up to 28 days between co - twins , 86% of the twin pairs were concordant for timing of emergence of the first tooth . the mean age of colonization was 12.7 6.1 months , with the earliest time of colonization observed at 2.4 months and the latest to colonize at just over 2.5 years . table 2 shows the overall proportion of twin pairs concordant for s. mutans presence , and additionally the breakdown of male and female twin pairs . allowing for a discrepancy of up to 12 months between co - twins , 93% of the 151 twin pairs figure 1 examines the relationship between tooth emergence timing and colonization timing . there was no significant association between timing of tooth emergence and timing of colonization . table 3 compares twins within a pair for their colonization status before the emergence of the first tooth . concordance for colonization prior to first tooth emergence was 15% ( 23 twin pairs ) . the covariates , birth weight and timing of first tooth emergence , were not significantly different between pre- and postemergence colonizers , with an average intrapair difference of 0.27 0.24 kg and 18 30 days , respectively . timing of s. mutans colonization was , unsurprisingly , significantly different between pre- and postemergence colonizers , with an average intrapair difference of 7.2 5.2 months . studies of twins have contributed significantly to our understanding of the role of genetic factors in the process of dental caries in humans [ 1719 ] . most previous studies of dental caries based on twins have employed the classical twin model in which comparisons are made between mz twin pairs who share the same genes and dizygotic ( dz ) twin pairs who share 50% of their genes on average . this model enables estimates to be made of the heritability of selected phenotypes , with values ranging from 0% ( no genetic contribution to observed variation ) to 100% ( all the variation can be explained by genetic factors ) . different researchers have focussed on different variables relating to the process of dental caries , with evidence of genetic influences being found for bacterial , dietary , and host factors [ 6 , 20 , 21 ] . there is also evidence , based on assessments of the genetic correlation between primary and permanent caries scores , that different genes may be involved in the carious process between dentitions . while estimates of heritability are important in establishing whether there is a significant genetic contribution to phenotypic variation , they are population - based statistics , and caution is needed in extrapolating findings to the individual . for example , even though the estimate of heritability for a given feature may be high , this does not necessarily mean that an environmental intervention can not have a major effect on the phenotype . another twin model that has been applied in a limited way to the study of dental caries in humans is the twins reared apart model . two studies based on twins in the minnesota study of twins reared apart have provided valuable insights into the important role of genetic influences on the carious process [ 23 , 24 ] . these studies looked at caries experience in adult twin pairs who had been separated around birth and then raised in different environments throughout their lives . despite their separation , the twin pairs showed remarkably similar patterns of dental caries experience as disclosed by the numbers of decayed , missing and filled teeth . the researchers noted that there were several variables , all of which are likely to have a genetic basis that could explain their findings including : similarities in salivary factors and oral microflora ; similarities in timing and sequence of tooth emergence ; similarities in dental morphology , arch dimensions , and dental spacing ; and dietary preferences . our previous studies of australian twins have confirmed that there is a significant genetic contribution to variation in timing of tooth emergence and various morphological features of both the primary and permanent dentitions [ 13 , 14 ] . the twin model that we have applied in the present study is the mz co - twin model which has several advantages for studies of complex diseases such as dental caries . for example , mz co - twins are matched for age and sex and have very similar dentitions from a developmental and morphological perspective , reflecting their similar genetic makeup [ 16 , 25 , 26 ] . we have , however , shown that mz co - twins are commonly discordant for the expression of certain dental features , such as missing and extra teeth , which reflects differences in environmental and/or epigenetic , influences between the co - twins [ 15 , 27 ] . the mz co - twin model therefore provides an opportunity to obtain new insights into the interactions between genetic , epigenetic and environmental influences on phenotypic variation . the mz co - twin model is extremely powerful because data from only a relatively small number of twin pairs are required to be examined to gain insight . this makes this particular twin model ideal for clinical studies where it is often difficult to recruit the large numbers of subjects who are otherwise required for studies based on the classical twin model . our approach to the use of the mz co - twin model has been to focus initially on the early stages of the carious process , that is , the initial colonization of caries - related microorganisms within the oral cavity . this approach is in contrast to many previously published studies which score the outcomes of the process , that is , decayed missing and filled teeth . it is clear that further studies are needed on genetic contributions to variability observed between individuals at all stages of the process of dental caries . however , we believe that focussing on the early stages may provide results that will have more immediate application in the prevention of the disease . by referring to the detailed information on general health , oral hygiene practices , and diet of the twins and their families in our study , we have been able to retrospectively explore potential factors that may have contributed to discordances between mz co - twins . for example , differences in the timing of initial colonization of decay - producing bacteria such as s. mutans , as well as exploring why some twin pairs or co - twins may have become colonized with s. mutans prior to the emergence of the first primary tooth and others afterwards . we acknowledge that s. mutans is not the only microorganism that is involved in the carious process , and that around 10% of individuals with rampant caries do not have detectable levels of s. mutans . we consider , however , that there is sufficient published evidence to focus on genetic and environmental influences relating to this microorganism in the first instance within the context of the ecological plaque hypothesis . further investigation of significant differences in measurable variables such as biologically meaningful birth weight differences between co - twins creates a unique environmental factor which may be contributing to discordance of other variables . fourteen twin pairs in our current sample exhibited a birth weight difference of 500 g or greater , possibly as a result of twin - twin transfusion syndrome ( ttts ) arising from vascular anastomoses in utero . such birth weight discordance may have significant effects on the future health and wellbeing of the lighter twin , as well as implications for the timing and processes of development that scale allometrically with body weight . ttts complicates traditional twin models as it is a function of the mz twinning process and hence reduces the mz correlation relative to the dz correlation , overestimating the contribution of the unique environment to phenotypic variance . the mean time of first tooth emergence in this sample was around 8 months of age , similar to our previously reported findings for the larger cohort , and significantly later , by approximately two months , than that commonly reported for singletons . this is likely to reflect an allometric relationship between tooth emergence timing and body weight as males were also heavier at birth , on average . however , this finding may also reflect fundamental differences in genetic and/or hormonal influences between sexes acting on the twins in utero or early postnatally . as reported in our recent papers [ 13 , 14 ] , emergence of the first tooth has a very high narrow - sense heritability estimate of 8796% , suggesting that the process of tooth emergence is under strong genetic control within a population . this is not to say that specific environmental ( e.g. , ttts ) or epigenetic factors can not give rise to significant discrepancies in tooth emergence timing within individual mz pairs . when tooth emergence timing in twin pairs in the current study was categorized as concordant / discordant , allowing an intrapair difference of up to 28 days , approximately 90% of the twin pairs were classified as concordant . when taken in light of our previous high estimates of heritability , this suggests that an intrapair difference of greater than one month is appropriate for ascertainment of mz twins markedly discordant for tooth emergence timing and for further analysis of unique environmental or epigenetic influences . the mean age of colonization ( 12.7 6.1 months ) calculated for our sample of twins is one of the first large - sample estimates of colonization timing reported in the literature as far as we are aware . at a population level , both distributions showed significant overlap , and there was no significant association between timing of first tooth emergence and timing of colonization ( see figure 1 ) . these two factors cast doubt on a model of colonization which requires a hard tooth surface to be present in the mouth prior to colonization , and this is emphasized by the fact that approximately 25% of our the individuals in our sample were colonized prior to tooth emergence . this result supports the work of wan et al . , who showed that colonization can occur in predentate singletons . it is a significant issue that needs to be considered when developing and analyzing models of early childhood caries aetiology . in a manner analogous to that for timing of emergence of the first tooth , when colonization timing in twin pairs in the current study was categorized as concordant / discordant , allowing an intrapair difference of up to 12 months , approximately 90% of the twin pairs were found to be concordant . when taken in light of our previous moderate- to high estimates of heritability for colonization timing , this suggests that an intrapair difference of greater than a year is appropriate for ascertainment of mz twins markedly discordant for colonization timing for further analysis of unique environmental or epigenetic influences . an exploration of our questionnaire material for feeding practices , tooth brushing habits , and general health may give further insight into factors influencing the timing of s. mutans colonization . the relationship between tooth emergence timing and colonization timing was examined further by comparing twins within pairs for their event sequence ( i.e. colonization before or after tooth emergence ) . a significant proportion ( 21% ) we have demonstrated that discordance was not due to birth weight discrepancies between twins , nor to marked intrapair differences in tooth emergence timing . it is likely that a range of genetic and nongenetic factors play a significant role in both the timing of emergence of the first tooth and when the oral cavity becomes colonized with s. mutans , and further multivariate modelling of this relationship in the larger cohort of twins is ongoing . a particularly exciting prospect for future studies of dental caries progression will be to carry out genomic and epigenomic scans of the mz co - twins who are discordant for expression of the disease or for factors known to be linked to the disease . already , studies have been performed showing that there can be differences in the epigenetic profiles of mz twin pairs , and that these differences can be associated with discordances in particular phenotypic features between the co - twins [ 27 , 32 ] . however , so far we are not aware of any studies of this type that are related to dental caries . a recent study has provided the first genome - wide scan for dental caries in a human population . these researchers were able to identify suggestive genetic loci for both low caries susceptibility and high caries susceptibility on chromosomes 5 , 13 , and 14 , as well as on the x chromosome . they also speculated that there may be a protective locus for caries on the x chromosome that might explain the tendency for a difference in caries experience between the sexes . there has also been a complex segregation analysis carried out recently on brazilian families that has indicated a dominant , major gene effect influencing resistance to dental caries . we believe that future studies combining the advantages of studying twins and their families with modern methods of genome scanning and segregation analyses offer great potential to identify key genetic risk factors for susceptibility to dental caries . it has generally been assumed in the past that dental caries is mainly determined by environmental factors , and so most of the strategies for preventing or managing the disease have focussed on modifications to that environment , including oral hygiene or diet alteration . however , dental caries continues to be a major public health issue , even in countries such as australia . there is a growing interest in the identification of risk factors that might predispose individuals to dental caries and also in identifying factors that might provide individuals with protection . it is highly likely that these factors will reflect the genetic makeup of host - related factors , including the nature of the oral biofilm . if our understanding of the development of the oral biofilm can be improved , it may be possible to adjust its ecology and thereby decrease the likelihood of children developing dental caries . clinical applications of findings from our project , focussing on preventive practices in young children during primary tooth emergence , promise to lead to reduced dental disease prevalence and significant reductions in health expenditure . hillman 's work with genetically modified mutans has been through the clinical trial stage and our findings on timing of s. mutans colonization will provide important evidence for the most appropriate timing of inoculation [ 35 , 36 ] . thus , information on colonization timing will be invaluable for developing strategies to prevent or delay infection with ms in young children .

findings are presented from a prospective cohort study of timing of primary tooth emergence and timing of oral colonization of streptococcus mutans ( s. mutans ) in australian twins . the paper focuses on differences in colonization timing in genetically identical monozygotic ( mz ) twins . timing of tooth emergence was based on parental report . colonization timing of s. mutans were established by plating samples of plaque and saliva on selective media at 3 monthly intervals and assessing colony morphology . in 25% of individuals colonization occurred prior to emergence of the first tooth . a significant proportion of mz pairs ( 21% ) was discordant for colonization occurring before or after first tooth emergence , suggesting a role of environmental or epigenetic factors in timing of tooth emergence , colonization by s. mutans , or both . these findings and further application of the mz co - twin model should assist in development of strategies to prevent or delay infection with s. mutans in children .

traumatic dentoalveolar injuries occur frequently in children and adolescents , affecting teeth , their supporting structures and adjacent soft tissues and contributing to major psychosocial and economic problems.1 various studies conducted among different populations have reported prevalence rates for traumatic dental injuries to be between 4.9%37%.27 one of the most serious traumatic dental injuries is avulsion , in which one or more teeth are completely knocked out of their alveolar sockets.8 avulsions constitute 0.5%16% of all traumatic dental injuries to permanent anterior teeth910 and most often involves the maxillary central incisor.11 although avulsion may occur at any age , the most common age for avulsion of permanent dentition is between 812 years of age , a time when the loosely structured periodontal ligament surrounding erupting teeth provides only minimal resistance to an extrusive force.8 prompt and appropriate emergency management is exceedingly important for the best long - term prognosis of teeth affected by avulsion , especially in young children.12 ideally , an avulsed tooth should be immediately replanted in its socket in order to avoid further damage to the periodontal membrane.8 the prognosis of a replanted tooth depends on the period of time elapsed between trauma and replantation , the type and condition of storage medium , the stage of root formation and the presence of contamination.1 therefore , it is essential that the lay population , including parents , caretakers and teachers , who are among those most often present when an injury occurs , are aware of the appropriate methods for managing avulsion.13 although numerous studies have been conducted in different countries to evaluate parental knowledge and attitudes regarding avulsed permanent teeth,1,12,15,16 to the best of our knowledge , few studies have been conducted among turkish parents.17 therefore , this study aimed to investigate parental knowledge and attitudes regarding avulsed permanent teeth and their emergency treatment in children in samsun , turkey . a total of 289 parents of children aged 612 years receiving care at the ondokuz mayis university pediatric dentistry clinic were included in the study . the questionnaire was designed to collect data on demographic characteristics ( age , sex , educational level and number of children ) ; previous training in traumatic dental injury and treatment ; and knowledge of avulsed permanent teeth and their immediate emergency management . data collected from the questionnaires was coded and statistical analysis performed using the software spss version 13.0 for windows ( spss inc . , chicago , il , usa ) . chi - square tests were used to identify differences in responses for different variables , with the level of significance set at p=.05 . more than half of participating parents ( 69.6% , n=201 ) were female and between age 3039 years ( 64.4% , n=186 ) . responses to questions related to parents perceived knowledge and attitudes regarding traumatic dental injuries are given in table 2 . most parents ( 74.7% ; n=216 ) reported that they had never received any information about traumatic dental injuries , although 19% ( n=55 ) of parents had obtained knowledge about traumatic dental injuries from a dentist . while the majority of parents ( 78.2% , n=226 ) felt they had inadequate information about traumatic dental injuries , 59.5% ( n=172 ) of the respondents said having information about traumatic dental injuries was very important . responses to questions related to specific knowledge of emergency management of avulsed tooth injuries are given in table 3 . the vast majority 90.7% ( n=262 ) of the respondents reported that they would not replant an avulsed tooth in its socket . when asked what type of media was best for storing an avulsed tooth until a dental professional could be reached , only 9% ( n=26 ) chose physiological media ( sterile saline , milk , saliva ) , whereas 67.1% ( n=194 ) chose non - physiological media ( tap water , ice storage , alcohol , dry storage ) and the remaining 23.9% ( n=69 ) of respondents said they did not know which storage media was best . only 5.9% ( n=17 ) of respondents were able to identify the correct procedure for cleaning an avulsed tooth , whereas 71.6% ( n=207 ) were unable to provide any answer regarding how to clean an avulsed tooth . when asked where they would take their children in the event of a traumatic dental injury , 46.0% ( n=133 ) moreover , most parents ( 68.2% , n=197 ) knew the optimal length of time within which to seek professional help for an avulsed tooth . no statistically significant differences were found in the number of correct responses by age , sex , level of educational or number of children . the prognosis for avulsed teeth is improved by prompt and appropriate treatment , which often depends upon the knowledge of non - professionals present at the site of an accident before professional dental care can be provided.8 given the many incidents that occur at home and school , parents and schoolteachers present in an emergency situation are fundamental to the provision of appropriate care to the injured child.18 although numerous studies have been conducted in various countries examining the knowledge levels of parents , caretakers , teachers and other school personnel ( nurses , coaches , etc . ) regarding correct emergency treatment of tooth avulsion,1,12,15,16,1921 very few studies in turkey have examined parental knowledge of the subject . therefore , this study aimed to assess the knowledge and attitudes of turkish parents regarding the emergency management of avulsed permanent teeth in children . in line with the findings of similar studies conducted in nigeria20 and singapore,19 the present study found parents have a low level of knowledge regarding the emergency treatment of tooth avulsion . this can be explained by the fact that most parents had not received any information about traumatic dental injuries . in one positive finding , parents indicated they were unsatisfied with their level of knowledge of dental trauma management and were interested in learning more . given that a lack of knowledge about emergency management procedures for avulsed teeth may severely affect the prognosis of an avulsed tooth , there is a clear need for educational campaigns to broaden the lay publics knowledge of emergency management procedures for avulsed teeth . as table 3 shows , very few respondents ( 9.3% ) said they would replant an avulsed tooth in the alveolar socket , which is in line with previous studies.20,21 few parents were able to identify the appropriate method for cleaning a dirty avulsed tooth prior to replantation , which is also in line with earlier studies.1,22 in the present study , nearly one - quarter of respondents stated that they would scrub a dirty avulsed tooth before replanting it in its socket , which , in fact , is known to severely decrease the chance of successful replantation . despite the anxiety involved in seeking emergency care for an injured child , nearly half of the respondents stated that they would bring their child to the faculty of dentistry following a traumatic dental injury . this may be explained by parents perceptions that an academic setting will provide more highly trained professional staff and more advanced technology than other types of facilities,1 which reflects two basic assumptions , namely , that the faculty of dentistry is prepared to handle this type of patient , and that a specialist will provide better care than a general dentist.23 studies of clinical outcomes of avulsed teeth have demonstrated that the longer the length of time elapsed between tooth avulsion and replantation , the greater the risk of replacement resorption and inflammatory root resorption.22 whereas appropriate replantation of an avulsed permanent tooth within 30 min has been shown to have a 90% chance of success,24 only a negligible chance ( 5% ) of long - term retention of an avulsed tooth exists if replantation occurs after 2 h.24 it is encouraging to note that 68.2% ( n=197 ) of the respondents knew that for optimal healing , professional help should be sought within 30 min . of a traumatic incident . parental knowledge on this subject is comparable to that reported previously by zhao and gong.25 the ideal storage medium should be capable of preserving cell vitality , adherence and clonogenic capacity26 and should be readily available at the site of the accident or easily accessible.27 if the tooth is transferred to a liquid medium such as the patient s own saliva , milk or saline within the first 15 minutes following avulsion , it is likely that some of the cells in the periodontal ligament and cementum will survive and play a role in regeneration.28 in the present study , rather than physiological media , most respondents believed tap water , ice , alcohol , or dry storage to be the best storage medium , and only a few were able to correctly identify sterile saline , milk , or saliva as the best storage medium . regardless of the age , education level or other factors , parental knowledge of tooth avulsion management was found to be low . while the important epidemiologic findings from this study may serve as a warning , they also represent a useful source of information for the support of education and prevention strategies and reduction of the overall cost of emergency and rehabilitative treatment . considering that many traumatic dental injuries occur at home and at school , the participation of parents and schoolteachers in the emergency situation is fundamental to providing appropriate care to the injured child . within the limitations of the present study , parents were found to have a low level of knowledge regarding procedures to follow in an emergency related to tooth avulsion and replantation . knowledge of emergency treatment of avulsed teeth should be increased by providing educational and preventive programs .

objective : the aim of this study was to examine parental knowledge and attitudes about avulsed permanent teeth and their emergency treatment in children.methods:a total of 289 parents of children aged 612 years receiving care at the ondokuz mayis university pediatric dentistry clinic were included in the study . questionnaires were used to collect data on parents demographic characteristics , previous training in traumatic dental injury and treatment and knowledge of avulsed permanent teeth and their emergency management . chi - square tests were used to identify differences in responses for different variables.results:more than half of the participating parents were female ( 69.6% ) and between the age of 3039 years ( 64.4% ) . most ( 90.7% ) reported that they would not replant the avulsed tooth in its socket , and most ( 68.2% ) gave correct answers regarding the optimum time for which an avulsed tooth should be left out of the mouth prior to replantation . however , most parents were not sufficiently informed about the appropriate method for cleaning a dirty avulsed tooth or transporting it to a dentist or doctor . none of the demographic characteristics or training status was found to have a statistically significant effect on the number of correct responses regarding appropriate management of avulsion injuries ( p>.05)conclusion : this study revealed that the parental level of knowledge about the emergency treatment of avulsion in children is inadequate and that educational campaigns are necessary to improve proper emergency management of dental avulsion .

the potential clinical benefits of a measure of brain encoding and plasticity in hearing aid users have driven a growing interest in aided cortical auditory evoked potentials ( caeps ) . a better understanding of the effects of hearing aids on brain function , and resulting behavior , may improve the current science underlying successful rehabilitation of hearing loss . caeps , a type of event - related electroencephalography ( i.e. , scalp - recorded electrical brain activity ) recorded 50300 ms following stimulus onset , are thought to reflect neural activity in reverberant thalamocortical circuits ( for a review see [ 1 , 2 ] ) . aided caeps , or potentials recorded when stimuli are presented via a hearing aid , have been proposed as a possible physiological measure of the effects of amplification on the brain . many studies have explored the potential use of aided caeps , demonstrating considerable variability in results across experiments and individual participants [ 321 ] . the variability across studies highlights the current uncertainty surrounding the clinical usefulness of aided caeps . much of the existing aided caep literature can be grouped into two general approaches : ( 1 ) a focus on physiological response detection , or ( 2 ) emphasis on physiological response discrimination . the physiological detection approach compares the caeps from an ( inaudible or barely audible ) unaided stimulus , to the response obtained from the same stimulus that has been processed by a hearing aid and delivered at a suprathreshold level . in this case , the unaided caep is often absent or weak , while the aided caep is often present with robust waveform morphology . the absence or presence of a response demonstrates a good correlation with inaudible and audible stimuli , amounting to a physiological correlate of detecting the presence of sound . historically , caeps have successfully been used to estimate behavioral thresholds ( approx . within 10 db of behavioral threshold ) of both normal - hearing and hearing - impaired populations [ 2224 ] . however , it remains to be established whether such strong correlations between physiological and behavioral thresholds are maintained in the case of aided caeps , in which the stimuli have been altered by hearing aid processing . in contrast to the physiological detection approach , the physiological discrimination approach compares caeps from two audible stimuli to determine differences between the waveforms ( e.g. , unaided versus aided conditions in normal - hearing individuals or two audible aided conditions with varied parameters ) . physiological discrimination is measured by specific differences in waveform morphology ( i.e. , differences in peak latencies and peak amplitudes ) between two present waveforms . the different focus of these two approaches contributes to the variability in the existing literature involving aided caeps . significant changes in waveform morphology ( i.e. , amplification effects ) were often found when individuals / groups were tested in studies that used a physiological detection approach , comparing inaudible to audible conditions ( e.g. , [ 8 , 14 , 17 ] ) ; whereas , amplification effects were often absent or small in studies that used a physiological discrimination approach comparing two suprathreshold responses ( e.g. , [ 3 , 4 , 20 ] ) . a comparison of these approaches is presented in figure 1 and the corresponding table 1 , where the examples in the left column ( a d ) demonstrate clear amplification effects obtained in a physiological detection approach ; in contrast , examples of a physiological discrimination approach displayed in the right column ( e h ) show small or absent amplification effects . it is noteworthy that many of the early publications highlighted case studies , and that despite the significant number of aided caep publications , only a limited subset displayed electrophysiological waveforms . in addition to considering the audibility of the signal , it is equally important to take into account the audibility of the underlying noise and its relationship to the signal ( i.e. , signal - to - noise ratio or snr ) . snr is a key contributor to both unaided and aided caep morphology [ 4 , 25 ] . accounting for snr is particularly important when establishing the effects of hearing aids on auditory processing because hearing aids both contribute circuit noise inherent to signal processing and because hearing aids amplify ambient environmental noise . when establishing the clinical utility of aided caeps , it is important to establish the effects of noise on caeps in situations where signal and noise are audible in both unaided and aided conditions or when two different aided conditions are being compared . clinicians might encounter problems when fitting a hearing aid if they assume that changes to the hearing aid should improve the morphology of the evoked response when in reality no change should occur . the purposes of this study are to characterize the existing aided caep literature relative to two potential clinical approaches , and to determine whether these approaches ( i.e. , physiological detection and physiological discrimination ) demonstrate clinical utility for encoding hearing - aid - processed signals . specifically , we asked two questions : will aided caeps be different for a near - threshold signal relative to a suprathreshold signal ( i.e. , physiological detection ) ? will aided caeps be different for two suprathreshold signals ( i.e. , physiological discrimination ) ? will aided caeps be different for a near - threshold signal relative to a suprathreshold signal ( i.e. , physiological detection ) ? will aided caeps be different for two suprathreshold signals ( i.e. , physiological discrimination ) ? we set out to answer these questions in the aided caep domain by recording hearing aid output and eliciting caeps with the recorded stimuli . caeps were recorded using hearing - aid - processed stimuli . in addition , a background noise masker was presented to the normal - hearing participants as a means of simulating the audibility factors that are present when an individual with hearing impairment is fit with a hearing aid . testing noise - masked normal - hearing participants allowed tight control of audibility ( i.e. , audibility of hearing - aid - processed signals and hearing aid noise ) while avoiding hearing - impairment - related confounds associated with recording caeps from individuals with hearing impairment . twelve individuals ( six women and six men ) participated in the study ( mean age = 22.1 years ; sd = 2.47 ) . all participants were right handed with normal hearing from 250 to 8000 hz ( 20 db hl ) , were in good general health , reported no significant history of otologic or neurologic disorders , and denied use of mood or sleep - altering medications . all participants provided informed consent , and all research complied with the regulations of the portland veterans affairs medical center institutional review board . signals were 1000-hz tones recorded from the output of two hearing aids : hearing aid a was a currently available digital hearing aid , hearing aid b was the same analogue hearing aid used in previous studies from our laboratory [ 3 , 4 ] . the frequency response of both hearing aids was matched in the test box of a hearing aid analyzer ( fonix 8000 , frye electronics , tigard , oregon ) to be within 3 db at frequencies between 200 and 5000 hz using a 45 db pure - tone sweep . the hearing aids were then placed on the right ear of the brel and kjr head and torso simulator ( type 4128c ) using stock foam earmolds with no venting . fully anechoic chamber ( reverberation time constant of 2 ms and background noise level of 10 db spl at 1000 hz ) . a 450-ms , 1000-hz tone with 9 ms rise / fall times and an interstimulus interval ( isi ) of 1900 ms was presented via a cambridge soundworks free - field speaker placed at 0 degrees azimuth at a distance of 1.5 meters from the hearing aid microphones . volume control wheels , available noise cancellation algorithms , and directional microphones were deactivated on both hearing aids . overall hearing aid gain and speaker output level electroacoustic analysis of the settings used in the three recordings revealed that attack and release times for hearing aid a were 5 ms and 225 ms , respectively ; using a 1000-hz tone , compression ratios were measured at 1.25 : 1 from 5075 db and 1 : 1 from 7590 db . hearing aid b attack and release times were 2.5 ms for both recordings ; a 1 : 1 compression ratio was found from 5060 db and 2 : 1 from 6090 db for recording 2 settings , and an essentially linear input / output function was measured for recording 3 settings . the goal was to record three signals that varied in signal - to - noise ratio ( snr ) . as indicated in table 2 , resulting snrs for the three recordings were 8 db , 11 db , and 23 db . recordings were approximately two minutes in length and consisted of continuous hearing aid noise and a total of 50 signal presentations . the use of an anechoic chamber for the recordings ensured that the hearing aid noise consisted entirely of circuit noise , as the environmental noise was negligible ( 10 db spl in the 1/3 octave band surrounding 1000 hz ) . the three recordings were then scaled in matlab ( version 7.0 , mathworks , natick , ma ) to create stimuli of varying absolute levels such that the audibility of the tonal signal and the underlying hearing aid noise varied systematically relative to the background noise masker ( see section 2.2.2 for details of background noise creation ) . a schematic depicting the hearing aid signal ( i.e. , 1000-hz tone ) and noise levels in relation to the background noise masker for each hearing aid condition is shown in figure 2 . for low - input recordings from hearing aid a and hearing aid b , four scaling factors were calculated that adjusted the level of the tone and hearing aid noise in approximately 10 db steps to produce the following presentation levels : near , a near - threshold level at which both tone and hearing aid noise were inaudible due to the noise masker amplitude ( intended to represent an inaudible unaided condition ) ; low , a level at which the signal was above the level of the noise masker but the hearing aid noise was below ; mid and high , two levels at which the signal and hearing aid noise were both audible but absolute signal level differed . due to the higher ratio of signal level to hearing aid noise obtained in the higher input level recording from hearing aid b , five stimulus levels were necessary in order to span the range of audible and inaudible signals . in total notice that , within each block of hearing aid conditions , the snr between the signal level and the hearing aid noise remains constant ( i.e. , approx . 11 db for hearing aid a ( recording 1 ) , 8 db for hearing aid b ( recording 2 ) , 23 db for hearing aid b ( recording 3 ) , while the overall stimulus output level changes in 10 db steps . however , the effective snr was much smaller for near and low conditions due to the background noise masker . levels displayed in figure 2 are 1/3 octave band values centered at 1000 hz measured with a brel and kjr 2260 investigator sound level meter fitted with a brel & kjr ear simulator ( number 4157 ) . the hearing aid noise spectra obtained from the three 59 db hearing aid condition presentations are represented in figure 3 ( measured with the sound level meter in 1/3 octave bands with center frequencies from 100 to 6300 hz ) along with the noise floor of the measurement system . to ensure that the lowest signal level presentations were below threshold , participants were asked to listen to each of the three lowest level conditions and report whether or not they could detect the tonal signal . this measure confirmed that the lowest stimulus levels for each of the three hearing aid recordings were either inaudible or barely audible to all participants . in order to simulate hearing - impaired thresholds and to control the audibility of the hearing - aid - processed signal and underlying hearing aid noise , a continuous background noise masker was created in matlab passing a gaussian white noise through a series of 1/3 octave band filters with center frequencies from 100 to 5000 hz . the output of the filters was adjusted to generate a noise masker with a spectrum matching the thresholds of a patient with a moderate , sloping hearing loss . the spectrum of the noise masker was verified with a spectrum analyzer in 1/3 octave bands . in addition , behavioral thresholds at octave and interoctave frequencies from 250 to 8000 hz were established using er-3a ( etymotic research , inc . the thresholds of four participants were established using 1 db steps in a 1-up , 2-down procedure while the background masker was played through the audiometer . mean behavioral thresholds for the four individuals were 11.0 , 15.75 , 22.25 , 24.25 , 28.0 , 34.75 , 44.75 , 43.25 , and 8.75 db hl at frequencies of 250 , 500 , 750 , 1000 , 2000 , 3000 , 4000 , 6000 , and 8000 hz , respectively . for each of the 13 conditions , a recorded 50-tone wav file was repeated three times , yielding a total of 150 tone presentations recording over approximately six minutes for each condition . both the noise masker and hearing aid noise were continuous throughout each block of trials , and all stimuli were presented in the right ear using an er-3a insert earphone and the stim2 system ( compumedics neuroscan , charlotte , nc ) . the presentation order of the three hearing aid conditions was randomized across subjects , and the various stimulus levels were randomized within each hearing aid condition to minimize order effects across participants . two - minute listening breaks were given between each condition , and subjects were offered a longer break after one hour of testing . acquisition sessions lasted three hours and consisted of consenting , audiometric testing , electrode placement , and caep acquisition . participants were seated comfortably in a double - walled sound attenuating booth , and they were instructed to ignore the auditory stimuli and to watch a closed - captioned movie of their choice . evoked potential activity was recorded using an electro - cap international , inc . horizontal and vertical eye movement was monitored with electrodes located inferiorly and at the outer canthi of both eyes . the recording window consisted of a 100-ms prestimulus period and a 700-ms poststimulus time . using scan 4.5 ( compumedics neuroscan , charlotte , nc ) , evoked responses were analog bandpass filtered online from 0.15 to 100 hz ( 12 db / octave roll off ) and converted using an analog - to - digital sampling rate of 1000 hz . trials with eye - blink artifacts were corrected offline using neuroscan software . this blink reduction procedure calculates the amount of covariation between each evoked potential channel and a vertical eye channel using spatial , singular value decomposition and removes the vertical blink activity from each electrode on a point - by - point basis to the degree that the evoked potential and blink activity covaried . after blink correction , trials containing artifacts exceeding 70 v were rejected from averaging . after artifact rejection , the remaining sweeps were averaged and filtered offline from 1 hz ( highpass filter , 24 db / octave ) to 30 hz ( lowpass filter , 24 db / octave ) . averages for 98% of conditions tested had more than 100 accepted trials ; the remaining 2% had between 70 and 100 accepted trials . n1 and each judge used temporal electrode inversion , global field power ( gfp ) traces , and even and odd sweep waveform versions ( to demonstrate replication ) for a given condition . peaks in the near conditions were very difficult to identify because of the electrophysiological noise . therefore , in order to quantify synchrony in the near - threshold conditions , the rectified area was measured in the time region from 40 to 300 ms as a representation of synchrony of the p1-n1-p2 complex . we fit the linear mixed model representation of the repeated measures analysis of variance ( anova ) . this model has the two - fold advantage of ( 1 ) being fit using maximum likelihood so that all observations are included in the analysis and not just observations for subjects with complete data , and ( 2 ) taking into account nonsymmetrical variances that may occur across conditions . main effects of hearing aid condition were tested for two contrasts : low versus mid and mid versus high . the mid versus high comparison directly tests the physiological discrimination approach while the low versus mid comparison verifies that when snr is changing , the aided caep is also likely to change . where main effects were found , post - hoc comparisons were made for each hearing aid condition . to test the effectiveness of the physiological detection approach , paired comparisons were completed on rectified area measures of near versus low for the three hearing aid conditions . area measures were used because of the large number of absent responses in the near condition . the current study investigated the ability of aided caeps to demonstrate amplification effects using both neural physiological response detection and physiological discrimination approaches . based on a review of literature , we hypothesized that aided caeps would show the most robust effect of amplification in neural response detection approaches which correlate with the difference in response to audible versus inaudible stimuli . in contrast , aided caep discrimination approaches that reflect the ability of the auditory system to represent differences between audible hearing - aid - processed stimuli were expected to show weak amplification effects . this study addressed the effects of amplification on audible and inaudible stimuli by comparing the near condition ( i.e. , aided caep responses to the lowest stimulus level at which the tone level was at or below the noise masker level ) to the low condition ( i.e. , aided caep responses to the stimulus level at which the tone was just audible above the noise masker ) . for hearing aid b ( recording 3 ) , two different low conditions were used ; however , to simplify the data analysis , all data for the two low conditions were averaged together to result in a measurement for one low condition . butterfly plots ( overlaid responses at all electrodes across the scalp ) and the global field power plots ( a quantification of simultaneous activity across the scalp ; ) calculated from the grand average for these two stimulus levels across subjects and hearing aid conditions are presented in figure 4 ( left ) . the butterfly plot of the near condition is shown in the top - left panel with the response of the cz electrode highlighted in blue . the butterfly plot of the low condition response is shown in the center - left panel with the cz electrode highlighted in dashed red . the bottom - left panel shows the gfp waveforms for the near and low conditions overlaid in blue and dashed red , respectively . in contrast , responses to the low condition result in visible peaks in activity across electrodes resulting in clearly identifiable peak waves in the gfp plot as well as the cz electrode response . n1 and p2 peaks were difficult to identify in many of the near conditions , resulting in large amounts of missing data ( approx . 50% of n1 and p2 peaks were not able to be identified ) , making statistical analysis using traditional peak latency and amplitude values difficult ; therefore , an area measure ( rectified area was calculated from 40 to 300 ms ) was used to provide an overall measure of synchrony in the p1-n1-p2 region of the waveform . figure 5 displays area values for the near and low conditions for all three hearing aid conditions . paired comparisons between near and low conditions generally demonstrated significantly higher areas for low conditions relative to near conditions : hearing aid a ( recording 1 ) : t = 2.077 , df = 11 , p = .062 ; hearing aid b ( recording 2 ) : t = 2.853 , df = 11 , p = .016 ; hearing aid b ( recording 3 ) : t = 4.225 , df = 11 , p = .003 . the discrimination task of the current study measured the ability of aided caep measures to reflect differences between two clearly audible stimuli ( i.e. , mid and high conditions ) . to demonstrate the main effect of signal level , butterfly and gfp plots were constructed from grand average responses across subjects and hearing aid recordings for mid and high conditions ( figure 4 , right ) . the top - right panel of figure 4 depicts the butterfly plot in response to the mid conditions with the cz electrode highlighted in orange . notice that a clear response waveform is present and the coherence between responses among electrodes on the butterfly plot . the center - right panel shows the butterfly plot in response to high conditions with the cz electrode highlighted in dotted green . again , a response is clearly present as expected given audibility of the signal . in the lower - right panel , gfp responses for the mid and high conditions are overlaid and plotted in orange and dotted green , respectively . notice that the gfp response between the two conditions is very similar with nearly identical peak amplitudes and latencies . this figure helps to highlight the difficulty in using aided caep measures to establish the brain 's ability to physiologically discriminate between two suprathreshold hearing - aid - processed stimuli . n1 and p2 peak amplitude and latency data for the low , mid , and high conditions are displayed in figure 6 with corresponding statistical analyses shown in table 3 . as mentioned above , the responses to the two low conditions for hearing aid b ( recording 3 ) were averaged together for latency / amplitude comparisons between the three hearing aid conditions . the results indicate no main effect of mid versus high conditions for n1 and p2 measures . however , comparisons of low versus mid conditions resulted in main effects of n1 and p2 latency and amplitude . results from previous literature indicate that caep responses are more sensitive to changes in snr than to changes in absolute signal level [ 4 , 25 ] . in the present study , a lack of significant change in n1 and p2 amplitudes or latencies in response to the mid and high conditions supports the idea that waveforms generally do not change when snr is held constant . to further quantify the effects of snr on aided caeps , we compared responses across low , mid , and high conditions . the low conditions had smaller snrs than the mid and high conditions because of the audible background noise masker . for both the mid and high conditions in all three hearing aid conditions , snr was dictated by the constant ratio of signal level to hearing aid noise within the 1000-hz band surrounding the signal tone . the snr for each hearing aid condition was 11 db for hearing aid a ( recording 1 ) , 8 db for hearing aid b ( recording 2 ) , and 23 db for hearing aid b ( recording 3 ) . therefore , the snr was constant at the two highest presentation levels within each hearing aid condition but varied between hearing aid conditions . if snr drives amplitude and latency changes in caeps , we would expect to see significant changes between the low and mid / high conditions , and potentially differences between hearing aids due to differences in snr . the amplitude and latency plots ( figure 6 ) generally indicate the expected changes in n1 amplitudes and latencies . this visual impression is confirmed by the significant main effect of low to mid level conditions on n1 and p2 amplitudes and latencies , which is not found in the comparison between mid to high level conditions ( table 3 ) . further , the effect of the difference in snr between hearing aid conditions was apparent when comparing the statistical significance between the low and mid level stimuli for each hearing aid condition . the hearing aid condition with the largest output snr ( hearing aid b , recording 3 ) was most likely to show significant differences in n1 and p2 amplitudes and latencies between low and mid level recordings , followed by the hearing aid with the second largest output snr ( hearing aid a , recording 1 ) . the hearing aid condition with the poorest snr ( hearing aid b , recording 2 ) was least likely to demonstrate significant differences in peak amplitudes or latencies . overall , these findings corroborate earlier reports of the significant influence of snr on caep recordings . the purpose of this study was to help clarify some of the variability that is seen in decades of aided caep research . this variability is shown in figure 1/table 1 where some studies demonstrate robust effects of amplification ( a d ) while others do not ( e h ) . we hypothesized that large portions of this variability can be explained by whether the signal and underlying noise are audible relative to the contrasting condition . two approaches , physiological detection ( i.e. , the absence versus presence of a caep ) and physiological discrimination ( i.e. , the differentiation of two present responses ) , were tested . physiological detection demonstrates robust amplification effects , while physiological discrimination demonstrates limited differences between mid and high conditions . these results are in agreement with those found by korczak and colleagues where both approaches can be identified in subsets of their data . korczak et al . compared unaided and aided caeps in two groups of individuals with hearing loss ( some with moderate hearing loss and some with severe hearing loss ) using stimuli presented at approximately 70 and 85 db hl . they found improved caeps only for the lower level stimulus or when the unaided stimulus was near threshold ( i.e. , physiological detection ; figure 1(d ) ) . interestingly , when both unaided and aided stimuli were likely above threshold ( i.e. , physiological discrimination ) , as was the case for the moderate hearing - impaired group using a 85-db stimulus , limited effects of amplification were found on n1 and p2 waves ( figure 1(h ) ) . the physiological detection approach appears to be a reasonable use of aided caeps because these measures are sensitive to differences in detectability of an inaudible or barely audible signal and a suprathreshold signal . our results , and the results of other past studies , demonstrate robust amplification effects when taking a detection approach [ 8 , 9 , 14 , 16 , 17 ] . this study simulates the process a clinician may use in fitting a hearing aid , in which hearing aid gain is increased in 10-db steps and the resulting caep is examined . in this scenario , figure 7 shows two representative individuals from the 12 participants and demonstrates the clinical process of increasing the gain of a hearing aid . the near curve shows an absent response in most cases ; whereas , the mid and sometimes low conditions show present responses . in contrast , these data and examples from the literature [ 3 , 4 , 9 , 15 , 20 ] demonstrate that approaching aided caeps from a physiological discrimination perspective is problematic , especially if background noise is audible , as is the case for the mid and high conditions in this study . for two individuals ( figure 7 ) , similarities between mid and high conditions are apparent and demonstrate the difficulty in differentiating between the caeps to two suprathreshold signals . importantly , statistical differences were found for comparisons between low and mid conditions ( see table 3 ) ; in these cases , differences reflect changes in snr as the hearing aid noise is not yet audible . therefore , the comparison between low and mid conditions can be considered a successful example of physiological discrimination . indeed , caeps have been used successfully as a measure of physiological discrimination for decades ; however , our understanding of aided caeps is still lacking . when evoking caeps with hearing - aid - processed stimuli , it remains unclear when a discrimination approach is valid ; it may be valid only for specific hearing aids , specific hearing aid settings , specific stimuli , or as in this study , specific conditions ( e.g. , low versus mid and not mid versus high ) . additional research is needed to delineate what hearing aid and stimulus interactions are affecting the evoked response . the audibility of a broadband stimulus and the underlying noise will vary depending on the hearing configuration of the individual being tested . the participants in this study were young normal - hearing individuals , and a noise masker was used to simulate thresholds that were comparable with a typical sloping hearing loss . however , even with tightly controlled audibility and a pure tone stimulus , variability across participants was found . figure 8 demonstrates how 10-db increments in signal level affect n1 latency and amplitude in the 12 individuals tested . testing hearing - impaired individuals with broadband stimuli would likely result in increased variability across participants because of varying etiologies of hearing loss and differences in threshold across frequencies . therefore , use of aided caeps in individuals in a clinical setting using a physiological discrimination approach is likely to result in considerable variability resulting from the many varying subject and stimulus factors . it should be noted that the method of scaling used in the design of this study to modify signal level is different than clinical hearing aid gain adjustments in that modification of gain can lead to a wide range of acoustic modifications to the signal . while snr has been shown to remain similar across gain settings in some hearing aids , snr can also vary significantly from one device and recording condition to another . these considerations are important when using a physiological discrimination approach ; while one hearing aid may show a physiological amplification affect , another device may not because of the specific acoustic features that are modified . this variability in outcomes severely limits the clinical usefulness of physiological discrimination approaches in aided caeps until more is understood about the interaction between hearing - aid - processed signals and their effects on the evoked response . as mentioned above , the problems related to the physiological discrimination approach likely result from a combination of subject and stimulus factors . snr and onset modification are two stimulus characteristics whose importance has been demonstrated in the literature ( e.g. , [ 3 , 4 , 12 , 25 , 2931 ] ) . first , the effects of snr are important to consider when recording evoked potentials at the level of the cortex , because cortical neurons are more sensitive to snr than to absolute signal level [ 32 , 33 ] . the audibility of underlying noise in the hearing - aid - processed signal , whether amplified ambient noise or circuit noise , must be considered when interpreting aided caeps . second , onset changes to the time waveform that result from hearing aid processing are also important . the n1-p2 caep is an onset response , meaning that it is generated when many cortical pyramidal cells fire synchronously to the onset of a stimulus . these neurons are especially sensitive to abrupt changes in amplitude or frequency ; therefore , hearing - aid modifications to the onset are important to consider [ 12 , 30 ] . these considerations are complicated when speech stimuli are used , because it becomes difficult to determine the snr across different portions of the speech signal , particularly in light of changes in compression across running speech . the results of this study seem to indicate that the contribution of stimulus factors can be minimized when the physiological detection approach is used . specific signal - processing modifications made by the hearing aid are less important when the comparison response waveform is absent . in contrast , subtle acoustic changes ( e.g. , modification of snr or onset characteristics ) are essential when comparing two audible signals in a physiological discrimination approach . to characterize acoustic changes , it is necessary to complete in - the - canal recordings of hearing - aid - processed signals . only then can measures of the important signal modifications be made and related to the resulting aided caeps . two approaches for using aided caeps , physiological detection and physiological discrimination , were tested to determine the clinical usefulness of each . results are in agreement with an analysis of the literature ( see figure 1 ) , and they demonstrate that physiological detection , or a determination of the presence of a response to an audible signal relative to the absent response of an inaudible signal , is likely a valid use of aided caeps and provides an indication of the encoding of the aided signal at the level of the auditory cortex . in contrast , the physiological discrimination approach ( i.e. , the comparison of waveforms that are generated by two audible signals ) can be problematic and difficult to interpret in individuals when using hearing - aid - processed stimuli . a more detailed understanding of how hearing aid processing modifies stimulus acoustics ( e.g. , snr and onset characteristics ) is needed before the physiological discrimination approach should be used for clinical decision making .

the clinical usefulness of aided cortical auditory evoked potentials ( caeps ) remains unclear despite several decades of research . one major contributor to this ambiguity is the wide range of variability across published studies and across individuals within a given study ; some results demonstrate expected amplification effects , while others demonstrate limited or no amplification effects . recent evidence indicates that some of the variability in amplification effects may be explained by distinguishing between experiments that focused on physiological detection of a stimulus versus those that differentiate responses to two audible signals , or physiological discrimination . herein , we ask if either of these approaches is clinically feasible given the inherent challenges with aided caeps . n1 and p2 waves were elicited from 12 noise - masked normal - hearing individuals using hearing - aid - processed 1000-hz pure tones . stimulus levels were varied to study the effect of hearing - aid - signal / hearing - aid - noise audibility relative to the noise - masked thresholds . results demonstrate that clinical use of aided caeps may be justified when determining whether audible stimuli are physiologically detectable relative to inaudible signals . however , differentiating aided caeps elicited from two suprathreshold stimuli ( i.e. , physiological discrimination ) is problematic and should not be used for clinical decision making until a better understanding of the interaction between hearing - aid - processed stimuli and caeps can be established .

pursuit of in vitro biomimetic organ growth has spurred a number of recent investigations of methods to generate three - dimensional ( 3d ) scaffolding structures and techniques for cellular seeding [ 13 ] . in vitro organs that have demonstrated functionality similar to natural organs have been produced previously by cell reseeding onto cadaver - derived , decellularized protein scaffolds . these results suggest that the development of a sufficiently complex 3d cell scaffold may allow the regrowth of organs de novo . cell scaffolding structures , commonly referred to as extracellular matrices ( ecm ) , should be constructed from benign compounds . scaffold materials will either decompose metabolically during cell propagation or be fully incorporated into the final organ . materials that have been suggested include ceramics , chitosan , collagen , peptides , polyethylene glycol ( peg ) , polysaccharides , and various synthetic biomaterials . for applications involving human hosts , material selection criteria must consider toxicity , antigenicity , mechanical strength , thermal stability , and porosity . it is a crystalline [ 911 ] , triple helical molecule and a favorable material for biomedical applications , since it is a biodegradable and biocompatible insoluble fibril with high mechanical strength and relatively low immunogenicity [ 1315 ] . gelatin is the incompletely denatured form of collagen and comprises variable - length peptides which have fibrillar structure but lack configurational order . in vivo use of gelatin has been successfully demonstrated by implantation in animal models , with results that suggest low toxicity and reduced antigenicity relative to collagen [ 17 , 18 ] . furthermore , gelatin is relatively inexpensive compared to collagen and its cell adhesion and proliferation characteristics are essentially indistinguishable . gelatin 's use in ecm is complicated by its lack of 3d structural integrity , lower melting temperatures , and rapid dissolution in water [ 16 , 20 ] . for use as cell scaffolds , recent studies have sought to increase the mechanical and thermal resiliency through compositing with various compounds [ 21 , 22 ] and by utilization of covalent cross - linking agents . many cross - linking agents , however , are toxic or immunogenic , for example , glutaraldehyde . one potential method of cross - linking is the application of uv radiation to generate covalent intermolecular bonds . demonstrated that application of uv - b to gelatin films greatly increased gel strength and viscosity . the melting characteristics of the irradiated gels , however , remained relatively unchanged and thus the conditions employed are insufficient to generate gels which are thermostable at physiological conditions . to improve the thermal stability of gelatin hydrogels , this study employs a combination of glucose and shorter uv - c radiation to cross - link gelatin . the utilization of sugars as a gelatin cross - linking agent has been previously investigated , and its usefulness in vivo without host toxicity has been successfully demonstrated . cross - linking between gelatin and both nonreducing and reducing sugars can be observed without catalysis ; however , due to weak ionic interactions , dissolution still occurs at physiological temperatures ( lower than 37c ) , albeit at a reduced rate . the maillard reaction pathway is a potential chemistry that can generate covalent bonds between reducing sugars and protein amine groups and produces physical changes in gelatin and other protein matrices [ 2729 ] . glycation end products are the resultant glycosylated proteins generated by maillard chemistry [ 30 , 31 ] . sugar cross - linking of gelatin molecules has been shown to increase stiffness and decrease solubility [ 31 , 32 ] . the present paper reports that ultraviolet ( uv - c ) radiation can provide the necessary energetic input required to cross - link gelatin , with increased yield in the presence of glucose . this is the first investigation to quantitatively describe the observed increased thermal stability using calorimetry . the cross - linked gelatin product demonstrates good thermal stability and has the potential for future 3d cell scaffold application . gelatin films were prepared using commercial gelatin powder type a ( fisher scientific cas number 9000 - 70 - 8 ) , glucose ( dextrose anhydrous ; fisher scientific , cas number 50 - 99 - 7 ) , and distilled deionized water . the films were prepared gravimetrically with a mettler ab-104s microbalance using 5 cm diameter aluminum weighing pans having a blend ratio of 2 : 1 : 2 w / w / w gelatin , glucose , and water . distilled and deionized water was heated to approximately 100c and added to the weighed gelatin and glucose . the mixture was quickly blended using a metal spatula until the material was homogenous and viscous . before solidifying samples having no added sugar were prepared using a 2 : 1 w / w ratio of gelatin and water . samples were irradiated with 27 j / cm ( 2700 uw for 2 hours and 46 minutes ) in a spectroline uv cross - linker model xl-1000 equipped with 254 nm bulbs . after irradiation , films were placed in 50 ml of distilled and deionized water then incubated at 45c for 24 hours . gelatin that was not successfully cross - linked after uv exposure solubilized in the distilled and deionized water . the remaining temperature - stable material was collected by decanting and triple rinsing with distilled water . nonirradiated control samples were hydrated in 50 ml of distilled deionized water and incubated at room temperature for 24 hours prior to refrigeration . to test for the effects of glucose on yield of cross - linked product , gelatin films were cast with and without glucose . the glucose containing films were prepared by homogenizing type a gelatin , glucose , and distilled water in a 2 : 1 : 2 w / v / w ratio . the glucose - deficient films were prepared by dissolving gelatin in distilled water in a 1 : 1 w / v ratio . both types of films were prepared simultaneously in triplicate by the addition of 5 ml of material to a six - well tissue culture plate ( costar cat number 3506 ) having a circular well surface area of 9.62 cm . the surface of the gels was flattened by first covering and placing the tissue culture plate in a 45c incubator . all gels were irradiated at 254 nm and were subsequently removed and submerged in 50 ml of distilled water in 100 ml erlenmeyer flasks and placed in a 45c incubator for 4 hours . the remaining solid material was kept by decanting the liquid and rinsed 3 times with 20 ml of water . the dry mass was calculated using an analytical balance and preweighed aluminum pan at 45c 12 hrs . a ta instruments multicell differential scanning calorimeter ( model mc dsc ) having a detection limit of 0.2 mw was utilized for determination of melting temperature profiles . gelatin films were lightly dried on weighing paper to remove excess water and approximately 0.3 g of sample was measured gravimetrically and placed into 1 ml hastelloy ampoules for testing . the temperature was then increased linearly at a rate of 0.5c / min and heat flow recorded having a resolution of 10 seconds from 10c to 90c . the upper limit of 90c was utilized to avoid complications with the measurement associated with the water phase change . the samples were then allowed to dry in the open ampoules for 24 hours at 45c . for accurate heat flow determination , calculations were performed utilizing gravimetrically determined dry samples mass . scanning electron microscopy ( sem ) was utilized to detect any visible small - scale surface structural differences in the uv - irradiated and nonirradiated samples . subsamples of approximately 25 mg were then placed on aluminum support stubs covered with carbon tape and sputter coated with gold / palladium in a hummer 6.2 sputter coater . uv - visible spectrophotometry was employed to detect differences in the transmission spectra between uv - irradiated and nonirradiated samples . the sample ratio was modified to a 2 : 1 : 16 w / w / w ratio of gelatin , glucose , and water to promote even spreading on fused silica windows . 100 l of liquefied sample was evenly spread on a 1 fused silica window . the nonirradiated gel - coated window the nonirradiated sample 's percent transmittance was then measured in a perkin elmer lambda 2 spectrophotometer from 190 nm to 1100 nm . the sample was then uv - irradiated with 27 j / cm and its percent transmittance was measured . the same sample was then placed inverted in a 45c water bath for an hour to remove the colored byproduct produced by the glycolytic reaction , dried in the incubator again , and remeasured . 100 micron - scale patterns were generated on the gels through selective irradiation with uv . patterns were generated and printed onto gel films with 10% wt ascorbic acid solution using a modified hp deskjet 1000 . the ascorbic acid functioned both as a photomask and as a potential free radical scavenger to prevent uv - induced cross - linking . the films were then exposed to 27 j / cm of uv and were immersed in 90c water for 5 minutes . irradiation of gelatin - sugar samples results in the formation of a colored hydrogel as seen in the photograph presented in figure 1(a ) . at the irradiation intensities employed in this investigation , figure 1(b ) is a photograph of the back of the two samples shown in figure 1(a ) . the nominal cross sectional thickness of the films on the slide is about 0.75 mm ( figure 1(c ) ) . examination of the film cross sections with a light microscope ( olympus bx43 ) suggests that the color change penetrates less than 20% of the film thickness . incubation of the irradiated material at 45c in an aqueous solution dissolves the uncolored portion , leaving only a thin sheet of hydrated thermostable product . the observed color of the irradiated sample is mostly removed upon incubation in water . this suggests that the compound responsible for the color change is a byproduct of the cross - linking reaction and is not strongly associated with the newly cross - linked molecular arrangement . addition of glucose increased the yield of cross - linked product ( welch 's t - test , p = 0.0181 ) . the mean dry mass of uv - irradiated glucose containing films was 0.0889 g with a standard deviation of 0.0263 g , while the mean dry mass of uv - irradiated glucose - deficient films was 0.0122 g with a standard deviation of 0.0021 g. comparison of the dry mass results for the glucose containing samples suggests that uv penetration and cross - linking occurs at a depth similar to what is visually seen by microscopy . given the 40 percent water content in glucose containing gelatins the dry mass of the recovered material would correlate with a depth penetration that is approximately 0.15 mm . in order to confirm and evaluate quantitatively the apparent thermostability of the uv - irradiated gelatin , samples were tested using differential scanning calorimetry ( dsc ) and compared to nonirradiated glucose containing controls . the gelatin polymers were hydrated using the protocol described in the preparation of ultraviolet treated samples prior to analysis with the mc dsc instrument in order to minimize errors associated with differences in water content . the actual dry weights of each sample , which were used to calculate heats of fusion from the dsc data , were determined postcalorimetry as described previously . figure 2 presents representative thermograms for triplicate replicates of the uv - irradiated gelatin - glucose samples and nonirradiated glucose containing controls . for comparison , figure 2 also includes a single thermogram for a nonirradiated gelatin sample with no added glucose ( figure 2 g ) . the seven traces shown in figure 2 are reported with slight vertical offsets for image clarity . the initial sharp descent in the curves reflects the transient state that occurs as heating begins following the 30-minute equilibration period at 10c , during which the sample needed to be constantly cooled . nonirradiated gelatin - glucose and gelatin ( only ) samples ( figure 2 ; a , b , c , and g ) all exhibited a sudden , sharp increase in negative ( endothermic ) heat flow as temperature rose above 34c . the average melting temperature of the nonirradiated gelatin - glucose controls was determined to be 34.60 0.84c from nine - replicate measurements . the average heat of fusion values were calculated using sample dry mass and by numeric integration of the chromatogram area . the nonirradiated samples had an average heat of fusion of 45.08 2.35 j / g . thermograms of the irradiated samples ( figure 2 ; d , e , and f ) exhibited no changes in heat flow over the measured temperature from 10c to 90c . the original conformations of the inserted irradiated samples are maintained , unlike nonirradiated controls that have melted and resolidified in the ampoule well . to visually illustrate the structural thermal stability of irradiated gelatin - glucose , samples were stained after hydration with a red food coloring dye and placed in an 85c water bath for 30 minutes . as seen in figures 3(a ) and 3(b ) , the nonirradiated samples were no longer visible , having dissolved completely within 1 minute . the stable physical structure of the irradiated samples is shown in figures 3(c ) and 3(d ) and did not change after 30 minutes of immersion . the nonirradiated sample 's surface is very smooth and nonporous ( figure 4(a ) ) while the irradiated sample 's surface appeared to be fibrillar and pockmarked ( figure 4(d ) ) . the irradiated sample appears to have a more stratified appearance , where increased density is observed at the surface interface which is directly exposed to ultraviolet irradiation ( figures 4(b ) and 4(c ) ) . the nonirradiated sample appears more homogenous with fewer surface features ( figure 4(a ) ) . as previously mentioned , the surface penetration of the uv is limited to micron - scale depths . cross - linking density may decrease with depth due to uv extinction . scanning uv - visible spectrophotometry figure 5 presents the percent transmittance of nonirradiated and uv - irradiated gelatin - glucose gels for wavelengths in the 1901100 nm range . the peak centered at approximately 280 nm in the nonirradiated sample is potentially attributed to a convolution of absorbance by phenylalanine and tyrosine , which are present in small amounts . following uv irradiation , the peak centered at approximately 280 nm greatly increases in magnitude and the reductions in transmittance at 280 nm and at 475 nm were thought to be related to the cross - linking mechanism . because some oxidation products of tyrosine absorb at around 475 nm , for example , dopachrome and aminochrome , it was posited that tyrosine oxidation was part of the potential cross - linking pathway . the peaks at 280 and 475 mm are still maintained after soaking irradiates samples in heated water . lastly , figure 6(a ) depicts a sample of selectively uv - irradiated gel at 40x magnification , following immersion in 90c water . prior work has demonstrated that uv radiation - induced cross - linking increases the mechanical strength of collagen and collagen - sugar systems [ 30 , 32 , 38 ] . gelatin is a denatured form of collagen and has similar chemical properties . unlike collagen , gelatin is easily solubilized in liquid suspensions and can be cast into complex two- and three - dimensional shapes . ionic interactions between the gelatin fibrils maintain a degree of structural integrity ; however , gelatin rapidly dissolves in excess water and melts at physiological temperatures , which complicates its use as a material for biomimetic scaffolding structures . based on the similarities in the chemical structure of gelatin and collagen , and by analogy to the results of ohan et al . , we posited that the application of uv radiation to a homogeneous gelatin - glucose substrate would promote cross - linking and , consequently , reduce solubility in aqueous media and enhance thermal stability . we were able to create a gelatin polymer that is stable at temperatures of at least 90c . due to the chemical similarity between collagen and gelatin , we expected that the same mechanisms would apply to gelatin . the first theory proposes that the collagen and sugar molecules undergo the maillard reaction to form cross - links with neighboring collagen molecules . one of the critical steps in the maillard reaction is glycation , where the amine group of the protein attacks the reactive carbonyl group of the sugar . glycation requires the sugar to be in its linear chain form in order to have access to the reactive carbonyl group ; typically , only 0.002% of glucose molecules are in the linear chain form . it has been hypothesized that uv irradiation generates free radicals which react with the sugar molecules to increase the concentration of linear chain form sugar , which in turn increases the rate of glycation and cross - link formation . another mechanism is based on the observation that uv can promote cross - linking in collagen without sugar . exposure to uv radiation at appropriate wavelengths generates free radicals on aromatic amino acids , for example , tyrosine and phenylalanine [ 38 , 39 ] , that can then form intermolecular bonds [ 38 , 40 ] . we posit that uv irradiation generates free radicals in solution which accelerate cross - linking between individual gelatin molecules . addition of antioxidant ( l - ascorbic acid ) inhibited the cross - linking process ( figure 6 ) , agreeing with the results of ohan et al . based on our current results , it is difficult to ascertain the mechanism by which the free radicals react with the gelatin and sugar . rather than one exclusive pathway for the formation of cross - links , multiple pathways can potentially exist and proceed in parallel . in the presence of sugar , gelatin undergoes the maillard reaction and also forms bonds between its radical aromatic residues . without sugar , the results from the spectrophotometric experiments offer a potential mechanism for cross - link formation between aromatic residues . the large decrease in transmittance following uv irradiation at 280 nm in figure 5 is thought to be caused by either an increase in tyrosine content or the formation of dityrosyl groups . typically , tyrosine , cysteine , tryptophan , and , to a much lesser extent , phenylalanine are assumed to be the primary contributors to absorbance at 280 nm . gelatin lacks tryptophan but has appreciable quantities of phenylalanine and a lesser amount of tyrosine . it is unlikely for phenylalanine to form under these conditions ; however , the conversion of phenylalanine into tyrosine as a result of uv irradiation with low efficiency has been documented . nevertheless , because the molar extinction coefficient of tyrosine at 280 nm in water is roughly an order of magnitude greater than that of phenylalanine in water , even partial conversion of phenylalanine to tyrosine would significantly reduce transmittance . even though the efficiency of phenylalanine conversion noted by ishimitsu et al . is relatively low , their experiment used free amino acids whereas the current experimental setup has phenylalanine as part of a protein chain . the intermolecular interaction between gelatin chains and the intramolecular interactions between constituent amino acids additionally , the absorbance peak that occurs at around 475 nm in the uv - irradiated samples may be indicative of dopachrome or aminochrome content , which are downstream products of tyrosine oxidation and cyclization [ 37 , 42 ] . while the generation of tyrosine from phenylalanine will not result in cross - link formation , two tyrosyl radical groups can react to form a covalently - bound dityrosyl group . ultraviolet radiation at 254 nm has been shown to generate tyrosine free radicals in aqueous solution . the formation of dityrosyl cross - links could also contribute to the increased absorbance at 280 nm . the results of rosei et al . indicate that dityrosine absorbs more strongly than tyrosine near 300 nm , with a primary peak at around 280 nm . in figure 5 , the peak at 280 nm also appears to have broadened towards longer wavelengths , which could indicate dityrosyl formation . experiments involving the uv irradiation of elastin have also proposed a similar mechanism for cross - linking . the formation of dityrosyl groups is dependent on the presence of free radicals in solution . the introduction of a free radical scavenger ascorbic acid as shown in figure 6 strongly inhibits the formation of cross - links . from these data , the cross - linking mechanism is free radical dependent and the covalent cross - links are low in density . dityrosyl bonds are durable and can withstand extreme conditions of prolonged incubation at 95c in 6 n hcl . based on the reported durability of dityrosyl cross - links , such covalent bonds could be responsible for the increased thermal stability of uv - irradiated gelatin . the addition of glucose to the reaction increased the yield of cross - linked product . it is possible that glucose increases cross - linking density , while not physically involved in polymer formation . aqueous solutions of glucose and other sugars have been documented to degrade upon irradiation with wavelengths < 300 nm releasing , peroxide species [ 47 , 48 ] . upon irradiation peroxide forms hydroxyl radicals that oxidize phenylalanine forming tyrosine . hydroxyl radicals can further oxidize tyrosine , resulting in radical production and the formation of a bityrosine covalent cross - link . further analysis is necessary to determine if this is one of the possible mechanistic pathways that results in increased thermostable polymer yield . we were able to achieve reasonably high resolution in our selective irradiation of gel samples ( figures 6(a ) and 6(b ) ) . the smallest feature sizes were about 500 microns in diameter when swollen in water at 36c . the feature sizes appeared to be limited by the resolution of the printhead , so it is likely that more specialized equipment could produce even finer details . nevertheless , our smallest achievable pore sizes are comparable to some of the sizes required for tissue growth . as opposed to conventional methods of pore formation , for example , electrospinning and freeze - drying , the position of every feature is planned beforehand in this method of selective irradiation . it could be possible to generate gelatin films with the same micron - scale geometry as native tissue scaffolds . native gelatin is easily degraded by proteases , possesses minimal antigenicity [ 52 , 53 ] , and is very soluble in aqueous solution . based on our results , cross - linking gelatin renders it insoluble at physiological temperatures ; prior work also correlates greater cross - linking density with reduced rate of enzymatic degradation . by selectively irradiating sugar - gelatin mixtures with uv application of heat and/or protease will then remove the nonirradiated sectors of the gel , allowing the rapid and economical generation of complex gel geometries for use in cell scaffolds . the current investigation has demonstrated the increased thermal stability and reduced water solubility of gelatin - sugar dispersions cross - linked by uv exposure . by increasing the melting temperature of gelatin , we have removed a major impediment for use of gelatin in tissue engineering applications . since increased cross - link density generally correlates with increased mechanical strength and resistance to enzymatic degradation , it is also proposed that the described methodology enhances gelatin 's ability to serve as a material for cell scaffold applications . the method of cross - link formation is posited to require the generation of free radicals and the formation of dityrosine between neighboring molecules . future work will investigate whether a causal link between tyrosine content and cross - link formation exists . through selective uv irradiation of gelatin - sugar dispersions , it should be possible to synthesize physiologically benign cell scaffolds with complex geometries for tissue engineering and possibly organ growth .

the effects of ultraviolet ( 254 nm ) radiation on a hydrated gelatin - glucose matrix were investigated for the development of a physiologically thermostable substrate for potential use in cell scaffold production . experiments conducted with a differential scanning calorimeter indicate that ultraviolet irradiation of gelatin - glucose hydrogels dramatically increases thermal stability such that no melting is observed at temperatures of at least 90c . the addition of glucose significantly increases the yield of cross - linked product , suggesting that glucose has a role in cross - link formation . comparisons of lyophilized samples using scanning electron microscopy show that irradiated materials have visibly different densities .

there are few issues in medicine less controversial than treatment of acute myocardial infarction . unlike in stable coronary syndromes there is no discussion whether patients should be better off in terms of prognosis and long term relief of symptoms after bypass surgery or coronary angioplasty or simply remaining on medical therapy , a difficult decision in individual cases that often leads to fierce discussions in our multidisciplinary meetings . for primary angioplasty there is unanimous consent that a direct immediate mechanical opening of the occluded artery with angioplasty is preferable to fibrinolysis and far preferable to no reperfusion treatment , as often occurs when patients have contraindications to fibrinolysis . this article reports the indications coming from guidelines supported by large trials , meta - analysis and large registries and reviews the reasons why this clear message has not led to a generalised application in most european countries , addressing the changes in clinical practice required to implement this winning strategy . the first esc guidelines clearly indicating that primary angioplasty was the treatment of choice when performed in good time , by an experienced team , were published in 2003 . retrospectively , we can ask ourselves whether it was timely enough since the first two large randomised trials using streptokinase . or rtpa . were published 10 years before , followed by a compelling meta - analysis of more than 3000 patients showing advantages in mortality . we must remember that at the time there was still heated discussion on the value of third generation fibrinolytics and questions coming from poorly interpreted registry data . the 2003 guidelines should be considered as highly innovative , because they gave the strongest possible recommendation ( class i , level of evidence a ) to a practice that many still felt to be inapplicable in most situations . in countries with an advanced health service it was seen as a distraction , or as an excuse to delay or prevent the application of fibrinolysis , the other treatment truly available everywhere within minutes from diagnosis . with minimal changes in subsequent updates , we must gratefully thank professor van der werf and his coauthors for having given an official approval to a therapy still felt experimental , or not practically applicable , by many at the time . this guideline faced the dilemma of defining an acceptable time delay of pci in order to remain superior to fibrinolysis . the issue is complicated by the basic knowledge that a minimal delay in the first hours after symptom onset causes much greater damages than the same delay after 6 or 9 h. an incorrect interpretation of some early pci trials suggested similar mortality , irrespective of the time delay after symptom onset when pci was used , a very different outcome than after fibrinolytics , that also become less effective on old thrombi . more recently this mistake has been corrected , based on the evidence of a worsening in outcome according to the time delay between symptoms onset and pci , more evident in high - risk patients . the 2008 guidelines have identified a cohort of patients with large anterior infarctions and low bleeding risk in whom a lower threshold ( no more than 90 minute delay ) should be used , while the general time delay has been prolonged to 2 h , including also the delay between first medical contact and the transport to the hospital door . despite its clear formulation , the application of primary angioplasty has been very slow , to the point that an immediate past - president of the esc , jean pierre bassand , felt the need to create a task force for promoting its implementation . the results of the survey , conducted in 2005 , observed a large gap between guideline recommendations and clinical practice , with none of the large european countries having greater than 50% application of primary angioplasty . the treatment looked suitable for small , well - organised countries such as the netherlands , belgium , czech republic or switzerland , but impractical elsewhere . this was the driving force of the first stent for life ( sfl ) initiative and a repeated survey led by petr widimsky in 2008 , showed that germany , poland , sweden , hungary , croatia , slovenia and others had come on board and much progress was made in france and italy too . no data could be obtained on the quality of the pci performed and , in particular , on the respect of the 90-minute door - to - balloon time suggested at the time . a large , ongoing survey is being coordinated by dr kristensen on behalf of the sfl group and data are expected to be concluded in 2013 . a major increase in penetration of primary angioplasty will emerge , probably showing the fastest pace of progress since its introduction . the biggest positive surprise is certainly the uk and we will not express more than admiration for a stunning performance moving from < 40% in 2006 to > 90% pci in 2011 . the uk has a unified national health care system and decisions taken in whitehall apply to the entire country , a situation very different from italy or france , where the patchy application mirrors different regional directives . hospitals in the uk respond to authorities with the power to decide which hospital should perform pci and regulate patient transfer accordingly . secondly , the uk had already developed an excellent system to timely administer fibrinolysis based on dedicated nurses in a&e . the system worked equally well when these resources have been allocated upstream , in the ambulance , with the personnel performing and interpreting the ecg and informing the closest primary pci centre of the arrival of a stemi patient . thirdly , auditing was fully implemented via the minap acs and bcis pci databases , offering the advantage of constant monitoring of quality indicators such as time delays and outcomes such as mortality . this advantage is shared with sweden and it is interesting to know that data from both countries indicate impressive mortality benefit after the implementation of the universal primary pci program , together with cost reduction mainly generated by shorter hospital stays and immediate patient triage . equally impressive results have been achieved in most of the 13 countries participating to the sfl initiative . it is obviously impossible to distinguish the natural process of diffusion of a new therapeutic treatment from the additional push the participation of sfl gave to the program . in italy and spain , wide regional differences remain , with large regions such as lombardy in italy and catalonia in spain close to universal application of primary angioplasty and others much further behind . similar considerations can be repeated for france with the additional complication in interpreting figures that pre - hospital thrombolysis is still occasionally applied in the country . it is seen not as an alternative to , but as a preparation for , angioplasty . characteristics specific to the country are the reluctance of the samu , the very efficient equipped ambulances with an anaesthetist on board , to play a passive role simply shipping the patient to the closest angioplasty centre where mechanical recanalization is performed . , at odds with all the other thrombolysis vs primary pci randomised comparisons because they saw superiority of thrombolysis within 2 h from symptoms onset must be interpreted in the context of a very atypical 80% use of angioplasty in the hours after fibrinolysis . unlike in assent iv . there were no signals of higher morbidity and mortality when pci immediately followed the administration of lytics , possibly because of the timely use with fibrinolysis of dual antiplatelet treatment . unlike almost all the other trials of facilitated pci ( finesse . , etc ) there was no excess of bleeding , possibly because of the frequent adoption of a radial approach . the real surprise , however , comes from the other countries participating in the sfl program , starting from a very low penetration and expected to be limited by an insufficient number of 24/7 primary pci centres and equipped mobile units for pre - hospital diagnosis . serbia , bulgaria , romania , turkey made impressive steps forward , thanks to the ability of their country champions to make primary angioplasty an absolute priority in receiving ample resources to meet the goals . the process of integration between ems and 24/7 hospitals to provide a seamless delivery of care throughout a geographical area irrespective of the time of the day , or day of the week , was already strongly advocated in the 2008 stemi guidelines . this point acquired dignity of a specific recommendation , however , only in the 2010 esc myocardial revascularisation guidelines . and in the last update of the acc / aha / scai stemi guidelines . again a class 1 level of evidence a was indicated , based on large series showing how individual changes in service organisation have impacted morbidity and mortality . the principle is the identification of a geographical area where delivery of care for stemi patients is provided by a single ems system , connected to well - identified 24/7 primary centres . the practical application sees enormous differences from country - to - country and region - to - region , in terms of dimension of population served , organisation of ems service and modalities of offer of the primary angioplasty service . first medical contact : a unified telephone number for emergency calls with an operator promptly available and able to identify symptoms possibly related to acute myocardial infarction , is the first essential requirement for a timely delivery of p - pci . in europe there is large variability in the education of the public to use such an emergency service , which is key to ensure the smoothest and most rapid access to primary angioplasty . occasionally patients rely on their gp and waste precious time to wait for him to be available to give advice , or come to see them . more often , relatives or neighbours believe they can speed up diagnosis and treatment by bringing the patient directly to the a&e . this is not only a dangerous exercise because of the possible complications arising en route , but also hardly the best way to immediately get the full attention of overcommitted staff in a crowded a&e department . it is almost certainly bound to lead to delays when compared to the ideal path described below . the operator on the line will probably have the most difficult job in the many steps leading to a successful recanalization : identify the symptoms of an acute cardiac ischaemic syndrome from the broken voice of an emotional relative or from the words of an old patient trying to minimise the severity of his / her symptoms to the outside world . constrictive chest pain with classical irradiation lasting more than 10 minutes is not the way symptoms always manifest . as shown in the article by chieffo et al . older women often only complain of the dyspnoea secondary to left ventricular failure , and many patients have pain limited to the jaw or the epigastrium and/or profuse vomiting and diaphoresis . all these symptoms , especially when they develop in a patient within an age group at risk , or in a patient with clear risk factors for cad , or with known cardiac pathology , should trigger the dispatch of an ambulance with the equipment and personnel able to perform and interpret / transmit an ecg , perform resuscitation , transfer him if needed to the closest primary pci centre available , not stopping in the a&e but bringing the patient directly to the cath lab . emergency medical system : the organisation of an efficient , yet affordable , emergency system is a complex task . the guidelines stress the word integration , which means that the intensive care specialists , nurses and drivers of the ambulance offering first physical contact with a doctor or a paramedic after the phone call , should feel like part of the team involved in a very special diagnosis and treatment facing one of the relatively few medical emergencies where a timely and appropriate treatment can make the difference between life and death . after having checked vital signs and pressure , when the suspect of an stemi is present , an ecg should be performed immediately and the interpretation obtained either directly by the ambulance personnel , using clear thresholds of abnormality to trigger a primary pci call , or via teletransmission to a dedicated specialist giving feedback as a first priority . there are advantages in having an expert central operator coordinating the entire metropolitan area , because he is aware of possible unusual circumstances ( road blocks , unavailability of the closest centre engaged in other calls ) and may suggest transfer to a different primary pci centre . if the ambulance personnel has an appropriate background knowledge , or has attended a well - structured training course on ecg interpretation targeted to the recognition of st - segment elevation , then this is a simple , but appealing , alternative and ensures the full attention of the ambulance crew towards this demanding choice . the experience in london is that courses organised and repeated in the main p - pci hospitals achieve their educational goal and help in creating the team approach and the strive for excellence these sick patients desperately need . a correct stemi diagnosis of 94% , as achieved in london , is even more valuable when you consider that most of the other diagnoses ( pericarditis , aortic dissection , etc ) still require urgent cardiological input . besides pain control , many other treatments can be started in the spacious modern well - equipped ambulances , provided protocols are well defined and the ambulance personnel is authorised to administer drugs . while nobody will argue for aspirin , decisions on the association of clopidogrel , prasugrel or ticagrelor and the use of pre - hospital thrombolysis when the anticipated delay is greater than 2 h , are obviously dependent on the duration of the transfer . 24/7 primary angioplasty hospital : in principle , the population served must be sufficient to maintain competency of the primary angioplasty centre . there has been a progressive and , at times , rapid decrease in the incidence of stemi , attributed to better control of risk factors . the decline of stemi is a quite recent phenomenon and is probably related to the almost complete abolition of the smoking habit in high - risk groups in some european countries . early recognition and treatment of new onset angina and nstemi is probably equally responsible , but more difficult to quantify . epidemiology of acute coronary syndrome ( acs ) is outside the scope of this paper , but it is important to realise that these rapid changes require equally rapid adjustments . a gross estimate of 1000 stemi per million inhabitants was initially used as average european prevalence to set the goal of the stent for life initiative to offer primary angioplasty to at least 600 patients out of a million inhabitants served . this estimate is probably applicable only in some eastern european countries such as ukraine or russia . northern european countries , with a historically very high prevalence of coronary artery disease ( cad ) , see only pockets of epidemics in more deprived areas , while densely - populated regions such as south - east england are down to 500 - 600 stemi cases / million / year . the rapid rise of obesity and diabetes limits the contraction of cad and determines a change in the groups at risk with women > 70 years of age becoming a more frequent target . assuming an average stemi population of 500 patients / million inhabitants which is probably closer to the current incidence in many european countries , a single hospital providing service should not have less than 250300,000 inhabitants in its served territory , in order to be able to provide 125150 stemi cases per year , as in some lucky areas of southern europe such as catalonia or some regions in southern france . in rural or mountainous areas lower basins can be an acceptable choice . the esc guidelines do not indicate minimal numbers to maintain competency of hospital and operators , and this is probably a reflection of the extreme geographical variations across europe that makes difficult to set meaningful thresholds . are of help but their targets are also very loose , with a minimum of 36 primary pci / year to maintain competency as a centre . more importantly , they reaffirm that a primary pci centre should have a reasonable volume of at least 400 pcis / year with each operator performing a minimum of 75 pci / year . these are numbers nobody can argue with : a pci centre with a volume below 400 cases / year makes no sense economically and is unlikely to offer adequate safety to patients when its personnel is exposed to an average of < 8 pci / week . this is especially so when the patient in question has an occluded artery that requires rapid recanalization and is prone to arrhythmias and haemodynamic complications . also the number per operator of 75 pci / year ( all pcis , not only primary angioplasties ) fits well these calculations because you need a minimum of 4 to 6 operators to maintain a sustainable rota and 400 - 450 pcis are sufficient to maintain this level , if evenly split among the operators . if this is a meaningful general rule , exceptions have to be accepted and sometimes encouraged . the operators ' and possibly the paramedics ' competency can be maintained performing angioplasties in a different centre and you may accept a centre with a suboptimal number of 75100 primary angioplasties / year if this is the only solution to ensure a timely offer of primary angioplasty in a homogeneous region sufficiently remote and this is compatible with the economic resources available for the health system . where possible , however , and this includes all the urban / metropolitan areas of europe and probably 2/3 of its population , the encouragement should be to move in the opposite direction and encourage the development of large primary angioplasty hubs with 300 - 500 patients / year . obviously , there are no possible randomised trials comparing outcomes in low volume and high volume centres but all data coming from matched comparisons suggest success increases and complications fall in larger volume centres . clearly there is an inherent cost in maintaining an on - call service which is relatively inactive , and this also plays a role in suggesting to limit the number of active primary pci centers . impersonal service , with the operator seen during the acute treatment not showing up thereafter , and occasional instances of excessive pressure to retransfer or discharge the patient are potential drawbacks to consider . much more difficult to accept , is that the hard work of the ems to quickly bring the patient for primary angioplasty is wasted because of unacceptable delays to the cath lab because of several simultaneous cases of primary angioplasty . this is a problem likely to occur especially at nights or during weekends if only one lab is used for primary angioplasty . a good balance can be maintained when various centres are active within the same metropolitan areas and there is a central allocation unit able to accommodate patients to the nearest hospital with a cath lab available to immediately start angioplasty . there is great creativity in the way the primary angioplasty service is organised across europe and the solutions are often a compromise to satisfy all of the hospitals involved , not necessarily those offering the most efficient or cost - efficient solution . in most european cities , all centers able to offer a round the clock service are allowed to offer primary angioplasty . the advantage is that ambulances are spoilt with choices and can shorten the drive to the hospital and the competition creates an incentive to raise standards . the disadvantage is that this often drives numbers below the minimal acceptable figures indicated above . a solution which is possibly acceptable in remote areas , but not in the heart of a large city . rotation of the hospitals on call for providing service is a possible alternative to maintain all hospitals involved , but is applicable only when the hospitals in question are strategically placed to meet the needs and not creating another problem of insufficient experience being built up in some of the participating centres . in order to maintain an active 24/7 service , centres may need to attract operators not routinely working in the hospital , which may create difficulties dealing with acute cases in a foreign environment . provisions after angioplasty : primary angioplasty has drastically changed the treatment and prognosis of stemi patients . the risks of acute arrhythmias and late ventricular tachycardia , mechanical complications , pericarditis and dressler 's syndrome , haemodynamic compromise , recurrence of angina or reocclusion have all become rare events . patient triage is performed during primary angioplasty and patients with multivessel disease are identified and scheduled for further treatment . the use of radial approach and the elimination of the most heroic antithrombotic and antiplatelet cocktails , substituted by weight - adjusted doses of less dangerous drugs , potentially allow rapid transfer after p - pci . transfer can be positive when it offers access to departments where the patient completes up - titration of beta - blockers and ace - inhibitors , receives detailed instructions on diet , pressure and cholesterol control , and the importance of physical activity . transfer can help to balance patient flow , compensating hospitals that have accepted not to promote p - pci facilities because they are redundant for the territory in question or they have transferred the patient after having performed diagnosis . conversely , staying in the same unit where angioplasty was performed , speaking with the operator and receiving reinforcement messages on the importance of double antiplatelet treatment , can work equally well or better if the situation allows . the important message to convey is that the stent is only the first of a series of measures to implement in order to transform the risk profile . again , multiple models have been developed , but it is essential that the patient can not be transformed into a postal package to move when and where convenient and that the patient 's safety and well - being must remain the centre of the treatment strategy . primary angioplasty can be performed in the vast majority of patients with st elevation myocardial infarction , with the possible exception of patients living in remote regions who are unable to reach the hospital within the first 90120 minutes after diagnosis . in all other areas , covering more than 85% of the european population , the poor penetration of primary angioplasty is caused by the lack of a coherent organisation , revolving around a coordination centre and a network of cleverly placed primary pci hospitals .

abstract : this manuscript is focused around two key messages from the current guidelines of the european society of cardiology on st elevation myocardial infarction ; the need to use primary angioplasty whenever timely and effectively applicable and the importance of organisational changes in the emergency system to implement this indication . based on a review of the trials motivating these guidelines and the successful experience of many european countries , practical indications are provided on the methods to overcome resistances and malpractices that prevent the delivery of optimal care in these critically ill patients .

while affiliative experiences may be rewarding , social situations and hierarchies may also be associated with negative affects such as anxiety . given the centrality of affiliative and social concerns in human life , the high prevalence of social anxiety is not surprising . furthermore , it is not surprising that social anxiety disorder ( sad ) is accompanied by significant distress and functional impairment . behavioral inhibition , characterized by socially fearful and avoidant behavior , is both a feature of sad and a key predictor for the development of this condition . both in daily life and in experimental work , such social fear and avoidance in sad manifests in terms of blushing , as well as avoidant or submissive responses to the eye gazes of others . blushing and gaze aversion in sad are arguably a pathological manifestation of an evolutionarily evolved submissive response that appears in primate dominance encounters . while rodents dominate by means of aggression , in primates subordinates avert their gaze when challenged by the threat stare of the dominant animal , so allowing the social hierarchy to be regulated nonaggressively . in sad , however , such social adaptations seem to have gone awry and socially fearful , avoidant , and submissive behaviors are generalized to all of human social interaction . animal research has established a pivotal role for the neuropeptide oxytocin ( oxt ) and the steroid testosterone ( t ) in the development and adaptive preservation of social hierarchies . such work , as well as placebo - controlled studies with single administrations of oxt and t in humans , leads to the hypothesis that imbalances in oxt and t systems may contribute to the pathogenesis of sad . in many species , including humans , the peptide hormone oxt and the steroid hormone t play a key role in the development and execution of social - emotional behavior , both in males and in females . depending on the relevant social context and environment , these social hormones act via , and interact with , all the main neurotransmitter systems ( that is , the serotoninergic , noradrenergic , and dopaminergic systems ) . the other key social hormone systems , the estrogen and the vasopressin systems , depend critically on t , as the most important estrogen , estradiol , is a metabolite of t , and vasopressin depends on t for its gene expression . since oxt depends on estradiol for its gene expression , all of our social behavior is ultimately- based upon t , a hormone that is ironically associated with antisocial behavior in some folk psychologies . although oxt and t possess opposite behavioral properties , for example in the domain of cognitive empathy , they are certainly not antagonistic hormones in their effects on brain and behavior . on the contrary , the oxt and t systems are intrinsically intertwined , jointly critical for the execution of sexual behavior , and have important and seemingly complementary anxiolytic / fear - reducing properties . oxt and t act , directly or indirectly , via other hormone or neurotransmitter systems , on all social - affective brain systems including the orbitofrontal cortex ( ofc ) , anterior cingulate cortex ( acq , amygdala , ventral striatum , hypothalamus , and brain stem . however , depending on biological predisposition , early adversity ( for example , abuse or neglect ) can disorganize the expression of social - emotional behaviors by creating imbalances in hormonal systems , and especially in the oxt and t systems . these hormonal imbalances have negative effects on the social - emotional brain and can produce socially fearful , avoidant , and submissive behaviors , with insensitivity for social rewards . early - life social experiences shape the expression of adult social behaviors via neuropeptide and steroid systems , and especially the oxt and t systems , which among others organize the expression of fear and aggression , and related approach and avoidance behaviors . notably , changes in the production and secretion of oxt and t in response to social events , and changes in the social brain 's sensitivity to these hormones , are fundamental mechanisms by which social experience affects later social behaviors . furthermore , the social - emotional behaviors governed by the oxt and t systems seem to play a major role in sad . there is some evidence that oxt and t levels measured in plasma or saliva are lowered in sad . however , saliva and plasma measures only coarsely- reflect brain oxt and t , as oxt does not cross the bloodbrain barrier , and t is produced not only by the gonads and adrenals , but also is a neurosteroid in the brain that is not measurable peripherally . what makes oxt and t of specific interest to sad are placebo - controlled oxt and t administration studies in humans , which suggest that these hormones can alleviate the core behavioral hallmarks of sad ( social tearfulness , avoidance , submissiveness ) , and increase sensitivity to social rewards , as described in more detail below . the robust evidence for acute behavioral plasticity in the oxt and t systems , with transient development of new social behaviors in adulthood in response to single administrations of oxt and t , potentially opens up significant therapeutic opportunities , particularly in light of one of the important scientific discoveries of recent decades , that of adult neuroplasticity . adult neuroplasticity refers to structural changes in brain regions and rewiring of brain pathways in adulthood , and social experiences . oxt and t not only develop and execute our social - emotional behavior , but they also are key players in adult neuroplasticity . importantly , oxt and t are able to open up new developmental windows in adulthood , and on these windows social experiences ( and thus psychosocial interventions ) can potentially act to bring lasting change ( figure 1 ) . in rodent research , the anxiolytic or ( social ) fearreducing properties of oxt and t have been well established . t elevations in rats increase social exploration , while decreasing anxiety , punishment , and avoidance . compared with intact male rats and/or gonad ectomized rats with replacement hormone , gonadectomized rats show more anxiety , fear , and freezing behavior on a large variety of tasks . importantly , anxiolytic / fear - reducing effects of oxt are often observed in social situations in rodents . notably , after being defeated in dominance interactions , rats display social avoidance , but not after administration of oxt . oxt acts on a localized population of oxt receptors in the amygdala to reduce fear behaviors . oxt also modulates activity within the hypothalamic - pituitary - adrenal ( hpa ) axis , augmenting hpa - axis activation immediately after stress exposure . this arguably facilitates system adaptation , and subsequently enhances the suppression of the iipa axis , which helps in the re - establishment of system homeostasis . t also has multiple controls over the iipa axis ; t can inhibit the hpa axis at the hypothalamic , pituitary , and adrenal levels . thus , oxt and t not only have numerous anxiolytic / fear reducing actions , especially in the social domain , but also have powerful control over hpa function and activity , particularly during stress . the evidence for anxiolytic and fear - reducing effects of oxt and t in humans is based upon recent studies with single intranasal administrations of oxt and single sublingual administrations of t. using a placebo - controlled single testosterone administration method developed for human females with an uniquely established quantity and time course of effect ( the tuiten method ) , the fear and stress - reducing properties of the steroid hormone testosterone in humans have been demonstrated repeatedly by van honk and colleagues . early human data on the relation between testosterone , fear , and anxiety predominantly involved questionnaires that index the conscious appraisal of anxious mood . however , testosterone does not acutely act on such emotions , but only on genuine fear behaviors . indeed , in the absence of any effects on anxiety , van honk et al showed significant reductions in vigilant responses to masked facial fear after testosterone administration . hermans et al measured baseline startle and fear - potentiated startle in a threat - of - shock paradigm , and showed that testosterone administration acutely reduced the fearpotentiated startle , but did not affect baseline startle . finally , hermans et al showed reductions in skin conductance and startle modulation in response to stress - inducing negative and threatening pictures in anxiety - prone participants . angry facial expressions are thought to have evolved in primates with a key function being social threat signaling in dominance encounters . in face - to - face challenges between primates , high levels of testosterone relate to social dominance in numerous species including humans . indeed , using an adapted emotional stroop task it was shown that vigilant responses ( enduring gazes ) to angry facial expressions are positively related to testosterone levels in both males and females . in these findings however , with a placebo controlled testosterone administration design ( the tuiten method ) , it was shown that t induces cardiac acceleration to exclusively angry faces in healthy women , indicating that the hormone plays a causal role in encourages dominance behavior . furthermore , using the same method and testing subjects in a social approach - avoidance task , enter et al showed reductions in avoidant responses to facial anger , suggesting that t counteracts submissive responses of eye and gaze aversion to facial anger . this finding is relevant to sad , because socially anxious subjects are strongly avoidant to facial anger in the same social approach - avoidance task . terburg et al developed an eye - tracking paradigm to measure true gaze aversion and staring endurance in unconscious face - to - face confrontations , with backwardly masked angry faces . backward masking is a phenomenon wherein the presentation of a target stimulus ( here the angry face ) is immediately followed ( in this case 30 milliseconds ) , by a masking stimulus with the same visual information but scrambled , which results in a failure to consciously perceive the target stimulus . with this paradigm it was shown that on the dominance - submissive spectrum , relatively high dominance traits predict a prolonged gaze to masked facial anger ( enduring gaze ) , while relatively low dominance traits predict gaze aversion . crucially , terburg et al next used acute t administration and showed that individual response patterns shifted from gaze aversion to staring endurance . t thus seems to lead to social dominance in humans ( and likely also in other mammals and in reptiles ) implicitly and nonconsciously . on the other hand , with their social approach - avoidance task , which operates at more explicit , conscious levels of processing , radke and coworkers not only showed that avoidant response to angry facial expressions are positively related to social anxiety levels , but also that t reduced this socially avoidant behavior . taken together , corresponding to the effects on baseline anxiety and cue - specific fear , t and oxt seem to influence dominance - submissive behaviors at different levels of processing . t operates on the most implicit nonconscious level of processing while oxt also acts on automatic behaviors but only at higher , more explicit , levels of information processing . furthermore , it has repeatedly been shown that oxt improves or increases the processing of happy facial expressions . these data are consistent with findings that intranasal oxt generally leads to increased attention to the eyes of others both in experimental and realistic conditions . interestingly , t and oxt may act in opposite ways in the case of facial happiness , as the happy face is also an appeasement signal . t may lead to social approaches in the context of competition eg , for dominance , but not in other situations . in contrast , oxt seems to make social interactions in general more rewarding , and the hormone therefore enhances the processing of facial happiness . in sum , oxt may increase attention to , and memory for , positive facial expressions , because the hormone increases the rewarding properties of social interactions . t especially induces vigilant attention and responsivity to angry facial expressions , because the angry face functions as a dominance signal in face - to - face interactions , thus social dominance is at stake and the hormone is on its guard . overall , oxt reduces background anxiety , and improves the recognition of , and attention to , positive facial expression . oxt also increases attention to the eye region of the face , a sign of interest in social others , and of the willingness to socialize . thus , the social peptide oxt seem to hold properties that make social interaction more rewarding . the social steroid t on the other hand reduces cue - specific and social fear , and submissiveness and facilitates vigilance and social approach but exclusively in competitive conditions : to compete , approach and defend status in social interactions . anxiety , social fear and avoidance , and impaired social reward processing all are behavioral hallmarks of sad , but the specific actions of oxt and t may suggest a personalized approach to their use in therapeutic interventions . that is , individuals suffering from sad and a lack of motivation to socialize may show a response to oxt , while socially fearful , submissive sad subjects who are eager to interact may show a response to t. importantly , as is clear from the earlier discussion , a range of validated behavioral , neuropsychological , and psychophysiological paradigms are available to assess putative behavioral differences in sad . it is important to note that the effects of oxt and t may depend upon various personal and situational factors . this represents another potential opportunity ; that is , for employing oxt and t administration in synergy with exposure to social experiences as part of an innovative combined treatment strategy in sad . to date , however , research with intranasal oxt in sad is still scarce , and published research in sad with t administration is nonexistent . one trial with intranasal oxt adjunctive to exposure therapy in sad showed improvement in the mental representation of the self . further , two pharmacological neuroimaging studies in patients with sad receiving intranasal oxt and placebo showed attenuation of amygdala activity to fear , and the modulation of medial frontal hyperactivity to sad faces . taken together these preliminary data are promising and provide a foundation for additional research on oxt administration in sad . the modern behavioral and psychophysiological paradigms tapping into social fearfulness , social avoidance , submissiveness , and social reward processing discussed above may be particularly useful in further investigations of the assessment and treatment of sad . research on oxt and t has suggested that these hormones alter such paradigms in overlapping but distinct ways . future research on pathogenesis may usefully focus on identifying subtypes of sad which respond differentially to oxt and t administration . ultimately , a better understanding of oxt and t , as well as the pathogenesis of sad may lead to interventional research which optimally integrates psychotherapy with adjunctive hormone administration .

social anxiety disorder ( sad ) is a highly prevalent and disabling disorder with key behavioral traits of social fearfulness , social avoidance , and submissiveness . here we argue that hormonal systems play a key role in mediating social anxiety , and so may be important in sad . hormonal alterations , often established early in development through the interaction between biological and psychological factors ( eg , genetic predisposition x early trauma ) , predispose to socially fearful , avoidant , and submissive behavior . however , whereas gene variants and histories of trauma persist , hormonal systems can be remodeled over the course of life . hormones play a key role during the periods of all sensitive developmental windows ( ie , prenatal , neonatal , puberty , aging ) , and are capable of opening up new developmental windows in adulthood . indeed , the developmental plasticity of our social brain , and thus of social behavior in adulthood , critically depends on steroid hormones such as testosterone and peptide hormones such as oxytocin . these steroid and peptide hormones in interaction with social experiences may have potential for reprogramming the socially anxious brain . certainly , single administrations of oxytocin and testosterone in humans reduce socially fearful , avoidant , and submissive behavior . such work may ultimately lead to new approaches to the treatment of sad .

methylenetetrahydrofolate reductase ( mthfr ) is an important enzyme in folate metabolism since it generates the main circulating form of folate , 5-methyltetrahydrofolate ( 5-methylthf ) , which can serve as a methyl donor for the remethylation of homocysteine to methionine and subsequently the generation of s - adenosylmethionine ( sam ) ( fig . is involved in lipid metabolism and also donates a methyl group to catechol - o - methyltransferase ( comt ) , which catalyzes synthesis of neurotransmitters in the brain , . increased levels of homocysteine , as a result of folate deficiency , have been linked to low levels of monoamine neurotransmitter levels in patients with depression . many polymorphisms of mthfr have been described in the human population and the most common is a polymorphism at base pair 677 , a conversion from c to t which results in a missense mutation causing an alanine to valine conversion . these individuals have reduced enzyme activity ( heterozygotes 65% and homozygotes 30% compared to controls ) and slightly elevated levels of plasma homocysteine , which is highly dependent on folate status , . individuals with the tt genotype have increased risk of developing cognitive impairment , late onset alzheimer 's disease , as well as depression and schizophrenia . another condition involved in mthfr deficiency is an inborn error of metabolism , which results in a severe deficiency in the enzyme and leads to homocystinuria . patients with a severe mthfr deficiency have significantly elevated levels of homocysteine and reduced levels of methionine and present with neurological and vascular complications , , . in order to investigate the effects of mthfr deficiency on neurological function in vivo , a knockout mouse model for mthfr mthfr mice model the polymorphism at base pair 677 , as they have elevated levels of plasma homocysteine when compared to wildtype mice but are phenotypically normal . whereas mthfr mice model the severe form of mthfr deficiency , these mice have significantly elevated levels of homocysteine and have motor and cognitive impairments . in brain tissue , both mthfr and mthfr mice have significantly reduced levels of dna methylation and s - adenosylhomocysteine and mthfr mice have reduced levels of 5-methylthf , and sam . additionally , behavioral studies have reported impairments in cognitive and motor function , . the purpose of this study was to investigate whether levels of monoamine neurotransmitters and amino acids are altered in different brain regions of mthfr - deficient mice . all experiments were approved by the landesamt fr gesundtheit und soziales berlin and performed in accordance with the german animal welfare act . at 3-months of age blood and brain tissue from c57bl/6 male mice was collected and processed for post - mortem neurochemical analyses using high performance liquid chromatography ( hplc ) as described previously . the levels of dopamine , serotonin , and their metabolites dihydroxyphenylacetic acid ( dopac ) and 5-hydroxyindoleacetic acid ( 5-hiaa ) in the amygdala , caudate putamen , cerebellum , dorsal raphe , hippocampus , medial prefrontal cortex , nucleus accumbens , and thalamus , amygdalae were measured by hplc with electrochemical detection . we worked with male mice in this study to avoid sex differences that have been previously described in mthfr mice and neurotransmitter analysis . blood samples taken from the same mice were centrifuged at 7000 g for 7 min at 4 c to obtain the plasma . hplc was used to measure plasma homocysteine concentrations by a diagnostic laboratory at charite university medicine ( labor 28 , berlin ) . one - way analysis of variance ( anova ) was used to compare genotype groups for each measurement followed by tukey 's post - hoc test if applicable . significance level was set at p 0.05 . mthfr and mthfr had significantly higher plasma homocysteine levels when compared to mthfr mice ( table 1 , p < 0.05 ) . serotonin levels were increased in mthfr in the amygdala when compared to mthfr mice ( table 1 , p < 0.05 ) . dopamine levels within the cerebellum were different between genotype groups ( table 1 , one - way anova , f ( 2 , 23 ) = 8.45 , p < 0.01 ) . 5-hiaa was increased in the cerebellum of mthfr ( table 1 , p < 0.05 ) and there was a genotype effect in the hippocampus ( table 1 , one - way anova , f ( 2 , 21 ) = 3.35 , p < 0.05 ) . glutamate levels were different between genotype groups within the amygdala ( table 1 , one - way anova , f ( 2 , 21 ) = 3.45 , p < 0.05 ) . mthfr mice had reduced levels of glutamate in the cerebellum ( table 1 , p < 0.05 ) compared to mthfr mice , and hippocampus ( table 1 , p < 0.05 ) compared to mthfr mice . cerebellar gaba levels were increased in mthfr and mthfr when compared to mthfr mice ( table 1 , p < 0.05 ) . while , gaba levels were reduced in hippocampus ( table 1 , p < 0.05 ) and thalamus ( table 1 , p < 0.05 ) of mthfr mice compared to mthfr . increased levels of plasma homocysteine have been linked to impaired monoamine synthesis as a result of reduced levels of sam . in the present study using a hyperhomocysteinemic mthfr - deficient mouse model we investigated levels of the monoamine neurotransmitters , dopamine , 5-ht , and their respective metabolites , as well as the amino acids , glutamate and gaba in different brain areas . we confirmed that mthfr ( ~ 14 m ) and mthfr ( ~ 70 m ) have significantly elevated levels of plasma homocysteine as previously reported in earlier work , , . we identified that mthfr have increased levels of serotonin in the amygdala , whereas mthfr have increased levels of cerebellar dopamine levels . levels of 5-hiaa are increased in mthfr mice in the cerebellum and reduced in the hippocampus of mthfr and mthfr mice . additionally , hippocampal levels of hva levels were reduced in mthfr mice . more significantly , in mthfr and mthfr mice glutamate and gaba levels were changed in the amygdala , cerebellum , hippocampus and thalamus . interestingly , when we examined whole brain levels of monoamine neurotransmitter and amino acid levels , no differences between genotype groups were observed ( data not shown ) . the cerebellum and hippocampus are susceptible to a deficiency in mthfr as previously described , , , . more specifically , the cerebellum has the highest levels of homocysteine , which may make it more vulnerable to damage via homocysteine and change levels of metabolism of dopamine , serotonin and amino acids , specifically glutamate and gaba . previous work has shown that homocysteine results in stimulation of the glutamate subunit on the n - methyl - d - aspartate receptor , it may be the increased levels of glutamate and gaba within the cerebellum are to counter the excitatory effects of homocysteine . on the other hand , deficiencies in mthfr have been shown to impair hippocampal function , . there is ongoing neurogenesis within the dentate gyrus during adulthood which may increase the demand for folate . in the present study we identified that glutamate is increased in amygdala and gaba is decreased in the thalamus as a result of severe mthfr deficiency , , a deficiency in mthfr does not result in significant changes of monoamine neurotransmitter levels in majority of brain areas measured . this could be a result of compensation in mthfr and mthfr mice , since the deficiency is present from conception . in the peripheral nervous system , choline metabolism can serve as an alternative methyl donor for remethylation of homocysteine to methionine via betaine s - homocysteine methyltransferase , however this enzyme is not highly expressed in brain tissue . we propose that synthesis of serotonin and dopamine may be compensated in the peripheral nervous system , since previous data has shown that sam levels are not changed in the liver of mthfr and mthfr mice possibly as a result of choline compensation . this study has a number of limitations , the first being that tetrahydrobiopterin ( bh4 ) which has been described as the rate limiting step required in the synthesis of monoamine neurotransmitters . interestingly , bh4 structurally resembles folate and has been described to be reduced in endothelial cells when increased levels of homocysteine are present . an additional study measuring levels of bh4 in brain tissue of mthfr - deficient mice may be beneficial in understanding how this deficiency affects monoamine neurotransmission . furthermore , studies measuring more monoamine neurotransmitters such as norepinephrine as well as catechol - a - methyltransferase ( comt ) levels in different brain areas may help dissect the effect of mthfr deficiency , elevated levels of plasma homocysteine and reduced sam levels on neurotransmitter synthesis in this mouse model . c57bl/6 mice have previously been shown to be less susceptible to mthfr deficiency in comparison to balb / c mice , therefore additional studies are may be warranted to investigate levels of monoamine neurotransmitter levels in balb / c mice .

methylenetetrahydrofolate reductase ( mthfr ) is an enzyme key regulator in folate metabolism . deficiencies in mthfr result in increased levels of homocysteine , which leads to reduced levels of s - adenosylmethionine ( sam ) . in the brain , sam donates methyl groups to catechol - o - methyltransferase ( comt ) , which is involved in neurotransmitter analysis . using the mthfr - deficient mouse model the purpose of this study was to investigate levels of monoamine neurotransmitters and amino acid levels in brain tissue . mthfr deficiency affected levels of both glutamate and -aminobutyric acid in within the cerebellum and hippocampus . mthfr/ mice had reduced levels of glutamate in the amygdala and -aminobutyric acid in the thalamus . the excitatory mechanisms of homocysteine through activation of the n - methyl - d - aspartate receptor in brain tissue might alter levels of glutamate and -aminobutyric acid .

chronic obstructive pulmonary disease ( copd ) is a chronic respiratory disease characterized by a progressive and persistent airflow obstruction . the airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs in response to noxious agents , including cigarette smoke , biomass fuels , and occupational agents.1,2 the prevalence of copd has been estimated to range from 4% up to 20% in adults over 40 years of age , with a considerable increase by age , particularly among smokers.37 in italy , the estimates of prevalence for copd from administrative database analysis range from 3.1% to 5.2% , while a more recent italian survey has estimated a prevalence ranging from 3.8% to 6.8%.8 the world health organization ( who ) suggested that copd was the fourth largest cause of death in 2011 , with three million deaths worldwide , representing 5.8% of total mortality.9 in italy , out of the 37,659 deaths that occurred for respiratory diseases in 2008 ( 6.5% of total mortality ) , 474 were related to asthma , whereas 20,786 ( about 50% ) were associated with copd ( considering chronic bronchitis , emphysema and other chronic obstructive pulmonary disease).10 in 2005 , copd , was the tenth largest cause of total disability - adjusted life years ( dalys ) lost worldwide,2 with 27,700 dalys lost by 2020 , copd will only be preceded by ischemic heart disease , severe depression , traffic accidents , and cerebrovascular disease.11 the economic burden of copd is considerable and will continue to grow as the number of elderly people continues to increase.12 the high impact of copd on the italian national health service ( inhs ) has been highlighted by several studies that demonstrate how patients with copd incurred relevant costs in charges to the inhs , which increase with disease severity and presence of comorbidities.1319 copd also has a significant impact on inhs resource consumption in terms of hospitalizations : according to the last available annual report on hospitalizations of the italian ministry of health ( referring to year 2011 ) , 58,930 hospitalizations for copd were reported ( 10.2% of the total hospital admissions for respiratory diseases ) , accounting for a total of 508,343 hospital days.20 in italy , direct health care costs account for more than 70% of total disease costs , and the major cost drivers are repeated hospitalizations , access to emergency departments , and frequent recourse to general practitioner and specialist consultations.21 a fixed - dose combination of inhaled corticosteroids and long - acting 2-agonists is the recommended treatment for the prevention of copd exacerbations in severe patients.2225 the present study was a cost consequences analysis that aimed to compare the major clinical outcomes ( copd hospitalizations and pneumonia - related hospitalizations ) derived from the pathos ( an investigation of the past 10 years health care for primary care patients with chronic obstructive pulmonary disease ) study ( clinical trial identifier nct01146392 ; clinicaltrials.gov ) with direct health care costs related to treatment with budesonide / formoterol or fluticasone / salmeterol for copd , from the inhs perspective . the cost consequences analysis estimates the costs as well as health consequences associated with one intervention compared with an alternative intervention for a health condition ; here , the results , in terms of costs and health consequences , were presented separately . the pathos study was a retrospective , observational , population - based , matched - cohort , register linkage study of copd patients conducted in sweden , with a follow - up period of 11 years , comparing the effectiveness of the treatment of copd patients with the budesonide / formoterol turbuhaler ( astrazeneca , sdertlje , sweden ) and fluticasone / salmeterol diskus ( glaxosmithkline , middlesex , uk ; referred to as budesonide / formoterol and fluticasone / salmeterol , respectively ) . the study design and main results of the pathos study have been already described elsewhere.26,27 in the pathos study , the treatment with budesonide / formoterol was more effective than fluticasone / salmeterol in preventing exacerbations and was associated with a significantly lower incidence rate of all clinical outcomes ( table 1 ) . in summary , budesonide / formoterol reduced the annual rate of moderate to severe exacerbations by 26% compared with fluticasone / salmeterol ( p<0.0001).26 the significant and clinically relevant reduction in favor of budesonide / formoterol was apparent for all types of exacerbation event ( eg , antibiotic use , oral steroid use , or hospital admission ) . indeed , use of budesonide / formoterol reduced rates of copd - related hospitalization by 29% ( p<0.0001 ) , with 34% fewer ( p<0.0001 ) hospital days due to copd exacerbation compared with fluticasone / salmeterol.26 moreover , the fluticasone / salmeterol group was associated with a 73% higher pneumonia rate ( p<0.001 ) and with 74% higher pneumonia - related hospital admissions than was the budesonide / formoterol group ( p<0.001 ) . in the fluticasone / salmeterol group , 7.4 hospitalizations per 100 patient / years were reported compared with 4.3 hospitalizations per 100 patient / years in the budesonide / formoterol group.27 in the pathos study , copd - related hospitalizations were identified by the international classification of disease , 10th revision clinical modification ( icd-10-cm ) code of j44 as primary diagnosis or j44.0/j44.1 as secondary diagnosis ; pneumonia was identified through the icd-10-cm code j10j18 ( patients admitted to hospital because of pneumonia were distinguished from outpatient episodes of care ) . for the purposes of the current study particularly , the pathos study revealed an intraclass difference between fixed - dose combination of inhaled corticosteroids and long - acting 2-agonists in terms of copd related hospitalizations ( 29.1% in the group treated with budesonide / formoterol versus [ vs ] fluticasone / salmeterol group ) and of pneumonia related hospitalizations ( 42% in the group treated with budesonide / formoterol vs fluticasone / salmeterol group ) . the mean collected dose over time reported in the pathos study for the two drugs was 568235 g / day for budesonide / formoterol ( as budesonide dosage ) and 783338 g / day for fluticasone / salmeterol ( as fluticasone dosage ) . for this cost consequence analysis , yearly cost of the pharmacological treatment per group was estimated based on an inhs perspective ; for these retail drugs , the approved reimbursement price after transient compulsory reductions was considered ( table 2 ) . the average yearly costs for the two drugs were estimated weighting the prices by the respective number of days of treatment delivered for each package available ( calculated using italian ims health data at july 2013 and average daily dosages reported by the pathos study ) . copd - related hospitalization cost was the mean diagnosis related group ( drg ) reimbursement tariff derived from an italian observational study15 and amounted to 3,218 . average pneumonia - related hospitalization cost amounted to 3,603 and was derived from a study conducted on hospital information systems of lazio region,28 italy . this was done because there is no italian registry that can provide such information . in order to support the findings of the base - case analysis , in which the most probable values of each variable were considered , we conducted different sensitivity analyses , which were designed to test the robustness of the results . sensitivity analyses are used to determine how different values of an independent variable will impact a particular dependent variable ( result ) under a given set of assumptions , and are used to test the robustness of the results of a model in the presence of uncertainty and to increase understanding of the relationships between input and output variables in a model . the variables tested in the sensitivity analysis were : average drug dosages , drugs prices ( considering the possible availability of generic medications ) , copd - related hospitalizations cost , and pneumonia - related hospitalizations cost . regarding mean dosages of the two drugs , we tested the results considering the highest and lowest values for the standard deviations reported in the pathos study , and the defined daily doses ( ddds ) reported by the who website29 for each formulation and strength . for drug prices , as generic versions of the fixed - dose combination budesonide / formoterol and fluticasone / salmeterol are expected in the near future ( end of 2013 for fluticasone / salmeterol and 2014 for budesonide / formoterol ) ( roggeri et al , unpublished data , 2014 ) , we tested the current findings considering the price reduction for generic drugs indicated as convenient for the inhs ( depending on the yearly expenditure of the drug ) as established by law 189 of november 8 , 2012 ( decreto balduzzi).30 in cases where the manufacturer of the generic drug accepts the price reduction indicated in the decreto balduzzi , the drug will be reimbursed and directly included in the lista di trasparenza ( list of maximum reimbursed costs for all generic drugs ) ; otherwise , if the manufacturer prefers a lower discount rate , a price negotiation with the health authorities is necessary . we calculated that the price reductions for which the price negotiation would not be necessary were 70% for fluticasone / salmeterol and 55% for budesonide / formoterol . we also tested the case of the availability of the generic drug only for fluticasone / salmeterol , to simulate the possibility of an early commercialization of this drug vs budesonide / formoterol ( this case could be realistic only for the period end 20132014 ) . in september 2013 , a new fixed - dose combination of fluticasone / salmeterol with a different inhalation device ( elpenhaler ; elpen pharmaceutical co. inc . , attica , greece ) was marketed and reimbursed by the inhs in italy for this reason it was not considered as generic of fluticasone / salmeterol diskus ; this new fixed - dose combination was considered as a reference for price for a sensitivity analysis . for pneumonia - related hospitalizations costs , in order to evaluate the impact of the costing of this outcome on the results of the present analysis , we consider as sensitivity analysis the lowest and the highest costs reported in the italian study conducted by merito et al28 ( corresponding to the cost associated to the age class > 85 years and of the age class 6574 years , respectively ) . the pathos study was a retrospective , observational , population - based , matched - cohort , register linkage study of copd patients conducted in sweden , with a follow - up period of 11 years , comparing the effectiveness of the treatment of copd patients with the budesonide / formoterol turbuhaler ( astrazeneca , sdertlje , sweden ) and fluticasone / salmeterol diskus ( glaxosmithkline , middlesex , uk ; referred to as budesonide / formoterol and fluticasone / salmeterol , respectively ) . the study design and main results of the pathos study have been already described elsewhere.26,27 in the pathos study , the treatment with budesonide / formoterol was more effective than fluticasone / salmeterol in preventing exacerbations and was associated with a significantly lower incidence rate of all clinical outcomes ( table 1 ) . in summary , budesonide / formoterol reduced the annual rate of moderate to severe exacerbations by 26% compared with fluticasone / salmeterol ( p<0.0001).26 the significant and clinically relevant reduction in favor of budesonide / formoterol was apparent for all types of exacerbation event ( eg , antibiotic use , oral steroid use , or hospital admission ) . indeed , use of budesonide / formoterol reduced rates of copd - related hospitalization by 29% ( p<0.0001 ) , with 34% fewer ( p<0.0001 ) hospital days due to copd exacerbation compared with fluticasone / salmeterol.26 moreover , the fluticasone / salmeterol group was associated with a 73% higher pneumonia rate ( p<0.001 ) and with 74% higher pneumonia - related hospital admissions than was the budesonide / formoterol group ( p<0.001 ) . in the fluticasone / salmeterol group , 7.4 hospitalizations per 100 patient / years were reported compared with 4.3 hospitalizations per 100 patient / years in the budesonide / formoterol group.27 in the pathos study , copd - related hospitalizations were identified by the international classification of disease , 10th revision clinical modification ( icd-10-cm ) code of j44 as primary diagnosis or j44.0/j44.1 as secondary diagnosis ; pneumonia was identified through the icd-10-cm code j10j18 ( patients admitted to hospital because of pneumonia were distinguished from outpatient episodes of care ) . for the purposes of the current study particularly , the pathos study revealed an intraclass difference between fixed - dose combination of inhaled corticosteroids and long - acting 2-agonists in terms of copd related hospitalizations ( 29.1% in the group treated with budesonide / formoterol versus [ vs ] fluticasone / salmeterol group ) and of pneumonia related hospitalizations ( 42% in the group treated with budesonide / formoterol vs fluticasone / salmeterol group ) . the mean collected dose over time reported in the pathos study for the two drugs was 568235 g / day for budesonide / formoterol ( as budesonide dosage ) and 783338 g / day for fluticasone / salmeterol ( as fluticasone dosage ) . for this cost consequence analysis , yearly cost of the pharmacological treatment per group was estimated based on an inhs perspective ; for these retail drugs , the approved reimbursement price after transient compulsory reductions was considered ( table 2 ) . the average yearly costs for the two drugs were estimated weighting the prices by the respective number of days of treatment delivered for each package available ( calculated using italian ims health data at july 2013 and average daily dosages reported by the pathos study ) . copd - related hospitalization cost was the mean diagnosis related group ( drg ) reimbursement tariff derived from an italian observational study15 and amounted to 3,218 . average pneumonia - related hospitalization cost amounted to 3,603 and was derived from a study conducted on hospital information systems of lazio region,28 italy . in order to support the findings of the base - case analysis , in which the most probable values of each variable were considered , we conducted different sensitivity analyses , which were designed to test the robustness of the results . sensitivity analyses are used to determine how different values of an independent variable will impact a particular dependent variable ( result ) under a given set of assumptions , and are used to test the robustness of the results of a model in the presence of uncertainty and to increase understanding of the relationships between input and output variables in a model . the variables tested in the sensitivity analysis were : average drug dosages , drugs prices ( considering the possible availability of generic medications ) , copd - related hospitalizations cost , and pneumonia - related hospitalizations cost . regarding mean dosages of the two drugs , we tested the results considering the highest and lowest values for the standard deviations reported in the pathos study , and the defined daily doses ( ddds ) reported by the who website29 for each formulation and strength . for drug prices , as generic versions of the fixed - dose combination budesonide / formoterol and fluticasone / salmeterol are expected in the near future ( end of 2013 for fluticasone / salmeterol and 2014 for budesonide / formoterol ) ( roggeri et al , unpublished data , 2014 ) , we tested the current findings considering the price reduction for generic drugs indicated as convenient for the inhs ( depending on the yearly expenditure of the drug ) as established by law 189 of november 8 , 2012 ( decreto balduzzi).30 in cases where the manufacturer of the generic drug accepts the price reduction indicated in the decreto balduzzi , the drug will be reimbursed and directly included in the lista di trasparenza ( list of maximum reimbursed costs for all generic drugs ) ; otherwise , if the manufacturer prefers a lower discount rate , a price negotiation with the health authorities is necessary . we calculated that the price reductions for which the price negotiation would not be necessary were 70% for fluticasone / salmeterol and 55% for budesonide / formoterol . we also tested the case of the availability of the generic drug only for fluticasone / salmeterol , to simulate the possibility of an early commercialization of this drug vs budesonide / formoterol ( this case could be realistic only for the period end 20132014 ) . in september 2013 , a new fixed - dose combination of fluticasone / salmeterol with a different inhalation device ( elpenhaler ; elpen pharmaceutical co. inc . , attica , greece ) was marketed and reimbursed by the inhs in italy for this reason it was not considered as generic of fluticasone / salmeterol diskus ; this new fixed - dose combination was considered as a reference for price for a sensitivity analysis . for pneumonia - related hospitalizations costs , in order to evaluate the impact of the costing of this outcome on the results of the present analysis , we consider as sensitivity analysis the lowest and the highest costs reported in the italian study conducted by merito et al28 ( corresponding to the cost associated to the age class > 85 years and of the age class 6574 years , respectively ) . the base case analysis showed that the treatment of copd patients with budesonide / formoterol was associated with a saving in total annual cost to the inhs of 27.6% vs treatment with fluticasone / salmeterol ( 1,311.60 per patient [ drugs and hospitalizations ] vs 1,811.50 per patient , respectively ) . all cost components , in absolute values , were lower in the budesonide / formoterol group : 195.10 per patient for drugs , 193.10 per patient for copd - related hospitalizations , and 111.70 per patient for pneumonia - related hospitalizations ( 22.5% , 28.6% , and 41.9% , respectively ) . several one - way sensitivity analyses were performed in order to test the robustness of the results ( table 3 ) . all but one of these cases confirmed the economic advantages associated with treatment with budesonide / formoterol . for all dosages considered in the sensitivity analysis , the treatment with budesonide / formoterol remained cost - saving , with a reduction per patient / year ranging from 12.9% to 28.1% . the availability of both generics at the prices indicated in the decreto balduzzi , would confirm the results of the base case analysis ( 21.8% per patient / year for budesonide / formoterol ) ; however , if only the generic of fluticasone / salmeterol were to be on the market ( and the price of budesonide / formoterol remains the actual price ) , the treatment with budesonide / formoterol would be associated with an increase in costs of 9.0% . the comparison of budesonide / formoterol with the new fixed - dose combination of fluticasone / salmeterol elpenhaler at the new negotiated price ( 29% vs fluticasone / salmeterol diskus ) suggests a savings , favoring budesonide / formoterol , of 17.6% for the inhs , confirming the robustness of the results . by varying the pneumonia - related hospitalization costs between the highest and lowest values reported above , treatment with budesonide / formoterol vs fluticasone / salmeterol could lead to an economic saving ( of 27.4% and 27.7% , respectively ) . several one - way sensitivity analyses were performed in order to test the robustness of the results ( table 3 ) . all but one of these cases confirmed the economic advantages associated with treatment with budesonide / formoterol . for all dosages considered in the sensitivity analysis , the treatment with budesonide / formoterol remained cost - saving , with a reduction per patient / year ranging from 12.9% to 28.1% . the availability of both generics at the prices indicated in the decreto balduzzi , would confirm the results of the base case analysis ( 21.8% per patient / year for budesonide / formoterol ) ; however , if only the generic of fluticasone / salmeterol were to be on the market ( and the price of budesonide / formoterol remains the actual price ) , the treatment with budesonide / formoterol would be associated with an increase in costs of 9.0% . the comparison of budesonide / formoterol with the new fixed - dose combination of fluticasone / salmeterol elpenhaler at the new negotiated price ( 29% vs fluticasone / salmeterol diskus ) suggests a savings , favoring budesonide / formoterol , of 17.6% for the inhs , confirming the robustness of the results . by varying the pneumonia - related hospitalization costs between the highest and lowest values reported above , treatment with budesonide / formoterol vs fluticasone / salmeterol could lead to an economic saving ( of 27.4% and 27.7% , respectively ) . the current study aimed to evaluate the possible consequences of the scenario described in the pathos study in the italian context . by using a specific statistical approach , we found that budesonide / formoterol combination treatment seems more advantageous than fluticasone / salmeterol combination treatment , in terms of health care cost savings , in copd populations . results of the pathos study , in terms of reduction of copd - related hospitalizations , emergency department visits , and medication utilization , in patients treated with budesonide / formoterol vs fluticasone / salmeterol confirmed the effectiveness data of a previous study conducted in canada.31 this propensity - matched , canadian copd cohort study of 1-year duration suggested that efficacy differences may exist between the inhaled corticosteroids and long - acting 2-agonists , in favor of budesonide / formoterol . this study highlights that the budesonide / formoterol group was significantly less likely to have an emergency department visit ( adjusted relative risk [ rr ] = 0.75 ) or hospitalization ( adjusted rr = 0.61 ) for copd and had fewer claims for prescriptions for tiotropium ( adjusted rr = 0.71 ) . as all clinical outcomes reported by the pathos study showed a higher effectiveness of the fixed - dose combination budesonide / formoterol , the present analysis could be considered conservative as only the most relevant outcomes were evaluated from the economic perspective . moreover , results of the pathos study showed a significant reduction in concomitant treatment with oral steroids , antibiotics , and tiotropium in patients treated with budesonide / formoterol ( p<0.0001 ) , which have not been costed in the present analysis . the base - case analysis results were confirmed by sensitivity analysis in which all economic parameters and drugs dosages were varied between upper and lower limits in order to test the robustness of the results ; the only case for which the treatment with budesonide / formoterol resulted in a slightly higher cost vs fluticasone / salmeterol was based on the availability of a generic version with a discount rate of 70% vs the actual reimbursed price of fluticasone / salmeterol diskus and budesonide / formoterol this case could be considered realistic only in the period between the patents expiration for the two drugs . the discount rates ( 70% for fluticasone / salmeterol and 55% for budesonide / formoterol ) applied in the sensitivity analysis for the two drugs were the minimum discounts , for which the generic manufacturer would not have to negotiate with health authorities in italy ; this means that manufacturers of generic versions could apply for the negotiation and obtain a lower discount rate ( higher price ) . in this view , the hypothesis of a 70% discount for fluticasone / salmeterol and 0% for budesonide / formoterol is the most disadvantageous condition for budesonide / formoterol ; even in this case , the slight increase in costs ( + 9.0% ) for the treatment with budesonide / formoterol is associated with significantly better clinical outcomes in terms of mortality , hospitalization rates , and use of concomitant medications . the major limitation of this investigation is that we used clinical outcomes from a study conducted in a real - world setting in sweden to apply to italian health care costs , thus assuming that the relative differences in effectiveness between the two treatments would be maintained among the two european countries . regardless , as described in an article published by the international society for pharmacoeconomics and outcomes research,32 there is an increasing demand from payers to demonstrate product effectiveness , not only in clinical trials but also , in a real - world setting . this need is driven by the fact that clinical trials are designed to test safety and efficacy and not to answer questions about the effectiveness of a drug in a real - life setting , which is the more relevant question for economic analysis . internationally , real - life data are increasingly used in the development of clear , evidence - based documentation demonstrating the effectiveness value of drugs . observational studies offer the opportunity to recruit a more representative patient population and assess clinically relevant end points , rather than the often more short - term surrogate end points . in the evaluation of this study limitation , it is important to consider that the pathos trial was one of the largest published observational studies in the respiratory area , conducted on more than 5,400 caucasian patients . furthermore , to our knowledge , the two treatments compared for efficacy and safety in the pathos study were not directly compared before in long - term randomized , double blind , double - dummy trials . an observational study conducted in italy could be useful to confirm the results of the present analysis . treatment of copd in a real - world setting with the fixed - dose combination of budesonide / formoterol led to a significant improvement in relevant clinical outcomes and in a simulation , to a possible reduction in direct health care costs charged to the inhs vs the fixed - dose combination fluticasone / salmeterol . sensitivity analysis supported the economic advantages , even once generic version of both drugs will become available .

purposefixed - dose combinations of inhaled corticosteroids and long - acting 2-agonists have proven to prevent and reduce chronic obstructive pulmonary disease ( copd ) exacerbations . the aim of this analysis was to explore the clinical consequences and direct health care costs of applying the findings of the pathos ( an investigation of the past 10 years health care for primary care patients with chronic obstructive pulmonary disease ) study to the italian context.patients and methodseffectiveness data from the pathos study , a population - based , retrospective , observational registry study conducted in sweden , in terms of reduction in copd and pneumonia - related hospitalizations , were considered , in order to estimate the differences in resource consumption between patients treated with budesonide / formoterol and fluticasone / salmeterol . the base case considers the average dosages of the two drugs reported in the pathos study and the actual public price in charges to the italian national health service , while the difference in hospitalization rates reported in the pathos study was costed based on italian real - world data.resultsthe pathos study demonstrated a significant reduction in copd hospitalizations and pneumonia - related hospitalizations in patients treated with budesonide / formoterol versus fluticasone / salmeterol ( 29.1% and 42% , respectively ) . in the base case , the treatment of a patient for 1 year with budesonide / formoterol led to a saving of 499.90 ( 195.10 for drugs , 193.10 for copd hospitalizations , and 111.70 for pneumonia hospitalizations ) corresponding to a 27.6% difference compared with fluticasone / salmeterol treatment.conclusiontreatment of copd with budesonide / formoterol compared with fluticasone / salmeterol could lead to a reduction in direct health care costs , with relevant improvement in clinical outcomes .

in about one - third of patients with an aneurysmal subarachnoid haemorrhage ( sah ) the haemorrhage extends into the parenchyma . patients with an intracerebral haematoma ( ich ) after aneurysmal rupture have , in general , a worse clinical condition , higher fatality rate and smaller chance of functional recovery . risk factors for intraparenchymal extension are largely unknown . if risk factors can be identified , these factors could be included in the risk balance of treating unruptured aneurysms . in addition to clinical characteristics , aneurysm characteristics such as location , shape and direction of blood flow may contribute to the risk of ich development . we aimed to identify risk factors for intraparenchymal extension by comparing clinical and aneurysm characteristics between patients with and without intraparenchymal extension in a large cohort of patients with sah . from a prospectively collected database of patients admitted with sah to our service , we retrieved all 310 patients who were admitted from january 2005 through december 2007 and who fulfilled the inclusion criteria for the current study : ( 1 ) patient s age was 18 years or higher at the time of diagnosis ; ( 2 ) confirmed diagnosis of aneurysmal sah by ct - scan and ct - angiography ; and ( 3 ) the initial ct - scan was performed within 72 h from the onset of clinical symptoms . patients with a clinical episode suggestive of rebleeding between admission and the initial ct - scan were excluded , as were patients with ct - angiography of poor quality or patients with multiple aneurysms in which the site of the ruptured aneurysm was unknown . the following clinical information was retrieved for all patients from the database : ( 1 ) age ; ( 2 ) gender ; ( 3 ) smoking status , with smoking defined as a current smoker or quit smoking less than 10 years ago ; ( 4 ) history of hypertension as diagnosed by physicians or the use of antihypertensive drug(s ) ; ( 5 ) history of sah defined as a previous episode of sah , with treatment of the ruptured aneurysm ; and ( 6 ) family history of sah , with positive family history defined as at least one first - degree relative with sah . we reviewed the admission ct - scans of these patients to determine the presence of ich independent from the clinical information . ich was defined as an intraparenchymal blood collection with a diameter 1 cm on plain ct - scan . then we sorted the patients by the time of diagnosis and retrieved the ct - angiograms of the latest 50 consecutive patients with and the latest 50 consecutive patients without an ich . in these 100 patients , we assessed location and shape of the aneurysm and direction of blood flow into the aneurysm . with 50 patients per category , we had sufficient power to detect a risk ratio of 1.8 for risk factors present in half of the study population . the location of the aneurysm was divided into ( 1 ) anterior communicating artery ( acom ) and the anterior cerebral artery ( aca ) ; ( 2 ) middle cerebral artery ( mca ) ; ( 3 ) internal carotid artery ( ica ) , including the posterior communicating artery ; and ( 4 ) posterior circulation . we categorized the shape of the aneurysms into spherical , oblong , oval and multilobed as described previously based on the maximal length and width of they aneurysm . maximal length is the largest distance measured between two points on the aneurysm which can be connected by a straight line . maximal width is defined as the length of the longest perpendicular straight line on the line representing the maximal length . a spherical aneurysm is defined as a width of 81100% of its maximal length , an oval aneurysm as a width of 6180% of its maximal length and an oblong aneurysm as a width of 60% or less of the maximal length . direction of blood flow is classified into three groups based on two angles measured at the point of the aneurysm , whereby a is defined as the angle made by the feeding vessel with the aneurysm and b the angle made by the feeding vessel with the main branching vessel . blood flow is defined as curved when a is smaller than b. thus , at the point of the aneurysm , a greater portion of the blood flows into the main branching vessel than into the aneurysm . when a is greater than b , and , therefore , more blood flows into the aneurysm than into the main branching vessel , blood flow is defined as straight . finally , when the difference between a and b is less than five degrees , the blood flows are considered equivalent . we calculated relative risks ( rrs ) with corresponding 95% confidence intervals ( 95%cis ) for the clinical and the aneurysmal characteristic . regarding to location of the aneurysm and the direction of blood flow , we compared each location and direction separately with the other locations and directions combined as reference respectively . for shape , we compared spherical with non - spherical and calculated rrs with 95%cis for oblong , oval and multilobed with spherical as reference in addition . the 310 included patients had a mean age of 57.9 years ( range 2288 ) . the mean age of the group that developed an ich was 56.8 years ( sd 12.8 ) , and of the group without ich it was 58.3 years ( sd 13.6 ) . the clinical and aneurysm characteristics and their risks for presence of an ich are shown in table 1 . rrs for ich of the clinical factors studied varied from 0.5 ( 95%ci 0.21.3 ) for positive family history to 1.2 ( 95%ci 0.71.8 ) for males and were statistically not significant . of all aneurysmal characteristics analysed , only the location of the aneurysm at the mca ( rr 1.8 95%ci 1.22.5 ) was statistically significantly associated with the presence of an ich.table 1the risk of developing an ich for all characteristics studiedno . of patients withno . of patients withoutrelative riskich ( % ) ich ( % ) ( 95% ci)clinical characteristics ( n = 310)n = 75n = 235gender male19 ( 25.3%)51 ( 21.7%)1.2 ( 0.71.8 ) female56 ( 74.7%)184 ( 78.3%)age 55 year41 ( 54.7%)131 ( 55.7%)1.0 ( 0.71.4 ) < 55 year34 ( 45.3%)104 ( 44.3%)smoking yes28 ( 37.3%)104 ( 44.3%)1.0 ( 0.61.6 ) * no23 ( 30.7%)84 ( 35.7% ) unknown24 ( 32.0%)47 ( 20.0%)hypertension yes40 ( 53.3%)145 ( 61.7%)0.9 ( 0.51.5 ) * no16 ( 21.3%)51 ( 21.7% ) unknown19 ( 25.3%)39 ( 16.6%)history of sah yes1 ( 1.3%)6 ( 2.6%)0.6 ( 0.13.8 ) * no70 ( 93.3%)227 ( 96.6% ) unknown4 ( 1.3%)2 ( 0.9%)family history of sah yes4 ( 5.3%)33 ( 14.0%)0.5 ( 0.21.3 ) * no35 ( 46.7%)130 ( 55.3% ) unknown36 ( 48%)72 ( 30.6%)aneurysm characteristics ( n = 100)n = 50n = 50location acom and aca21 ( 42.0%)19 ( 38.0%)1.1 ( 0.71.6 ) mca21 ( 42.0%)8 ( 16.0%)1.8 ( 1.22.5 ) ica6 ( 12.0%)15 ( 30.0%)0.5 ( 0.31.0 ) posterior circulation2 ( 4.0%)8 ( 16.0%)0.4 ( 0.11.3)shape spherical9 ( 18.0%)8 ( 16.0% ) non - spherical0.9 ( 0.61.5 ) oblong14 ( 28.0%)10 ( 20.0%)1.1 ( 0.61.9 ) oval17 ( 34.0%)13 ( 26.0%)1.1 ( 0.61.9 ) multilobed10 ( 20.0%)19 ( 38.0%)0.7 ( 0.31.3)blood flow straight26 ( 52.0%)22 ( 44.0%)1.2 ( 0.81.7 ) curved15 ( 30.0%)17 ( 34.0%)0.9 ( 0.61.4 ) equivalent9 ( 18.0%)11 ( 22.0%)0.9 ( 0.51.5 ) * relative risk calculated based on less than 310 patients because data were not available for all the risk of developing an ich for all characteristics studied * relative risk calculated based on less than 310 patients because data were not available for all the location of the aneurysm is a risk factor for presence of an ich in patients with aneurysmal sah : aneurysms originating from the mca are associated with a higher risk than aneurysms at other locations . other aneurysmal and clinical characteristics we studied were not associated with the presence of an ich . the absence of clinical factors as determinants for ich from aneurysmal rupture is in line with previous studies . a study of 512 patients with sah from japan found no significant relationship between age and gender with ich . in another prospective series of 1,076 patients , again , no differences were found for age , gender or hypertension between the group with or without ich . one study did find a difference in age between patients with ( 59 years ) and those without ( 52 years ) , but this study was restricted to patients with mca aneurysms . since we had not pre - specified this analysis beforehand , we have not performed the subgroup analyses . our finding that mca aneurysms have a higher risk for ich than aneurysms at other locations has been previously suggested [ 2 , 6 ] . most other aneurysmal characteristics studied had no or only little influence on the risk of ich , with point estimates of the rr close to one . because small increased or decreased rrs have no clinical relevance in deciding to treat an unruptured aneurysm or not , larger studies will not be embarked upon with enthusiasm . we did not routinely use rotational digital subtraction angiography in our study population because in clinical practice treatment decisions and actual treatment are usually done on ct- or conventional angiography in our centre . we could , therefore , not reliably assess the presence of blebs on aneurysms , and did not include this characteristic in our set of determinants . another potential limitation of our study is the selection of patients for the assessment of radiological factors . we did not randomly retrieve patients , but selected patients on date of admission , starting with the last admitted patients . we have chosen this method because of its efficiency , as many older ct - angiograms were not easy to access . since the retrieval of this subset of patients was done retrospectively and independent from clinical or other radiological factors , we believe this type of retrieval has not introduced any bias . the proportion of patients with ich from a ruptured aneurysm in our studies is comparable to proportions in other studies , which supports the external validity of our study [ 7 , 8 ] . most of the other clinical and radiological characteristics we studied , are easily examined in daily practice , but have no predictive power for the presence of ich . they can , therefore , not be used in clinical practice in the risk balance for treatment of unruptured aneurysms . other aneurysmal characteristics , such as pulsatility and intraluminal flow patterns may be related to risk of ich in case of ruptures , but for assessment of these characteristics , more sophisticated tools are needed than available in daily practice .

intracerebral haematoma ( ich ) occurs in one - third of patients with aneurysmal subarachnoid haemorrhage ( sah ) and is associated with poor prognosis . identification of risk factors for ich from aneurysmal rupture may help in balancing risks of treatment of unruptured aneurysms . we assessed potential clinical and aneurysmal risk factors for ich from aneurysmal rupture . in all 310 sah patients admitted to our service between 2005 and 2007 , we compared clinical risk factors ( gender , age , smoking , hypertension , history of sah and family history ) of patients with and without an ich . from the latest admitted , 50 patients with and 50 without ich , we compared the location , shape and direction of blood flow of the aneurysms on ct - angiography . relative risks ( rrs ) of ich were 1.2 ( 95% confidence interval , ci):0.71.8 ) for males , 1.0 ( 95%ci:0.71.4 ) for age 55 year , 1.0 ( 95%ci:0.61.6 ) for smoking , 0.9 ( 95%ci:0.51.5 ) for hypertension , 0.6 ( 95%ci:0.13.8 ) for history of sah and 0.5 ( 95%ci:0.21.3 ) for family history of sah . rrs of ich were 1.8 ( 95%ci:1.22.5 ) for mca aneurysms , 0.5 ( 95%ci:0.31.0 ) for ica aneurysms , 0.4 ( 95%ci:0.11.3 ) for posterior circulation aneurysms , and 0.7 ( 95%ci:0.31.3 ) for multilobed aneurysms . the rrs of other aneurysmal characteristics varied between 0.9 and 1.2 . patients with mca aneurysms are at a higher risk of developing ich . the other aneurysmal or clinical factors have no or only minor influence on the risk of ich after rupture and are , therefore , not helpful in deciding on treatment of unruptured aneurysms .

we recently diagnosed a case of subcutaneous pythiosis in an 8-month - old german shepherd dog originally from bamako , mali , a country in northwestern africa . the dog was born in that region and had lived in mali until his infection required special treatment . when the cutaneous lesion became chronic and unresponsive to surgical and medical treatments , the owners brought the dog to france for further examination and diagnosis . at the physical examination , the dog had a large ( 15 cm in diameter ) , ulcerative and draining , single , cutaneous lesion on the right side of the hip ( figure 1 ) . the cutaneous lesion had appeared in may 2003 , and despite several surgical excisions and oral administration of antimicrobial agents , the lesion progressed . in september 2003 , several skin biopsy specimens were excised from the lesions . histopathologic examination showed a pyogranulomatous inflammation with numerous broad ( 39 m ) , irregular , septate hyphae ( figure 2 ) . the hyphae were easily observed on gomori methenamine silver and periodic - acid schiffstained sections , but cultures from biopsy samples failed to grow on sabouraud dextrose agar . unique cutaneous lesion on the right side of the hip in an 8 month - old german shepherd . the lesion had appeared 4 months before this image was taken and had rapidly evolved into a large ulcer with draining tracts . presence of numerous , broad ( 39 m in diameter ) , irregular , septate hyphae in a pyogranulomatous dermatitis ( gomori methenamine silver stain ) . , genomic dna was extracted from biopsy specimens collected from the infected tissues ( dneasy tissue kit , qiagen , valencia , ca , usa ) and was further subjected to internal transcribed spacer ( its ) and 5.8s rrna gene sequencing by using primers its1 and its4 ( 5 ) . sequencing reaction was performed in a 10-l volume containing 50 ng of sample dna , 4 pmol of primers , and 4 l of bigdye mix ( applied biosystems , foster city , ca , usa ) . the unique polymerase chain reaction product was analyzed on an abi prism genetic analyzer ( applied biosystems ) . ay683444 ) , which was closely related to its and 5.8s rrna sequences of p. insidiosum ( genbank accession no . ay151157 - 79 ) ( blastn , national center for biotechnology information , bethesda , md , usa ) . the dog was treated with oral ketoconazole ( 10 mg / kg / day ) for 5 weeks . however , antimicrobial therapy was not sufficient to shrink the cutaneous lesion to a size that could permit surgery , and the dog was finally killed in december 2003 . a necropsy was not performed . pythiosis most commonly affects young and large breed ( > 20 kg ) dogs ( 6,7 ) . outdoor and hunting dogs , which are likely to be in contact with swampy water , are at higher risk of contracting pythiosis . the dog in this report was living in a large park and had no access to a swamp . the dog had swum in the niger river a few weeks before the cutaneous lesion appeared . however , the owners did not recall any trauma , puncture , or wound at the site of the infection . bamako is located in the sudanese climatic region with an average annual rainfall of 55 inches . in that region , the year is divided into 2 major seasons : a cool and dry season from november to february and a rainy season from june to september . to identify more precisely the etiologic agent of the disease , we conducted a complete phylogenetic analysis of the its sequence obtained from the dog s infected tissues and other pythium spp . representative its and 5.8s rrna sequences were obtained from genbank and initially aligned with clustal x version 1.63b ( institut de genetique et de biologie moleculaire et cellulaire , strasbourg , france ) and then by visual optimization . to infer phylogenetic relationships among pythium isolates of our dataset , we conducted neighbor - joining and maximum parsimony ( 8) analyses using paup 4.0b9 software ( 9 ) . evaluation of statistical confidence in nodes was based on 1,000 bootstrap replicates ( 10 ) . its and 5.8s rrna sequences from other oomycetes ( lagenidium giganteum , genbank accession no . the total number of 264 characters was constant , 214 were variable but parsimony uninformative , and 364 were parsimony informative . congruent phylogenetic trees in terms of branching and clustering of taxa were generated with the neighbor - joining ( figure 3 ) and maximum parsimony methods . in each of these topologies , its and 5.8s rrna sequences from p. insidiosum isolates were aligned into 3 clusters previously described by schurko et al . ( 4 ) . inside and between these clades , the sequence ay683444 , corresponding to the canine case , was very different from all other oomycetes sequences ( genetic distances varied from 25.0% to 48.0% ) . although the dog s its sequence clearly could be grouped with the other p. insidiosum clusters , the branching associated this sequence with the p. insidiosum strains was supported with only 60% bootstrap values . sequences from other pythium species formed 2 distinct groups . in the first group ( p. dissotocum , p. myriotylum , p. volutum , p. vanterpoolii , and p. porphyrae ) , genetic distances varied from 7.1% to 19.9% . in the second group ( p. acanthicum , p. hydnosporum , p. oligandrum , and p. periplocum ) , genetic distances varied from 1.8% to 6.2% . this finding suggests that the causative agent of the disease represents a new species within the genus pythium . because the isolation in pure culture of the etiologic agent was not successful , specific nutritional requirements might account for the failure of the isolation of this particular strain . this report indicates that more cases of pythiosis in animals or humans from africa could be expected in the near future . evolutionary tree of 37 internal transcribed spacer ( its ) and 5.8s rrna sequences from oomycetes . are 23 sequences from pythium insidiosum from america ( ) and asia / australia ( ) . the phylogram presented resulted from bootstrapped data sets ( 3 ) using parsimony analysis ( heuristic search option in paup 4.0 ) . this tree was identical to the consensus of 17 most parsimonious trees generated from the branch and bound algorithm in paup 4.0 . the percentages above the branches are the frequencies with which a given branch appeared in 1,000 bootstrap replications . the evolutionary distance between two sequences is obtained by summing the lengths of the connecting branches along the horizontal axis , using the scale on the bottom . lagenidium giganteum ( ay151183 ) , saprolegnia parasitica ( ay310504 ) , and s. salmonis ( ay647193 ) were chosen as outgroups .

we report the first case of pythiosis from africa in an 8-month - old dog with a chronic and ulcerative cutaneous lesion . the etiologic agent belonged to the genus pythium . phylogenetic analysis placed the isolate in a sister group to the other p. insidiosum strains . however , the isolate may belong to a new pythium species .

molecular oxygen ( o2 ) is required for aerobic metabolism to maintain intracellular bioenergetics and serves as an electron acceptor in many biochemical reactions . ambient air contains 21% of o2 ; however , most mammalian tissues exist at 29% o2 . hypoxia , usually defined as 2% o2 , occurs in a variety of pathological conditions , including stroke , tissue ischemia , inflammation , and tumor growth . cells have developed a number of essential mechanisms to cope with the stress of hypoxia . among these coping mechanisms helix per - arnt - sim ( bhlh - pas ) family protein that forms a heterodimer with its and subunits and functions as a transcription factor . while hif-1 is constitutively active , hif-1 is regulated by oxygen status . in well - oxygenated states , constitutively synthesized hif-1 undergoes continuous degradation . during this process , two proline sites ( pro402 and pro564 ) in the oxygen - dependent degradation ( odd ) domain are hydroxylated by prolyl hydroxylase . of the three prolyl hydroxylases ( phd1 , phd2 , and phd3 ) identified in mammalian cells , phd2 is known to play a major role . under normoxic conditions , phd2 uses molecular oxygen and 2-oxoglutarate ( -ketoglutarate ) as cosubstrates for proline hydroxylation of hif-1. hydroxylated hif-1 is then recognized by the tumor suppressor protein von hippel hypoxic o2 levels limit the ability of phd2 to function and result in enhanced stability of hif-1. stabilized hif-1 forms a heterodimer with its subunit and translocates into the nucleus where it binds to its target gene promoters known as hypoxia response elements ( hres ) and activates gene transcription . hif-1 is a main regulator of hypoxia , activating several hundred genes involved in angiogenesis ( vegf ) , glucose transport ( glut1 ) , glycolytic pathways ( ldh - a ) , ros signals ( inos ) , erythropoiesis ( epo ) , and a variety of other processes . tumors adapt to hypoxia by increasing angiogenesis and metastatic potential , altering apoptosis , and regulating metabolism . these adaptations mediated by hif-1 make tumors both more aggressive and treatment - resistant , resulting in poor clinical outcomes . immunohistochemical analyses have revealed that hif-1 is overexpressed in many human cancers and is closely associated with patient mortality . overexpression is not only involved in tumor progression but also associated with resistance to radiation and chemotherapy . due to the importance of hif-1 in tumor development and progression , a considerable amount of effort has been made to identify hif-1 inhibitors for treatment of cancer . saururus cernuus l. ( saururaceae ) , an aquatic plant found throughout the eastern half of the united states , has a long history of medicinal use in the treatment of tumors by both native americans and early colonists . the dineolignans manassantin a ( 1 ) , manassantin b ( 2 ) , 4-o - demethylmanassantin b ( 3 ) , and manassantin b1 ( 4 ) are natural products isolated from saururus cernuus ( figure 1 ) . structure of dineolignans from saururus cernuus . in broad connection with our interest in using natural products to understand the signaling pathways in biology , we devised a convergent synthetic route to 1 and 2 that would be easily amenable to the synthesis of analogues for biological studies . after completing the synthesis of 1 and 2 , we determined the effect of these manassantins on hif-1 and vegf expression . these studies revealed that manassantin a ( 1 ) is a potent inhibitor of hif-1 activity . western blots of 4t1 cells ( murine mammary carcinoma cells ) exposed to 1 under hypoxic conditions showed that hypoxia - induced hif-1 expression was significantly inhibited at concentrations between 10 and 100 nm . evaluation of 1 in the national cancer institute s 60-cell line screen showed that 1 possesses strong differential cytotoxicities against several breast , leukemia , and melanoma cell lines ( see the supporting information for details ) . encouraged by the potential of manassantins as novel therapeutics for cancer , we prepared a series of manassantin analogues to define the structural requirements for hif-1 inhibition . herein , we describe our efforts toward the synthesis and sar study of manassantin analogues to guide the design of structurally simplified manassantins for anticancer therapeutic development . manassantins a ( 1 ) and b ( 2 ) consist of two regions ( figure 2 ) : a tetrahydrofuran core region and a side chain region . since we demonstrated that the 2,3-cis-3,4-trans-4,5-cis - configuration of the tetrahydrofuran core is critical for hif-1 inhibition , we envisioned the preparation of a series of analogues with modification of the substituents and the length of the side chains in an effort to establish a more comprehensive sar and identify manassantin analogues with reduced structural complexity . the manassantin analogues could be easily prepared from the known tetrahydrofuran core 5(24 ) following the convergent synthetic approach shown in figure 2 . we anticipated that sar studies would identify structural motifs and minimal structural requirements required for hif-1 inhibition . such information will allow structural modifications to increase specificity , decrease off - target effects , and improve drug performance . first , we prepared an extended side chain analogue ( ma02 ) to gain insights into the importance of side chain length in hif-1 inhibition . the synthesis of ma02 started with the preparation of tosylate 10 and -bromoketone 12 for coupling reactions ( scheme 1 ) . benzyl ( bn)-protection of the known trans - m - propenyl guaiacol ( 6 ) followed by asymmetric dihydroxylation of 7 provided diol 8 . selective ddq - oxidation of the benzylic hydroxyl group of 9 followed by tosylation successfully afforded 10 . -bromoketone 12 was prepared by treatment of the commercially available 3,4-dimethyoxyacetophenone ( 11 ) with br2 . bemp - mediated coupling of tetrahydrofuran core 5(24 ) and tosylate 10 and bn - deprotection provided ma01 in excellent overall yield . final coupling of ma01 with 12 completed the synthesis of ma02 . as shown in scheme 2 , a truncated analogue ( ma03 ) was prepared by monoalkylation of 5 with the known tosylate 14(24 ) ( 48% ) followed by methylation ( k2co3 , mei , 93% ) . stereoselective reduction of ma03 with polymer - bound bh4 afforded the desired truncated analogue ( ma04 , 4-o - methylsaucerneol ) in 80% yield , which constitutes the first synthesis of 4-o - methylsaucerneol . we hypothesized that the c7 and c7 hydroxyl groups could play an important role by providing hydrogen bonds to the binding target(s ) or influencing the conformation of the side chains via intramolecular hydrogen bonds with the neighboring ether oxygen atom . to test this hypothesis , we synthesized the corresponding keto analogue ( ma05 ) by coupling 5 and 14 . the c7/c7 methoxy analogue ( ma06 ) was prepared by methylation of manassantin a ( 1 ) with nah and mei ( 79% ) ( scheme 3 ) . to define the role of c8/c8 methyl groups and reduce the structural complexity of manassantin side chains , we prepared c8/c8 desmethyl analogues ( ma07ma09 ) and c8/c8 desmethyl and c7/c7 deoxo analogue ( ma10 ) ( scheme 4 ) . asymmetric corey bakshi shibata reduction of ma07 afforded ma08 as a single diastereomer ( 82% ) , which was further converted to ma09 by methylation with nah and mei . to further reduce the complexity of manassantins , we removed both c8/c8 methyl and c7/c7 hydroxyl groups and prepared ma10 by coupling 5 with the commercially available 4-(2-bromoethyl)-veratrole ( 16 ) . in addition to reducing the structural complexity , removal of the benzylic hydroxyl groups would reduce the number of hydrogen - bonding donors and influence pk / pd parameters by improving permeability and absorption and removing the potential metabolism site ( e.g. , phase-2 conjugation ) . previously , it was reported that a reduction in potency was observed with the replacement of the c4 methoxy group in the distal phenyl rings of manassantins a ( 1 , figure 1 ) and b ( 2 ) with a hydroxyl group as in 4-o - demethylmanassantin b ( 3 ) . to characterize the role of the methoxy groups on the distal phenyl rings , we prepared the c3/c3 hydroxy analogue ( ma01 ) ( the intermediate to ma02 , scheme 1 ) and phenyl analogues ( ma11 and ma12 ) ( scheme 5 ) . asymmetric dihydroxylation of the commercially available ( e)-1-propenyl - benzene ( 17 ) provided diol 18 in 92% yield . selective ddq - oxidation of the benzylic hydroxyl group of 18 followed by tosylation smoothly proceeded to afford tosylate 20 . bemp - mediated coupling of 5 with 20 successfully provided ma11 , which was reduced stereoselectively to give ma12 . after the completion of the analogue synthesis , we performed a dual luciferase - reporter assay to assess the effect of manassantin analogues ( ma01ma12 ) on hif-1 activity . we used hek-293 t cells ( human embryonic kidney cells ) transiently transfected with both pgl3 - 5hre - luc ( pgl3-hre - luc ) and prl - sv40 encoding renilla luciferase ( pren - luc ) vectors to investigate the effect of ma01ma12 on hif-1 transactivation activity . we generated a reporter vector , pgl3-hre - luciferase plasmid containing five copies of hre sequences identical to that in the human vegf promoter gene . the dual luciferase - reporter assay was used as an initial in vitro test to identify active compounds for further evaluation . t cells were seeded in a 96-well plate at a density of 5 10 cells / well . after 24 h incubation , cells were treated with hypoxic conditions ( 1% o2 ) and serially diluted compounds ( 1 and ma01ma12 ) for 24 h. to measure the firefly luminescence signals , dual - glo reagent was added , and the luminescence signals were measured by using a dual - color luminescence detection system . the luciferase signals were normalized to the activity of renilla luciferase and quantified as relative light units ( rlu ) ( see the supporting information for details ) . none of the analogues tested was as potent as manassantin a ( 1 ) , but several manassantin analogues reduced the luciferase signal to the maximum percent inhibition level relative to that observed under normoxic conditions ( table 1 ) . first , the extended analogue ma02 was almost inactive , suggesting the importance of the side chain length of manassantins in hif-1 inhibitory activity . the truncated analogue ma04 ( 4-o - methylsaucerneol ) , although less potent than 1 , still showed strong inhibition of hif-1 activity ( ic50 = 0.32 m ) . previously , zhou and co - workers reported that ma04 is less potent than 1 due to the absence of one side chain phenylpropyl unit , which was further confirmed by our study . although ma04 possesses lower potency relative to 1 , the reduced molecular weight , hydrophobicity , and structural complexity obtained by removing one of the two side chains of 1 may allow for improvement of other crucial parameters in drug discovery ( see the supporting information for details ) . ligand efficiency ( le ) has been utilized as a method for improving the overall performance of molecules by providing an index of how effectively a molecule uses its structural features to bind and inhibit its target . ligand efficiency indices compare molecules according to their average binding energy per atom or potency of inhibition per atom . this concept has been extended to include other properties such as lipophilicity , molecular mass , and polar surface area . these measures are typically focused on in vitro binding affinity and not on in vivo properties . since overemphasis on potency can often produce large molecules with poor drug properties , optimizing ligand efficiency can be a very important metric in lead optimization . when the le and physicochemical properties of ma04 were calculated using the activity in our dual luciferase - reporter assay ( see the supporting information for details ) , ma04 showed better ligand efficiency index ( lei , ma04 = 0.16 vs 1 = 0.15 ) and binding efficiency index ( bei , ma04 = 11.74 vs 1 = 10.84 ) than 1 . moreover , ma04 compared more favorably than 1 in other physicochemical property assessments ( e.g. , clogp , number of rotatable bonds ) . although multiple parameters ( e.g. , cell permeability ) can impact cellular activity such that theoretical considerations of le may not be directly applicable , the potency of ma04 is significant given the improvement in many other parameters associated with good drug properties . analogues with the c7/c7 keto groups ( ma03 and ma05 ) showed lower activities than analogues with hydroxyl groups at the same positions ( ma03 vs ma04 , ma05 vs 1 ) , suggesting that the c7/c7 hydroxyl groups are important to the potency of 1 . methylation of the c7/c7 hydroxyl groups ( ma06 and ma09 ) also reduced the potency ( ma06 vs 1 , ma09 vs ma08 ) . removal of the c9/c9 methyl groups lowered the activity of the analogues ( ma08 vs 1 , ma09 vs ma06 ) , although in the c7/c7 keto analogues , ma07 demonstrated good activity ( ic50 = 0.35 m ) compared with ma05 ( ic50 = 0.91 m ) . the lack of the c9/c9 methyl groups and displacement of the c7/c7 hydroxyl groups with keto groups in the structure of ma07 dramatically simplifies the chemistry . analogues ( ma11 and ma12 ) without the methoxy groups in the distal phenyl rings were significantly less potent than 1 , however , they showed ic50 values in the submicromolar range . given that methoxy groups in a phenyl ring can be metabolically labile , this result is noteworthy because it provides sar information to guide optimization of pk parameters and to help design affinity probes for target identification studies . one of the most critical aspects of drug discovery programs driven by natural products is the identification of structurally simple and synthetically more accessible compounds , which ultimately aids the preparation of compounds in large quantities for preclinical and clinical studies . none of the analogues that we prepared and tested was as potent as 1 , but several analogues with reduced structural complexity such as ma04 warrant further biological evaluations in this regard . general toxicity of manassantin analogues was examined using the mtt assay , a standard colorimetric cytotoxicity assay . t cells were seeded in a 96-well plate and treated with serially diluted 1 and ma01ma12 ( 0.00510 m ) for 24 h under hypoxia . the absorbance of the formazan was detected at 540 nm . up to the highest concentration examined ( 10 m ) , cells had > 80% survival rate in hek-293 t ( see the supporting information for details ) . following the initial dual luciferase - reporter assay , we used western blots to further examine the down - regulation of hif-1 by manassantin analogues . we selected a subset of manassantin analogues ( ma04 , ma07 , and ma11 ) based on their potency ( from luciferase - reporter assay ) , lack of general toxicity ( from mtt assay ) , and structural simplicity . after incubating hek-293 t cells under hypoxia ( 1% o2 ) for 24 h with or without compounds , total protein lysate was extracted . compounds ma04 , ma07 , and ma11 at 1 m significantly down - regulated the expression level of hif-1 in hek-293 t cells under hypoxia ( figure 3a ) . to determine whether ma04 , ma07 , or ma11 regulates hif-1 , a transcription partner of hif-1 , we performed western blot analysis with antibody against hif-1. compounds ma04 , ma07 , and ma11 did not regulate hif-1 as in the case of 1 , suggesting that ma04 , ma07 , and ma11 regulate hif-1 but not hif-1. manassantin a ( 1 ) , ma04 , ma07 , and ma11 inhibit the expression of hypoxia - induced hif-1 and hif-1 target genes . ( a ) hek-293 t cells were treated with 1 m of 1 , ma04 , ma07 , or ma11 under hypoxia for 24 h. hif-1 , hif-1 , and cdk6 expression levels were determined by western blot analysis with -actin as an internal control ( n , normoxia ; h , hypoxia ) . ( b ) relative expression of vegf mrna was determined by real - time pcr after treatment of cells with 1 m of 1 , ma04 , ma07 , or ma11 under hypoxic conditions for 24 h. relative expression of vegf was normalized against -actin ( n , normoxia ; h , hypoxia ) . to further confirm the effects of ma04 , ma07 , and ma11 on hif-1 activity , we determined the effect on hif target genes at the transcriptional level . upon treatment of ma04 , ma07 , or ma11 at 1 m , the protein level of cyclin - dependent kinase 6 ( cdk6 ) , which is a hif-1 target gene that regulates cell cycle progression , was decreased under hypoxic conditions ( figure 3a ) . vegf ( a key angiogenic factor and a main target for hif-1 ) mrna levels were significantly reduced by ma04 and to a lesser extent by ma07 or ma11 ( figure 3b ) . all together , these data demonstrate that ma04 , ma07 , and ma11 are inhibitors of hif-1 activity . these compounds may serve as potential lead compounds for future in vivo animal and preclinical studies for novel anticancer drug development . among various methods for molecular target identification , small molecule affinity chromatography exploits the ability of small molecule probes to specifically bind to their molecular targets . this approach has led to the discovery of important drug targets such as histone deacetylases and splicing factor sf3b . in particular , the approach can be very effective when a probe possesses an electrophile or a photo - cross - linking group to form a covalent linkage to its target proteins . due to our strong interest in establishing the modes of action of manassantins , we designed and synthesized photo - cross - linking probes for future molecular target identification studies . among the commonly used photophores , the ( 3-trifluoromethyl)phenyldiazirine group is most popular owing to its wavelength for activation , the size of the photophore , cross - linking yields , side reactions , and stability of labeled products . the carbene species generated from the diazirine group are strongly electrophilic and immediately insert into the target protein . on the basis of the sar analysis described above , we designed two complementary photo - cross - linking probes ( ma13 and ma14 ) by incorporation of a ( 3-trifluoromethyl)phenyldiazirine group as the cross - linking group and an alkyne or a biotin as the handle for protein isolation ( scheme 6 ) . these photo - cross - linking probes ( ma13 and ma14 ) were easily prepared as illustrated in scheme 6 . after the preparation of ma13 and ma14 , we evaluated the activity of ma13 and ma14 in the dual luciferase - reporter assay as described above . ma13 and ma14 showed ic50 values of 0.73 m and 2.32 m ( see the supporting information for details ) , respectively , which was in accordance with our sar analysis . after further biological evaluations of ma13 , we plan to use ma13 in a small molecule affinity pull - down experiment side - by - side with other target identification approaches such as global gene expression and energetics - based proteomics . under hypoxia , tumors increase angiogenesis and metastatic potential , alter apoptosis , and regulate metabolism to cope with the stress of hypoxia . these adaptations make tumors more aggressive and treatment - resistant resulting in poor patient prognosis . hif-1 is a main regulator of these adaptions through the activation of several hundred genes involved in angiogenesis , glucose transport , glycolytic pathway , ros signals , erythropoiesis , and other processes . due to the importance of hif-1 in tumor development and progression , we have been exploring manassantins a ( 1 ) and b ( 2 ) , potent inhibitors of hif-1 activity isolated from saururus cernuus , as effective therapeutics for cancers . the sar study of manassantin analogues provided valuable insights into the role / function of the side chains of manassantins and their effects on hypoxia signaling . among the analogues tested , ma04 , ma07 , and ma11 with reduced structural complexity down - regulated hypoxia - induced hif-1 protein stabilization and reduced the levels of cdk6 and vegf under hypoxia . moreover , two photo - cross - linking probes ( ma13 and ma14 ) for molecular target identification were designed and synthesized on the basis of our sar analysis . we expect that our findings will be an important basis for the future design of new manassantin analogues and optimization of drug - like properties of analogues for novel therapeutic development for cancers . all reactions were conducted in oven - dried glassware under nitrogen . unless otherwise stated all reagents were purchased from sigma aldrich , acros , or fisher and were used without further purification . all solvents were acs grade or better and used without further purification except tetrahydrofuran ( thf ) , which was freshly distilled from sodium / benzophenone each time before use . analytical thin layer chromatography ( tlc ) was performed with glass backed silica gel ( 60 ) plates with fluorescent indication ( whatman ) . visualization was accomplished by uv irradiation at 254 nm or by staining with p - anisaldehyde solution . flash column chromatography was performed by using silica gel ( particle size 230400 mesh , 60 ) . all h nmr and c nmr spectra were recorded with a varian 400 ( 400 mhz ) and a bruker 500 ( 500 mhz ) spectrometer in cdcl3 by using the signal of residual chcl3 as an internal standard . all nmr values are given in ppm , and all j values are in hz . electrospray ionization ( esi ) mass spectrometry ( ms ) was recorded with an agilent 1100 series ( lc / msd trap ) spectrometer and performed to obtain the molecular masses of the compounds . infrared ( ir ) absorption spectra were determined with a thermo - fisher ( nicolet 6700 ) spectrometer . optical rotation values were measured with a rudolph research analytical ( a21102 api/1w ) polarimeter . to a cooled ( 0 c ) solution of the known trans - m - propenyl guaiacol ( 6 ; 867 mg , 5.28 mmol ) in dry dmf ( 13 ml , 0.4 m ) were added nah ( 60% dispersion in mineral oil , 304 mg , 7.92 mmol ) and bnbr ( 0.63 ml , 5.28 mmol ) . after stirring for 15 min , the reaction was quenched by addition of h2o and extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 20/1 ) to afford 7 as a white powder ( 1.22 g , 91% ) : h nmr ( 400 mhz , cdcl3 ) 7.50 ( d , j = 8.0 hz , 2h ) , 7.41 ( t , j = 8.0 hz , 2h ) , 7.35 ( d , j = 7.2 hz , 1h ) , 6.98 ( s , 1h ) , 6.92 ( d , j = 7.2 hz , 1h ) , 6.85 ( d , j = 8.4 hz , 1h ) , 6.34 ( d , j = 15.6 hz , 1h ) , 6.07 ( dq , j = 6.8 , 15.6 hz , 1h ) , 5.19 ( s , 2h ) , 3.89 ( s , 3h ) , 1.89 ( d , j = 6.8 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 148.9 , 148.2 , 137.2 , 131.1 , 130.6 , 128.5 , 127.4 , 127.3 , 123.6 , 119.2 , 111.8 , 111.5 , 71.0 , 55.9 , 18.3 ; ir ( neat ) 2932 , 1510 , 1258 , 1136 , 1024 , 962 cm ; hrms ( esi ) m / z 255.1389 [ ( m + h ) , c17h18o2 requires 255.1380 ] . to a cooled ( 0 c ) solution of ad - mix- ( 5.06 g , 1.4 g / mmol of substrate ) and meso2nh2 ( 344 mg , 3.62 mmol ) in t - buoh / h2o ( 1/2 , 31.5 ml ) was added dropwise alkene 7 ( 920 mg , 3.62 mmol ) in etoac / t - buoh ( 1/2 , 16 ml ) . the reaction was quenched by addition of sodium sulfite ( 5.42 g , 1.5 g / mmol of substrate ) , diluted with etoac , and stirred for 30 min at 25 c . the combined organic layers were washed with 2 n koh and brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/1 ) to afford 8 as a colorless oil ( 834 mg , 80% ) : h nmr ( 400 mhz , cdcl3 ) 7.42 ( d , j = 7.2 hz , 2h ) , 7.35 ( t , j = 7.2 hz , 2h ) , 7.29 ( d , j = 7.2 hz ) , 6.876.85 ( m , 3h ) 4.21 ( d , j = 7.6 hz , 1h ) , 3.86 ( s , 1h ) , 3.72 ( dq , j = 6.4 , 14.0 hz , 1h ) , 2.71 ( br s , 2h ) , 0.94 ( d , j = 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 149.4 , 147.9 , 136.9 , 133.7 , 128.4 , 127.8 , 127.5 , 119.9 , 113.0 , 111.6 , 79.1 , 72.1 , 71.0 , 55.9 , 18.7 ; ir ( neat ) 3383 , 2970 , 1733 , 1513 , 1256 , 1134 , 1021 cm ; hrms ( esi ) m / z 311.1244 [ ( m + na ) , c17h20o4 requires 311.1254 ] . to a solution of diol 8 ( 834 mg , 2.89 mmol ) in dioxane ( 29 ml , 0.1 m ) was added ddq ( 1.31 g , 5.78 mmol ) at 25 c . the reaction mixture was heated to 60 c and stirred for 2 h at the same temperature . the reaction mixture was cooled to 0 c , quenched by addition of saturated aqueous sodium thiosulfate and saturated aqueous nahco3 , diluted with ch2cl2 , and stirred vigorously for 1 h. the layers were separated , and the aqueous layer was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford 9 as a yellow oil ( 762 mg , 92% ) : h nmr ( 400 mhz , cdcl3 ) 7.567.52 ( m , 2h ) , 7.477.45 ( m , 2h ) , 7.39 ( dd , j = 7.2 , 8.0 hz , 2h ) , 7.33 ( d , j = 7.6 hz , 1h ) , 6.93 ( d , j = 8.4 hz , 1h ) , 5.20 ( s , 2h ) , 5.04 ( q , j = 7.2 hz , 1h ) , 3.96 ( s , 3h ) , 1.37 ( d , j = 7.2 hz ) ; c nmr ( 125 mhz , cdcl3 ) 200.7 , 154.2 , 148.3 , 136.5 , 128.7 , 128.2 , 127.5 , 126.1 , 123.8 , 113.5 , 110.8 , 71.1 , 68.9 ; ir ( neat ) 3459 , 2976 , 1667 , 1594 , 1512 , 1263 , 1114 , 1016 , 861 cm ; hrms ( esi ) m / z 287.1269 [ ( m + h ) , c17h18o4 requires 287.1278 ] . to a cooled ( 0 c ) solution of hydroxyketone 9 ( 750 mg , 2.62 mmol ) in pyridine ( 25 ml , 0.1 m ) was added ts2o ( 1.28 g , 3.93 mmol ) . after stirring for 40 min at the same temperature , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford 10 as a white powder ( 935 mg , 81% ) : h nmr ( 400 mhz , cdcl3 ) 7.71 ( d , j = 8.4 hz , 2h ) , 7.54 ( dd , j = 2.0 , 8.8 hz , 1h ) , 7.467.41 ( m , 3h ) , 7.367.32 ( m , 2h ) , 7.307.28 ( m , 1h ) , 7.22 ( d , j = 8.4 hz , 2h ) , 6.86 ( d , j = 8.8 hz , 1h ) , 5.69 ( q , j = 7.2 hz , 1h ) , 5.12 ( s , 2h ) , 3.90 ( s , 3h ) , 2.36 ( s , 3h ) , 1.48 ( d , j = 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 192.9 , 154.7 , 148.3 , 145.0 , 136.5 , 133.6 , 129.8 , 128.6 , 128.1 , 127.9 , 127.6 , 126.5 , 123.9 , 113.4 , 110.7 , 77.2 , 71.0 , 56.2 , 21.6 , 18.9 ; ir ( neat ) 2961 , 1684 , 1594 , 1515 , 1265 , 1175 , 1013 , 932 cm ; hrms ( esi ) m / z 441.1356 [ ( m + h ) , c24h24o6s requires 441.1366 ] . to a cooled ( 0 c ) solution of the known bis - phenol tetrahydrofuran core 5(24 ) ( 54 mg , 0.16 mmol ) in dry ch2cl2 ( 2 ml , 0.08 m ) was added dropwise 2-tert - butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine ( bemp , 0.09 ml , 0.31 mmol ) . the resulting mixture was stirred at the same temperature for 10 min before tosylate 10 ( 207.2 mg , 0.47 mmol ) in ch2cl2 ( 2 ml , 0.23 m ) was added . after stirring for 30 min at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford 13 as a white foam ( 133 mg , 96% ) : h nmr ( 400 mhz , cdcl3 ) 7.827.76 ( m , 4h ) , 7.46 ( d , j = 7.6 hz , 4h ) , 7.36 ( dd , j = 7.2 , 8.0 hz , 4h ) , 7.31 ( dd , j = 2.4 , 7.2 hz , 2h ) , 6.89 ( d , j = 8.4 hz , 2h ) , 6.82 ( dd , j = 2.0 , 10.0 hz , 2h ) , 6.766.73 ( m , 2h ) , 6.69 ( dd , j = 1.6 , 8.4 hz , 2h ) , 5.365.32 ( m , 4h ) , 5.17 ( s , 4h ) , 3.92 ( s , 6h ) , 3.83 ( s , 6h ) , 2.232.17 ( m , 2h ) , 1.64 ( d , j = 6.8 hz , 6h ) , 0.62 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 197.5 , 154.2 , 150.0 , 147.9 , 145.8 , 136.6 , 135.6 , 128.6 , 128.0 , 127.6 , 127.3 , 123.9 , 118.5 , 115.7 , 113.9 , 110.6 , 110.5 , 83.4 , 78.3 , 70.8 , 56.0 , 44.0 , 19.1 , 14.8 ; ir ( neat ) 2961 , 1684 , 1593 , 1509 , 1265 , 1137 , 1020 , 867 cm ; hrms ( esi ) m / z 898.4169 [ ( m + nh4 ) , c54h56o11 requires 898.4161 ] . to a stirred solution of bis - benzyl ether 13 ( 29 mg , 0.03 mmol ) in etoac ( 2 ml , 0.02 m ) was added 10% pd c ( 6 mg , 20 wt % ) . the resulting mixture was stirred under h2 atmosphere at 25 c for 8 h. the reaction mixture was then filtered through celite with etoac and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 3/2 ) to afford ma01 as a white foam ( 22.1 mg , 96% ) : [ ]d = 19.9 ( c 0.73 , chcl3 ) ; h nmr ( 400 mhz , cdcl3 ) 7.737.68 ( m , 4h ) , 6.87 ( dd , j = 1.6 , 8.4 hz , 2h ) , 6.80 ( dd , j = 1.6 , 6.0 hz , 2h ) , 6.776.73 ( m , 2h ) , 6.67 ( dd , j = 1.6 , 8.4 hz , 2h ) , 5.71 ( br s , 2h ) , 5.40 ( ddd , j = 6.8 , 6.8 , 6.8 hz , 2h ) , 5.34 ( d , j = 6.0 hz ) , 3.93 ( s , 6h ) , 3.83 ( s , 6h ) , 2.202.16 ( m , 2h ) , 1.67 ( dd , j = 2.4 , 6.8 hz , 6h ) , 0.61 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 197.8 , 151.1 , 150.0 , 146.0 , 145.6 , 135.8 , 128.3 , 122.7 , 118.6 , 116.4 , 116.1 , 115.2 , 110.8 , 110.1 , 83.6 , 78.2 , 56.2 , 44.1 , 19.2 , 14.8 ; ir ( neat ) 3404 , 2962 , 1684 , 1608 , 1509 , 1272 , 1139 , 1028 , 866 cm ; hrms ( esi ) m / z 701.2951 [ ( m + h ) , c40h44o11 requires 701.2956 ] . to a cooled ( 0 c ) solution of bis - phenol ma01 ( 44 mg , 0.06 mmol ) in dry ch2cl2 ( 1.5 ml , 0.04 m ) was added dropwise bemp ( 0.04 ml , 0.13 mmol ) . the resulting mixture was stirred at the same temperature for 10 min before the known bromide 12(33 ) ( 65 mg , 0.25 mmol ) in ch2cl2 ( 1.5 ml , 0.17 m ) was added . after stirring for 1 h at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/2 ) to afford ma02 as a yellow foam ( 52.6 mg , 80% ) : [ ]d = 7.9 ( c 0.66 , chcl3 ) ; h nmr ( 400 mhz , cdcl3 ) 7.867.82 ( m , 2h ) , 7.61 ( dd , j = 2.0 , 6.4 hz , 4h ) , 7.54 ( s , 2h ) , 6.90 ( dd , j = 6.0 , 8.8 hz , 4h ) , 6.79 ( dd , j = 1.6 , 8.8 hz , 2h ) , 6.73 ( dd , j = 4.8 , 8.4 hz , 2h ) , 6.686.64 ( m , 2h ) , 5.405.31 ( m , 8h ) , 3.953.92 ( m , 18h ) , 3.80 ( s , 6h ) , 2.192.15 ( m , 2h ) , 1.65 ( d , j = 6.8 hz , 6h ) , 0.60 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 197.4 , 192.2 , 154.2 , 154.0 , 149.8 , 149.4 , 147.5 , 145.8 , 135.7 , 127.8 , 127.4 , 124.7 , 122.7 , 118.5 , 116.0 , 113.8 , 110.9 , 110.7 , 110.4 , 110.3 , 83.4 , 78.3 , 71.1 , 56.2 , 44.0 , 19.1 , 14.8 ; ir ( neat ) 2932 , 1685 , 1595 , 1513 , 1266 , 1134 , 1021 , 870 cm ; hrms ( esi ) m / z 1074.4497 [ ( m + nh4 ) , c60h64o17 requires 1074.4482 ] . to a solution of the known phenol 15(24 ) ( 32 mg , 0.06 mmol ) in acetone ( 3 ml , 0.02 m ) was added k2co3 ( 16.6 mg , 0.12 mmol ) at 25 c . after stirring for 1 h at 40 c , the reaction mixture was cooled to 25 c and treated with mei ( 0.02 ml , 0.24 mmol ) at the same temperature . after stirring for 2 h at reflux , the reaction mixture was cooled to 25 c , diluted with etoac , and rinsed with saturated aqueous nh4cl . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/1 ) to afford ma03 as a white foam ( 31 mg , 93% ) : [ ]d = 22.5 ( c 0.13 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 7.847.80 ( m , 1h ) , 7.687.66 ( m , 1h ) , 6.876.67 ( m , 7h ) , 5.445.36 ( m , 3h ) , 3.93 ( s , 3h ) , 3.91 ( s , 3h ) , 3.87 ( s , 3h ) , 3.86 ( s , 3h ) , 3.85 ( s , 3h ) , 2.252.19 ( m , 2h ) , 1.71 ( d , j = 6.8 hz , 3h ) , 0.65 ( dd , j = 6.4 , 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 197.9 , 153.7 , 149.8 , 149.1 , 148.7 , 148.0 , 146.4 , 145.9 , 135.8 , 134.0 , 127.6 , 123.7 , 118.6 , 115.9 , 111.4 , 110.9 , 110.7 , 110.2 , 109.7 , 109.0 , 83.6 , 78.5 , 56.1 , 44.2 , 19.3 , 14.9 ; ir ( neat ) 2960 , 1682 , 1593 , 1512 , 1261 , 1136 , 1023 , 799 cm ; hrms ( esi ) m / z 551.2631 [ ( m + h ) , c32h38o8 requires 551.2639 ] . to a stirred solution of ma03 ( 31 mg , 0.06 mmol ) in meoh ( 2 ml , 0.02 m ) was added polymer - suppported borohydride ( 2 mmol bh4/g resin , 560 mg , 1.12 mmol ) . the reaction mixture was stirred with gentle agitation at 25 c for 72 h. the polymer beads were then removed by filtration , and the filtrate was purified by column chromatography ( silica gel , hexanes / etoac , 4/1 ) to afford ma04 as a colorless oil ( 25 mg , 80% ) : [ ]d = 78.3 ( c 0.52 , chcl3 ) ; h nmr ( 400 mhz , cdcl3 ) 7.006.82 ( m , 9h ) , 5.45 ( d , j = 6.0 hz , 2h ) , 4.64 ( d , j = 8.4 hz , 1h ) , 4.154.08 ( m , 1h ) , 3.92 ( s , 3h ) , 3.89 ( s , 3h ) , 3.88 ( s , 6h ) , 3.87 ( s , 3h ) , 2.222.31 ( m , 2h ) , 1.17 ( d , j = 6.4 hz , 3h ) , 0.71 ( d , j = 6.4 hz , 3h ) , 0.69 ( dd , j = 6.4 , 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 150.7 , 149.1 , 149.0 , 148.8 , 148.0 , 146.6 , 136.8 , 134.0 , 132.7 , 120.1 , 118.8 , 118.5 , 111.0 , 110.9 , 110.3 , 110.2 , 109.7 , 84.3 , 83.6 , 83.5 , 78.5 , 56.0 , 44.2 , 17.2 , 14.9 ; ir ( neat ) 3453 , 2930 , 1510 , 1256 , 1138 , 1027 , 811 , 733 cm ; hrms ( esi ) m / z 570.3072 [ ( m + nh4 ) , c32h40o8 requires 570.3061 ] . to a cooled ( 0 c ) solution of manassantin a ( 25 mg , 0.03 mmol ) in thf ( 2 ml , 0.02 m ) were added nah ( 60% dispersion in mineral oil , 10.5 mg , 0.27 mmol ) and mei ( 0.02 ml , 0.34 mmol ) . after stirring for 1 h at the same temperature , the reaction was quenched by addition of h2o and extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford ma06 as a colorless oil ( 23 mg , 79% ) : [ ]d = 49.9 ( c 0.3 , chcl3 ) ; h nmr ( 400 mhz , cdcl3 ) 6.976.77 ( m , 12h ) , 5.42 ( d , j = 6.4 hz , 2h ) , 4.534.46 ( m , 2h ) , 4.32 ( d , j = 6.4 hz , 2h ) , 3.90 ( s , 6h ) , 3.89 ( s , 6h ) , 3.85 ( s , 6h ) , 3.29 ( s , 6h ) , 2.282.22 ( m , 2h ) , 1.06 ( d , j = 6.4 hz , 6h ) , 0.68 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 150.5 , 149.2 , 148.9 , 146.8 , 135.1 , 131.4 , 120.5 , 118.7 , 116.6 , 110.8 , 110.6 , 86.6 , 83.7 , 79.0 , 57.3 , 56.2 , 56.1 , 44.2 , 16.5 , 15.0 ; ir ( neat ) 2933 , 1508 , 1271 , 1028 cm ; hrms ( esi ) m / z 778.4153 [ ( m + nh4 ) , c44h56o11 requires 778.4161 ] . to a cooled ( 0 c ) solution of the known bis - phenol 5 ( 90 mg , 0.26 mmol ) in dry dmf ( 2 ml , 0.13 m ) was added nah ( 60% dispersion in mineral oil , 30 mg , 0.78 mmol ) . after stirring for 5 min , the reaction mixture was treated with the known bromide 12(33 ) ( 271 mg , 1.04 mmol ) in dmf ( 4 ml , 0.26 m ) and stirred for 40 min . the resulting mixture was allowed to warm to 25 c for 2 h. at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/1 ) to afford ma07 as a colorless oil ( 90 mg , 47% ) : [ ]d = 25.7 ( c 0.4 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 7.67 ( dd , j = 2.0 , 8.4 hz , 2h ) , 7.59 ( s , 2h ) , 6.906.73 ( m , 8h ) , 5.40 ( d , j = 6.0 hz , 2h ) , 5.28 ( s , 4h ) , 3.95 ( s , 6h ) , 3.93 ( s , 6h ) , 3.89 ( s , 6h ) , 2.262.20 ( m , 2h ) , 0.66 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 193.5 , 153.9 , 149.5 , 149.3 , 146.6 , 135.7 , 128.0 , 122.9 , 118.5 , 114.5 , 110.6 , 110.4 , 110.2 , 83.5 , 72.4 , 56.2 , 56.1 , 44.2 , 14.9 ; ir ( neat ) 2931 , 1689 , 1512 , 1261 , 1163 , 1021 cm ; hrms ( esi ) m / z 701.2959 [ ( m + h ) , c40h44o11 requires 701.2956 ] . to a cooled ( 20 c ) solution of ( r)-(+)-2-methyl - cbs - oxazaborolidine ( 0.69 mg , 2.5 mol ) and bh3sme2 ( 5 l , 0.05 mmol ) in toluene ( 1 ml , 0.05 m ) was added dropwise ketone ma07 ( 35 mg , 0.05 mmol ) in toluene ( 2 ml , 0.03 m ) . after stirring for 1 h at the same temperature , the reaction was quenched by addition of meoh followed by 1 n hcl . the aqueous layer was extracted with etoac , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/2 ) to afford ma08 as a colorless oil ( 29 mg , 82% ) : [ ]d = 32.9 ( c 0.4 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 7.016.80 ( m , 12h ) , 5.44 ( d , j = 6.0 hz , 2h ) , 5.05 ( dd , j = 2.8 , 9.2 hz , 2h ) , 4.15 ( dd , j = 3.2 , 10.4 hz , 2h ) , 3.96 ( t , j = 10.0 hz , 2h ) , 3.894 ( s , 6h ) , 3.89 ( s , 6h ) , 3.87 ( s , 6h ) , 3.30 ( br s , 2h ) , 2.312.23 ( m , 2h ) , 0.69 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , etoac ) 149.9 , 149.2 , 148.9 , 146.9 , 135.8 , 132.3 , 118.8 , 118.7 , 115.6 , 111.1 , 110.2 , 109.5 , 83.5 , 76.5 , 72.2 , 56.0 , 44.2 , 14.9 ; ir ( neat ) 3481 , 2922 , 1514 , 1262 , 1027 , 810 cm ; hrms ( esi ) m / z 722.3523 [ ( m + nh4 ) , c40h48o11 requires 722.3535 ] . to a cooled ( 0 c ) solution of alcohol ma08 ( 24 mg , 0.03 mmol ) in dry dmf ( 3 ml , 0.01 m ) were added nah ( 60% dispersion in mineral oil , 20.5 mg , 0.55 mmol ) and mei ( 0.04 ml , 0.68 mmol ) . after stirring for 1 h at the same temperature , the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/3 ) to afford ma09 as a white foam ( 23 mg , 92% ) : [ ]d = 43.2 ( c 0.38 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 6.956.75 ( m , 12h ) , 5.41 ( d , j = 5.6 hz , 2h ) , 4.60 ( dd , j = 3.2 , 7.6 hz , 2h ) , 4.18 ( dd , j = 8.0 , 10.8 hz , 2h ) , 4.02 ( dd , j = 3.6 , 6.8 hz , 2h ) , 3.90 ( s , 6h ) , 3.88 ( s , 6h ) , 3.86 ( s , 6h ) , 3.35 ( s , 6h ) , 2.262.20 ( m , 2h ) , 0.66 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 149.6 , 149.3 , 149.0 , 135.0 , 131.4 , 119.6 , 118.5 , 114.1 , 111.1 , 110.5 , 109.9 , 83.6 , 82.2 , 74.0 , 57.2 , 56.1 , 56.0 , 44.2 , 14.9 ; ir ( neat ) 2932 , 1513 , 1261 , 1137 , 1028 cm ; hrms ( esi ) m / z 750.3846 [ ( m + nh4 ) , c42h52o11 requires 750.3848 ] . to a solution of the known bis - phenol 5 ( 20 mg , 0.06 mmol ) in dry dmf ( 1.5 ml , 0.04 m ) were added k2co3 ( 32 mg , 0.23 mmol ) and 3,4-dimethoxyphenethyl bromide ( 143 mg , 0.58 mmol ) at 25 c . the reaction mixture was heated to 120 c and stirred for 30 h at the same temperature . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 3/1 ) to afford ma10 as a yellow oil ( 18 mg , 44% ) : [ ]d = 6.79 ( c 0.28 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 6.876.82 ( m , 12h ) , 5.43 ( d , j = 6.0 hz , 2h ) , 4.19 ( t , j = 7.2 hz , 4h ) , 3.883.87 ( m , 18h ) , 3.10 ( t , j = 7.2 hz , 4h ) , 2.292.22 ( m , 2h ) , 0.68 ( d , j = 6.0 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 149.2 , 149.0 , 147.8 , 147.3 , 134.5 , 130.9 , 121.1 , 118.6 , 112.9 , 112.6 , 111.4 , 110.3 , 83.6 , 70.2 , 56.2 , 56.1 , 55.9 , 56.2 , 56.1 , 55.9 , 44.2 , 35.6 , 14.9 ; ir ( neat ) 2928 , 1507 , 1229 , 1138 , 1026 cm ; hrms ( esi ) m / z 690.3632 [ ( m + nh4 ) , c40h48o9 requires 690.3637 ] . to a cooled ( 0 c ) solution of ad - mix- ( 5.4 g , 1.4 g / mmol of substrate ) and meso2nh2 ( 367 mg , 3.85 mmol ) in t - buoh / h2o ( 1/2 , 30 ml ) was added dropwise trans--methylstyrene 17 ( 0.5 ml , 3.85 mmol ) in t - buoh ( 10 ml ) . the reaction was quenched by addition of sodium sulfite ( 5.78 g , 1.5 g / mmol of substrate ) , diluted with etoac , and stirred for 30 min at 25 c . the combined organic layers were washed with 2 n koh and brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 3/1 ) to afford 18 as a colorless oil ( 539 mg , 92% ) : h nmr ( 400 mhz , cdcl3 ) 7.337.23 ( m , 5h ) , 4.25 ( d , j = 7.6 hz , 1h ) , 4.02 ( br s , 2h ) , 3.77 ( dq , j = 6.4 , 13.6 hz , 1h ) , 0.95 ( d , j = 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 141.2 , 128.4 , 128.0 , 127.0 , 79.5 , 72.1 ; ir ( neat ) 3362 , 2973 , 1454 , 1265 , 1128 , 1036 , 870 cm ; hrms ( esi ) h2o , c9h12o2 requires 135.0804 ] . to a solution of diol 18 ( 539 mg , 3.54 mmol ) in dioxane ( 35 ml , 0.1 m ) was added ddq ( 1.6 g , 7.08 mmol ) at 25 c . the reaction mixture was heated to 80 c and stirred for 12 h at the same temperature . the reaction mixture was cooled to 0 c , quenched by addition of saturated aqueous sodium thiosulfate and saturated aqueous nahco3 , diluted with ch2cl2 , and stirred vigorously for 1 h. the layers were separated , and the aqueous layer was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford 19 as a colorless crystal ( 308 mg , 58% ) : h nmr ( 400 mhz , cdcl3 ) 7.92 ( dd , j = 1.2 , 8.4 hz , 2h ) , 7.637.58 ( m , 1h ) , 7.49 ( dt , j = 1.6 , 7.6 , 14.0 hz , 2h ) , 5.16 ( q , j = 7.2 hz , 1h ) , 3.73 ( br s , 1h ) , 1.44 ( d , j = 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 202.6 , 134.1 , 133.5 , 128.9 , 69.5 , 22.4 ; ir ( neat ) 3455 , 2979 , 1679 , 1597 , 1268 , 1126 , 1025 , 968 cm ; hrms ( esi ) m / z 133.0647 [ ( m + h h2o ) , c9h10o2 requires 133.0648 ] . to a cooled ( 0 c ) solution of hydroxyketone 19 ( 308 mg , 2.05 mmol ) in pyridine ( 20 ml , 0.1 m ) was added ts2o ( 1 g , 3.08 mmol ) . after stirring for 30 min at the same temperature , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford 20 as a white powder ( 437 mg , 70% ) : h nmr ( 400 mhz , cdcl3 ) 7.87 ( dd , j = 1.2 , 8.4 hz , 2h ) , 7.75 ( dd , j = 1.2 , 8.4 hz , 2h ) , 7.617.56 ( m , 1h ) , 7.45 ( dt , j = 1.6 , 8.0 , 13.6 hz , 2h ) , 7.26 ( d , j = 8.0 hz , 2h ) , 5.78 ( q , j = 7.2 hz , 1h ) , 2.40 ( s , 3h ) , 1.59 ( d , j = 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 194.9 , 145.2 , 134.0 , 133.8 , 133.6 , 129.9 , 128.9 , 128.0 , 77.5 , 21.7 , 18.8 ; ir ( neat ) 2960 , 1699 , 1597 , 1360 , 1175 , 1017 , 919 cm ; hrms ( esi ) m / z 305.0836 [ ( m + h ) , c16h16o4s requires 305.0842 ] . to a cooled ( 0 c ) solution of the known bis - phenol 5 ( 30 mg , 0.09 mmol ) in dry ch2cl2 ( 1.5 ml , 0.06 m ) the resulting mixture was stirred at the same temperature for 10 min before tosylate 20 ( 106 mg , 0.35 mmol ) in ch2cl2 ( 1.5 ml , 0.23 m ) was added . after stirring for 30 min at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 4/1 ) to afford ma11 as a colorless oil ( 45.5 mg , 86% ) : [ ]d = 27.57 ( c 0.5 , chcl3 ) ; h nmr ( 400 mhz , cdcl3 ) 8.118.08 ( m , 4h ) , 7.587.54 ( m , 2h ) , 7.45 ( dd , j = 6.0 , 7.2 hz , 4h ) , 6.826.77 ( m , 4h ) , 6.706.66 ( m , 2h ) , 5.46 ( dq , j = 4.0 , 6.8 hz , 2h ) , 5.35 ( d , j = 6.0 hz , 2h ) , 3.82 ( s , 6h ) , 2.222.16 ( m , 2h ) , 1.70 ( d , j = 7.2 hz , 6h ) , 0.62 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 199.3 , 150.0 , 145.9 , 136.0 , 134.8 , 133.5 , 129.1 , 128.7 , 118.6 , 116.7 , 116.5 , 110.7 , 83.5 , 78.5 , 56.1 , 44.2 , 30.0 , 19.0 , 14.9 ; ir ( neat ) 2962 , 1697 , 1508 , 1269 , 1221 , 1142 , 1034 , 966 cm ; hrms ( esi ) m / z 626.3107 [ ( m + nh4 ) , c38h40o7 requires 626.3112 ] . to a stirred solution of 11 ( 42 mg , 0.07 mmol ) in meoh ( 4.5 ml , 0.02 m ) was added polymer - suppported borohydride ( 2 mmol bh4/g resin , 690 mg , 1.38 mmol ) . the reaction mixture was stirred with gentle agitation at 25 c for 48 h. the polymer beads were then removed by filtration , and the filtrate was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford ma12 as a colorless oil ( 23 mg , 54% ) : [ ]d = 62.14 ( c 0.4 , etoac ) ; h nmr ( 400 mhz , cdcl3 ) 7.417.28 ( m , 10h ) , 7.006.81 ( m , 6h ) , 5.46 ( d , j = 6.0 hz , 2h ) , 4.70 ( d , j = 8.0 hz , 2h ) , 4.184.09 ( m , 3h ) , 3.93 ( s , 6h ) , 2.322.26 ( m , 2h ) , 1.17 ( dd , j = 2.0 , 6.4 hz , 6h ) , 0.72 ( d , j = 6.4 hz , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 150.7 , 146.6 , 140.2 , 136.7 , 128.5 , 128.2 , 127.5 , 119.0 , 118.8 , 110.3 , 84.3 , 83.5 , 78.8 , 56.0 , 44.3 , 17.1 , 15.0 ; ir ( neat ) 3481 , 2964 , 1508 , 1258 , 1036 cm ; hrms ( esi ) m / z 630.3447 [ ( m + nh4 ) , c38h44o7 requires 630.3425 ] . to a cooled ( 78 c ) solution of ( e)-1-bromo-4-(prop-1-en-1-yl)benzene ( 340 mg , 1.73 mmol ) in dry thf ( 15 ml , 0.12 m ) was added dropwise n - buli ( 2.3 m in hexane , 1.13 ml , 2.59 mmol ) . the resulting mixture was stirred for 2 h at the same temperature before methyl fluoroacetate ( 0.23 ml , 2.24 mmol ) in dry thf ( 9 ml ) was added dropwise . after stirring for 1.5 h at the same temperature , the reaction was quenched by addition of saturated aqueous nh4cl , and the resulting mixture was extracted with et2o . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes ) to afford ketone 22 ( 337 mg , 90% ) : h nmr ( 400 mhz , cdcl3 ) 8.00 ( d , j = 8.0 hz , 2h ) , 7.46 ( d , j = 8.4 hz , 2h ) , 6.516.43 ( m , 2h ) , 1.95 ( d , j = 5.2 hz , 3h ) . to a solution of 22 ( 337 mg , 1.57 mmol ) in pyridine / etoh ( 2:1 , 6 ml ) was added hydroxylamine hydrochloride ( 142 mg , 2.05 mmol ) at 25 c . after stirring for 1.5 h at reflux , the solvents were removed under reduced pressure . the residue was extracted with etoac / h2o , dried over anhydrous na2so4 , and concentrated in vacuo . the crude residue was employed in the next step without further purification . to a solution of the crude oxime ( 348 mg , 1.52 mmol ) in pyridine ( 6 ml , 0.25 m ) was added tscl ( 434 mg , 2.28 mmol ) at 25 c . after stirring for 2 h at reflux the residue was extracted with etoac / h2o , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 20/1 ) to afford tosyloxime 23 as a yellow powder ( 555 mg , 92% for 2 steps ) : h nmr ( 400 mhz , cdcl3 ) 7.91 ( dd , j = 8.4 , 14.4 hz , 2h ) , 7.427.36 ( m , 6h ) , 6.38 ( dd , j = 4.0 , 9.6 hz , 2h ) , 2.48 ( d , j = 5.6 hz , 3h ) , 1.92 ( d , j = 5.2 hz , 3h ) . to a cooled ( 78 c ) solution of 23 ( 550 mg , 1.43 mmol ) in ch2cl2 ( 10 ml , 0.14 m ) was bubbled nh3 gas . the resulting mixture was stirred in a sealed tube for 5 min at 78 c and slowly warmed to 25 c . after stirring for 12 h at the same temperature , the reaction mixture was cooled to 78 c , and nh3 was evaporated for 6 h at 25 c . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 10/1 ) to afford diaziridine 24 ( 296 mg , 90% ) : h nmr ( 400 mhz , cdcl3 ) 7.537.49 ( m , 2h ) , 7.377.32 ( m , 2h ) , 6.436.36 ( m , 1h ) , 6.30 ( dq , j = 6.4 , 19.2 hz , 1h ) , 2.77 ( br s , 1h ) , 2.20 ( br s , 1h ) , 1.911.86 ( m , 3h ) . to a solution of 24 ( 290 mg , 1.27 mmol ) in dry et2o ( 10 ml , 0.13 m ) was added a freshly prepared ag2o ( 883 mg , 3.81 mmol ) at 25 c . after stirring for 1 h at the same temperature , the reaction mixture was filtered through celite . the crude residue was employed in the next step without further purification . to a cooled ( 0 c ) solution of ad - mix- ( 1.5 g , 1.4 g / mmol of substrate ) and meso2nh2 ( 103 mg , 1.08 mmol ) in t - buoh / h2o ( 1/2 , 12 ml ) was added dropwise the crude alkene ( 244 mg , 1.08 mmol ) in t - buoh ( 4 ml , 0.27 m ) . the reaction was quenched by addition of sodium sulfite ( 1.55 g , 1.5 g / mmol of substrate ) . the resulting mixture was diluted with etoac and stirred for 30 min at 25 c . the combined organic layers were washed with 2 n koh and brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 3/2 ) to afford diol 25 ( 255 mg , 77% for 2 steps ) : h nmr ( 400 mhz , cdcl3 ) 7.38 ( d , j = 8.4 hz , 2h ) , 7.18 ( d , j = 8.4 hz , 2h ) , 4.40 ( d , j = 7.2 hz , 1h ) , 3.81 ( m , 1h ) , 2.42 ( br s , 2h ) , 1.07 ( d , j = 6.4 hz , 3h ) . to a cooled ( 0 c ) solution of 25 ( 100 mg , 0.38 mmol ) in ch2cl2 ( 10 ml , 0.04 m ) was added dmdo ( 0.1 m in acetone , 8.5 ml , 0.85 mmol ) . after stirring for 18 h at the same temperature , the residue was purified by column chromatography ( silica gel , hexanes / etoac , 4/1 ) to afford the corresponding hydroxy ketone s1 ( 29 mg , 29% ) : h nmr ( 400 mhz , cdcl3 ) 7.95 ( d , j = 8.8 hz , 2h ) , 7.31 ( d , j = 8.0 hz , 2h ) , 5.14 ( dq , j = 6.4 , 6.8 hz , 1h ) , 3.65 ( d , j = 6.8 , 1h ) , 1.44 ( d , j = 6.8 hz , 3h ) . to a cooled ( 0 c ) solution of s1 ( 29 mg , 0.11 mmol ) in pyridine ( 3 ml , 0.04 m ) was added ts2o ( 55 mg , 0.17 mmol ) . after stirring for 30 min at the same temperature , the reaction was quenched by addition of saturated aqueous nh4cl and the resulting mixture was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 5/1 ) to afford tosylate 26 as a white powder ( 36 mg , 76% ) : h nmr ( 400 mhz , cdcl3 ) 7.91 ( d , j = 8.8 hz , 2h ) , 7.72 ( d , j = 8.4 hz , 2h ) , 7.26 ( dd , j = 8.4 , 8.4 hz , 4h ) , 5.67 ( q , j = 6.8 hz , 1h ) , 2.42 ( s , 3h ) , 1.58 ( d , j = 6.8 hz , 3h ) . to a cooled ( 0 c ) solution of the known bis - phenol 5 ( 35 mg , 0.1 mmol ) in dry ch2cl2 ( 1 ml , 0.1 m ) was added dropwise bemp ( 0.03 ml , 0.1 mmol ) . the resulting mixture was stirred at the same temperature for 10 min before tosylate 10 ( 45 mg , 0.1 mmol ) in ch2cl2 ( 1 ml , 0.1 m ) was added . after stirring for 30 min at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl and extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/1 ) to afford phenol 27 ( 25 mg , 40% ) : h nmr ( 400 mhz , cdcl3 ) 7.827.76 ( m , 2h ) , 7.47 ( d , j = 7.2 hz , 2h ) , 7.37 ( dd , j = 7.2 , 7.6 hz , 2h ) , 7.31 ( d , j = 7.6 hz , 1h ) , 6.89 ( dd , j = 6.0 , 8.4 hz , 2h ) , 6.83 ( dd , j = 2.0 , 13.2 hz , 2h ) , 6.776.68 ( m , 3h ) , 5.54 ( s , 1h ) , 5.39 ( dd , j = 5.2 , 6.0 hz , 2h ) , 5.34 ( dd , j = 4.4 , 6.8 hz , 1h ) , 5.18 ( s , 2h ) , 3.93 ( s , 3h ) , 3.89 ( s , 3h ) , 3.84 ( s , 3h ) , 2.242.20 ( m , 2h ) , 1.64 ( d , j = 6.8 hz , 3h ) , 0.670.64 ( m , 6h ) . to a cooled ( 0 c ) solution of 27 ( 18 mg , 0.03 mmol ) in dry ch2cl2 ( 2 ml , 0.02 m ) were added imidazole ( 12 mg , 0.18 mmol ) and tbdpscl ( 0.02 ml , 0.09 mmol ) . after stirring for 10 h at the same temperature , the reaction was quenched by addition of saturated aqueous nh4cl , and the resulting mixture was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 3/1 ) to afford the corresponding ether s2 as a white foam ( 24 mg , 95% ) : h nmr ( 400 mhz , cdcl3 ) 7.817.68 ( m , 6h ) , 7.477.29 ( m , 11h ) , 6.89 ( d , j = 8.4 hz , 1h ) , 6.826.66 ( m , 5h ) , 6.53 ( d , j = 8.4 hz , 1h ) , 5.345.31 ( m , 3h ) , 5.17 ( s , 2h ) , 3.93 ( s , 3h ) , 3.82 ( s , 3h ) , 3.54 ( s , 3h ) , 2.192.12 ( m , 2h ) , 1.64 ( d , j = 6.8 hz , 3h ) , 1.11 ( s , 9h ) , 0.59 ( dd , j = 6.4 , 16.0 hz , 6h ) . to a stirred solution of s2 ( 102 mg , 0.12 mmol ) in etoac / etoh ( 3/1 , 12 ml , 0.1 m ) was added 10% pd / c ( 30 mg , 30 wt % ) . the resulting mixture was stirred under h2 atmosphere at 25 c for 8 h. the reaction mixture was then filtered through celite with etoac and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford phenol 28 ( 62 mg , 67% ) : h nmr ( 400 mhz , cdcl3 ) 7.747.68 ( m , 6h ) , 7.407.29 ( m , 6h ) , 6.88 ( dd , j = 2.0 , 8.4 hz , 1h ) , 6.81 ( dd , j = 2.0 , 6.0 hz , 1h ) , 6.75 ( dd , j = 4.4 , 8.4 hz , 1h ) , 6.706.66 ( m , 3h ) , 6.556.52 ( m , 1h ) , 5.61 ( s , 1h ) , 5.39 ( m , 1h ) , 5.32 ( d , j = 4.8 hz , 2h ) , 3.95 ( s , 3h ) , 3.84 ( s , 3h ) , 3.54 ( s , 3h ) , 2.182.12 ( m , 2h ) , 1.68 ( dd , j = 2.0 , 6.4 hz , 3h ) , 1.11 ( s , 9h ) , 0.59 ( dd , j = 6.4 , 13.2 hz , 6h ) . to a cooled ( 0 c ) solution of 28 ( 20 mg , 0.03 mmol ) in dry dmf ( 2 ml , 0.01 m ) were added propargyl bromide ( 0.02 ml , 0.03 mmol ) and k2co3 ( 5.5 mg , 0.04 mmol ) . after stirring for 1 h at 25 c , the reaction was quenched by addition of water , and the resulting mixture was extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford the corresponding ether s3 as a white foam ( 14 mg , 67% ) : h nmr ( 400 mhz , cdcl3 ) 7.887.84 ( m , 2h ) , 7.69 ( dd , j = 1.6 , 8.0 hz , 4h ) , 7.407.30 ( m , 6h ) , 6.90 ( d , j = 8.0 hz , 1h ) , 6.81 ( dd , j = 1.6 , 9.2 hz , 1h ) , 6.77 ( dd , j = 3.6 , 8.0 hz , 1h ) , 6.706.65 ( m , 3h ) , 6.53 ( dd , j = 1.6 , 8.4 hz , 1h ) , 5.37 ( dq , j = 4.4 , 6.8 hz , 1h ) , 5.335.30 ( m , 2h ) , 4.80 ( d , j = 2.4 hz , 2h ) , 3.93 ( s , 3h ) , 3.84 ( s , 3h ) , 3.54 ( s , 3h ) , 2.51 ( s , 1h ) , 2.192.12 ( m , 2h ) , 1.71 ( d , j = 6.8 hz , 3h ) , 1.11 ( s , 9h ) , 0.59 ( dd , j = 6.4 , 15.6 hz , 6h ) . to a solution of s3 ( 14 mg , 0.02 mmol ) in dry thf ( 1 ml , 0.02 m ) was added tbaf ( 1.0 m in thf , 0.04 ml , 0.04 mmol ) . after stirring for 5 min at 25 c , the reaction was quenched by addition of saturated aqueous nh4cl , and the resulting mixture was extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford the corresponding phenol s4 ( 8 mg , 80% ) : h nmr ( 500 mhz , cdcl3 ) 7.797.84 ( m , 2h ) , 6.926.68 ( m , 7h ) , 5.52 ( s , 1h ) , 5.405.36 ( m , 3h ) , 4.81 ( d , j = 2.0 hz , 2h ) , 3.93 ( s , 3h ) , 3.89 ( s , 3h ) , 3.85 ( s , 3h ) , 2.51 ( s , 1h ) , 2.242.18 ( m , 2h ) , 1.71 ( d , j = 7.0 hz , 3h ) , 0.670.64 ( m , 6h ) . to a cooled ( 0 c ) solution of s4 ( 8 mg , 0.01 mmol ) in dry ch2cl2 ( 1 ml , 0.01 m ) was added dropwise bemp ( 0.004 ml , 0.01 mmol ) . the resulting mixture was stirred at the same temperature for 10 min before tosylate 26 ( 7 mg , 0.02 mmol ) in ch2cl2 ( 0.5 ml , 0.04 m ) was added . after stirring for 30 min at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl , and the resulting mixture was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford ma13 as a colorless oil ( 5 mg , 45% ) : h nmr ( 400 mhz , cdcl3 ) 8.15 ( dd , j = 4.8 , 8.4 hz , 2h ) , 7.887.84 ( m , 2h ) , 7.23 ( d , j = 8.4 hz , 2h ) , 6.90 ( d , j = 8.4 hz , 1h ) , 6.826.76 ( m , 4h ) , 6.706.66 ( m , 2h ) , 5.405.33 ( m , 4h ) , 4.80 ( d , j = 2.4 hz , 2h ) , 3.93 ( s , 3h ) , 3.84 ( s , 3h ) , 3.80 ( s , 3h ) , 2.51 ( s , 1h ) , 2.212.18 ( m , 2h ) , 1.69 ( dd , j = 6.8 , 16.0 hz , 6h ) , 0.630.60 ( m , 6h ) ; ir ( neat ) 2960 , 1684 , 1509 , 1264 , 1025 cm ; hrms ( esi ) m / z 823.2803 [ ( m + na ) , c44h43f3n2o9 requires 823.2813 ] . to a solution of 28 ( 15 mg , 0.02 mmol ) in dry dmf ( 2 ml , 0.01 m ) was added k2co3 ( 6 mg , 0.04 mmol ) at 25 c . after stirring for 30 min at the same temperature , the reaction mixture was cooled to 0 c and treated with tert - butyl ( 2-[2-(2-bromoethoxy)ethoxy]ethyl)carbamate ( 7.5 mg , 0.02 mmol ) and tbai ( 0.8 mg , 0.002 mmol ) . after stirring for 16 h at 25 c , the reaction was quenched by addition of water , and the resulting mixture was extracted with etoac . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 1/2 ) to afford the corresponding phenol s5 ( 5 mg , 34% ) : h nmr ( 400 mhz , cdcl3 ) 7.82 ( dd , j = 6.0 , 8.4 hz , 1h ) , 7.70 ( s , 1h ) , 6.896.67 ( m , 7h ) , 5.54 ( s , 1h ) , 5.415.38 ( m , 3h ) , 5.15 ( br s , 1h ) , 4.23 ( dd , j = 4.8 , 4.8 hz , 2h ) , 3.913.89 ( m , 5h ) , 3.89 ( s , 3h ) , 3.85 ( s , 3h ) , 3.72 ( dd , j = 4.0 , 4.8 hz , 2h ) , 3.63 ( dd , j = 4.0 , 5.2 hz , 2h ) , 3.54 ( dd , j = 4.8 , 5.2 hz , 2h ) , 3.31 ( d , j = 4.8 hz , 2h ) , 2.242.18 ( m , 2h ) , 1.70 ( d , j = 6.8 hz , 3h ) , 1.42 ( s , 9h ) , 0.65 ( dd , j = 6.8 , 7.2 hz , 6h ) . to a cooled ( 0 c ) solution of s5 ( 24 mg , 0.03 mmol ) in dry ch2cl2 ( 2 ml , 0.02 m ) was added dropwise bemp ( 0.03 ml , 0.1 mmol ) . the resulting mixture was stirred at the same temperature for 10 min before tosylate 26 ( 39 mg , 0.1 mmol ) in ch2cl2 ( 1 ml , 0.1 m ) was added . after stirring for 30 min at 0 c , the reaction was quenched by addition of saturated aqueous nh4cl , and the resulting mixture was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , hexanes / etoac , 2/1 ) to afford boc - protected amine 29 ( 26 mg , 79% ) : h nmr ( 400 mhz , cdcl3 ) 8.15 ( dd , j = 4.8 , 8.4 hz , 2h ) , 7.847.79 ( m , 1h ) , 7.70 ( s , 1h ) , 7.23 ( d , j = 8.4 hz , 2h ) , 6.87 ( d , j = 8.4 hz , 1h ) , 6.826.74 ( m , 4h ) , 6.706.65 ( m , 2h ) , 5.425.30 ( m , 4h ) , 5.06 ( br s , 1h ) , 4.23 ( dd , j = 4.8 , 4.8 hz , 2h ) , 3.913.88 ( m , 5h ) , 3.84 ( s , 3h ) , 3.80 ( s , 3h ) , 3.71 ( dd , j = 4.4 , 5.2 hz , 2h ) , 3.63 ( dd , j = 4.0 , 4.8 hz , 2h ) , 3.54 ( dd , j = 4.8 , 5.2 hz , 2h ) , 3.31 ( d , j = 4.8 hz , 2h ) , 2.222.16 ( m , 2h ) , 1.68 ( dd , j = 6.8 , 10.8 hz , 6h ) , 1.41 ( s , 9h ) , 0.630.59 ( m , 6h ) . to a cooled ( 0 c ) solution of 29 ( 26 mg , 0.03 mmol ) in dry ch2cl2 ( 1 ml , 0.03 m ) was added dropwise tfa ( 0.5 ml ) . after stirring for 40 min at the same temperature , the solvent was removed under reduced pressure . the crude residue was employed in the next step without further purification ; the residue was dissolved in dry dmf ( 1 ml , 0.03 m ) , and the mixture was treated with et3n ( 0.009 ml , 0.06 mmol ) followed by biotin n - hydroxysuccinimide ester ( 9 mg , 0.03 mmol ) in dmf ( 1 ml , 0.03 m ) at 0 c . after stirring for 15 h at 25 c , the reaction was quenched by addition of water , and the resulting mixture was extracted with ch2cl2 . the combined organic layers were washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo . the residue was purified by column chromatography ( silica gel , ch2cl2/meoh , 10/1 ) to afford ma14 as a colorless oil ( 28 mg , 99% for 2 steps ) : h nmr ( 400 mhz , cdcl3 ) 8.198.11 ( m , 2h ) , 7.877.79 ( m , 1h ) , 7.71 ( s , 1h ) , 7.257.20 ( m , 2h ) , 6.916.65 ( m , 7h ) , 6.55 ( br s , 1h ) , 5.69 ( br s , 1h ) , 5.425.30 ( m , 4h ) , 5.074.90 ( m , 2h ) , 4.474.41 ( m , 1h ) , 4.374.32 ( m , 1h ) , 4.284.20 ( m , 2h ) , 3.933.88 ( m , 5h ) , 3.84 ( s , 3h ) , 3.80 ( s , 3h ) , 3.733.69 ( m , 2h ) , 3.663.62 ( m , 2h ) , 3.583.54 ( m , 2h ) , 3.433.40 ( m , 2h ) , 3.133.05 ( m , 1h ) , 2.972.89 ( m , 1h ) , 2.762.63 ( m , 2h ) , 2.222.12 ( m , 4h ) , 1.831.66 ( m , 8h ) , 1.401.37 ( m , 2h ) , 0.630.59 ( m , 6h ) ; ir ( neat ) 2162 , 1506 , 1148 , 650 cm ; hrms ( esi ) m / z 1120.4541 [ ( m + h ) , c57h68f3n5o13s requires 1120.4559 ] . t cells were seeded on 96-well plates at a density of 5 10 cells / well and allowed to attach for 24 h. cells were transfected with pgl3 - 5hre - vegf - luciferase plasmid containing five copies of hres in human vegf promoter and prl - sv40 plasmid encoding renilla luciferase using vivamagic transfection reagent ( vivagene , seongnam , korea ) , according to the manufacturer s instructions . cells were treated with hypoxic conditions ( 1% o2 ) and serially diluted compounds for 24 h after transfection . luciferase reporter assay was performed using the dual - glo luciferase assay system ( promega , madison , wi , usa ) according to the manufacturer s instructions . briefly , an equal volume of dual - glo reagent to culture medium was added to each well , and the mixture was incubated for 10 min . the firefly luminescence was measured using a microplate reader ( infinite m200 pro , tecan , mnnedorf , switzerland ) . dual - glo stop & glo reagent was added , and the mixture was incubated for 10 min before the measurement of renilla luminescence . cells were seeded on 96-well plates at a density of 1 10 cells / well , and then incubated for 24 h. after cells were treated with various concentrations of compounds for 24 h under hypoxia , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt , amresco ) solution was added to each well , and the mixture was incubated for another 4 h at 37 c . the resulting formazan crystals were dissolved in dmso ( 100 l / well ) and the absorbance of the plate was read with a microplate reader ( infinite m200 pro , tecan , mnnedorf , switzerland ) at 540 nm . proteins were extracted from the cells using pro prep ( intron biotech , south korea ) . cell extracts were separated by sds - page and transferred to a nitrocellulose membrane ( whatman , maidstone , england ) . the membranes were blocked with 5% skim milk in tris - buffered saline ( tbs ) containing 0.1% tween-20 for 30 min at room temperature . after blocking , membranes were incubated with specific primary antibodies overnight at 4 c , followed by incubation with horseradish peroxidase - conjugated mouse- or rabbit - igg for 1 h at room temperature . membranes were then incubated with horseradish peroxidase - conjugated antibody mouse or rabbit immunoglobulins at room temperature and developed through west pico chemiluminescent substrate ( pierce , woburn , ma , usa ) . total rna was extracted from cells using trizol reagent ( invitrogen , carlsbad , ca ) , and cdna synthesis was performed using mmlv reverse transcriptase . real - time pcr was performed using a sybr green pcr master mix ( applied biosystems , ca , usa ) . each pcr reaction contained cdna with 10-fold dilution and gene - specific primers . the thermal cycle used was 2 min at 50 c , 10 min at 95 c , 40 cycles of 15 s denaturation at 95 c with 1 min annealing at 60 c . the mean cycle threshold ( ct ) values were calculated for the gene expression with normalization to -actin as an internal control .

to cope with hypoxia , tumor cells have developed a number of adaptive mechanisms mediated by hypoxia - inducible factor 1 ( hif-1 ) to promote angiogenesis and cell survival . due to significant roles of hif-1 in the initiation , progression , metastasis , and resistance to treatment of most solid tumors , a considerable amount of effort has been made to identify hif-1 inhibitors for treatment of cancer . isolated from saururus cernuus , manassantins a ( 1 ) and b ( 2 ) are potent inhibitors of hif-1 activity . to define the structural requirements of manassantins for hif-1 inhibition , we prepared and evaluated a series of manassantin analogues . our sar studies examined key regions of manassantin s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug - like properties . our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development . analogues ma04 , ma07 , and ma11 down - regulated hypoxia - induced expression of the hif-1 protein and reduced the levels of hif-1 target genes , including cyclin - dependent kinase 6 ( cdk6 ) and vascular endothelial growth factor ( vegf ) . these findings provide an important framework to design potent and selective hif-1 inhibitors , which is necessary to aid translation of manassantin - derived natural products to the clinic as novel therapeutics for cancers .

the cerebellum is considered to have a regular arrangement of neuronal cell types produced by the geometric organization of their somata and dendritic trees . in terms of their dendritic arbors , purkinje cells ( pcs ) are the most prominent cell type in the cerebellar cortex . obersteiner reported that these display consistent orientation of dendritic branching with respect to the transverse direction of cerebellar folia . parallel fibers , which are one of the main sources of input to pcs , were described by santiago ramon y cajal as lying perpendicular to pc dendritic arborizations . in addition to its regularity , the cerebellum is also highly foliated , giving rise to three distinct gross anatomical subdivisions : sulcus , apex , and bank [ 25 ] . in the sagittal plane , these subdivisions are easily distinguished , since the folia are divided by fissures of varying lengths , creating valleys and summits , separated by less convoluted regions . pc density in the apex is greater than in the sulcus [ 2 , 3 ] . studies of sparsely labeled golgi preparations reported differences in pc branching structure according to their anatomical location . pcs in the sulcus often branch from the somata at an angle of nearly 180 , whereas those in the apex branch tens of microns into the molecular layer . a climbing fiber innervation study demonstrated different connectivity patterns to pcs in the sulcus , indicative of a specialized functional role . more recently , sudarov and joyner showed that mouse cerebellar foliation is initiated by formation of anchoring centers , created by an inward accumulation of granule cell precursors ( gcps ) and giving rise to subdivision of the sulcus . finally , at the single cell level , adult sulcus pcs were shown to retain multiplanar dendritic arbor arrangements . investigations of morphological , and to a lesser extent , functional relationships of dendritic arbors between cerebellar subdivisions have necessarily focused on single cells . however , determining spatial parameters of neighboring dendritic arbors is important for understanding functional connectivity , particularly of afferent parallel fiber input , across individual pcs residing in different cerebellar subdivisions . population - level morphological studies have been difficult to execute for several reasons . only within the last decade has it become possible to label all neighboring neurons of specific cell type with a fluorescent label strong enough to withstand high resolution confocal imaging . faster scanning speeds , enabling more efficient acquisition times , have been developed only within the last five years . lastly , classical methods for tracing dendritic morphology are so time - consuming that it is impossible to reconstruct enough cells for appropriate analyses of dendritic morphologies across anatomical regions of interest . newer methods are being developed [ 79 ] , although few explore the local architecture of neuronal populations [ 10 , 11 ] and their connectivity to different cell types [ 12 , 13 ] . this study investigated morphological relationships of dendritic arbors of neighboring pcs in the two most distinct cerebellar regions , sulcus and apex . unfortunately , to date , no automated algorithm to reliably reconstruct neighboring dendritic arbors has become available . however , with neurolucida software , devised for reconstructing neurons , we adapted a tool that is normally used to draw cell bodies . this was used to efficiently trace dendritic trees labeled with green fluorescent protein ( gfp ) of l7-tau - gfp mice under control of the l7 promoter . the adapted contour protocol allowed us to capture full pc dendritic geometries in one - fourth the time that would have been required with classical tracing methods . these reconstructions permitted quantitative comparative analyses of small populations of neighboring dendritic arbors that hitherto have been impossible . we show that distinctly different dendritic tree geometries in the sulcus produce greater arbor field overlap among neighboring pcs than in the apex . these findings shed light on functional connectivity of pc dendritic arbor afferent input , relationships between cerebellar cortex subdivisions , and the relationship between developmental plasticity and attainment of adult cerebellar architecture . l7-tau - gfp mice were euthanized in the laboratory of michael husser , university college london ( ucl ) . at postnatal day 12 ( p 12 ) ( young ) and p 150 ( old ) , mice were deeply anesthetized using a cocktail of ketamine / xylazine ( 80/5 mg / kg , i.p . ) . transcardial perfusion employed a syringe pump at a perfusion rate of 12 ml / min . perfusion solution contained 60 ml saline - containing heparin ( 10 u / ml ) and 4060 ml 4% paraformaldehyde in 0.1 m phosphate buffer ( pb ) ( ph 7.4 ) . this procedure complied with the protocol approved by the ucl animal care committee and with animal care guidelines of the uk home office . following this perfusion , brains were removed , postfixed overnight with the same fixative , and transferred to a 0.01 m pb solution containing 0.9% sodium chloride ( pbs , ph 7.4 ) . brains were stored in pbs in order to avoid overfixation and possible tissue shrinkage during shipment . fixed brains were then shipped to the okinawa institute of science and technology graduate university ( oist ) , where they were prepared for confocal microcopy in the laboratories of bernd kuhn and jeff wickens . at oist , brains were sliced into 80 m sagittal sections using a leica vibratome . sections containing the cerebellar vermis were collected in cold 0.01 m pbs , mounted onto glass slides , air dried , and cover - slipped using antifade prolong ( invitrogen ) mounting media . to collect samples in a random and systematic manner , pc dendritic arbors from the same locations in apex regions of lobule viii and sulcus regions of lobules viii and ix were imaged from both young and old cerebella ( figures 1(a)1(e ) ) . cerebellar sagittal sections were imaged with an lsm 710 carl zeiss confocal microscope equipped with a 63x ( na 1.46 , oil ) objective lens and argon ( 458 nm , 488 nm , and 514 nm ) lasers . image acquisition parameters were as follows : an x , y - size of 0.22 m / pixel ( dimensions x : 224.7 m , y : 224.7 m ; image size 1024 1024 ) and a z - step size of 0.25 m . high - resolution confocal image acquisition enabled us to unambiguously resolve dendrites of neighboring pcs in the parasagittal plane , or through the depth of the sagittal slice ( figure 1(f ) ) . digitized image stacks were acquired with zen imaging software ( zeiss ) . utilizing the continuous contour tracing function of neurolucida ( mbf bioscience ) , a surface contour was traced around the outer edge of each pc dendritic tree using a wacom cintiq video tablet and digital pen . neurolucida 's continuous contour tracing function does not require discontinuous insertion of points but adds points automatically as the experimenter drags the pen along the edge of the dendritic membrane , comparable to drawing on a piece of paper . digitally , an enclosed hollow contour that accurately delineates the geometry and spatial boundaries of the dendritic arbor was obtained ( figure 1(g ) ) . these steps were repeated for each dendritic tree and soma in the sulcus and apex of the folia to develop a reconstructed population of neighboring dendrites and somata ( figure 1(h ) ) . all traced pc trees were edited to insure that contours were traced tightly around dendritic trees for the sake of consistency . this contour tracing technique is considerably more efficient than classical reconstruction methods that require the experimenter to adjust the diameter of the cursor at each step and to insert a bifurcation node at each branch point on the dendritic tree . because approximately 10 pcs are found in the sulcus of an 80 m sagittal cerebellar slice , 10 2 pcs were traced from both sulcus and apex of four mice , for a total of 88 pc dendritic trees . the number of pcs traced per sample was determined by tracing a reference pc in the middle of the sulcus and then reconstructing neighboring pcs within that sulcus subdivision . pcs with dendrites cut off by the confocal image stack ( 224 m by 224 m ) were excluded . since morphological differences between dendritic trees of sulcus and apex are strikingly different , approximately 20 pcs from each age group and anatomical subdivision were sufficient statistically . two important requirements for using student 's t - test to compare group means are that samples have normal distributions and equal sizes . since the number of pcs in each experimental condition was almost equal ( 10 2 pcs ) , student 's t - test was used to compare means between sulcus and apex pcs for both young and old developmental groups . in cases involving crossed dendritic branches from the same pc , the nonparametric mann - whitney u test was used because samples did not possess normal distributions and not all pc arbors displayed crossings , rendering sample sizes ( number of pcs per group ) unequal . in order to unambiguously resolve distances between dendrites of neighboring cells , high - resolution confocal imaging in the z - dimension ( depth of the sagittal slice ) is critically important . this relatively large separation is because bergmann glia , basket cells , and other interneurons need to be accommodated as well . in addition , pc dendritic diameters average 0.52.2 m for spiny distal dendrites and 3.45 m for smooth proximal dendrites . given these dimensions , with fine optical sectioning a recent study reported that pc dendrites touch each other ; however , in that study , optical sectioning in the z - dimension of the sagittal slice ( mediolateral plane ) was set to 1 m , so that pc dendrites appeared to be sticking to each other . confocal acquisition with a small z - step size can take time , leading to tissue bleaching if fluorophore expression is not stable and intense . in this study , using a fast laser scanning confocal microscope ( zeiss lsm 710 ) and l7-tau - gfp transgenic mice that strongly express gfp , we were able to unambiguously resolve dendrites of neighboring pcs without bleaching . image stack acquisitions took approximately 20 min . traced pc dendritic contours containing geometric information in the form of x , y , z coordinates were analyzed with the polygon clipper function ( sebastian hlz , matlab central file exchange ) and with trees 1.15 ( matlab toolbox ) . in addition , we wrote custom code to extract information about the number of primary and secondary dendrites of each ramified arbor ( figures 2(e)2(h ) ) , the dendritic field overlap of neighboring arbors ( figures 3(b ) and 3(c ) ) , the surface area encompassed by the traced contour of each dendritic arbor ( figure 4(a ) ) , and intracellular dendritic branch crossings ( figures 5(c ) and 5(d ) ) . shared dendritic field is the overlap area of two neighboring dendritic field arbors ( figure 3(b ) ) . then , tetrahedrons representing dendritic arbors were clustered , based on their depth within the tissue section . this step facilitated pairing of nearby tetrahedrons ( pc dendritic arbors ) and avoided accidentally overlapping pc arbors that were actually well separated in the z - dimension . next , overlapping areas of neighboring pc dendritic arbors were computed within each designated cluster . to eliminate variable arbor size as a confounding factor , areas of dendritic field overlap were normalized to the average total dendritic contour of each pc pair ( ( area of cell 1 + area of cell 2)/2 ) . intracellular dendritic branch crossings were computed by detecting sites where branches within the same dendritic tree intersected within a 2d projection . anatomical , physiological and gene expression studies have reported that cerebellar subdivisions function as partitioned modules [ 1924 ] . various studies have demonstrated gene expression boundaries that subdivide the cerebellar cortex into four transverse zones : anterior , central ( cza and czb subdivisions ) , posterior , and nodular , as well as additional topographic units such as the pc stripy bands or microzones [ 2328 ] . a well - established anatomical landmark for describing cerebellar sagittal sections is the lobules ( lobules i - x ) , which are separated by fissures and folds that give rise to sulcus and apex ( figure 1(a ) ) . while the sulcus and apex are prominent topographical subdivisions , their pc dendritic cytoarchitecture and functional diversity have received little attention [ 3 , 6 ] . previous work has shown that timing of pc dendritic growth is different depending on maturation of specific lobules . the formation of cerebellar lobules is under strong genetic control , as confirmed by the absence of lobulation in cxcr4-deficient mice , where granule cell precursors ( gcps ) fail to migrate properly to the internal granule cell layer , leading to a disorganized and irregular arrangement of pc dendritic trees . distinct functional roles of different cerebellar lobules have been demonstrated by recent work that identified different firing patterns between lobules iii - v and lobule x . in addition , human subject fmri studies point specifically to lobule viii as being involved in timing of complex motor movement [ 32 , 33 ] . in order to elucidate functions of neighboring pcs in different topological units , we used sagittal cerebellar sections near the midline to acquire confocal image stacks of gfp - expressing pc dendritic arbors in the sulcus and apex of the posterior cerebellum ( figure 1(a ) ) . for anatomical consistency , we imaged pc arbors from the sulcus between lobules viii and ix and the apex of lobule viii in both young and old mice ( figures 1(a)1(e ) ) . the contour tracing method extracted geometric information about pc dendritic arbor shape ( figure 1(g ) ) by imaging pc dendrites with high - resolution confocal microscopy to resolve distances ( m ) between neighboring dendrites ( figure 1(f ) ) . determination of dendritic arbor morphology enables us to understand the remarkably variable geometry of neuronal shape and to better understand how neighboring neurons gather information together . to quantify geometric arbor shape differences between pc dendrites of sulcus and apex pc arbors with two primary dendrites give rise to strikingly different geometric morphologies compared to arbors with only one . dendritic arbors with only one primary dendrite are the typically portrayed pc shapes with a branching structure sitting atop a stalk - like main process ( figures 2(b ) and 2(d ) ) . conversely , dendritic arbors with two primary dendrites diverge from each other within the sagittal plane , with one side of the arbor often more ramified than the other ( figures 2(a ) and 2(c ) ) . dendritic arbors with two main primary dendrites usually populate the sulcus region of young and old mice ( figures 2(a ) and 2(c ) ) . these morphological differences are especially obvious in young mice when dendritic arbors are less well developed ( figure 2 , panels ( a ) and ( b ) versus panels ( c ) and ( d ) ) . in young mice , 72% of dendritic arbors in the sulcus have two primary dendrites , whereas in the apex , only 28% do ( figure 2(f ) ) . this morphological difference is statistically significant in young mice ( p < 0.05 , t - test ) , while less pronounced in old mice , where 55% of the sulcus pc arbor population has two primary dendrites ( figures 2(f ) and 2(h ) ; n = 88 , p = 0.6 ) . while dendrites were not traced in the cerebellar bank in this study , pcs with two primary dendrites are less abundant in the bank than in the sulcus . profound geometric differences of pc arbors in the sulcus imply a differential dendritic architecture of neighboring pcs dependent on anatomical location . sulcus pc arbors are also more frequently innervated by multiple climbing fibers relative to pc arbors of the apex and bank , further underscoring the uniqueness of the sulcus . in addition , sudarov and joyner demonstrated that sulcus formation is associated with a peculiar aggregation of migrating gcps . this aggregation , unique to the sulcus , is known as an anchoring center and seems to initiate invagination of the folia , giving rise to a fissure , which later becomes the concave sulcus . this process takes place as gcps migrate toward the internal granule cell layer , leaving their axons behind . the axons then bifurcate to become the future parallel fibers , synapsing with dendritic arbors . thus , from a cytoarchitectural point of view , the peculiar gcps aggregation in the sulcus may influence the developing pc dendritic architecture . future anatomical connectivity and functional studies are needed to determine whether neighboring sets of sulcus pcs and their afferents perform different downstream functions relative to their apex and bank counterparts . how does the geometry of a sulcus pc arbor with two primary dendrites spreading along the sagittal plane influence neighboring dendritic arbors ? such overlap has functional implications because individual mouse pc dendrites receive approximately 100,000 excitatory inputs from unmyelinated parallel fibers . these glutamatergic inputs are axons that run roughly perpendicular to the pc arbors ( figure 3(a ) ) . parallel fibers are roughly 1.53 millimeters long and may contact a few hundred pc arbors , forming en passant synapses with neighboring pcs . the dendritic field shared by two neighboring pcs is likely to receive the same parallel fiber input ( figure 3(b ) ) . next , we calculated the extent of this shared area in order to understand whether neighboring pc arbor geometries of sulcus and apex might give rise to different overall dendritic architectures , implying functional differences . contour reconstructions permitted accurate representation of pc arbor geometries and were processed with an original algorithm that detected dendritic colocalization ( space between branches was not analyzed ) . the algorithm performed the analysis in pairwise fashion and yielded the shared dendritic field as a surface area index ( figure 3(b ) ) . this index was normalized to eliminate variable arbor size as a confounding factor and was used to compare the developing apex and sulcus ( figure 3(c ) ) . neighboring pc arbors in young sulcus display greater dendritic field overlap than their counterparts in the apex ( figure 3(c ) ; n = 88 , p < 0.05 , t - test ) . in contrast , in old mice , the shared dendritic field area between neighboring arbors was not significantly different between sulcus and apex ( n = 88 , p = 0.09 ) . these results suggest a location - dependent developmental switch and dendritic developmental plasticity , where the ontogenetic effect on neighboring arbors potentially sharing similar afferent input depends on the dendritic architecture ( figure 3(c ) ) . it is well accepted that while single parallel fibers have a weak effect on the postsynaptic firing probability of pcs , the cumulative input of many fibers is the driving force responsible for pc activity [ 35 , 36 ] . in the context of neighboring pcs , shared dendritic fields are very likely innervated by the same parallel fiber bundle , potentially promoting synchronous activity . depending on the extent of dendritic field sharing , afferent input may regulate specific sets of pcs differently . in turn , pcs provide information to targeted cerebellar nuclei [ 37 , 38 ] , which are thought to regulate learned cerebellar movement behaviors , especially during development . future experiments will attempt to determine whether sulcus pcs project to cerebellar nuclei with different connectivity patterns than those of the apex . to identify possible additional pc morphological characteristics specific to the sulcus , we analyzed pc arbor surface area and width in sulcus and apex . contoured traces , tightly drawn around dendritic trees , enabled us to compute arbor surface areas . mean dendritic arbor surface areas of young sulcus ( mean 2272 m 840 ; range 9123761 m ) and young apex ( 2334 m 888 ; 10504142 m ) did not differ significantly , and this insignificance persisted in old mice ( sulcus , 3905 m 1680 , 10706578 m ; apex , 4232 m 1773 , 14847324 m ) ( figure 4(b ) ) . next , we calculated the lateral span of pc arbors ( figure 4(c ) ) in order to explain the observed increase of shared dendritic field between neighboring young pcs . the upper portion of the arbors was measured from the left - most to the right - most branch tip ( figure 4(c ) ) . average arbor widths , like surface areas , did not differ significantly between sulcus and apex pcs in either young or old animals ( young sulcus , 124 m 18 ; young apex , 117 m 28 , old sulcus , 137 m 31 , and old apex , 141 m 45 ) . as expected , during maturation , both surface area and widths of older dendritic arbors are significantly larger than those of young mice ( figures 4(b ) and 4(d ) , surface areas , n = 88 , p < 0.001 ; widths , p < 0.05 , t - test ) . dendritic tree width for old relative to young sulcus arbors only approached statistical significance ( figure 4(b ) , n = 88 , p = 0.08 ) . the small lateral growth is not surprising because pcs arbors grow first wider and then taller . population morphometric means obscure strikingly different individual pc arbor geometries ( figures 2 and 4 ) . previous reports on population means have asserted geometric uniformity in the cerebellar cortex [ 2 , 40 ] , while individual neuron level work points to varying dendritic morphologies playing a functional role in the propagation of action potentials , enrichment of computational power , or correlation with electrophysiological development . notably , both apex arbor surface areas and widths often exceeded those of sulcus arbors ( figures 4(b ) and 4(d ) , open circles ) . this suggests that apex arbors are less spatially restricted , enabling more extensive ramification in the sagittal plane relative to sulcus arbors . the greater sulcus dendritic field overlap derives not from broader arbors , but from relative positions of extremely variable individual arbors , factors that are essential in order to understand functional connectivity between parallel fibers and pcs . why are the most profound geometric differences of sulcus and apex pc arbors more prominent in young animals ? how can the high frequency ( 72% ) of young pc arbors with two primary dendrites be explained ? what implication might these shapes have on shared dendritic overlap among young neighboring arbors ? first , relative to the sulcus , the apex is less spatially restrictive to dendritic arbor growth . secondly , p12 constitutes a developmental time window of active synaptogenesis , where the external granule cell layer is still present near the pial surface , and granule cells have not finished migrating through the molecular layer to make synaptic connections with postsynaptic pc dendrites . at this stage , young dendritic arbors are in a stage of rapid growth , which may permit them to probe the environment more freely , invading the space of neighboring arbors within the spatial constraints of sulcus and apex . the combination of limited space and rapid dendritic growth may explain the significantly greater shared dendritic fields of young sulcus arbors . conversely , pc arbors in old mice have completed their growth and although they experience more crowding in the sulcus , they exhibit similar amounts of shared dendritic field when compared to apex dendritic arbors ( figure 3(c ) , p = 0.1 ) . a plausible explanation for this is that a compensatory structural change occurs in sulcus pc arbors . to better understand the effect of spatial constraints on sulcus dendritic arbor development , we designed an algorithm to detect branch crossings of individual pc arbors ( figure 5(c ) ) . there is a statistically significant increase in the number of intracellular branch crossings in old sulcus pc arbors relative to those of young sulcus ( figure 5(d ) , n = 82 total pcs , p < 0.05 , mann - whitney u test ) . adult sulcus pc arbors have more dendritic branch crossings than adult apex pcs ( p < 0.05 , mann - whitney u test ) . differences in the number of intracellular branch crossings between sulcus and apex are less pronounced in young mice ; however , their arbors are immature . these results imply that adult sulcus arbors may compensate for the space limitations of the sulcus by developing a compact dendritic arbor within the sagittal plane . previous work concluded that pc dendritic branch crossings provide evidence for dendritic arbor multiplanarity , retained into adulthood . we observed that intracellular branch crossings almost always occurred between small , distal dendrites ~0.51.75 m in diameter . however , in 3d space , crossed branches of the same arbor did not contact each other , and , as could be readily visualized by rotating the pc , the small interbranch distances usually did not extend beyond the plane of the main dendritic trunk . primary dendrites reportedly range from 4 to 6 m in thickness [ 11 , 17 ] and from 8 to 9 m , including the 24 m - space between neighbors . our measurements of primary dendrite thickness agree well with those cited above ( results not shown ) . in addition , we measured the thickness of the entire pc arbor ( its depth in the mediolateral plane ) ( figures 5(a ) and 5(b ) ) . sulcus trees tend toward greater thickness than those of apex ( figure 5(b ) ) . some sulcus arbors attain nearly 40 m thickness , suggesting that they may be multiplanar . this is most likely due to unusually large branches in the mediolateral plane , rather than to smaller branch crossings . these results suggest that space limitations within the sagittal plane of the sulcus cause dendritic branches to expand into the mediolateral plane . because our study focused mainly on the sagittal dimension , we did not intend initially to explore the planarity of pc trees . dendritic planarity would be best addressed with coronally cut slices or slices transverse to the length of the lobule . intracellular branch crossings and shared dendritic fields are structural attributes that occur without interdendrite contact ; thus , it seems likely that pc dendrites do not experience strong steric hindrances in intact brain . due to its organized anatomy and well - studied circuitry , the cerebellum is an ideal site for drawing functional inferences from neuronal geometry . by combining thin optical slicing on a fast scanning confocal microscope with an adapted software tool to trace fluorescent dendrites , we demonstrated that spatial constraints of the sulcus alter pc dendritic arborization relative to that of the apex . first , the majority of young sulcus arbors have two primary dendrites that diverge in the sagittal plane . second , young neighboring sulcus arbors overlap to a greater degree . third , adult sulcus dendrites manifest more intraarbor branch crossings and tend toward narrower arbors , resulting in more compact structure sagittally . finally , this morphological plasticity is ontogenetic .

foliation divides the mammalian cerebellum into structurally distinct subdivisions , including the concave sulcus and the convex apex . purkinje cell ( pc ) dendritic morphology varies between subdivisions and changes significantly ontogenetically . since dendritic morphology both enables and limits sensory - motor circuit function , it is important to understand how neuronal architectures differ between brain regions . this study employed quantitative confocal microcopy to reconstruct dendritic arbors of cerebellar pcs expressing green fluorescent protein and compared arbor morphology between pcs of sulcus and apex in young and old mice . arbors were digitized from high z - resolution ( 0.25 m ) image stacks using an adaptation of neurolucida 's ( mbf bioscience ) continuous contour tracing tool , designed for drawing neuronal somata . reconstructed morphologies reveal that dendritic arbors of sulcus and apex exhibit profound differences . in sulcus , 72% of the young pc population possesses two primary dendrites , whereas in apex , only 28% do . spatial constraints in the young sulcus cause significantly more dendritic arbor overlap than in young apex , a distinction that disappears in adulthood . however , adult sulcus pc arbors develop a greater number of branch crossings . these results suggest developmental neuronal plasticity that enables cerebellar pcs to attain correct functional adult architecture under different spatial constraints .

many models have been put forward as an attempt to explain and counteract the real - life outcomes of several different inflammatory events in which neutrophil leukocytes play an outstanding role . trauma , infection , hemorrhage , the response to both elective and emergency surgical interventions , and other pathological processes are incredibly prevalent in the human population . such conditions are often complicated by nefarious immune responses that arise from these events that are , at least partially , mediated by neutrophils [ 14 ] , since these are cells known for their central role in the mechanisms of inflammation in mammals [ 5 , 6 ] . logically , there is an ongoing effort to explain the inflammatory dynamics arising from these types of insults , as a first step in the direction of modulating and perhaps coordinating such responses in order to improve outcomes , reduce hospitalization times , and even prevent death . the two - hit or multiple - hit hypothesis is a model that explains how sequential insults can synergically contribute to an inappropriate immune response in which mods / mof ( multiple organ dysfunction syndrome / multiple organ failure ) is often the endpoint . as a broad definition , the two - hit model hypothesizes that an initial inflammation - triggering event , such as pancreatitis , trauma , burns , excessive bleeding , or elective surgery , can set in motion a priming condition for the immune system that can cause limited expression of sirs ( systemic inflammatory response syndrome ) or other mild effects if left alone . additional hits or insults ( e.g. , second - look laparotomy , infection , further blood loss , or ischemic injury during the process of aneurysm repair ) are capable of causing an extraordinary and exaggerated immune response that can evolve to mods / mof and death . ischemic preconditioning or ipc , on the other hand , does not represent an actual attempt to explain the inflammatory processes involved in sirs / sepsis and its continuum of mods / mof . rather , it is a collection of techniques that make use of the dynamics of the inflammatory response to generate a modulatory effect over these events . ipc is a demonstrable , observable , reproducible phenomenon in which a nonlethal , mild , and often cyclic ischemic event has the capacity to protect organs and tissues from a secondary , prolonged , and otherwise deleterious ischemic event , mitigating the response to ischemia and the ischemia - reperfusion injury ( iri ) . incidentally , the predominance of information in the literature about both models has a marked timeline difference . throughout the late 1980s and early 1990s the two - hit model was considered a good standard as an intuitive and empirical explanation for some real - life chronologically based events seen in trauma and septic patients . conversely , while the manipulation of the inflammatory response through exposure to controlled ischemic scenarios was already underway in the late 1980s , it was not until recently that ipc was shown to have clinical and surgical applications that far surpass those initially conceived and has , not without merit , been increasingly present in the literature . the shift in the literature and the seemingly obvious difference as to how the two models treat a first inflammatory event , on one hand as a first / priming hit , and on the other hand as a protective / beneficial insult , have led to the notion that one model is capable of debunking the previous theory . in the following pages , we shed some light on some of the paradoxes regarding the coexistence of both models and try to reconcile both theories as simultaneously valid answers to some very different questions . sometime by the late 1990s the two - hit model soared to a unique position as a theory that successfully explained and accounted for many of the bedside events that accompanied trauma patients , which by the nature of their injuries were often exposed to sequential insults . another relevant well - known real - life example of the application of the two - hit model is the correction of ruptured aortic aneurysms , which requires imposing a second long - duration dry ischemic event for the actual repair of the initial naturally occurring hemorrhagic injury . a host of experimental models has also been developed to mimic the events of multiple or sequential hits in order to further understand the processes involved in the augmentation of the inflammatory response . one example of those models is from the researchers that in 1998 demonstrated that neutrophil recruitment to the lung was increased when hemorrhagic shock ( first hit ) was followed by inoculation of lps ( second hit ) if compared to a single - hit process . another group , on the following year , demonstrated the same marked increase in pmn recruitment to the lung when subjects were exposed to a second hit with direct lung injury from lps and immune complexes after a septic event that served as the first hit . nevertheless , examples of multiple - hit models that failed to induce the anticipated augmented immunological response can also be found in the literature . for instance , an american study from 2000 in which subjects were exposed to intratracheal injection of acid with or without previous induction of sepsis by clp ( cecal ligation and puncture ) could not demonstrate a synergistic or even additive effect on the inflammatory response . such study compared a two - hit insult versus a single - hit murine model , although their evaluation was limited to the number of pmn and the concentration of albumin present after bal ( bronchoalveolar lavage ) . this goes to show that the type and dynamics of the insult are to be considered when experimental models are designed to reproduce the inflammatory effects of multiple - hit insults . as a rule , patients can be exposed to a variety of first - hit events , such as trauma itself or any number of hemorrhagic , ischemic , or infectious insults . during their hospital stay , patients are exposed to second , third , or further sequential events ( e.g. , laparotomy , fluid replacement therapy , blood transfusions , fracture repair surgeries , infection via catheter , or other sources ) . the mechanics behind it is that the first hit serves as a priming event that sets the patient towards the establishment of sirs ( systemic inflammatory syndrome ) . sirs in itself is a fairly straightforward diagnosis , consisting in the identification of two or more of the following criteria : ( a ) body temperature below 36c or above 38c ; ( b ) heart rate higher than 90 bpm ; ( c ) respiratory rate in excess of 20 mpm or paco2 lower than 32 mmhg ; and ( d ) total white blood cell count above 12.000 mm or below 4.000 mm or the presence of over 10% band forms . if the first insult is by any chance infectious in nature , sirs is loosely termed sepsis . after the establishment of sirs , a secondary , seemingly trivial , insult can jumpstart a detrimental organic response that can culminate in potentially lethal conditions such as mods / mof . depending on the type of sequential hits , the path following sirs ( which in this text is generally considered being end result of the first hit ) is somewhat dualistic in nature . a first , anti - inflammatory state can ensue , which is called cars ( compensatory anti - inflammatory response syndrome ) that , in and of itself , can be dangerous since it predisposes the body to infection that can in turn serve as one of the following hits . during cars , immunosuppression occurs via impairment of t - cell function that can deteriorate the pathophysiological cascade and lead to infection , sepsis , mods / mof , and death [ 1824 ] . a second proinflammatory state is triggered depending on the nature of the sequential events ( additional hits ) . major surgery , ir - like injury by fluid reperfusion in a previously hypovolemic patient , and infection by loss of gut barrier are some of the events that can serve as second or sequential hits . cytokines and other molecular markers for both the anti- and proinflammatory states can be simultaneously found in patients facing hospitalization from several causes of sirs / sepsis / mods / mof . this observation implies that these two biological conditions are not so clearly separated in real - life biological conditions but rather serve as a dynamic modulatory system that ideally keeps both the suppression and augmentation of the inflammatory response in check throughout the clinical evolution of the patient . additional hits tend to throw this system out of balance , causing the inflammatory response and clinical presentation to escalate . regardless of the type of insult , the two - hit model postulates that the inflammatory response to the additional challenges is generally exaggerated , since the body has already been primed by the first event , or hit . the complex molecular cascades and the serious remote injuries triggered by this proinflammatory state are responsible for the potentially fatal state of mods / mof . the two - hit model clearly shows that severely injured or ill patients are commonly more easily susceptible to the sequential insults , which singly or cumulatively lead to their unfavorable outcomes , observation which indeed can be easily correlated to common clinical outcomes . the molecular pathways that explain the two - hit model are incredibly complex and , to this date , there has not been a definitive flow of events identified . one can easily understand why this is the case , since the variable nature of the first and subsequent events imposes a humongous challenge to the definition of the underlying inflammatory biochemistry behind the two - hit model theory . for that reason , any discussion of the events occurring during the progression of the multiple inflammatory hits has to take into account the specifics of every single type of insult . below we discuss some general molecular and physiological aspects underlying the two - hit or multiple - hit inflammatory events . after an initial insult the immune system is affected at a cellular level as the inflammatory cells become easily susceptible and primed to any sequential stimulus / insult and are therefore further activated by a minor sequential exposure , allowing mildly injurious stimuli to synergistically set off the inflammatory machinery and cause tissue damage [ 2528 ] both locally and remotely . the immune response induced by the first hit can be traumatic in nature and may be limited locally as in monotrauma or it may be a massive systemic immune activation as in polytrauma [ 18 , 21 , 2931 ] . different trauma - related inflammatory actors have been recently characterized among which the complement system stands out as a key mediator [ 20 , 21 , 24 , 3237 ] . when the complement system is activated by any of its three pathways , it plays a pivotal role in eliminating foreign pathogens by opsonization / phagocytosis ( c3b , c4b ) and chemotaxic attraction of leukocytes ( c3a , c5a ) and also directly lysing the pathogens through the membrane attack complex ( mac , c5b-9 ) [ 35 , 3840 ] . the anaphylatoxins like c3a and c5a recruit phagocytes and polymorphonuclear leukocytes ( pmn ) to the site of injury as these anaphylatoxins are strong chemoattractants for phagocytes and also induce the degranulation of mast cells , basophils , and eosinophils [ 35 , 39 , 40 ] . it is clear from clinical and experimental studies that after trauma the complement system gets activated both locally and at the injury site , as well as systemically [ 4247 ] . tissue damage and cell injury cause the release of alarmins , which are non - pathogen - derived danger signals capable of activating the innate immune responses . these include annexins , heat - shock proteins ( hsps ) , defensins , and classical markers of tissue injury like s100 protein and high mobility group box-1 ( hmgb1 ) nuclear protein [ 48 , 49 ] . alarmins correlate with the heterogenic innate immune inflammatory molecules and pathogen - associated molecular patterns ( pamps ) , which are recognized by the immune system as foreign molecules because of their characteristic molecular pattern [ 50 , 51 ] . together , alarmins and pamps form a large family of damage - associated molecular patterns ( damps ) and are recognized by immune cells that express multiligand receptors , such as toll - like receptors ( tlrs ) , on their surfaces . therefore damps are capable of activating innate immune responses after trauma either when the traumatic injury is a standalone event or when the traumatic event is further complicated by infection [ 24 , 48 ] . it is reasonable to assume that similar triggers are present when the first and sequential hits are not traumatic in nature . whatever the cause of the insult , it is generally accepted that cytokinemia or cytokine storm is of major importance during the biological responses inside the two - hit model . cytokines are molecules of low molecular weight that are secreted by immune cells and serve as mediators for the communication between leukocytes , interlinking innate and adaptive immune responses . traumatic tissue injuries induce the expression of proinflammatory cytokines , such as tumor necrosis factor ( tnf ) and interleukin- ( il- ) 1 , il-6 , il-8 , il-12 , il-15 , and il-18 [ 5254 ] . in addition to other biological roles , cytokines also activate neutrophils which are key players in the early inflammatory response to trauma or other first - hit insults . neutrophils are pulled away from the circulation to the site of injury by chemotactic molecules , such as complement anaphylatoxins and chemotactic cytokines , called chemokines , most notably il-8 [ 39 , 56 ] . several studies have tracked cytokine production during inflammatory conditions , demonstrating their primary role in tissue damage by cellular priming / activation and also in the pathophysiology of sirs . in particular , tumor necrosis factor- ( tnf- ) , il-1 , il-6 , and il-8 have been consistently present during these observations . nevertheless inconsistent results in terms of levels of tnf- , il-1 , and il-6 have prevented a definitive association between high concentration of these agents and the risk for development of mods / mof [ 57 , 58 ] . in an ideally regulated immune response , neutrophils play an important role in the defense and repair of injured tissues . pmn priming for cytotoxicity covers a wide range of physiologic responses , like degranulation of enzymes , superoxide anion generation , lipid mediator ( ltb4 ) and cytokine ( il-8 ) production , decreased selectin expression ( l - selectin ) , enhanced integrin expression ( cd11b / cd18 ) , cellular elongation , reduced deformability , and delayed apoptosis [ 5963 ] in addition to other cellular events such as adhesion , rolling , and ultimately diapedesis [ 20 , 64 , 65 ] . neutrophil priming results from the preexposure of the cell to priming molecules like platelet activating factor ( paf ) , anaphylatoxin c5a , granulocyte macrophage - colony stimulating factor ( gm - csf ) , ltb4 , substance p , il-8 , interferon , tnf- , lps , l - selectin cross - linking , and cd18 cross - linking [ 20 , 6668 ] , which could arise from exposure to first - hit events [ 66 , 69 ] . some investigators have suggested that circulating monocytes and tissue macrophages also become primed after severe injury [ 7072 ] . despite the beneficial effects of neutrophils in host defense , a dysfunction in priming and subsequent cellular activation such response leads to tissue injury of previously healthy sites via the local release of toxic metabolites and enzymes that may lead to acute respiratory distress syndrome ( ards ) , mods / mof , secondary blood - brain barrier dysfunction , and brain edema after traumatic brain injury [ 21 , 68 , 7379 ] . aside from circulating cytokines and signaling molecules , the response of the endothelium and its relationship with immune cells are central in understanding the process through which sequential insults can cause tissue injury . endothelial cells are obviously present in virtually every single organ and are in constant contact with immune molecular and cellular mediators . therefore , rather than simply being a passive pipe - like structure , the endothelium serves as an important and complex immune agent and its dysfunction is closely associated with increased morbidity in sirs and its complications via an increase of uncontrolled vascular permeability . in addition , microvascular changes caused by a first hit such as hemorrhagic shock have been implicated as one of the mechanisms through which a second hit such as infection is able to cause an exaggerated systemic inflammatory response that is more likely to trigger mods / mof . central to the role of the endothelium in immune responses is the glycocalyx , a thin and complex structure of proteoglycans , glycosaminoglycans , glycoproteins , and other soluble molecules , which serves as a dynamic interface through which the vascular bed communicates with the flowing blood through continuous shedding and synthesis of this layer [ 81 , 82 ] . functions of the glycocalyx vary widely , from conferring the outer surface of the endothelium an overall negative charge to regulating vascular permeability and fluidic balance , all the way to preventing erroneous and inadvertent adhesion of leukocytes and platelets to the vascular wall by mechanically shielding molecules such as intercellular adhesion molecule 1 , vascular cell adhesion molecule 1 , and selectins [ 81 , 83 ] . the binding of cytokines in the glycocalyx also plays an important role in enclosing and effectively hiding these molecules from circulating leukocytes cell surface receptors . loss of glycocalyx function has been observed under many inflammatory processes such as diabetes , atherosclerosis , sepsis , and ir injury due to , amongst other things , changes in the interaction of the exposed vascular bed and circulating leukocytes and increased vascular permeability [ 81 , 82 ] . endothelial cells express innate immune receptors , such as toll - like receptors , which can trigger intracellular inflammatory responses through mediators such as mapk and nf-b that can ultimately modulate vascular permeability and coagulation . it is even conceivable that thrombi inside the microvasculature may have a role in mechanically preventing infectious agents from spreading . albeit never actually dormant , the overt activation of the endothelium can be triggered as a natural and physiological response to the stimulation of the innate immune response via reactive oxygen signaling dominance due to an uncoupled state of the endothelial nitric oxide synthase ( enos ) or as a pivotal part of many different disease processes involved mainly in cardiovascular illnesses . in healthy and undisturbed endothelium , cell junctions are constantly regulated to preserve overall vascular barrier integrity while allowing for the passage of small molecules and immune cells that are responsible for tissue surveillance [ 86 , 87 ] . activated neutrophils and their interaction with the endothelium , involving neutrophil adhesion and subsequent transendothelial migration , play a crucial role in sirs pathophysiology and are closely related to endothelial dysfunction , associated with loss of functional intracellular contact sites . this loss of integrity of cell - to - cell contact results in tissue edema and impairment of microcirculation and ultimately leads to organ dysfunction [ 8890 ] . microvascular endothelium also has an integral role in postinjury priming of the innate inflammatory response . neutrophil priming agents such as lps and tnf cause endothelial activation by stimulating the expression of adhesion molecules ( e.g. , icam and vcam ) on the vascular bed that can account for tissue damage - driven cell migration [ 9193 ] . the process of neutrophil activation involves complement dependent and complement independent mechanisms . when the blood comes in contact with the activated endothelium it strongly activates the complement and clotting cascades which in turn causes neutrophil activation via the anaphylatoxins c3a , c5a , and c5b9 , as previously discussed . neutrophil adhesion to the endothelium is carried out by cytokines such as il-1 and paf but also by adenosine , prostacyclin , and camp . endothelial activation causes the high expression of adhesion molecules or activation of constitutively expressed molecules like icam-1 , leukocyte function - associated antigen-1 ( lfa-1 ) , or e - selectin [ 95 , 96 ] . the endothelial activation by cytokines leads to the upregulation of adhesion molecules and can be accompanied by the expression of anaphylatoxins c3a and c5b , paf , and ltb4 . tnf- and for example , neutrophil degranulation induced by tnf- leads to vascular endothelium architecture destruction by proteolytic enzymes such as elastase , gelatinase , and collagenase and subsequently to the disruption of vascular wall integrity as elastase degrades the endothelial homodimeric cadherin - cadherin binding . the overall result of this process is augmented permeability , adhesion , and migration of pmn and other leukocytes to locally or remotely affected tissues and organs , causing tissue damage , organ dysfunction , and ultimately mods / mof [ 9193 ] . the combination of these cellular , molecular , and vascular phenomena needs to be constantly and ever so delicately balanced and regulated . the sheer simplicity of the reasoning behind the two - hit argument is that sequential and superimposed insults tend to challenge this fragile environment and are capable of synergistically rerouting the inflammatory response to a tissue injury - driven path that can be ultimately recognized through the progression of the complex sirs / sepsis / ards / mods / mof continuum , and possibly death . as early as 1986 , researches were already noticing the protective effects that short - term nonlethal ischemic cycles could have over the heart if it were to be later exposed to a prolonged , potentially fatal ischemic event and in 1990 a similar phenomenon had already been identified in the brain of gerbils . this basic mechanism itself can be established in a variety of different organs and systems , and the initial protection - inducing ischemia can be of varying types and profiles . some aspects of preconditioning through ischemia can even be naturally occurring as it is seen in patients that suffer from cerebral transient ischemic attacks ( tias ) and do not incur in structural damage to neurons but rather are actually protected from subsequent major episodes demonstrating that ipc - like mechanisms can be adaptive in nature . in general terms , ipc can be achieved by relatively gentle and often cyclic ischemic events . a tissue that undergoes an ischemic event is more likely to survive a subsequent prolonged deprivation of oxygen . ideal duration and proportion of these initial insults of preconditioning are species- and tissue - specific . for example ipc can be achieved in rats during one to three cycles of ir [ 100 , 101 ] , while in rabbits a single 5 min cycle of ir is sufficient and in dogs a 2.5 min single event of ir has been proven to be effective . processes involved during reperfusion of severe ischemic injuries are a consequence of a complex sequence of events leading to changes in capillary permeability , neutrophil recruitment , complement activation , and generation of reactive oxygen species , similar to other inflammation - driven insults . an early or classic stage of protection , independent of protein syntheses , begins almost immediately after the mild ischemic insult and can be sustained for up to 3 hours . a second or delayed phase lasts for up to 24 hours after the first preconditioning hit and is based on synthesis of new proteins and altered gene expression . aside from local protection of tissues close to the preconditioned area , remote or distant protection of organs ( ripc , or remote ischemic preconditioning ) can also occur . in broader terms , this notion means that exposing various tissues or organs ( such as limbs ) to mild preconditioning ischemic events can cause other distant organs and tissues to be more resilient to following ischemic insults , even though the latter were not directly preconditioned in the first place . this had already been demonstrated in 1993 when mcclanahan and colleagues showed in a preliminary report that myocardial infarct size was reduced if rabbits were previously exposed to a brief transient occlusion of the renal artery . in 1996 gho et al . proved that brief occlusion of adjacent coronary arteries , left renal artery , and anterior mesenteric artery protected the myocardium from a subsequent prolonged ischemic event . a murine model subject to two hours of ischemia of the hind limb showed significant protection against local ( leg skeletal muscle ) and distant ( intestinal injury and lung infiltrates ) organ injury by a subsequent severe ischemic event . due to its effect on decreasing neutrophil infiltrates in the lung [ 109111 ] and because of the systemic attenuation of subsequent inflammatory responses , ipc could be a tool for modulating systemic inflammation and for preventing local and distant organ injury or even sirs / sepsis or ards . another clinical application of remote or distant ipc is the protection that it delivers against acute kidney injury following major cardiac surgery , demonstrated by the reduced rate in which patients subject to the procedure demand renal replacement therapy . the possibility of remotely achieving ipc is pivotal in a number of conditions . during heart surgery , for instance , the repetitive clamping and declamping of major blood vessels to induce local ipc on the heart can cause the formation of emboli in addition to the risk that short repetitive ischemic episodes can be traumatic in nature to the organ itself . therefore , the possibility of triggering remote systemic protection against ischemia in organs such as the heart is invaluable [ 113 , 114 ] . the molecular mechanisms through which ipc or ripc protection occurs are still unclear and not a single definite pathway has been established . it seems that the protective state of ipc is achieved through a combination of humoral , neural , and systemic components [ 106 , 115 ] . principle mediators are adenosine , reactive oxygen species ( ros ) , nf-b , bradykinin , opioids , angiotensin , endocannabinoids , and nitric oxide ( no ) that alter cellular metabolism via atp - sensitive k ion channels and receptors that direct transcription of survival proteins and activation of intracellular kinases that ultimately protect against oxidative stress [ 105 , 115123 ] . the role of the endothelium seems to be central during the development of ischemic resistance through ipc . local and systemic endothelial function was proven to be greatly enhanced when human subjects were exposed to daily short - term limb ischemia for a period of 7 days , with measurable improvements in resting skin microcirculation and brachial artery function assessed by fmd ( flow - mediated dilation ) , effect which lingered after the late phase of ischemic protection was over . another study has shown that ipc is capable of protecting tight junctions both functionally and structurally on hearts that would otherwise suffer from cell - to - cell collapse and edema when exposed to severe ir injury . moreover , the role of endothelial nitric oxide synthase ( enos ) and its no output has been implicated as relevant for the ischemic preconditioning both in early and late ipc [ 27 , 124 , 125 ] . the reasoning behind the triggering mechanisms of ipc is that short preconditioning insults generate enough tissue damage upon reperfusion as to cause the release of adenosine ( from the breaking of atp molecules ) , bradykinin , and ros [ 105 , 126 ] . these substances trigger a molecular cascade that begins in the translocation and activation of protein kinase c and culminates in the phosphorylation of hsp27 via p38 mapk and the opening of atp - sensitive k channels . the role of organelles such as mitochondria and their function in regulating intracellular ca / k exchange has also been implicated in the mechanisms of ipc protection in neuronal tissues , since the artificial opening of atp - sensitive k channels can mimic some of the effects of ipc . many molecular mechanisms of ipc have the inhibition of mptp ( mitochondrial permeability transition pore ) channels as their endpoint , which , if opened , mediate cell death by atp depletion and mitochondrial swelling [ 115 , 129 ] . activation of pkc measured by its intracellular translocation and the role of several kinases such as pi3 , erk / mapk , and jak / stat are pivotal in conferring the state of protection during ipc [ 115 , 121 , 129 ] . furthermore , ipc can attenuate or eliminate o2 production and its effects by suppressing endothelin-1 ( et-1 ) secretion via the opening of these mitochondrial katp channels prior to subsequent ischemic events , since et-1 generation is related to increased production of superoxide anion and endothelial dysfunction with increased p - selectin expression and neutrophil adhesion . nevertheless , due to the complexity of these mechanisms , results generally lack consistency when it comes to defining pathways through which ipc occurs . for example , in two separate knockout models , no produced by an increased expression of nf-b has been shown to be relevant in the ischemic protection of the heart , while the same role could not be demonstrated in the protection of the intestines and brain tissue . the suppression of tnf- and bax ( both involved in apoptotic stimulation ) and the stimulation of cellular survival mechanisms such as phosphorylation of erk-1/2 and akt simultaneous to the upregulation of bcl-2 have been identified in ipc of the myocardium as mechanisms through which cell survival surpasses the rate of apoptosis after ir injury . once again some studies have suggested a protective self - regulatory effect that the secretion of tnf- could have on the stimulation of nf-b and on the suppression of proinflammatory proteins during subsequent ischemic injury . other recently unveiled novel molecular mediators of ipc have risen to the stage as possible targets in the investigation of the protective cascades of preconditioning . a study from 2013 showed that concentrations of hif1- and procaspase-3 were higher in patients receiving ripc by upper limb ischemia before cardiopulmonary bypass . these same patients had higher right atrial tissue and systemic concentrations of il-1 , il-8 , and tnf- , indicating the direct influence of ripc in the modulation of apoptosis and inflammation . the role of cx43 ( connexin 43 ) has also been established in both local and remote ipc , which attenuates the ischemia - induced dephosphorylation of cx43 that would otherwise cause the mechanical , chemical , and electrical instabilities in cardiomyocytes gap junctions by opening of the cx43 hemichannels [ 135 , 136 ] . levels of mrna 144 were found to be increased after ripc and the exogenous intravenous administration of mrna 144 was capable of mimicking the protective effect of ripc in rescuing tissue from ir injury . furthermore , increased activation of akt and p44/42mapk and decreased levels of mtor were observed after the administration of mrna 144 , suggesting that the molecule could serve as a biomarker for the efficacy of a conditioning procedure [ 115 , 137 ] . ipc as a technique has very practical applications . it can potentially be used as therapeutic preparation for surgical procedures that require some kind of ischemic episode , like in organ transplants . another very important application of the model for real - life pathologies is the identification of novel molecular targets that are involved in ir injury and in the protection against said injuries . the modulation of some of these molecular targets and pathways can serve as treatment for naturally occurring ischemic insults seen in strokes , in thromboembolic injuries , and in acute myocardial infarction , to name a few . as stated before , neither ipc nor the multiple - hit hypotheses are particularly new ideas , but it was not until the beginning of the century that more and more research has focused on one of the two models . during this paradigm shift , the idea that the two - hit model can satisfactorily account for many real - life pathological processes has been somewhat pushed aside . it is logical to assume that , because of its nature , ipc is a mechanism that can be spontaneously found in the body and its artificial mimicking consists of a way to tap into the underlying responses of the immune system upon inflammatory insults and can serve as a tool for modulating these responses . therefore , the two - hit model and ipc are not competing models in principle . while the former serves as a tool to explain the intricate processes that can be brought about via multiple sequential inflammatory insults which occur during a host of disease processes , such as trauma , hemorrhagic shock , and sepsis , the latter is an active investigation of techniques and strategies that can modulate the inflammatory response to a number of ischemic insults . nevertheless , there is an obvious paradox surrounding the subject : how can an inflammatory , ischemic insult drive the triggering of a protective mechanism to a subsequent ischemic insult , while the two - hit model states that a priming insult , including ischemia , should prepare the body to an exaggerated response to a second hit or insult , which incidentally can also be ischemic in nature . to our knowledge , there are no definitive answers to this conundrum . that the initial preconditioning ischemic event should not be harsh enough to cause serious tissue injury while maintaining enough potency to trigger the molecular mechanisms relevant to preconditioning seems reasonable and intuitive , but , other than that , no clear differentiation has been established concerning the nature of the first event in each model . researchers have already mapped protocols to ensure that certain ischemic events are limited in intensity and duration so that they would not serve as priming insults for the host while still holding their beneficial effects [ 100103 ] , but aside from this intuitive distinction little is known concerning what makes an insult behave in either a priming or protective manner . during this review many pathways , examples , and applications of both the two - hit and ipc models were explored , and little juxtaposition was found . the goal behind this text was exactly that of reconciling both models and demonstrating that each of the seemingly contradictory perspectives has a lot to offer as platforms to better understand and intervene in some very real and relevant medical situations , while maintaining the fact that both theories are not mutually exclusive .

inflammatory cascades and mechanisms are ubiquitous during host responses to various types of insult . biological models and interventional strategies have been devised as an effort to better understand and modulate inflammation - driven injuries . amongst those the two - hit model stands as a plausible and intuitive framework that explains some of the most frequent clinical outcomes seen in injuries like trauma and sepsis . this model states that a first hit serves as a priming event upon which sequential insults can build on , culminating on maladaptive inflammatory responses . on a different front , ischemic preconditioning ( ipc ) has risen to light as a readily applicable tool for modulating the inflammatory response to ischemia and reperfusion . the idea is that mild ischemic insults , either remote or local , can cause organs and tissues to be more resilient to further ischemic insults . this seemingly contradictory role that the two models attribute to a first inflammatory hit , as priming in the former and protective in the latter , has set these two theories on opposing corners of the literature . the present review tries to reconcile both models by showing that , rather than debunking each other , each framework offers unique insights in understanding and modulating inflammation - related injuries .

since the rate of population growth increases and food sources are not sufficient , birth control is considered as one of the important solution in order to control the growth rate of population . , the researchers have strived to produce effective contraceptive methods with few side effects to provide a better birth control . acceptability of a contraceptive method depends upon many interacting factors , like : characteristics of the method , demographic and socioeconomic variables pertaining to the population of clients ( 1 ) . injectable or implants contraception reduces the need of daily consumption ( e.g. combinational pills ) or depends upon sexual intercourse ( e.g. condoms , etc ) . today , nearly 16 million women worldwide use progestin - only or combined injectable contracetives ( 2 ) . the growing use of injectable contraceptives can be attributed to its desirable characteristic , inexpensive , not related to coitus , easy to use , noninvasive , reversible and long - acting effect . depo medroxyprogesterone acetate ( dmpa ) was presented as a contraceptive method in the usa in 1992 . however , some problems as amenorrhea , headache and depression were also reported ( 5 ) . cyclofem is consisted of 25 mg medroxy progesterone acetate and 5 mg estradiol cypionate , being used as microcrystalline suspension by deep and intramuscular injection into the gluteal or deltoid muscles . the injection should be done in the first five days of menstrual , or after delivery before the end of sixth week . the purpose of this study was to monitor the side effects of two injectable methods ( dmpa , cyclofem ) after using for six months . this cross - sectional descriptive study was conducted at samen - alaemeh clinic , kermanshah , iran and high risk pregnancy research center , imam reza hospital , kermanshah university of medical sciences , kermanshah , iran from july 2009 to september 2010 . the study was reviewed and approved by the ethics committee of the kermanshah university of medical sciences . eligible women are recruited among clients seeking family planning services at the two selected centers . based on the previous studies , the reported complications about dmpa and cyclofem were 24.6% and 43.5% , respectively . by assuming the study power 90% and alpha = 0.05 , sample size in each group was 125 people.(6 ) the inclusion criteria were women aged 18 - 40 years and no contraindication for using of cyclofem and dmpa . the exclusion criteria were : history of menstrual problem , breast feeding , pregnancy , previous injectable contraceptive , and history of other diseases , like diabetics , blood pressure , goiter , etc . about 125 women were in one - month cyclofem injection , while 125 people were in 3-month injection of dmpa . the procedure ensured that the number of individuals allocated to each study group was equal . participants were visited by a psychologist at the selected center , and were subjected to beck depression inventory ( bdi ) , so they were excluded from the study if they had suffered from anxiety , depression , etc . the first injection was applied during the first five days of the menstrual cycle . if the participants remained with the study for six months , they would be psychologically re - evaluated , and their mood changes would be recorded . follow up visits were performed every 30 days for both group . after first injection information on demographic and personal characteristics were obtained from a self - administered questionnaire , completed by each participant after entering to the study . after six months , they were asked to have any problems , such as weight changes , menstrual problem ( increase or decrease bleeding ) , amenorrhea , dyspareunia , vaginal dryness , painful sexual intercourse , headache , bone pains , as well as other questions , like their reasons for using / not using the methods . the mean age in dmpa group was 32.15.2 , while in cyclofem group was 29.95.6 . the mean parity in dmpa group was slightly greater than cyclofem group , 2.39 1.24 vs. 1.51 , respectively . in this study , the weight gain , breast tenderness , bone pain , headache , vaginal dryness , mood changes and menstrual pattern change variables were studied as side effects of dmpa and cyclofem methods . the most important side effects in dmpa group were menstrual cycle problems , like amenorrhea ( 74.4% ) , weight gain ( 48% ) , bone pains ( 24% ) , and vaginal dryness ( 10.4% ) . however , in cyclofem group , the side effects were reported as menstrual problem , like bleeding reduction 47(37.6% ) , headache ( 14.4% ) and breasts tenderness ( 20% ) ( table 1 ) . the comparison of side effects of two methods in this study , menstrual change was the most common discontinuation reason in dmpa and cyclofem groups ( about 25% in each group ) . weight gain ( 18.6% ) and bone pain ( 23.25% ) in dmpa group , whereas headache ( 10.4% ) and mood changes ( 8% ) in cyclofem group were also reported for discontinuation reasons ( table 2 ) . depo medroxy progesterone acetate ( dmpa ) is used by most of women in iran for a long time ; however , cyclofem is newly applied for contraception . in our study , the mean age and parity in women using dmpa were more than women using cyclofem ( p = 0.0008 , p = 0.0001 , respectively ) . although dmpa is considered as a long acting birth control , it has not been identified as a successful contraception because of delay in the return of fertility and limited acceptability , especially among young and low - parity women . ruminjo et al . in their study in 2005 have showed that the mean age and parity was similar in both groups ( combined and progestin - only injectable ) ( 6 ) . the weight gain has always been a major concern among the dmpa consumers . in current study , 48% of dmpa users gained weight after six months , but this was less in cyclofem group . guazzelli , bahamondes , bonny and seymor have demonstrated that cyclofem has a few effects on weight gain , but they have agreed on weight gain among dmpa users , especially among young women ( 812 ) . our study showed that cyclofem could considerably lead to breast tenderness , but it was not too serious for the participants to discontinue the method . some studies have also reported the increase of breast tenderness in cyclofem users ( 13 ) . although , one study has reported the reduction of breast pain using cyclofem ( 8) . according to the published studies , dmpa , as a pure progesterone , avoids the production of estrogen by suppressing ovaries and reducing blood estradiol level , leading to osteopenia ( 1417 ) . in recent study , bone density was not measured , but bone pains as pelvis , waist and legs were investigated . about 24% of the women of our study who used dmpa seriously suffered from bone pains , so stopped using it . some studies emphasized measuring bone density for the women over 40 years old using dmpa for two years , ( 20 ) . however , in our study , bone pain was less in cyclofem users ( 14.4% ) . in similar studies , cyclofem users experience less bone pains , so this method has no influence on bone density ( 8 , 21 ) . headache was observed among the women using cyclofem in our study , even leading to discontinuation ( 10.4% ) . study by guazzeli in 2007 has showed that headache is one of the discontinuation reasons of cyclofem ( 8) . vaginal dryness was less investigated , but in current study , dmpa in comparison to cyclofem significantly increased vaginal dryness in users ( 0.05 ) . vaginal dryness is not a considerable reason for discontinuation of method , and it can be explained by time restriction ( six months ) . mood reduction was observed more among cyclofem users with a rate of 8% for discontinuation . but in other studies , they did not observed any mood change , depression , or affective problems among young women using dmpa for 12 months ( 2224 ) . in our study , amenorrhea in dmpa group and bleeding reduction in cyclofem were the most common menstrual problems . dmpa , used by millions women in all over the world , causes menstrual problems , namely amenorrhea , even through subcutaneous injection ( 2426 ) . although it seems to be a good method to regulate menstrual cycle , in our study , there were many complaints about menstrual problems , causing to discontinuation . according to hall in 1998 , bahamondes and et al . observed that despite having estrogen , the cyclofem does n't cause endometrial hyperplasia ( 28 ) . provided endometrial biopsy for the dmpa and cyclofem users , and they observed not only reduced endometrial vasculature in both groups , but also endometrial atrophy . so , she concluded that there was no difference between cyclofem and dmpa ( 29 ) . in our study , hajikazemi et al showed the continuance rate after six months for dmpa was about 39% ( 5 ) . in kenia , the continuance rate after 12 months for dmpa and cyclofem were 75% and 56.5% , respectively . also , in mexico and china , the continuance rate after 12 months for cyclofem were 26% and 81% , respectively ( 3031 ) . this indicates that these two methods are more applicable for other races than iranian women ( 8) . cyclofem and dmpa may be used as highly effective , safe and convenient methods for long term reversible contraception . weight gain and bone pain in dmpa users , while headache and breast tenderness in cyclofem users were also observed as the most important side effects .

objectiveto compare side effects between users of two kinds of injectable contraceptives ( depo - medroxy progesterone acetate and cyclofem).materials and methodsthis cross - sectional descriptive study included 250 women , aged 18 - 40 years , using depo - medroxy progesterone acetate ( dmpa ) or cyclofem . the volunteers were examined six months after using contraceptive , and they were asked about following symptoms : weight changes , irregular bleeding , dysparounia , vaginal dryness , headache , breast pain , bone pain , and discontinuation reason . the data were analyzed by statistical methods.resultsthe important side effects of dmpa were : irregular bleeding ( 93.60% ) , weight gain ( 48% ) , bone pain ( 24% ) and vaginal dryness ( 10.40% ) , while the side effects in the cyclofem group were : irregular bleeding ( 65.60% ) , headache ( 14.4% ) and breast sensitivity ( 20% ) . bleeding pattern changes were the most important problem leading to discontinuation of both contraceptive methods in our participants.conclusionthe results of the study showed that the most important problem in both groups was change in bleeding pattern . proper consulting by a trained expert reduces the high amount of discontinuation and their failure rates .

although extranodal non - hodgkin 's lymphoma ( nhl ) in the oral cavity is considered to be relatively rare , lymphomas , a cancer of lymphoid tissue ( either b- or t - lymphocyte ) are the second most common malignant tumor in the head and neck after squamous cell carcinoma . hodgkin 's lymphoma ( hl ) and nhl are the two main classifications of lymphomas , with the latter being the most common according to the most recent american cancer society update . reed - sternberg cells , the binucleated or multinucleated giant cells characteristic of hl , are essential for the diagnosis of hl . nhl accounts for about 4% of all cancers in the usa . the estimate of new cases of nhl in 2015 is 71,850 people ( 39,850 males and 32,000 females ) . on the other hand , only 9,050 new cases ( 3,950 females and 5,100 males ) of hl are estimated to occur this year . nhl commonly arises in the lymph node , but extranodal involvement can occur . a retrospective study by urquhart and berg on 311 patients diagnosed with nodal and extranodal lymphoma in the head and neck found that about 23% of extranodal lymphomas were nhl , while only 4% were hl . lymphomas in the oral cavity are usually extranodal and are found either in soft tissue or centrally in bone . the most common intraoral location is the gingiva followed by the palate , while the most frequent locations in the head and neck are the tonsils followed by the parotid gland . a 54-year - old male presented with a history of root canal treatment to tooth no . 30 , followed by periapical surgery . at the time of periapical surgery , a periodontal problem was noted on tooth no . two weeks after the surgery , the patient presented with right posterior swelling that distorted the right cheek . 32 was noticed , which had a clinical appearance of a pyogenic granuloma . there was a class iii mobility to tooth no . the buccal mucosal tissue and gingiva was firm extending to the bicuspid and the vestibule . 32 was extracted , and the associated gingival lesion was removed . during the extraction , a large area of necrotic bone in the socket of tooth the radiographic findings were that of a radiolucent destruction of bone with ill - defined margins around tooth no . 1 ) . the differential diagnosis based on clinical and radiographic finding was an acute abscess , acute osteomyelitis or a malignant tumor such as an osteosarcoma . the first was during the apical surgery and the second was 2 weeks later when tooth no . the histopathology of the second specimen showed multiple fragments of a squamous mucosa , soft tissue and bone with a dense lymphoid infiltrate ( fig . the lymphocytes were intermediate - to - large and atypical with a moderate amount of cytoplasm . the nuclei had finely clumped chromatin with variably prominent nucleoli , and there were abundant mitotic figures throughout the lesion ( fig . a panel of immunostains was performed , with cells exhibiting positivity for cd20 ( fig . interphase fluorescence in situ hybridization ( fish ) showed no evidence of c - myc / igh translocation . based on the histopathology and immunohistochemical findings , the lesion was diagnosed as a high - grade b - cell lymphoma . after squamous cell carcinoma , lymphoma is the second most common neoplasm in the head and neck region . diffuse large b - cell lymphoma ( dlbcl ) a retrospective review stated that , out of 381 cases of head and neck nhl , 100 of which were extranodal , 16 patients ( 16% ) had jaw bone involvement , most of which were dlbcl . unexplained tooth mobility , bone pain or discomfort , and radiographic changes can be observed in most of these cases . an incorrect diagnosis and subsequent delayed treatment can lead to bone expansion , cortical bone perforation and intraoral soft tissue mass formation . radiographically , an ill - defined radiolucency with irregular margins can be detected in advanced cases . the median age is 64 years , and men are slightly more affected than women . microscopically , dlbcls consist of large tumor cells with large nuclei that are more than twice the size of lymphocytes . centroblasts have round or irregular nuclei and membrane - bound nucleoli , while immunoblasts have round nuclei and centrally located prominent nucleoli . multiagent chemotherapy together with anti - cd20 ( rituximab ) is usually the treatment of choice for dlbcl . the diagnosis of lymphoma in the jaws is difficult and often delayed for many reasons . clinically , lymphoma may cause vague pain and discomfort , which might be easily misdiagnosed as periapical lesion developed from an odontogenic infection . radiographic changes in early stages of the tumor may be subtle and usually are only detected late in the disease as an ill - defined radiolucency that might resemble a dental abscess . histologically , nhls are characterized by a proliferation of lymphocytic appearing cells easily mistaken for a periapical granuloma or cyst . the clinician must be aware of the jaw tumor symptoms , which can help in early detection and treatment . rapid local destruction of the bone without obvious odontogenic infection is a key characteristic of jaw tumors . persistent jaw pain and tooth mobility caused by rapid bone destruction may be an early indication of malignancy . in advanced cases , swelling , pain , lip numbness and pathologic fracture are also associated with a primary bony lesion . on the other hand therefore , clinicopathologic correlation is crucial to reach the correct diagnosis in clinically suspicious cases such as a malignant tumor in the jaw . the submitted documents represent original research that has not been published previously either in whole or in part in any form , and is not currently submitted for publication elsewhere .

in the oral cavity , extranodal non - hodgkin 's lymphoma can occur in the periapical region either in the maxilla or mandible . also , it can mimic inflammatory lesions that arise around the teeth apices such as periapical granuloma , radicular cyst and osteomyelitis . misdiagnosis of lymphomas in the jaws may reduce the chance of successful treatment and worsen the prognosis . therefore , any growth of periapical tissue must be submitted for histopathological evaluation to avoid a delay in the diagnosis . we present a case of extranodal non - hodgkin 's lymphoma of a 53-year - old male in the right posterior mandible that was initially misdiagnosed as a reactive periapical lesion . this case illustrates the importance for both the pathologist and the clinician of considering malignant lesions such as lymphoma in the differential diagnosis of periapical radiolucency .

synoptic reporting of all tumor excisions is required by the college of american pathologists ( cap ) . the cap specifically requires that each element in a synoptic report be reported in a required data element ( rde ) pair consisting of the element and the corresponding response ( cap laboratory accreditation process checklist question anp . however , recent studies suggest that specific formatting features are associated with not only the accuracy of the information entered into the report , but also user preference , accuracy of information retrieval , and speed of information retrieval . currently , cap requires that each element of the gleason grading system ( primary pattern , secondary pattern , tertiary pattern , and total score ) be reported on separate lines as separate and distinct rdes , despite the fact that in the biopsy setting these elements are reported on one line as primary pattern + secondary pattern = total score with tertiary pattern on a separate line if and only if it is present . whether these or other formats might affect the reader 's experience is not known . we sought to determine if such differences in reporting formats would affect the speed or accuracy of information retrieval by nonpathologists reading synoptic reports of prostatectomy specimens . to test the accuracy and speed of identification of specific data elements in a synoptic report , a python script was written that provided instructions and a test platform for these quizzes . specifically , the participant is shown a specific phrase that may or may not be in a synoptic report ( with exceptions listed below ) . when the user presses enter the synoptic report appears on the screen and the timer starts . the user then examines the report to determine if the phrase is or is not present . if it is present , they enter the number two , if it is not they enter 1 and then press return . the program automatically records the time and whether the answer was correct , and this data is then transferred to a comma separated values file for further analysis . we constructed our synoptic report for a radical prostatectomy using the checklist from cap , [ figure 1 ] for the standard format . all elements were identical except for changes in the reporting of gleason grade ( primary grade , secondary grade , tertiary grade , and total score ) . the formats were tested in three different quizzes , each given in sequence and at the same sitting . the first quiz contained 36 total questions and compared the standard format [ figure 1 ] with a format in which if there was no tertiary grade the line concerning tertiary grade was omitted [ figure 2 ] . in the standard format , the response for tertiary grade could be 3 , 4 , 5 , or not applicable . , where ? could be 3 , 4 , 5 , or not applicable . standard format synoptic report tertiary grade omitted because it was not applicable / present quiz two contained 32 questions and compared the standard format [ figure 1 ] with a biopsy format , [ figure 3 ] , where gleason grades are reported as primary pattern + secondary pattern = total score on one line and tertiary pattern is on a separate line . although this format is taken from and labeled as biopsy format , for this quiz , the total score was calculated as it is for prostatectomies where primary plus secondary regardless of the tertiary pattern ( also regardless of whether it was < 5% or > 5% of tissue ) which was reported separately . the question for this part could pertain to any of the 4 elements ( primary pattern , secondary pattern , total score , and tertiary pattern ) , and the answers could be any of 3,4,5,6,7,8,9 , or 10 ( not applicable was not included ) . in order for the participant to know which format to expect , 16 questions with the standard format were presented and then 16 questions with the biopsy format . within each group , the order of questions were randomized for each participant , but each participant got the exact same questions for each group . biopsy format synoptic report quiz three contained 32 questions and compared the standard format [ figure 1 ] with a biopsy format , [ figure 4 ] , where gleason grades are reported as primary pattern + secondary pattern + ( tertiary pattern ) - > total score on one line . the question for this part could pertain to any of the four elements ( primary pattern , secondary pattern , total score , and tertiary pattern ) , and the answers could be any of 3,4,5,6,7,8,9 , or 10 ( not applicable was not included ) . in order for the participant to know which format to expect , 16 questions with the standard format were presented and then 16 questions with the biopsy format . within each group , the order of questions were randomized for each participant , but each participant got the exact same questions for each group . they were all nonpathologists and included , five cancer registrars , four mds ( all internists ) , four non - md medical personnel ( all laboratory technologists ) , and three nonmedical personnel ( administrative assistants , other professionals ) . we specifically excluded pathologists from this testing , since we wanted to measure the performance of a user other than a pathologist . similarly , although we have no evidence that different types of readers perform differently on these tests than any other type of reader , like pathologists we did not include urologists to ensure that their particular set of knowledge and experience did not influence the results . although , previous studies have suggested that in this type of test format there were no significant differences between these different users in terms of accuracy or time to retrieval of information , this was again tested in the current study . previously we had noted that participants got faster from quiz 1 to quizzes 2 and 3 as they got practice with the test . as a result , no comparison was made between the three quizzes , and all three quizzes were always taken in the same order . in addition , there was a wide range of speed for the different users . in order to allow comparison between these uses , as a result , the normalized time for the standard format was the control with a normalized time of one , and the time for all other formats was in comparison with that time . statistical analysis was performed using a t - test or fishers exact test as appropriate with a significance threshold of 0.05 . as shown in table 1 , the nonmedical group had a significantly higher accuracy rate than the cancer registrars ( p = 0.02 ) . as a result , any significant findings related to accuracy using grouped data also underwent subset analysis . there was no significant difference in normalized times between any of the four user groups , [ table 2 ] . accuracy for different users compared to cancer registrars normalized time for all questions for different users compared to cancer registrars when tertiary grade was omitted whenever the result was not applicable , the accuracy of classification by the readers was significantly lower than when tertiary grade was listed [ 72 vs. 97% , p < 0.001 table 3 ] . 0.001 ) were evaluated as well as when all other users were evaluated ( 70% vs. 97% , p < 0.001 ) . in addition , when tertiary grade was omitted , the time to answer the question increased significantly ( 63% , p < 0.001 ) [ table 4 ] . accuracy for different formats compared to standard format normalized time compared to standard format when the standard format ( all four elements each on a separate line ) was compared with the biopsy format ( primary + secondary = total , with tertiary on a separate line ) , there was no difference in accuracy of data extraction ( 98 vs. 97% , p = 0.56 ) , however , the time needed to extract the data decreased 18% with the biopsy format ( p < 0.001 ) . nevertheless , no user preferred the biopsy format . when the standard format was compared with a single line format ( primary + secondary + ( tertiary ) - > total ) , the accuracy increased from 97% to 100% ( p = 0.02 ) using the single line format . subset analysis showed that the group with the biggest difference were the registrars ( 6/7 errors with the standard format ) . the time to extract the data also decreased 24% ( p < 0.001 ) with the single line format . nine users preferred the standard format and seven the single line format , even though they were more accurate and faster with the single line format . the data in this report suggest three major conclusions : removing not applicable elements from a synoptic report can reduce both the accuracy and speed of information retrieval , combining all elements of gleason grading on one line significantly improves the accuracy and speed of information retrieval from synoptic reports , and users preferences may not always correlate with either accuracy or speed . these results were similar across a wide variety of different users , just as they were in our previous studies . . one of the original intents of synoptic reporting was to have one data element per line , making automated data extraction easier . however , as the tools available for data extraction have evolved , and as the field has moved to data extraction before generating the pathology report , this restriction may not be as important as it has been in the past . in particular with gleason grading , the use of + and = signs makes regular expression extraction of multiple pieces of information from a single line both easy and routine . in this context , other factors , such as end users ability to quickly and correctly extract information from the report may be of increased value . it is not a surprise that removing elements form a synoptic report reduces the speed of identification of such elements . it has previously been documented that having a reproducible format where all elements are always included in exactly the same order has been associated with faster information identification . when not applicable elements are missing , the reader has to first ensure that indeed they are missing before they can then conclude that they are not applicable . however , we were surprised that removing items from the synoptic format also reduced the accuracy of the response . nevertheless , in constructing synoptic reports , there are many opportunities where omitting not applicable elements would reduce the length of the report , a feature that has previously been shown to be associated with decreased error in constructing the synoptic report . whether the benefit in terms of error by making the report shorter outweigh the reduced accuracy of interpretation by leaving out this information is not clear at this time . it is also not a surprise that combining all parts of gleason grading into one line is associated with faster information retrieval . however , in the standard format , the information ( response ) in those four separate lines consists only of a number and the label that explains the number ( rde ) is separated from it on the other side of the page . in discussing this with the participants , they felt that the reason that the single line was faster was not just because it was a single line , but also because they could simply look down the right - hand side of the report ( the responses ) to find the grading section without referring to any rde . this was because the format of the grading information had a structure that was easily identifiable , unique from all other responses in the report , and conveyed information without having to refer to the listed rde . thus , having a unique structure to the response and not just the rde is a key part of improving the speed of information retrieval from a synoptic report . this hypothesis is also consistent with our previous studies that examined the effect of different formats . nevertheless , the format of reporting gleason patterns / grades and scores has been the subject of considerable discussion previously . most importantly , the reasons one may prefer one format over another is multifaceted , and the information in the current study is just one aspect of this discussion . the information we present may be of value in future discussions by pathologists and organizations such as the international society of urologic pathologists who make recommendations using all of the different aspects that affect reporting of gleason grading / scoring . at present , though , current recommendations make a distinction between reporting formats for biopsies and prostatectomies . specifically , in the biopsy specimen if the tertiary pattern is high grade then that is reported as the secondary pattern rather than the true secondary pattern . this is done to ensure that the presence of this tertiary pattern is not overlooked by either clinicians or other readers , who may simply omit or skip the presence of a high - grade tertiary pattern if it is only reported in a note . while the rationale for this practice is clear , it does bring up two issues . secondary pattern or a high - grade tertiary pattern , this form of reporting is subject to information loss . specifically , if one reads a report with a gleason score of 3 + 5 , one can not tell from the report whether there are only two patterns ( 3 and 5 ) present in this biopsy , or whether this is a biopsy with patterns 3 , 4 and a tertiary pattern 5 . this may in part explain some of the controversy associated with assigning gleason score 3 + 5 cases in the newly proposed gleason groups . more importantly , there is an assumption in this analysis that the only other way to report tertiary grades is as a separate note . as our study clearly shows , this is not true this study uses just one of many possible options for reporting all four elements on one line . while the order is intuitive ( primary secondary tertiary and total in that order ) the signs that are used to distinguish between the different patterns are not and could be changed if different signs were identified that could convey more information than the signs we have used here . obviously using a plus signs and an equal sign implies that the numbers will add up , and in this format that is not true , and is the reason why we put tertiary patterns in parentheses . using a grade of 0 may also be easier to interpret than using the phrase not applicable or finally , recent studies have also suggested that the percentage of high - grade tumor should also be reported as this may also be prognostically significant as well as gleason groups . whether and how this information may be incorporated into a single line format is not clear at this time . nevertheless , the need to begin reporting gleason groups is an opportunity to re - address the most effective format for reporting all elements in gleason grading . first data recognition and user preference are just a few elements of the user interaction with a synoptic report . indeed , several of the nonmedical participants noted that while they could perform the study , they really did not understand the meaning of the phrases they were looking for . this in part explains why we were unable to find a consistent difference in the performance between our different user groups this test measures the users ability to read and identify information , regardless of their understanding of that information . other measures that focused on comprehension would most likely find significant differences between these different types of users . different report formats for gleason grading significantly affect users speed , accuracy , and preference ; users do not always prefer either speed or accuracy . the data and study design we describe may be useful in deciding between different format options .

context : the format of a synoptic report can significantly affect the accuracy , speed , and preference with which a reader can retrieve information.objective:the objective of this study is to compare different formats of gleason grading / score in synoptic reports of radical prostatectomies.methods:the performance of 16 nonpathologists ( cancer registrars , mds , medical non - mds , and nonmedical ) at identifying specific information in various formatted synoptic reports using a computerized quiz that measured both accuracy and speed.results:compared to the standard format ( primary , secondary , tertiary grades , and total score on separate lines ) , omitting tertiary grade when not applicable reduced accuracy ( 72 vs. 97% , p < 0.001 ) and increased time to retrieve information 63% ( p < 0.001 ) . no user preferred to have tertiary grade omitted . both the biopsy format ( primary + secondary = total score , tertiary on a separate line ) and the single line format ( primary + secondary + ( tertiary ) - > total score ) were associated with increased speed of data extraction ( 18 and 24% , respectively , p < 0.001 ) . the single line format was more accurate ( 100% vs. 97% , p = 0.02 ) . no user preferred the biopsy format , and only 7/16 users preferred the single line format.conclusionsdifferent report formats for gleason grading significantly affect users speed , accuracy , and preference ; users do not always prefer either speed or accuracy .

there has been a dramatic increase in the prevalence of diabetes mellitus in recent years . the chronic effects of diabetes may manifest in macro- and microvascular complications that are the major causes of morbidity and mortality in patients with diabetes . diabetic nephropathy ( dn ) , one of the microvascular complications , is a leading cause of death from kidney failure [ 2 , 3 ] . apart from haemodynamic factors , hyperglycaemia has been shown to be an underlying cause of pathogenesis in dn . the damaging effects of hyperglycaemia have been partly attributed to increased cellular glucose uptake in cells that are not protected from high ambient glucose levels . several investigators have associated the expression of cip / kip cyclin - dependent kinase ( cdk ) inhibitors , p21 and p27 , with glomerular hypertrophy [ 57 ] . it has been proposed that p21 and p27 may be involved in hypertrophy independently of their cell cycle regulatory properties ( monkawa 2002 ) . furthermore , the induction of p21 and p27 is also required for senescent arrest , a molecular signature of hypertrophic changes in the early stages of the development of diabetic kidney disease . the fact that p21 null mice do not develop glomerular hypertrophy supports the importance of p21 in dn . the activation of the mammalian target of rapamycin ( mtor ) , a serine / threonine kinase , plays a pivotal role in the pathologic forms of hypertrophy in the kidneys [ 1012 ] . these complexes have two different scaffolding proteins , raptor and rictor . by interacting with distinct downstream targets , these scaffolding proteins connect mtor to different signalling pathways , resulting in discrete functional roles . the raptor - mtor protein complex is rapamycin sensitive ; it integrates extracellular and intracellular signals originating from growth factors , hormones , and nutrients . this complex plays a key role in regulating the cellular response to nutrients by phosphorylating the downstream target proteins , p70s6 kinase1 ( s6k ) and initiation factor 4e . studies on skeletal muscle cells have shown that , through a negative feedback mechanism , the activation of the mtor pathway may lead to insulin resistance . furthermore chronic rapamycin treatment in rats induced the expression of hepatic gluconeogenic enzymes , which may adversely affect glucose levels in a diabetic state . on the other hand , it has been shown by several investigators that the inhibition of the mtor signalling pathway has a therapeutic potential for the treatment of dn [ 13 , 16 ] . mtor is also regulated by amp - activated protein kinase ( ampk ) , a sensor of intracellular amp levels . mammalian ampk is a heteromeric complex consisting of one catalytic subunit and regulatory , subunits . through a conformational change in the subunit , amp facilitates the phosphorylation of thr-172 on the subunit by various upstream kinases , including ca - calmodulin - dependent kinase , tgf--activated kinase 1 , and lkb1 serine / threonine kinase . the antidiabetic drug metformin depletes cellular atp levels by blocking mitochondrial respiratory complex i. in turn , the elevated amp levels induce the activation of ampk . nevertheless , it has been suggested that metformin may activate ampk through an amp - independent mechanism . although ampk activation results in the inhibition of mtor signalling , recent findings also suggest that metformin may abolish mtor activation independently of ampk . apart from its insulin - sensitising properties , metformin may have several beneficial effects in various clinical settings . according to clinical studies in both type 1 and type 2 diabetes , several studies have shown that excess glucose increases cell size in various cell types through the activation of akt - mtor signalling ; however , the mechanism leading to glucose - induced mtor activation has not been well defined . it has been suggested that the hypertrophic changes induced by hyperglycaemia may be the consequence of mtor activation through autocrine tgf- signalling . in addition , mtor activity has also been associated with increased expression of the glucose transporter 1 ( glut1 ) in mesangial cells . however , saturation of glucose uptake in mesangial cells has been reported to occur at 30 mm , indicating that hyperglycaemia can induce mtor in the absence of increased glut1 expression . the aim of this study was to compare the inhibitory effects of rapamycin and metformin on proliferation and cell growth in the context of high glucose - induced ampk / mtor signalling . we have observed differential effects of metformin and rapamycin in several ampk / mtor - related aspects with relevance to dysregulated cell growth and cell cycling in dn . human embryonic kidney ( hek293 ) cell line was maintained in minimum essential media ( invitrogen ) supplemented with 10% fbs , 1% pen / strep ( invitrogen ) , and 1% mem nonessential amino acid solution . the cells were cultured at 37c in a 95% air/5% co2 environment and passaged every 3 - 4 days at subconfluence . conditionally immortalised human podocyte cells ( courtesy of professor saleem , bristol ) were cultured in rpmi 1640 medium containing 11 mm d - glucose , 10% foetal bovine serum 1% penicillin / streptomycin . briefly , the cell line was generated by isolating podocyte cells from a normal human kidney specimen and transfected with a temperature sensitive simian virus-40 large t - antigen . the cells proliferate at a permissive 33c , then culturing the cells at 37c switches off t - antigen expression , allowing the cells to assume a native phenotype . in the experiments , the cells were cultured with different concentrations of d - glucose ( sigma - aldrich ) . where appropriate , mannitol ( sigma - aldrich ) was used to control osmotic effects . metformin ( sigma - aldrich ) stock solution was prepared in phosphate buffered saline ( pbs ) . eight pgipz lentiviral shrna constructs against human amp - activated , alpha2 catalytic subunit ( prkaa2 ) , and a nonsilencing construct were purchased from open biosystems . pgipz plasmids were stored in bacterial cultures of e. coli ( prime plus ) in lb lennox ( 5 g nacl / l ) with 8% glycerol , 100 g / ml carbenicillin , and 25 g / ml zeocin . isolation of plasmid dna was done by plasmid midi kit ( qiagen ) according to the protocol supplied by the manufacturer . stable cell lines were generated by transfecting hek293 cells with 2 g / ml plasmid dna in 24 well plates using arrest - in transfection reagent ( open biosystems ) . selection for stably transfected cells was done in medium supplied with 8 g / ml puromycin . the cell suspension was loaded in 96 well microtitre plates at 1 10/ml density and allowed to grow until 50% confluence when the cells were exposed to experimental conditions . cell proliferation was determined using the cell titre96 aqueous non - radioactive cell proliferation assay ( promega ) . the proliferation assay is an mts - based method that spectrophotometrically measures the conversion of tetrazolium salt into a formazan product . the quantity of this product is directly proportional to the number of living cells in culture . flow cytometry was performed to analyse cell cycle distribution and cell size in hek293 cells . after treatment , the cells were trypsinised and washed with pbs by centrifuging them twice at 200 g for 5 minutes ( min ) . the cells for cell cycle analysis were fixed in cold saline gm and 90% ethanol ( 1 : 3 ratio ) and stored at 80c . before analysis , the cells were centrifuged at 500 g for 5 min , resuspended in facs buffer ( pbs , 2% fcs , 10 mm sodium azide ) , and treated with 100 g / ml rnase a ( sigma ) . dna was stained with 50 g / ml propidium iodide ( sigma ) for 1 hour ( h ) at room temperature ( rt ) , and the percentage of 1 10 cells in the g1/g0 , s , and g2/m phases cells was determined . the cells for cell size analysis were resuspended in facs buffer and relative forward side scatter determined on a becman - coulterton epics xl.mcl flow cytometer running expo32 adc software ( 10,000 events ) . in order to investigate the viability of the cells , propidium iodide floating and adherent cells were harvested by trypsinisation , pelleted by centrifugation at 200 g for five minutes , and then washed twice with facs buffer . the pellet was resuspended in facs buffer with 20 g / ml propidium iodide , incubated on ice for 10 minutes , and analysed for dye inclusion on a bd accuri c6 flow cytometer . cell proteins from at least two independent experiments were extracted by addition of lysis buffer containing 20 mm tris - hcl ( ph 7.4 ) , 150 mm nacl , 1 mm edta , 1% tx100 , and 1 tablet/10 ml phosstop phosphatase inhibitor ( roche ) . the suspension was centrifuged at 14000 g and the supernatant containing cellular protein was collected . for western blotting , a 12.5% sodium dodecyl sulphate polyacrylamide gel was run under standard conditions , loading 25 g of total protein in each lane . the gel was placed in transfer buffer and set up for transfer onto a polyvinylidene fluoride membrane at 250 ma overnight . the membrane was rinsed in tris - buffered saline immersed in blocking buffer ( 2% bsa ) for 1 h then incubated with primary antibodies ( p - mtor ser , p - ampk thr , p - s6k thr , p21 , 1 : 1000 , and cell signalling ) overnight at 4c . after rinsing in wash buffer , the membrane was incubated with horseradish peroxidase - conjugated secondary antibody ( cell signalling ) for 1 h at 1 : 5000 dilution at rt . an enhanced chemiluminescence kit ( amersham ) was used for detection of the bands . in order to control protein loading , total - mtor , and -actin antibodies after exposure to 8 mm metformin for 24 h , the cells were fixed with 4% paraformaldehyde in pbs for 15 min at rt . the slides were washed in pbs , permeabilized , and blocked with 0.2% triton x-100 , 10% fcs , 125 mm l - lysine , and 10% sodium azide for 30 min . the slides were washed and incubated with the primary antibody at 1 : 400 dilution ( p21 , cell signalling ) overnight . the cells were rinsed and subsequently incubated with alexa fluor 568 ( invitrogen ) goat anti - mouse secondary antibody at 1 : 500 and dapi at 1 : 1000 dilutions for 1 h at rt . the slides were analysed by fluorescent microscopy ( nikon eclipse 80i microscope and a nikon digital colour camera ) . total protein / cell number ratio was used to determine whether the alteration of cell growth in response to high glucose treatment was accompanied by cell hypertrophy . for the experiments , hek293 cells were cultured in minimal essential medium . at the end of the treatment period , the cells were trypsinised and washed twice with pbs and counted in a haemocytometer chamber . the cells then were lysed to measure the total protein content by the bca protein assay ( thermo scientific ) . the total protein / cell number ratio expressed as g/10 cells was used as a hypertrophy index . normality of distribution was verified by shapiro - wilk test and homogeneity of variance by levene 's test . unless otherwise indicated , statistical significance of differences was calculated by t - test or one - way anova . in order to characterise the effects of metformin and rapamycin on hek293 cell proliferation , a dose - response study was carried out , using the mts assay . in other studies the effects of metformin and rapamycin have already been compared by using the alamar blue metabolic assay . the investigators have found a concentration - dependent difference between the effects of metformin and rapamycin on proliferation . in order to test whether their result could be reproduced by using the mts assay , the two drugs were used in the same concentration ranges . the results show that over a 24 h period , metformin and rapamycin inhibited cell growth in hek293 cells in a dose - dependent manner ( figure 1 ) . however , increasing concentrations of metformin inhibited proliferation in a linear fashion , whereas increasing the concentration of rapamycin in the range of 10500 nm did not cause a significant change in cell proliferation . although zakikhani et al . used lower serum concentration and longer incubation time ; the findings presented here are in line with their results , showing that the inhibitory effect of metformin on cell proliferation is concentration - dependent and , at higher concentrations , more pronounced than that of rapamycin . furthermore , 20 m compound c ( dorsomorphin ) , a research compound widely used for the inhibition of ampk had an opposing effect on metformin - induced decrease in proliferation . the purpose of this experiment was to investigate how cell cycling is affected by metformin and rapamycin treatments , and whether cell cycle changes are modulated by high glucose concentration . the effect of metformin on cell growth and proliferation has been investigated by various cell culture studies [ 17 , 27 , 28 ] . in these studies the use of these relatively high concentrations of metformin may be attributed to the low uptake of this drug by immortalised cell lines . in the context of the inhibition of hepatic gluconeogenesis , the use of metformin in the millimolar range has been shown to be physiologically relevant . the inhibitory effect of 8 mm metformin on cell cycle progression has been demonstrated in a cell culture study of breast cancer cells . in susceptible cells this concentration caused an approximately 50% reduction in cell number over a 24 h culture period . as demonstrated in figure 1 , at 8 mm concentration , metformin caused an approximately 50% reduction in proliferation , suggesting that our findings may be comparable to earlier studies . in this experiment metformin was used at 8 mm and its effect on cell cycling was compared to that of rapamycin . the effect of rapamycin on proliferation has also been studied in vascular smooth muscle cells . the inhibitory effect of rapamycin on proliferation has been shown in a range of concentrations ( 1100 ng / ml ) . it has also been reported that rapamycin did not affect the viability of cultured cells in this concentration range . in order to maximise its effect on cell cycling , in this experiment rapamycin it has also been shown by several investigators that after 24 to 72 h high glucose induces cellular changes implicated in the development of diabetic nephropathy , including increased protein synthesis , proliferation , and the expression of tgf-. in these studies , up to 72 h culture period , high glucose did not have a significant effect on viability [ 3235 ] . in order to study whether high glucose influences cell cycling , 30 mm d - glucose treatment was used for 72 h. in order to control the osmotic effects of high glucose , the normal culture media containing 5.5 mm d - glucose was supplemented with 24.5 mm mannitol . the results of cell cycle analysis show that both metformin and rapamycin increased the percentage of cells in the g0/g1 phase ( figure 2(a ) ) . in comparison , the effect of metformin on cell cycle inhibition was higher than that of rapamycin . in the high glucose treated conditions , however , metformin - induced cell cycle arrest was abrogated . in contrast , high glucose did not have an effect on rapamycin - induced cell cycle arrest . the probability that osmotic effects played a role in the reversal of metformin - induced cell cycle arrest by high glucose is low , as mannitol at an equimolar concentration to high glucose , did not influence cell cycling in the same way . nevertheless , mannitol treatment caused a significant decrease in the g2/m phase in the metformin - treated condition . since apoptosis can be induced through g0/g1 arrest , it was important to test whether cell cycle arrest mediated by metformin and rapamycin could have been associated with increased apoptosis . to investigate this , the results indicate that neither metformin nor rapamycin caused a statistically significant increase in the percentage of pre - g0/g1 cells ( figure 2(b ) ) . hek293 cells express both the 1 and 2 isoforms of ampk ; however , it is the 2 subunit that is primarily involved in ampk activation induced by reduced atp synthesis . according to a widely accepted view , the cellular effects of metformin involve the inhibition of the mitochondrial electron transport chain ; therefore , it was expected that metformin would exert its effects in an ampk2-dependent manner [ 21 , 42 ] . in order to investigate the involvement of ampk in cell cycle regulation , the alpha2 isoform of ampk was knocked down in hek293 cells by a shrna - mediated approach . ampk2 knockdown was validated by measuring phosphorylation of threonine 172 on both isoforms ( 1 , 2 ) of the alpha subunit ( figures 3(a ) and 3(b ) ) . in contrast to the nonsilencing condition , in ampk2 knockdown cells metformin did not induce cell cycle arrest in the g0/g1 phase ( figures 3(c ) and 3(d ) ) . ampk2 deficiency had a comparable effect to high glucose treatment in reversing metformin - induced cell cycle arrest . relative to cycling cells , g0/g1 arrested cells have a reduced cell size phenotype . in order to investigate if cell size measurements correlate with the cell cycle experiments , the size of hek293 cells was measured by flow cytometry . this cell type has been reported to have an intrinsic variation in cell size at various stages of confluence . nevertheless , in our study no significant differences were detected in the size of hek293 cells up to 60% confluence level ( data not shown ) . metformin caused approximately 20% decrease in cell size , which was at a comparable level to the effect of rapamycin ( figure 4(a ) ) . in the combination conditions with high glucose , the effects of the drugs on cell size were reversed , with a more pronounced effect in the metformin - treated condition . the dependence of metformin on ampk in cell size regulation was also investigated . in line with the results presented in figure 4(a ) , metformin caused a decrease in cell size in the control , nonsilencing condition ( figure 4(b ) ) . in ampk2-deficient cells metformin inhibited cell size to the same extent as it did in control cells . as expected , high glucose reversed the effect of metformin on cell size in both control and ampk2-deficient cells . the role of ampk was further studied by using compound c. some reports have suggested that compound c may promote cell death through apoptosis [ 44 , 45 ] . to investigate the toxic effects of compound c , although the viability of cells was not significantly affected by the treatments , cell size was measured on the propidium iodide - negative cell population . compound c reversed metformin - induced cell size reduction in a concentration - dependent manner , with a maximal effect of compound c at 20 m ( figure 4(c ) ) . in order to investigate whether the effect of compound c on cell size could be associated with corresponding changes in cell cycle , cell cycle analysis was done on cells exposed to the same experimental conditions described in figure 4(c ) . at 20 m , compound c blunted the effect of metformin on inducing an increase in the g0/g1 phase of the cell cycle ( figure 4(d ) ) . an increased cell size can correlate with increased protein content . as an index of high glucose - induced hypertrophy the results show that high glucose caused a 25% increase in total protein synthesis over a culture period of two days ( figure 5(a ) ) . in contrast , metformin did not exert an observable inhibitory effect on total protein synthesis in high glucose - treated cells . the inhibitory effects of rapamycin and metformin on mtor signalling were assessed by measuring the phosphorylation level of mtor and s6k at ser2448 and thr389 , respectively ( figure 5(b ) ) . incubating hek293 cells with 8 mm metformin and 100 nm rapamycin for 24 h reduced mtor and s6k phosphorylation , with more pronounced effects on s6k . in contrast to the combination condition with rapamycin , 30 mm d - glucose pretreatment had an opposing effect on mtor inhibition by metformin . in order to investigate whether high glucose opposes metformin - induced mtor inhibition via ampk , as a marker of its activation , we measured the phosphorylation level of threonine 172 on both isoforms ( 1 , 2 ) of the alpha subunit . as expected , ampk activation resulted in a concomitant decrease in s6k phosphorylation ( figure 5(c ) ) . in the range of 1530 mm , d - glucose inhibited ampk phosphorylation and at 25 mm concentration d - glucose treatment opposed the effect of metformin on ampk / s6k signalling . the expression of p21 was investigated in the context of ampk / mtor / s6k signalling by western blotting . metformin treatment caused a decrease in p21 expression in a dose - dependent manner ( figure 6(a ) ) . as expected , metformin caused downregulation of both p - s6k and cyclin d1 with more pronounced effects in the 58 mm concentration range . high glucose treatments ( 1530 mm ) increased the expression of p21 , cyclin d1 , and p - s6k and at 25 mm concentration blunted the inhibitory effects of metformin . similar to high glucose , compound c had an opposing effect of metformin - induced ampk activation , s6k dephosphorylation , and p21 downregulation ( figure 6(b ) ) . the inhibitory effect of metformin on p21 expression was also confirmed in hek293 cells stably expressing shrna against ampk2 ( figure 6(c ) ) . in ampk2-deficient cells , metformin - induced ampk activation was reduced . correspondingly , reciprocal changes in mtor phosphorylation could also be observed . in contrast , in the ampk2 knockdown condition the inhibitory effect of metformin on p21 expression was less pronounced . the expression of p21 can be regulated by the proteasome and recently it has been suggested that ampk activation may inhibit the function of the proteasome [ 4648 ] . to investigate whether metformin - induced downregulation of p21 is proteasome - dependent , the proteasome inhibitor , carbobenzoxy - leu - leu - leucinal ( mg132 ) , was used in control and ampk2-deficient cells . in both control and knockdown cells , the downregulation of p21 was prevented by mg132 treatments ( figure 6(d ) ) . in order to confirm the above western blot results and obtain information about changes in p21 localisation , immunofluorescence microscopy was performed on metformin and high glucose treated hek293 cells . as expected , metformin treatment decreased the expression of p21 and high glucose treatment reversed metformin - induced p21 downregulation ( figure 7 ) . in addition , high glucose treatment enhanced the nuclear compartmentalisation of p21 . mtor plays a central role in cell cycle regulation . furthermore , rapamycin downregulates p21 in mouse fibroblasts . in order to investigate whether mtor inhibition plays a role in p21 regulation , the effect of rapamycin on p21 expression the inhibition of the mtor signalling pathway was demonstrated by the reduced level of s6k phosphorylation . this finding was also confirmed in conditionally immortalised human podocytes that expressed p21 in a metformin - sensitive but rapamycin - insensitive manner ( figure 9 ) . we have found that in hek293 cells high glucose opposes the negative effects of metformin and rapamycin on proliferation , cell size , and protein synthesis , parameters that are widely associated with structural changes early in the development of dn . it has also been observed that high glucose differentially affects mtor - related phenotypes induced by metformin and rapamycin . contrary to our expectations , we have found that metformin inhibits p21 expression in a concentration - dependent manner , independently of its effect on mtor signalling . high glucose , ampk2-deficiency , and compound c overcome the inhibitory effect of metformin on p21 expression , suggesting that ampk may play a role in the regulation of this cyclin - dependent kinase inhibitor . early cellular changes in the development of dn involve hyperplasia and hypertrophy in both the tubular and mesangial compartments . these cellular changes have been attributed to increased cellular glucose uptake in cells that are not protected from high ambient glucose levels [ 52 , 53 ] . in the past decades a considerable interest has been devoted to cell cycle regulatory proteins in glomerular hypertrophy [ 5457 ] . these studies have suggested that the initial pathological changes in the diabetic kidney are associated with low - grade proliferation followed by cellular hypertrophy . in vitro and in vivo studies have shown that the absence of p21 prevents the development of hypertrophy associated with the diabetic kidney [ 56 , 58 ] . it has been shown that the expression of p21 gene ( cdkn1a ) is robustly induced in both type 1 and type 2 diabetic animals . according to a widely held view however , the role of p21 as a cell cycle inhibitor has been challenged by the finding that p21 may serve as an assembly factor for cyclin d - cdk4 complex formation . the cyclin d - cdk4 complex is required for cell cycle progression and is known to be ubiquitous among different cell types [ 62 , 63 ] . furthermore , several studies have suggested that increased cyclin d1 expression is associated with renal and cardiac hypertrophy , which may be attributed to increased stabilisation of p21 [ 6466 ] . in this study , high glucose opposed the inhibitory effects of metformin on both cyclin d1 and p21 expression . in addition , these effects of high glucose correlated with the promotion of cell cycle progression in metformin treated cells , suggesting that high glucose - induced expression of cyclin d1 and p21 may be linked by a common mechanism . the results of this study suggest that inhibition of ampk may be the underlying mechanism by which high glucose induces p21 expression , which in turn may stimulate proliferation and cell growth . the involvement of ampk in the regulation of proliferation has been reported in other studies [ 26 , 27 , 67 ] . in contrast to the 1 isoform , ampk2 has been shown to respond to stress - related conditions , such as hypoxia and glucose deprivation . furthermore , in ampk2 knockout mice the antihypertrophic effects of metformin are known to be attenuated . these findings suggest that energy - depleting agents , such as metformin or aicar , exert their antihypertrophic effects through activation of this isoform . however , both drugs may have some limitations in the interpretation of their cellular actions . it has been reported that , in a time- and concentration - dependent manner , aicar may act as an atp analogue due to the increase in ztp , its triply phosphorylated form . furthermore , it has been suggested that aicar may also mediate ampk - independent processes by regulating amp - sensitive enzymes , such as glycogen phosphorylase . amp - independent activation of ampk by metformin has also been reported [ 72 , 73 ] . however , a recent study has demonstrated that aicar - induced ampk activation downregulates p21 expression in retinoblastoma cells . similarly , despite inhibition of cell cycling , aicar treatment reduced p21 levels in myoblast cultures . the results of this latter study also show that inhibition of ampk with compound c associates with the reversal of aicar - induced loss of p21 expression . our finding that compound c abrogates metformin - induced cell cycle arrest is in line with the concept that ampk inhibition promotes proliferation . furthermore , in experiments studying metformin - induced cellular mechanisms , compound c has been successfully used for blunting ampk activation [ 76 , 77 ] . in accordance with these studies , our results also show that compound c opposes the effects of metformin on the ampk / s6k pathway and p21 expression . evidence suggests that metformin may lower the risk of cancer in patients with diabetes . in cancer studies the antiproliferative effect of metformin has been attributed to its ability to induce cell cycle arrest through an ampk - dependent mechanism [ 17 , 27 , 29 ] . according to a suggested mechanism , through the activation of ampk , metformin downregulates cyclin d1 , leading to the release of sequestered cdk inhibitors , p27 and p21 . in turn p27 and p21 may associate with e / cdk2 complexes and inhibit cell cycling at the g1/s checkpoint . on the other hand , in melanoma cells p21 expression our results show that both metformin and rapamycin inhibit proliferation in hek293 cells by inducing cell cycle arrest in the g0/g1 phase . interestingly , high glucose pretreatment abrogated the inhibitory effect of metformin on cell cycling but did not affect rapamycin - induced cell cycle inhibition . these findings suggest that increased p21 expression is not required for cell cycle inhibition by metformin and rapamycin . the involvement of cell cycle regulatory proteins in the development of dn has long been suggested [ 5 , 56 , 8082 ] . these studies have suggested that glomerular hypertrophy is a consequence of increased cyclin - dependent kinase inhibitor expression - mediated cell cycle arrest . the expression of p21 is generally associated with regulation of cell cycle , apoptosis , and ameliorating dna damage . in relation to the ampk / mtor signalling pathway , the induction of p21 as a senescence marker has been described with relevance to age - related diseases . accumulating evidence indicates that senescence may play an important role in the development of dn . premature senescence has been observed in fibroblasts and proximal tubular cells isolated from patients with dn [ 8587 ] . downregulation of connexin 43 , a gap junction protein , has been reported in podocytes of diabetic patients as well as in high glucose treated glomerular mesangial cells that showed increased expression of senescence markers , such as p21 , p27 , and -galactosidase staining [ 82 , 88 ] . furthermore , high glucose induces cyclin d1 expression , and interestingly , increased cyclin d1 expression is associated with senescence in fibroblasts . diabetes contributes to vascular ageing , and endothelial cell senescence is induced by high glucose or advanced glycosylated end products . in a study on rat kidney proximal tubule cells , an increase in the expression of p21 and p27 was associated with a phenotypic transition to senescence . however , it has been shown that dedifferentiation of cells in the kidney may contribute significantly to the pool of proliferating cells [ 92 , 93 ] . in turn , dedifferentiated cells responding to mitogenic factors can progress to senescent arrest . overexpression of fibronectin , one of the extracellular matrix proteins induced in diabetic nephropathy , is one of the characteristics of senescence cells . furthermore , proteasomal protein degradation is reduced in senescent cells , which can be a contributing factor for increased hypertrophy . the involvement of apoptosis in diabetic nephropathy has been indicated by several investigators [ 9698 ] . it has been widely acknowledged that apart from senescence , damaged cells rely on apoptosis to evade tumour formation . it has been suggested that apoptotic cell loss may be a process of normal tissue homeostasis regulating mesangial cell populations in enlarged glomeruli [ 100 , 101 ] . have demonstrated using diabetic rats that apoptosis may differentially affect glomerular cells in small and large glomeruli . they also showed that cellular hypertrophy was responsible for the differences in size , as the expression level of fibronectin , an extracellular matrix marker , was the same in both small and large glomeruli . increased expression of proapoptotic proteins was found in large glomeruli , suggesting that apoptosis may selectively operate in a more hypertrophied cellular environment . interestingly , upregulation of cyclin d1 , p21 , and p27 was only detected in smaller glomeruli , indicating that the higher expression of these proteins is associated with the initiation of glomerular hypertrophy . inhibition of mtor by rapamycin has been found to prevent the permanent loss of proliferative potential that is characteristic of cellular senescence . since metformin also inhibits the mtor signalling pathway , a similar effect of metformin to that of rapamycin could be expected in the prevention of senescent transformation . however , in contrast to rapamycin , chronic exposure to lower doses of metformin has been shown to promote cancer - protective senescence in other studies [ 40 , 103 ] . it has been proposed that rapamycin regulates proliferation by preventing the formation of cyclin - dependent kinase complexes . mtor activation by growth factors or high energy levels induces p21 that in turn facilitates the assembly of these complexes . in this study metformin inhibited p21 in both hek293 cells and human podocytes , suggesting that energy depletion may affect the expression of this cell cycle inhibitor . the underlying factor that determines the cellular actions of ampk may be its level of activation [ 84 , 105 ] . however , a major limitation to understanding the role of ampk in the diabetic kidney is the lack of studies investigating cell - specific differences in ampk expression . cells in the medulla predominantly depend on glycolytic metabolism , whereas tubular cells in the cortex depend on oxidative metabolism [ 107 , 108 ] . glycolytic flux is known to be reduced during the process of cellular senescence ; therefore one can envisage that senescence - induced metabolic disturbances may differentially impact on cell populations in the kidney [ 109 , 110 ] . immortalised cells may have a relatively high glycolytic flux ; therefore , it is possible that in our experiments high glucose exerted its effects through enhanced glycolysis . in preclinical studies , the doses of metformin are much higher than the level of the drug reported to accumulate in tissues after oral administration ; therefore , it is difficult to extrapolate the results of these studies to a clinical setting because of the differential and possibly poor expression of octs in immortalised cell lines . nevertheless , these studies show that metformin may have beneficial effects in pathologic states , including cancer and inflammation [ 17 , 112 , 113 ] . several molecular pathways in these diseases are also relevant to the aetiology of diabetic nephropathy , suggesting that metformin may have a therapeutic potential for the prevention of dn . indeed , by modulating the expression of proinflammatory genes , metformin has been shown to ameliorate dn in rats and ampk activation by metformin reduced renal hypertrophy in diabetic rats [ 114 , 115 ] . in summary , the above indicates that metformin can reduce the expression of p21 and ampk may play a role in the underlying mechanism . it can also be inferred from these results that p21 is not required for metformin - induced cell cycle arrest . this finding lends support to other studies that look beyond the role of p21 as a cell cycle inhibitor [ 74 , 75 ] . metformin has implications for the treatment of both diabetes and cancer and p21 has different , poorly understood roles in both diseases . recently , energy sensing pathways have been investigated in the context of both diabetic complications and cancer [ 116118 ] . in addition , reentry of differentiated cells into the cell cycle as well as genetic polymorphism in the p21 gene has been implicated in alzheimer 's disease [ 119 , 120 ] .

the mammalian target of rapamycin ( mtor ) pathway plays an important role in the development of diabetic nephropathy and other age - related diseases . one of the features of dn is the elevated expression of p21waf1/cip1 . however , the importance of the mtor signalling pathway in p21 regulation is poorly understood . here we investigated the effect of metformin and rapamycin on mtor - related phenotypes in cell lines of epithelial origin . this study reports that metformin inhibits high glucose - induced p21 expression . high glucose opposed metformin in regulating cell size , proliferation , and protein synthesis . these effects were associated with reduced ampk activation , affecting downstream mtor signalling . however , the inhibition of the mtor pathway by rapamycin did not have a negative effect on p21 expression , suggesting that metformin regulates p21 upstream of mtor . these findings provide support for the hypothesis that ampk activation may regulate p21 expression , which may have implications for diabetic nephropathy and other age - related pathologies .

the study included 546 healthy women randomly recruited from a national colonoscopy screening for crc in poland ( 20 ) . biopsies of normal mucosa from the cecum and sigmoid colon were collected in rnalater ( applied biosystems , lucerne , switzerland ) and stored at 80c . information on the use of aspirin , hrt , and cigarette smoking was collected using a self - administered questionnaire under the assistance of a study nurse ( table 1 ; supplementary materials , available online ) . clinical characteristics of the study population * regular use defined as two or more tablets per week for 1 or more months . non - user : women who indicated that they did not use two or more aspirin tablets per week for 1 or more months ( minimum level ) . short - term user : women who indicated that they used two or more aspirin tablets per week for less than 2 years . long - term user : women who indicated that they used two or more aspirin tablets per week for 2 or more years . || hormone replacement therapy defined as estrogen therapy and/or oral contraceptive for 1 or more years . non - user : women who indicated that they did not have hormone replacement therapy for 1 or more years ( minimum level ) . # aged < 50 : women who indicated that they did had hormone replacement therapy for 1 or more years before age 50 years . * * aged 50 years : women who indicated that they did have hormone replacement therapy for 1 or more years after the age of 50 years . body mass index : height ( cm ) and weight ( kg ) were self - reported , and body mass index was calculated ( kg / m ) from these variables . cigarette smoking was defined as one or more cigarettes per day for one or more years . nonsmoker : women who indicated that they did not smoke one or more cigarettes per day for 1 or more years ( minimum level ) . |||| short - term smoker : women who indicated that they smoked one or more cigarettes per day for less than 20 years . long - term smoker : women who indicated that they smoked one or more cigarettes per day for 20 or more years . bisulfite - converted genomic dna of 1092 samples was used to measure human mutl homolog 1 ( hmlh1 ) and o - methylguanine dna methyltransferase ( mgmt ) promoter methylation levels ( percentage of methylated alleles ) by locus normalized quantitative methylation specific polymerase chain reaction ( ln - qmsp ) . genome - wide assessment of dna methylation was done on infinium humanmethylation27 beadchip arrays ( illumina , san diego , ca ) , interrogating methylation at 27578 cpgs distributed in the promoters of 14475 coding genes . details on quantitation of methylation levels are available in the supplementary materials ( available online ) . all primary data generated in the study were deposited in the ncbi gene expression omnibus ( geo ; http://www.ncbi.nlm.nih.gov/geo/ ; accession no . expression data for 32 colon adenomas / normal tissues were obtained from geo ( accession no . the data were median normalized , log2 transformed , and analyzed using the r / bioconductor limma package . h3k27me3 is a polycomb repressive mark of interest because its presence in stem cells has been shown to predispose genomic sequences to aberrant dna methylation during aging and carcinogenesis ( 8,23,24 ) . an h3k27me3 profile in human embryonic stem cells was obtained from gse13084 ( 25 ) . five hundred base - pair windows symmetrically positioned around the illumina infinium humanmethylation27 cpgs were analyzed for enrichment of h3k27me3 . log - linear multivariable regression models were applied to investigate the interaction of age - related dna methylation changes with lifestyle factors by considering age as a linear predictor and the four lifestyle factors of aspirin use , hrt use , bmi , and smoking as categorical predictors . to analyze methylation interaction with age , corresponding interaction terms were introduced in the model . to investigate the interaction of age with polyps , separate multivariable regression models ( adjusted for the interaction of the four lifestyle factors with age ) were done by considering age as a linear predictor and polyps as categorical predictors ( either as one group [ any polyp ] or classified as tubular adenomas and serrated lesions , including both hyperplastic polyps and sessile serrated adenomas ) . separate calculations were done for each colonic location ( proximal and distal colon ) and gene . for data presentation , log - linear regression coefficients were back transformed and used to calculate rates of change in methylation per 10-year increase in age in respective lifestyle strata . the fold difference in the rate of change in methylation between two categories full details and analyses are given in the supplementary materials ( available online ) and supplementary tables 24 ( available online ) . the association between lifestyle factors and occurrence of polyps was examined by means of logistic multivariable regression models adjusted for age , aspirin use , hrt use , bmi , and smoking but without age interaction ( supplementary table 5 , available online ) . median regression analysis was conducted as supplementary analysis to evaluate the impact of extreme values on the results . the differential analysis of methylation data was performed on the m value ( log2 ratios of methylated and unmethylated probes ) using routines implemented in the r / bioconductor limma package ( 26 ) . false - discovery rate ( fdr)adjusted p values for multiple comparison were calculated using the benjamini and hochberg approach . cpgs were considered statistically significantly differentially methylated with age when displaying an fdr - adjusted p value less than .05 . cpgs were considered statistically significantly hypermethylated in colorectal cancer vs normal when displaying fdr - adjusted p value less than .0001 and fold change difference in m values greater than 2 . p values for the difference in median rate of dna methylation change were calculated by wilcoxon rank sum test . cancer relevance of identified age - related hypermethylation targets was addressed using the tumor - associated genes ( tag ) database ( 27 ) . the study included 546 healthy women randomly recruited from a national colonoscopy screening for crc in poland ( 20 ) . biopsies of normal mucosa from the cecum and sigmoid colon were collected in rnalater ( applied biosystems , lucerne , switzerland ) and stored at 80c . information on the use of aspirin , hrt , and cigarette smoking was collected using a self - administered questionnaire under the assistance of a study nurse ( table 1 ; supplementary materials , available online ) . clinical characteristics of the study population * regular use defined as two or more tablets per week for 1 or more months . non - user : women who indicated that they did not use two or more aspirin tablets per week for 1 or more months ( minimum level ) . short - term user : women who indicated that they used two or more aspirin tablets per week for less than 2 years . long - term user : women who indicated that they used two or more aspirin tablets per week for 2 or more years . || hormone replacement therapy defined as estrogen therapy and/or oral contraceptive for 1 or more years . non - user : women who indicated that they did not have hormone replacement therapy for 1 or more years ( minimum level ) . # aged < 50 : women who indicated that they did had hormone replacement therapy for 1 or more years before age 50 years . * * aged 50 years : women who indicated that they did have hormone replacement therapy for 1 or more years after the age of 50 years . body mass index : height ( cm ) and weight ( kg ) were self - reported , and body mass index was calculated ( kg / m ) from these variables . cigarette smoking was defined as one or more cigarettes per day for one or more years . nonsmoker : women who indicated that they did not smoke one or more cigarettes per day for 1 or more years ( minimum level ) . |||| short - term smoker : women who indicated that they smoked one or more cigarettes per day for less than 20 years . long - term smoker : women who indicated that they smoked one or more cigarettes per day for 20 or more years . bisulfite - converted genomic dna of 1092 samples was used to measure human mutl homolog 1 ( hmlh1 ) and o - methylguanine dna methyltransferase ( mgmt ) promoter methylation levels ( percentage of methylated alleles ) by locus normalized quantitative methylation specific polymerase chain reaction ( ln - qmsp ) . genome - wide assessment of dna methylation was done on infinium humanmethylation27 beadchip arrays ( illumina , san diego , ca ) , interrogating methylation at 27578 cpgs distributed in the promoters of 14475 coding genes . details on quantitation of methylation levels are available in the supplementary materials ( available online ) . all primary data generated in the study were deposited in the ncbi gene expression omnibus ( geo ; http://www.ncbi.nlm.nih.gov/geo/ ; accession no . bisulfite - converted genomic dna of 1092 samples was used to measure human mutl homolog 1 ( hmlh1 ) and o - methylguanine dna methyltransferase ( mgmt ) promoter methylation levels ( percentage of methylated alleles ) by locus normalized quantitative methylation specific polymerase chain reaction ( ln - qmsp ) . genome - wide assessment of dna methylation was done on infinium humanmethylation27 beadchip arrays ( illumina , san diego , ca ) , interrogating methylation at 27578 cpgs distributed in the promoters of 14475 coding genes . details on quantitation of methylation levels are available in the supplementary materials ( available online ) . all primary data generated in the study were deposited in the ncbi gene expression omnibus ( geo ; http://www.ncbi.nlm.nih.gov/geo/ ; accession no . expression data for 32 colon adenomas / normal tissues were obtained from geo ( accession no . the data were median normalized , log2 transformed , and analyzed using the r / bioconductor limma package . h3k27me3 is a polycomb repressive mark of interest because its presence in stem cells has been shown to predispose genomic sequences to aberrant dna methylation during aging and carcinogenesis ( 8,23,24 ) . an h3k27me3 profile in human embryonic stem cells was obtained from gse13084 ( 25 ) . five hundred base - pair windows symmetrically positioned around the illumina infinium humanmethylation27 cpgs were analyzed for enrichment of h3k27me3 . log - linear multivariable regression models were applied to investigate the interaction of age - related dna methylation changes with lifestyle factors by considering age as a linear predictor and the four lifestyle factors of aspirin use , hrt use , bmi , and smoking as categorical predictors . to analyze methylation interaction with age , corresponding interaction terms were introduced in the model . to investigate the interaction of age with polyps , separate multivariable regression models ( adjusted for the interaction of the four lifestyle factors with age ) were done by considering age as a linear predictor and polyps as categorical predictors ( either as one group [ any polyp ] or classified as tubular adenomas and serrated lesions , including both hyperplastic polyps and sessile serrated adenomas ) . separate calculations were done for each colonic location ( proximal and distal colon ) and gene . for data presentation , log - linear regression coefficients were back transformed and used to calculate rates of change in methylation per 10-year increase in age in respective lifestyle strata . the fold difference in the rate of change in methylation between two categories was defined as the methylation rate ratio ( mrr ) . full details and analyses are given in the supplementary materials ( available online ) and supplementary tables 24 ( available online ) . the association between lifestyle factors and occurrence of polyps was examined by means of logistic multivariable regression models adjusted for age , aspirin use , hrt use , bmi , and smoking but without age interaction ( supplementary table 5 , available online ) . median regression analysis was conducted as supplementary analysis to evaluate the impact of extreme values on the results . the differential analysis of methylation data was performed on the m value ( log2 ratios of methylated and unmethylated probes ) using routines implemented in the r / bioconductor limma package ( 26 ) . false - discovery rate ( fdr)adjusted p values for multiple comparison were calculated using the benjamini and hochberg approach . cpgs were considered statistically significantly differentially methylated with age when displaying an fdr - adjusted p value less than .05 . cpgs were considered statistically significantly hypermethylated in colorectal cancer vs normal when displaying fdr - adjusted p value less than .0001 and fold change difference in m values greater than 2 . p values for the difference in median rate of dna methylation change were calculated by wilcoxon rank sum test . cancer relevance of identified age - related hypermethylation targets was addressed using the tumor - associated genes ( tag ) database ( 27 ) . log - linear multivariable regression models were applied to investigate the interaction of age - related dna methylation changes with lifestyle factors by considering age as a linear predictor and the four lifestyle factors of aspirin use , hrt use , bmi , and smoking as categorical predictors . to analyze methylation interaction with age , corresponding interaction terms were introduced in the model . to investigate the interaction of age with polyps , separate multivariable regression models ( adjusted for the interaction of the four lifestyle factors with age ) were done by considering age as a linear predictor and polyps as categorical predictors ( either as one group [ any polyp ] or classified as tubular adenomas and serrated lesions , including both hyperplastic polyps and sessile serrated adenomas ) . separate calculations were done for each colonic location ( proximal and distal colon ) and gene . for data presentation , log - linear regression coefficients were back transformed and used to calculate rates of change in methylation per 10-year increase in age in respective lifestyle strata . the fold difference in the rate of change in methylation between two categories was defined as the methylation rate ratio ( mrr ) . full details and analyses are given in the supplementary materials ( available online ) and supplementary tables 24 ( available online ) . the association between lifestyle factors and occurrence of polyps was examined by means of logistic multivariable regression models adjusted for age , aspirin use , hrt use , bmi , and smoking but without age interaction ( supplementary table 5 , available online ) . median regression analysis was conducted as supplementary analysis to evaluate the impact of extreme values on the results . the differential analysis of methylation data was performed on the m value ( log2 ratios of methylated and unmethylated probes ) using routines implemented in the r / bioconductor limma package ( 26 ) . false - discovery rate ( fdr)adjusted p values for multiple comparison were calculated using the benjamini and hochberg approach . cpgs were considered statistically significantly differentially methylated with age when displaying an fdr - adjusted p value less than .05 . cpgs were considered statistically significantly hypermethylated in colorectal cancer vs normal when displaying fdr - adjusted p value less than .0001 and fold change difference in m values greater than 2 . p values for the difference in median rate of dna methylation change were calculated by wilcoxon rank sum test . cancer relevance of identified age - related hypermethylation targets was addressed using the tumor - associated genes ( tag ) database ( 27 ) . we investigated a cohort of 546 healthy women aged 50 to 80 years for relationships between gene promoter methylation in the colonic mucosa and the reported use of aspirin , reported hrt , bmi , and smoking ( table 1 ) . we first measured hmlh1 and mgmt promoter methylation as percentage of methylated alleles by ln - qmsp in a total of 1092 normal biopsies obtained from cecum ( proximal ) and sigmoid ( distal ) colon . log - linear multivariable regression analysis showed that methylation at the hmlh1 promoter statistically increased with age in the proximal colon ( rate per 10 years of age = 2.1% ; p < .001 ) ( figure 1a ; supplementary table 2 , available online ) . by contrast , the mgmt promoter did not show an overall age - dependent methylation change ( figure 1a ; supplementary table 3 , available online ) unless lifestyle factors were taken into account ( figure 1b ; supplementary table 3 , available online ) ; long - term aspirin use was associated with a more than 50% suppressed rate of methylation when compared with nonuse ( proximal colon : mrr = 0.44 , 95% ci = 0.27 to 0.70 , p = .004 ; distal colon : mrr = 0.47 , 95% ci = 0.31 to 0.73 , p = .005 ) , whereas hrt after the age of 50 years and long - term smoking slightly stimulated mgmt methylation in the proximal colon ( hrt : mrr = 1.63 , 95% ci = 1.05 to 2.53 , p = .07 ; smoking : mrr = 1.51 , 95% ci = 1.01 to 2.26 , p = .09 ) . notably , women with serrated lesions had a 48% higher rate of mgmt promoter methylation in the distal colon compared with women with no polyps ( mrr = 1.48 ; 95% ci = 1.12 to 1.95 ; p = .02 ) ( figure 1c ; supplementary table 4 , available online ) , and the risk of serrated lesions was reduced in long - term aspirin users ( or = 0.36 ; 95% ci = 0.16 to 0.74 ; p = .03 ) but increased in long - term smokers ( or = 2.67 ; 95% ci = 1.72 to 4.15 ; p < .001 ) ( figure 1d ; supplementary table 5 , available online ) . no association with lifestyle and/or the occurrence of polyps was found for hmlh1 promoter methylation ( supplementary table 2 , available online ) . association of human mutl homolog 1 ( hmlh1 ) and o - methylguanine dna methyltransferase ( mgmt ) promoter methylation with lifestyle factors and polyps . a ) hmlh1 and mgmt promoter methylation in proximal ( cecum ) and distal ( sigmoid ) colon as percentages of methylated alleles ( pmas ) determined by locus normalized quantitative methylation specific polymerase chain reaction ( ln - qmsp ) . for presentation shown are median ( lines ) and mean ( black circles ) pmas with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . p values are derived from log - linear multivariable regression analysis ( supplementary table 3 , available online ) representing significance of the difference in two regression lines . methylation rate ratios ( mrrs ) and p values are derived from log - linear multivariable regression ( supplementary table 4 , available online ) . odds ratios ( ors ) and p values are derived from logistic multivariable regression analysis ( supplementary table 5 , available online ) . bmi = body mass index ; ci = confidence interval ; hrt = hormone replacement therapy . next , we analyzed genome - wide dna methylation in 178 normal colon samples from our cohort ( illumina infinium humanmethylation27 beadchip ) . we analyzed 20025 promoter - associated cpgs with multivariable regression adjusted for colonic location , aspirin use , hrt , bmi , and smoking and identified 1713 cpgs gaining and 343 losing methylation with age ; 219 cpgs acquired methylation exclusively in the proximal , and 416 acquired methylation exclusively in the distal colon ( figure 2a ) . when stratified for lifestyle factors , age - dependent hypermethylation was suppressed in aspirin users ( 43 cpgs in users vs 1355 in nonusers ) and/or in women reporting hrt ( 1 cpg in users vs 1377 in nonusers ) but promoted in individuals with a high bmi ( 554 cpgs in high bmi vs 144 in normal bmi ) ( figure 2b ) . whereas aspirin use , hrt , and bmi modulated age - dependent dna methylation changes along the entire colon , the effect of smoking was confined to the proximal colon ( 180 cpgs in smokers vs 39 in nonsmokers ) ( supplementary figure 2 , a and b , available online ) . the median rate of age - dependent methylation change at hypermethylated cpgs ( median rate = 1.6% ; range = 0.07%6.97% ) was 56% higher in the distal colon than in the proximal colon ( mrr = 1.56 ; p = 910 ) ( figure 2c ) . the rate was 48% lower in aspirin users and 47% lower in women reporting hrt compared with non - users ( aspirin : mrr = 0.52 , p = 110 ; hrt : mrr = 0.53 ; p = 510 ) ( figure 2d ) , whereas a high bmi was associated with a 27% increased methylation rate in the distal colon ( mrr = 1.27 ; p = 510 ) , and smoking was associated with a 400% increased rate in the proximal colon ( mrr = 4.67 ; p = 110 ) but a 33% decreased rate in the distal colon ( mrr = 0.66 ; p = 810 ) ( supplementary figure 2 , a and b , available online ) . genome - wide dna methylation and its association with lifestyle factors . a and b ) numbers of age - associated differentially methylated cpgs in all samples or when stratified by colon location ( proximal [ cecum ] vs distal [ sigmoid ] ) and lifestyle factors ( aspirin : nonuser vs user [ long - term ] ; hormone replacement therapy [ hrt ] : nonuser vs user [ aged 50 years ] ; body mass index [ bmi ] : normal vs high [ > 25kg / m ] ; smoking : nonsmoker vs smoker [ long - term ] ) . numbers of samples tested in each category are indicated at the bottom of each bar . c and d ) ten - year rates of dna methylation change for cpgs showing age - associated hypermethylation in all samples or when stratified by colon location and lifestyle factors . shown are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . e ) concordance of probes showing suppression of age - associated methylation by aspirin - use and hrt or promotion of age - associated methylation by a high bmi and long - term smoking . f ) enrichment of age - associated differentially methylated sites marked by histone 3 lysine 27 tri - methylation ( h3k27me3 ) in human embryonic stem cells ( hescs ) . barplots indicate percentages of age - associated hypermethylated ( age - hyperm ) and hypomethylated ( age - hypom ) cpgs either marked by h3k27me3 ( positive ) or not ( negative ) , or the enrichment of cpgs marked by h3k27me3 in lifestyle modulated age - related hypermethylation ( bottom ) . odds ratios and p values according to fisher exact test . density plots on the right show the rate of change in age - hyperm and age - hypom probes at h3k27me3 positive ( gray line ) and negative ( black , dashed line ) cpgs . examining potential interactions between these lifestyle effects , we found statistically significant overlaps between cpgs showing suppression of age - related methylation by aspirin use or hrt ( or = 72 ; 95% ci = 62 to 83 ; p < 210 ) ( figure 2e ) and , less pronounced , between cpgs showing bmi- or smoking - stimulated hypermethylation ( or = 14 ; 95% ci = 11 to 19 ; p = 210 ) . although the methylation - retarding effect of aspirin use was apparent in the entire study population , it appeared more effective in individuals with a high bmi ( 52% ; mrr = 0.48 ; p = 210 ) compared with individuals with a normal bmi ( 22% ; mrr = 0.78 ; p = .0001 ) or in smokers ( 79% ; mrr = 0.21 ; p = 310 ) compared with nonsmokers ( 31% ; mrr = 0.69 ; p = 210 ) ( supplementary figure 2c , available online ) . hrt use retarded methylation in individuals with a high bmi only ( 70% ; mrr = 0.30 ; p = 910 ) , whereas its effect on smoking remained unclear because of limited statistical power . to investigate whether cpgs prone to age - dependent methylation are distinguished by specific genomic features , we classified array cpg probes with regard to coincidence of h3k27me3 in human embryonic stem cells . regions surrounding age - hypermethylated cpgs were statistically significantly enriched for domains marked by h3k27me3 in stem cells compared with total h3k27me3 marked cpgs on the array ( or = 4.84 ; 95% ci = 4 to 5 ; p = 410 ) ( figure 2f ) , and the median rate of age - dependent methylation was 53% higher at these sites compared with sites depleted for h3k27me3 ( mrr = 1.53 ; p = 410 ) . aspirin use suppressed hypermethylation selectively at h3k27me3 marked sequences ( user vs nonuser : or = 0.37 ; 95% ci = 0.2 to 0.8 ; p = .004 ) , whereas a high bmi promoted methylation at these sites ( high vs normal bmi : or = 1.6 ; 95% ci = 1.1 to 2.5 ; p = .02 ) . the association of hypermethylation with h3k27me3 modification was less in smokers ( smoker vs nonsmokers : or = 0.59 ; 95% ci = 0.5 to 0.8 ; p = .0003 ) , indicating that smoking promotes methylation in cpg - rich regions other than the typical polycomb targets . to address the cancer relevance of these age- and lifestyle - modulated dna methylation changes in the healthy colon , we compared methylation data of 59 crcs ( female patients ) included in a genome - wide methylation study ( 21 ) with those of our healthy mucosa samples . we identified 1709 cpgs statistically significantly hypermethylated and 1441 cpgs hypomethylated in the crcs ( figure 3a ) . half of the cpgs showing age - dependent hypermethylation in the healthy mucosa ( n = 856 of 1713 ; or = 20 ; 95% ci = 18 to 23 ; p < 210 ) coincided with sites hypermethylated in crc ( figure 3b ; age cancer ) . at these loci , the median rate of methylation gain per 10 years was doubled ( mrr = 2.21 ; p = 210 ) compared with age - methylated sites only ( figure 3b ; age - only ) , and they were most highly enriched for polycomb regions ( or = 3.67 ; 95% ci = 3.0 to 4.5 ; p = 110 ) . age - hypermethylated cpgs also coincided partially with genes transcriptionally downregulated ( n = 296 of 1287 ) ( figure 3c ) in colonic adenomas ( 22 ) , and 174 genes showed all features ( ie , age- and crc - dependent hypermethylation and transcriptional downregulation in adenomas ) ( figure 3d ) . a ) differences in dna methylation between 59 crc samples ( 21 ) of female patients and 178 normal biopsies . plotted are difference in log2-fold change ( fc ) in dna methylation on the x - axis with false discovery rate ( fdr)adjusted p values ( calculated by moderated t statistics ; -1 log10 scale ) on the y - axis . statistically significantly hypermethylated in crc are highlighted in red ( n = 1709 ; fdr - adjusted p < .0001 ; fc > 2 ) . b ) intersection between age - related hypermethylated cpgs in healthy mucosa and cpgs hypermethylated in tumor samples . barplots indicate percentages of cpgs marked by histone 3 lysine 27 tri - methylation ( h3k27me3 ) in each intersection ( age - only age cancer , age - only , cancer - only ) . are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . c ) intersection between 1287 age - related hypermethylated genes ( n = 1713 cpgs ) in the normal colon mucosa and genes downregulated ( fdr - adjusted p .05 ) in colon adenomas ( 22 ) . d ) intersection between genes statistically significantly hypermethylated over age in the normal colon mucosa , genes hypermethylated in crc samples , and genes downregulated in colon adenomas . odds ratios ( ors ) and associated p values were calculated according to the fisher exact test . p values for the difference in median rates of dna methylation change were calculated according to the wilcoxon rank sum test . importantly , cancer - relevant methylation change in the normal colonic mucosa was influenced by lifestyle . a statistically significant fraction of promoters showing either aspirin- or hrt - suppressed methylation ( aspirin : n = 549 of 1080 , or = 15 , 95% ci = 13 to 17 , p = 110 ; hrt : n = 612 of 996 , or = 25 , 95% ci = 22 to 30 , p < 2.210 ) or bmi- or smoking - promoted methylation ( high bmi : n = 209 of 510 , or = 6.8 , 95% ci = 6 to 8 , p = 910 ; smoking in proximal colon : n = 49 of 177 , or = 3.3 , 95% ci = 2 to 7 , p = 110 ) in the aging colon coincided with sites hypermethylated in crc ( figure 4a ) . these included genes controlling key aspects of carcinogenesis ( supplementary figures 36 , available online ) such as cell cycle regulation ( cdkn2a ) , dna repair ( mgmt ) , apoptosis ( dapk1 ) , cell invasion ( cdh1 ) , and wnt ( wnt16 ) and ras signaling ( rassf1 ) ( 2,2830 ) . moreover , the rates of methylation change at promoters of established tumor - associated genes ( 27 ) were statistically significantly reduced in aspirin users or in women reporting hrt compared with nonusers ( aspirin : mrr = 0.53 , p < .001 ; hrt : mrr = 0.54 , p < .001 ) ( figure 4b ) but increased in smokers vs nonsmokers or in individuals with high bmi vs normal bmi ( smoking in proximal colon : mrr = 4.0 , p < .001 ; high bmi : mrr = 1.57 , p = .004 ) . a ) concordance between genes showing modulation of age - associated methylation by lifestyle parameters and hypermethylation in colorectal cancer ( crc ) . enrichment in each category is calculated over the percentage of 11872 genes ( n = 20025 cpgs ) present on the illumina array ( total ) . odds ratios ( ors ) and p values were calculated according to the fisher exact test . b ) median rates of dna methylation on 664 annotated tumor - associated genes ( tag database ) ( 27 ) . shown are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . c ) mode for the modulation of crc risk by lifestyle factors ( 41 ) . inherent dna methylation instability generates epigenetic mosaicism ( gray cells ) in the aging colonic epithelium . some methylation changes will affect transcription of genes controlling carcinogenesis , eventually contributing to the evolution of premalignant ( gray cell cluster ) and cancer cells ( red cell cluster ) . lifestyle factors are capable of negatively ( blue arrow ) or positively ( green arrow ) influencing progressive age - related methylation change , thereby connecting lifestyle with cancer risk . bmi = body mass index ; hrt = hormone replacement therapy ; or = odds ratio . we first measured hmlh1 and mgmt promoter methylation as percentage of methylated alleles by ln - qmsp in a total of 1092 normal biopsies obtained from cecum ( proximal ) and sigmoid ( distal ) colon . log - linear multivariable regression analysis showed that methylation at the hmlh1 promoter statistically increased with age in the proximal colon ( rate per 10 years of age = 2.1% ; p < .001 ) ( figure 1a ; supplementary table 2 , available online ) . by contrast , the mgmt promoter did not show an overall age - dependent methylation change ( figure 1a ; supplementary table 3 , available online ) unless lifestyle factors were taken into account ( figure 1b ; supplementary table 3 , available online ) ; long - term aspirin use was associated with a more than 50% suppressed rate of methylation when compared with nonuse ( proximal colon : mrr = 0.44 , 95% ci = 0.27 to 0.70 , p = .004 ; distal colon : mrr = 0.47 , 95% ci = 0.31 to 0.73 , p = .005 ) , whereas hrt after the age of 50 years and long - term smoking slightly stimulated mgmt methylation in the proximal colon ( hrt : mrr = 1.63 , 95% ci = 1.05 to 2.53 , p = .07 ; smoking : mrr = 1.51 , 95% ci = 1.01 to 2.26 , p = .09 ) . notably , women with serrated lesions had a 48% higher rate of mgmt promoter methylation in the distal colon compared with women with no polyps ( mrr = 1.48 ; 95% ci = 1.12 to 1.95 ; p = .02 ) ( figure 1c ; supplementary table 4 , available online ) , and the risk of serrated lesions was reduced in long - term aspirin users ( or = 0.36 ; 95% ci = 0.16 to 0.74 ; p = .03 ) but increased in long - term smokers ( or = 2.67 ; 95% ci = 1.72 to 4.15 ; p < .001 ) ( figure 1d ; supplementary table 5 , available online ) . no association with lifestyle and/or the occurrence of polyps was found for hmlh1 promoter methylation ( supplementary table 2 , available online ) . association of human mutl homolog 1 ( hmlh1 ) and o - methylguanine dna methyltransferase ( mgmt ) promoter methylation with lifestyle factors and polyps . a ) hmlh1 and mgmt promoter methylation in proximal ( cecum ) and distal ( sigmoid ) colon as percentages of methylated alleles ( pmas ) determined by locus normalized quantitative methylation specific polymerase chain reaction ( ln - qmsp ) . for presentation shown are median ( lines ) and mean ( black circles ) pmas with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . p values are derived from log - linear multivariable regression analysis ( supplementary table 3 , available online ) representing significance of the difference in two regression lines . methylation rate ratios ( mrrs ) and p values are derived from log - linear multivariable regression ( supplementary table 4 , available online ) . odds ratios ( ors ) and p values are derived from logistic multivariable regression analysis ( supplementary table 5 , available online ) . bmi = body mass index ; ci = confidence interval ; hrt = hormone replacement therapy . next , we analyzed genome - wide dna methylation in 178 normal colon samples from our cohort ( illumina infinium humanmethylation27 beadchip ) . we analyzed 20025 promoter - associated cpgs with multivariable regression adjusted for colonic location , aspirin use , hrt , bmi , and smoking and identified 1713 cpgs gaining and 343 losing methylation with age ; 219 cpgs acquired methylation exclusively in the proximal , and 416 acquired methylation exclusively in the distal colon ( figure 2a ) . when stratified for lifestyle factors , age - dependent hypermethylation was suppressed in aspirin users ( 43 cpgs in users vs 1355 in nonusers ) and/or in women reporting hrt ( 1 cpg in users vs 1377 in nonusers ) but promoted in individuals with a high bmi ( 554 cpgs in high bmi vs 144 in normal bmi ) ( figure 2b ) . whereas aspirin use , hrt , and bmi modulated age - dependent dna methylation changes along the entire colon , the effect of smoking was confined to the proximal colon ( 180 cpgs in smokers vs 39 in nonsmokers ) ( supplementary figure 2 , a and b , available online ) . the median rate of age - dependent methylation change at hypermethylated cpgs ( median rate = 1.6% ; range = 0.07%6.97% ) was 56% higher in the distal colon than in the proximal colon ( mrr = 1.56 ; p = 910 ) ( figure 2c ) . the rate was 48% lower in aspirin users and 47% lower in women reporting hrt compared with non - users ( aspirin : mrr = 0.52 , p = 110 ; hrt : mrr = 0.53 ; p = 510 ) ( figure 2d ) , whereas a high bmi was associated with a 27% increased methylation rate in the distal colon ( mrr = 1.27 ; p = 510 ) , and smoking was associated with a 400% increased rate in the proximal colon ( mrr = 4.67 ; p = 110 ) but a 33% decreased rate in the distal colon ( mrr = 0.66 ; p = 810 ) ( supplementary figure 2 , a and b , available online ) . a and b ) numbers of age - associated differentially methylated cpgs in all samples or when stratified by colon location ( proximal [ cecum ] vs distal [ sigmoid ] ) and lifestyle factors ( aspirin : nonuser vs user [ long - term ] ; hormone replacement therapy [ hrt ] : nonuser vs user [ aged 50 years ] ; body mass index [ bmi ] : normal vs high [ > 25kg / m ] ; smoking : nonsmoker vs smoker [ long - term ] ) . numbers of samples tested in each category are indicated at the bottom of each bar . c and d ) ten - year rates of dna methylation change for cpgs showing age - associated hypermethylation in all samples or when stratified by colon location and lifestyle factors . shown are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . e ) concordance of probes showing suppression of age - associated methylation by aspirin - use and hrt or promotion of age - associated methylation by a high bmi and long - term smoking . f ) enrichment of age - associated differentially methylated sites marked by histone 3 lysine 27 tri - methylation ( h3k27me3 ) in human embryonic stem cells ( hescs ) . barplots indicate percentages of age - associated hypermethylated ( age - hyperm ) and hypomethylated ( age - hypom ) cpgs either marked by h3k27me3 ( positive ) or not ( negative ) , or the enrichment of cpgs marked by h3k27me3 in lifestyle modulated age - related hypermethylation ( bottom ) . odds ratios and p values according to fisher exact test . density plots on the right show the rate of change in age - hyperm and age - hypom probes at h3k27me3 positive ( gray line ) and negative ( black , dashed line ) cpgs . examining potential interactions between these lifestyle effects , we found statistically significant overlaps between cpgs showing suppression of age - related methylation by aspirin use or hrt ( or = 72 ; 95% ci = 62 to 83 ; p < 210 ) ( figure 2e ) and , less pronounced , between cpgs showing bmi- or smoking - stimulated hypermethylation ( or = 14 ; 95% ci = 11 to 19 ; p = 210 ) . although the methylation - retarding effect of aspirin use was apparent in the entire study population , it appeared more effective in individuals with a high bmi ( 52% ; mrr = 0.48 ; p = 210 ) compared with individuals with a normal bmi ( 22% ; mrr = 0.78 ; p = .0001 ) or in smokers ( 79% ; mrr = 0.21 ; p = 310 ) compared with nonsmokers ( 31% ; mrr = 0.69 ; p = 210 ) ( supplementary figure 2c , available online ) . hrt use retarded methylation in individuals with a high bmi only ( 70% ; mrr = 0.30 ; p = 910 ) , whereas its effect on smoking remained unclear because of limited statistical power . to investigate whether cpgs prone to age - dependent methylation are distinguished by specific genomic features , we classified array cpg probes with regard to coincidence of h3k27me3 in human embryonic stem cells . regions surrounding age - hypermethylated cpgs were statistically significantly enriched for domains marked by h3k27me3 in stem cells compared with total h3k27me3 marked cpgs on the array ( or = 4.84 ; 95% ci = 4 to 5 ; p = 410 ) ( figure 2f ) , and the median rate of age - dependent methylation was 53% higher at these sites compared with sites depleted for h3k27me3 ( mrr = 1.53 ; p = 410 ) . aspirin use suppressed hypermethylation selectively at h3k27me3 marked sequences ( user vs nonuser : or = 0.37 ; 95% ci = 0.2 to 0.8 ; p = .004 ) , whereas a high bmi promoted methylation at these sites ( high vs normal bmi : or = 1.6 ; 95% ci = 1.1 to 2.5 ; p = .02 ) . the association of hypermethylation with h3k27me3 modification was less in smokers ( smoker vs nonsmokers : or = 0.59 ; 95% ci = 0.5 to 0.8 ; p = .0003 ) , indicating that smoking promotes methylation in cpg - rich regions other than the typical polycomb targets . to address the cancer relevance of these age- and lifestyle - modulated dna methylation changes in the healthy colon , we compared methylation data of 59 crcs ( female patients ) included in a genome - wide methylation study ( 21 ) with those of our healthy mucosa samples . we identified 1709 cpgs statistically significantly hypermethylated and 1441 cpgs hypomethylated in the crcs ( figure 3a ) . half of the cpgs showing age - dependent hypermethylation in the healthy mucosa ( n = 856 of 1713 ; or = 20 ; 95% ci = 18 to 23 ; p < 210 ) coincided with sites hypermethylated in crc ( figure 3b ; age cancer ) . at these loci , the median rate of methylation gain per 10 years was doubled ( mrr = 2.21 ; p = 210 ) compared with age - methylated sites only ( figure 3b ; age - only ) , and they were most highly enriched for polycomb regions ( or = 3.67 ; 95% ci = 3.0 to 4.5 ; p = 110 ) . age - hypermethylated cpgs also coincided partially with genes transcriptionally downregulated ( n = 296 of 1287 ) ( figure 3c ) in colonic adenomas ( 22 ) , and 174 genes showed all features ( ie , age- and crc - dependent hypermethylation and transcriptional downregulation in adenomas ) ( figure 3d ) . a ) differences in dna methylation between 59 crc samples ( 21 ) of female patients and 178 normal biopsies . plotted are difference in log2-fold change ( fc ) in dna methylation on the x - axis with false discovery rate ( fdr)adjusted p values ( calculated by moderated t statistics ; -1 log10 scale ) on the y - axis . cpgs statistically significantly hypermethylated in crc are highlighted in red ( n = 1709 ; fdr - adjusted p < .0001 ; fc > 2 ) . b ) intersection between age - related hypermethylated cpgs in healthy mucosa and cpgs hypermethylated in tumor samples . barplots indicate percentages of cpgs marked by histone 3 lysine 27 tri - methylation ( h3k27me3 ) in each intersection ( age - only age cancer , age - only , cancer - only ) . tumor vs age - only hypermethylated loci . shown are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . c ) intersection between 1287 age - related hypermethylated genes ( n = 1713 cpgs ) in the normal colon mucosa and genes downregulated ( fdr - adjusted p .05 ) in colon adenomas ( 22 ) . d ) intersection between genes statistically significantly hypermethylated over age in the normal colon mucosa , genes hypermethylated in crc samples , and genes downregulated in colon adenomas . odds ratios ( ors ) and associated p values were calculated according to the fisher exact test . p values for the difference in median rates of dna methylation change were calculated according to the wilcoxon rank sum test . importantly , cancer - relevant methylation change in the normal colonic mucosa was influenced by lifestyle . a statistically significant fraction of promoters showing either aspirin- or hrt - suppressed methylation ( aspirin : n = 549 of 1080 , or = 15 , 95% ci = 13 to 17 , p = 110 ; hrt : n = 612 of 996 , or = 25 , 95% ci = 22 to 30 , p < 2.210 ) or bmi- or smoking - promoted methylation ( high bmi : n = 209 of 510 , or = 6.8 , 95% ci = 6 to 8 , p = 910 ; smoking in proximal colon : n = 49 of 177 , or = 3.3 , 95% ci = 2 to 7 , p = 110 ) in the aging colon coincided with sites hypermethylated in crc ( figure 4a ) . these included genes controlling key aspects of carcinogenesis ( supplementary figures 36 , available online ) such as cell cycle regulation ( cdkn2a ) , dna repair ( mgmt ) , apoptosis ( dapk1 ) , cell invasion ( cdh1 ) , and wnt ( wnt16 ) and ras signaling ( rassf1 ) ( 2,2830 ) . moreover , the rates of methylation change at promoters of established tumor - associated genes ( 27 ) were statistically significantly reduced in aspirin users or in women reporting hrt compared with nonusers ( aspirin : mrr = 0.53 , p < .001 ; hrt : mrr = 0.54 , p < .001 ) ( figure 4b ) but increased in smokers vs nonsmokers or in individuals with high bmi vs normal bmi ( smoking in proximal colon : mrr = 4.0 , p < .001 ; high bmi : mrr = 1.57 , p = .004 ) . a ) concordance between genes showing modulation of age - associated methylation by lifestyle parameters and hypermethylation in colorectal cancer ( crc ) . enrichment in each category is calculated over the percentage of 11872 genes ( n = 20025 cpgs ) present on the illumina array ( total ) . odds ratios ( ors ) and p values were calculated according to the fisher exact test . b ) median rates of dna methylation on 664 annotated tumor - associated genes ( tag database ) ( 27 ) . shown are median ( lines ) and mean ( black circles ) rates with interquartile ranges ( boxes ) , 1.5 times the interquartile ranges ( whiskers ) , and extreme values ( gray lines ) . c ) mode for the modulation of crc risk by lifestyle factors ( 41 ) . inherent dna methylation instability generates epigenetic mosaicism ( gray cells ) in the aging colonic epithelium . some methylation changes will affect transcription of genes controlling carcinogenesis , eventually contributing to the evolution of premalignant ( gray cell cluster ) and cancer cells ( red cell cluster ) . lifestyle factors are capable of negatively ( blue arrow ) or positively ( green arrow ) influencing progressive age - related methylation change , thereby connecting lifestyle with cancer risk . bmi = body mass index ; hrt = hormone replacement therapy ; or = odds ratio . we report that lifestyle factors that are known modulators of crc risk have widespread effects on the stability of gene promoter methylation in the aging colonic mucosa . we found age - associated dna hypermethylation to be suppressed by regular aspirin use and hrt use but accelerated by long - term smoking and a high bmi , which is concordant with the effects of these factors on crc risk . statistically significant subsets of affected promoters were associated with genes controlling key aspects of carcinogenesis and with chromatin features known to predispose to hypermethylation in cancer , accentuating the cancer relevance of the lifestyle - modulated dna methylation change in the normal colon . these findings provide an important resource for understanding the interplay between lifestyle exposure and aging in the modulation of epigenetic ( in)stability in colonic epithelial cells and , thereby , in the evolution of crc methylomes . our data extend the well - established relationships between aging and gene promoter hypermethylation ( 9,31,32 ) by providing a functional link to cancer . comparing genome - wide methylation data of crc ( 21 ) with those of normal aging mucosa allowed us to discriminate between age - only , cancer - only and age cancer hypermethylation . importantly , the rate of methylation change at age cancer hypermethylated sites was statistically significantly higher than at loci showing age - only methylation , and aspirin use and hrt use suppressed whereas high bmi and smoking promoted methylation in a large fraction of these promoters . the identification of this specific subgroup of gene promoters , displaying age - associated and cancer - associated hypermethylation modulated by lifestyle factors , is important for several reasons . first , the targets provide insight into molecular pathogenesis of crc and how lifestyle factors may exert their effects on crc risk . second , age - associated hypermethylation at such targets in the normal colonic mucosa implies a risk for crc . cancer hypermethylated cpgs may serve as biomarkers for risk prediction and disease progression because their methylation can be monitored in the healthy mucosa . third , unlike genetic mutations , epigenetic alterations are reversible and the identification of gene promoters at which hypermethylation can be modulated by lifestyle will help develop tailored methylation and thus cancer - preventive strategies . future investigations will thus have to be directed toward establishing biomarker panels with methylation thresholds predicting cancer risk in healthy individuals and to evaluate the clinical potential of methylation - suppressive medication . numerous studies have shown that aspirin use is associated with a lower incidence of colonic neoplasia , metastatic crc , and death due to crc ( 3336 ) . although the genetic makeup of a tumor is likely to play a role in this context ( 37 ) , our methylation profiling of the normal colonic mucosa adds to the understanding of this cancer protective effect , showing that regular aspirin use stabilizes dna methylation at promoters of genes controlling critical cancer pathways . this is likely to retard carcinogenesis at different stages , including metastasis , in the colon and other tissues . this insight will thus be instrumental in identifying both patients and healthy individuals who will benefit from aspirin use . first , we collected normal mucosa samples from an unselected screening cohort , providing for an unbiased analysis of methylation profiles . second , the age - related and lifestyle - modulated methylation changes observed in the promoters of the two dna - repair and tumor - suppressor genes hmlh1 and mgmt are an important proof of concept because the pathogenic consequence of their epigenetic silencing is well known . third , the comparison of our genome - wide methylation data in normal mucosa of a female screening cohort with those of women with crc enabled us not only to exclude sex - associated methylation variance but also to identify the gene promoters at which age - related hypermethylation is relevant for crc development . residual confounding by measured or unmeasured additional factors can not be excluded . to limit biological variation , we investigated only women and , therefore , our findings will have to be validated by independent studies including men . genome - wide methylation array is largely restricted to cpg islands , thereby limiting the detection of methylation globally . we conclude that the aging colonic mucosa undergoes changes in gene promoter methylation with rates modulated by lifestyle . the highly variable methylation rates at individual cpgs are consistent with an underlying inherent instability of dna methylation rather than an outgrowth of subpopulations of tissue cells ; a concept supported by many other studies ( 3840 ) . we propose that this epigenetic instability contributes to the aging of the colonic epithelium ( 41 ) , generating opportunities for the evolution of early cancer - initiating cells ( figure 4c ) showing patterns of aberrant dna methylation typically observed in malignant tissues ( 2 ) . the finding that lifestyle factors specifically modulate the rate of the underlying dna methylation change provides a novel paradigm for how the environment modulates cancer risk . this work was supported by the swiss cancer league ; association for international cancer research ( aicr-110247 ) ; stanley thomas johnson foundation ; and opo foundation .

backgroundaberrant dna methylation in gene promoters is associated with aging and cancer , but the circumstances determining methylation change are unknown . we investigated the impact of lifestyle modulators of colorectal cancer ( crc ) risk on the stability of gene promoter methylation in the colonic mucosa.methodswe measured genome - wide promoter cpg methylation in normal colon biopsies ( n = 1092 ) from a female screening cohort , investigated the interaction of lifestyle factors with age - dependent increase in methylation with log - linear multivariable regression , and related their modifying effect to hypermethylation in crc . all statistical tests were two-sided.resultsof 20025 promoter - associated cpgs analyzed , 1713 showed statistically significant age - dependent methylation gains . fewer cpgs acquired methylation in users of aspirin ( 2 years ) and hormonal replacement therapy ( hrt age 50 years ) compared with nonusers ( 43 vs 1355 ; 1 vs1377 , respectively ) , whereas more cpgs were affected in smokers ( 20 years ) and individuals with a body mass index ( bmi ) of 25kg / m2 and greater compared with control groups ( 180 vs 39 ; 554 vs 144 , respectively ) . fifty percent of the cpgs showing age - dependent methylation were found hypermethylated in crc ( odds ratio [ or ] = 20 ; 95% confidence interval [ ci ] = 18 to 23 ; p < 21016 ) . these loci gained methylation with a higher median rate compared with age - only methylated sites ( p = 21076 ) and were enriched for polycomb regions ( or = 3.67 ) . importantly , aspirin ( p < .001 ) and hrt use ( p < .001 ) reduced the methylation rate at these cancer - related genes , whereas smoking ( p < .001 ) and high bmi ( p = .004 ) increased it.conclusionslifestyle , including aspirin use , modulates age - associated dna methylation change in the colonic epithelium and thereby impacts the evolution of cancer methylomes .

adjacent segment degeneration ( asd ) is not a rare condition nowadays due to the fact that spinal surgery is more widely performed . this condition is defined as the recurrence of symptoms associated with the degeneration at the free segment above a fusion after symptom - free period . the incidence of symptomatic asd is reported to be up to 30% . in all asd patients , the incidence of recurrent disc herniation has been reported to be around 5 to 18% in patients after open lumbar surgery [ 28 ] . recurrent disc herniation is thought to be the major cause of surgical failure after open lumbar surgery , especially after microdiscectomy procedure . the generally accepted surgical management for recurrence of lumbar herniated disc is repeated open lumbar surgery ; however , approach associated complications attributed to tissue scarring and adjacent segment degeneration caused by further damage to the vertebral motion segments should be considered [ 3 , 911 ] . furthermore , other than repeated open lumbar surgery , there may be other options to be considered . the endoscopic posterolateral transforaminal approach through intact tissue can avoid repeated damage to the posterior and paraspinal structures , making percutaneous endoscopic lumbar discectomy ( peld ) a potential alternative to open lumbar surgery . peld has already shown encouraging results for lumbar herniated disc [ 8 , 1220 ] ; however , researches comparing clinical outcomes of repeated lumbar surgery by means of peld with repeated open lumbar surgery are rare . in our study , we attempted to compare the surgical results of peld and repeated open lumbar surgery for asd and recurrent lumbar disc herniation . from december 2011 to november 2013 , we retrospectively collected forty - three patients who received repeated lumbar surgeries due to asd or recurrent lumbar disc herniation . inclusion criteria were ( 1 ) undergoing open lumbar surgery previously with or without fusion / fixation , ( 2 ) occurrence of recurrent or adjacent lumbar radicular pain subsequent to a painless period of at least four weeks , ( 3 ) herniated disc at the original level or the adjacent level , and ( 4 ) failed nonsurgical treatment for longer than six weeks [ 8 , 21 ] . there were 26 men and 17 women , and the mean age of the patients was 55.9 years ( range 2580 years ) ( figure 1 ) . based on the surgical approaches , these patients were assigned to the following two groups . group a included patients who received peld , and group b consisted of patients who underwent repeated open lumbar surgery for asd and had recurrence of disc herniation . six neurosurgeons were involved in our study , and the decision of surgical approach depended on each surgeon 's expertise and preference . among them , only one performed peld for asd and recurrence of disc herniation . in the open lumbar surgery group , the steps involved in the procedure were described to each patient before their operation . the surgeries were performed under general anesthesia in the prone position on a radiolucent operating table . a midline skin incision was made and paravertebral muscles were divided . scar tissue was carefully removed to identify the previous laminotomy edges , and the surgeon was cautious not to tear the dura mater . various surgical procedures were performed including removal of implants , microdiscectomy , laminotomy or laminectomy , and discectomy with / without fusion . each wound was closed in layers after adequate decompression of the nerve roots . in the peld group , patients were also informed of every step of the operation . the surgeries were carried out under local anesthesia in the prone position on a radiolucent operating table . during the entire procedure patients were able to communicate with the operator . the skin incision was made approximately 10 to 12 cm lateral from the midline . an 18-gauge spinal needle was inserted under fluoroscopic guidance after infiltrating the entry point with local anesthetics . the ideal target point of the spinal needle was the intersection of the posterior vertebral line on the lateral image and the medial pedicular line on the anteroposterior image . after the introduction of the spinal needle into the target disc space , the subsequent steps were as follows : we ( 1 ) inserted the guide wire through the spinal needle , ( 2 ) removed the spinal needle , ( 3 ) made a skin incision at the entry point , ( 4 ) inserted a tapered cannulated obturator along the guide wire , ( 5 ) inserted the obturator into the disc space with hammering after touching the annulus , and ( 6 ) inserted a oval - shaped , bevel - ended working cannula into the disc space along the obturator and then removed the obturator . the endoscope was inserted through the cannula and the disc was removed using endoscopic forceps working from the central to the lateral portion of the disc space on the anteroposterior image ( figure 2 ) . after targeted fragmentectomy , the endoscope was retracted , the incision was sutured , and a sterile dressing was applied [ 14 , 15 ] . operative reports and medical charts were reviewed to obtain pre- and postoperative clinical data including operating time , total blood loss , and admission days . pain was measured by the 10-point visual analogue scale ( vas ) scoring ( 0 to 10 ) before and after surgery . complications were categorized into major and minor using the classification scheme described by carreon et al . . minor complications were considered not to have affected recovery significantly , whereas major complications were considered to have caused a negative effect on a patient 's recovery . statistical methods were used to compare the patient demographic data and clinical outcomes of the two groups . the student t - test was used on continuous parameters and the chi - square statistic test was used on categorical parameters . in the present study , p value < 0.05 was considered to be statistically significant . group a ( peld ) included 18 patients and group b ( repeated open lumbar surgery ) included 25 patients . there was no statistically significant difference in mean age at the time of surgery ( 57.4 in group a and 54.9 in group b , p = 0.597 ) and sex ratio ( male : female , 12 : 6 in group a and 14 : 11 in group b , p = 0.697 ) ( table 1 ) . in group a , the mean blood loss ( minimal ) was significantly less than that in group b ( 303.2 ml , p < 0.0001 ) . mean operating time was shorter significantly in group a ( 79.06 minutes ) as compared with group b ( 206.44 minutes , p < 0.0001 ) . mean hospital stay was shorter significantly in group a ( 1.89 days ) as compared with group b ( 12.28 days , p < 0.0001 ) ( table 2 ) . no complications were encountered in the peld group , but three minor complications occurred in the open surgery group in the perioperative period . after operation , immediate postoperative pain improvement in vas was 3.5 in the peld group as compared with 0.56 in the open lumbar surgery group ( p < 0.0001 ) ( table 2 ) . peld is a form of minimally invasive surgery and has many advantages over conventional open lumbar surgery . these advantages include performing the surgery under local anesthesia , preserved normal posterior and paraspinal structures , less postoperative pain , and early discharge . for repeated lumbar surgery , not needing to go through the scar tissue could also be one significant advantage that is worth highlighting . there are some studies which have discussed the use of peld for patients with recurrence of disc herniation ; however , there are still few relative discussions about the use of peld for patients with asd . a prospective cohort study performed by hoogland et al . evaluated a series of 262 patients who received peld for recurrence of disc herniation . both leg pain and back pain improved significantly , and the results of their surgeries were rated as excellent or good in 85.71% of the patients at 2-year follow - up . in another retrospective study , xia et al . the mean vas score decreased significantly , and based on the macnab criteria 81.4% of the patients met excellent or good outcomes at a mean follow - up duration of 31 months . in our study , we compared the surgical results of peld with those of repeated open lumbar surgery . peld yielded clinical outcomes superior to repeated open lumbar surgery in terms of shortened operative time and admission days , decreased total blood loss , and lessened postoperative pain . because normal paraspinal structures are preserved during peld , it was thought to be superior to open lumbar surgery in terms of postoperative back pain . during repeated open lumbar surgery , dissection of paraspinal muscles and further destruction of posterior vertebral elements , such as medial facet joint and lamina , may heighten the risk of postoperative back pain [ 1 , 10 , 11 ] . manchikanti et al . reported that the prevalence of facet joint related chronic low back pain in postoperative group who received lumbar laminectomy was 32% . in our study , postoperative pain is significantly lower in the peld group . as with peld for primary lumbar herniated disc , peld for recurrence of disc herniation as well had many advantages over repeated open lumbar surgery in that it could be carried out under local anesthesia , and the operating times as well as hospital stay were significantly shorter . regarding postoperative spinal stability , discectomy through transforaminal route has advantages over that through posterior route . as mentioned above , paravertebral and posterior spinal structures such as lamina , facet joint , ligaments and muscles could be preserved by means of transforaminal approach . osman et al . reported one comparative study regarding postoperative stability of transforaminal and posterior approach using cadavers . after transforaminal decompression , there was minimal anatomic damage to the spine and there was no flexibility change . in contrast , there was significant increase in extension and axial rotation flexibility after the posterior decompression . choi et al . suggested that the atrophy and weakness of paraspinal muscles was one of the possible predisposing factors for further instability and dysfunction . in terms of paraspinal muscle atrophy , peld was considered to have an advantage for asd and recurrence of disc herniation since repeated open lumbar surgery had a higher chance of further damaging paraspinal muscles and innervating dorsal rami . suggested that the influences of second surgeries on paraspinal muscles were not significant because denervation atrophy of paraspinal muscles had already significantly progressed after the first open lumbar surgery . epidural adhesion and scar tissue often make repeated open lumbar surgery difficult and thus increase the risk of nerve root injury or intraoperative incidental durotomy [ 3 , 911 , 20 ] . the incidence of intraoperative incidental durotomy was reported to be up to 20% in patients who received repeated open lumbar surgery [ 8 , 25 , 26 ] . it was suggested that intraoperative incidental durotomy during lumbar surgery could result in poorer clinical outcomes and long - term neurological sequelae . in contrast , considering repeated lumbar surgery by means of peld , some studies reported encouraging outcomes . lee et al . reported that there was no case of intraoperative incidental durotomy or cerebrospinal fluid leakage after surgery in 43 , 262 , and 23 consecutive patients , respectively . in our study , there was also no intraoperative incidental durotomy in peld group , whereas intraoperative incidental durotomy occurred in two patients ( 8% ) in the group of repeated open lumbar surgery . injury of nerve root during repeated lumbar surgery may result in long - term neurological sequelae and poorer clinical outcome . choi et al . reported that one ( 2.9% ) of 35 patients developed permanent foot drop after repeated open lumbar surgery . also reported that there was one ( 3.4% ) patient who developed persistent voiding disturbance along with dysesthesia after repeated open lumbar surgery in 28 consecutive patients . . reported three patients ( 1.1% ) with nerve root irritation and xia et al . reported one patient ( 2.3% ) with transient dysesthesia , but no patient suffered permanent injury of nerve root in either series . reported that there was no case of nerve root injury after peld in 23 consecutive patients . in our study , there was no nerve root injury observed in both peld and repeated open lumbar surgery group . although our study showed promising outcomes , the relatively small number of patients and the retrospective study design of this study should be taken into consideration when interpreting our results . for patients with asd and recurrent disc herniation , both peld and repeated open lumbar surgery showed favorable clinical outcomes in the present study . furthermore , peld had several advantages over repeated open lumbar surgery in terms of what follows : ( 1 ) the procedure being possibly performed under local anesthesia , ( 2 ) significantly shortened operating time , ( 3 ) significantly less mean blood loss , ( 4 ) no complications related to surgical approach , such as dural tear and/or nerve root injury , ( 5 ) less postoperative discomfort , and ( 6 ) significantly decreased number of admission days . overall , the peld group achieved the goal of early mobilization and a shorter postoperative period to normal daily activity . in the long run , the increased use of this type of procedure may reduce the economic burden of this disease for patients , their families , and society in general .

objective . the goal of the present study was to examine the clinical results of percutaneous endoscopic lumbar discectomy ( peld ) and open lumbar surgery for patients with adjacent segment degeneration ( asd ) and recurrence of disc herniation . methods . from december 2011 to november 2013 , we collected forty - three patients who underwent repeated lumbar surgery . these patients , either received peld ( 18 patients ) or repeated open lumbar surgery ( 25 patients ) , due to asd or recurrence of disc herniation at l3 - 4 , l4 - 5 , or l5-s1 level , were assigned to different groups according to the surgical approaches . clinical data were assessed and compared . results . mean blood loss was significantly less in the peld group as compared to the open lumbar surgery group ( p < 0.0001 ) . hospital stay and mean operating time were shorter significantly in the peld group as compared to the open lumbar surgery group ( p < 0.0001 ) . immediate postoperative pain improvement in vas was 3.5 in the peld group and 0.56 in the open lumbar surgery group ( p < 0.0001 ) . conclusion . for asd and recurrent lumbar disc herniation , peld had more advantages over open lumbar surgery in terms of reduced blood loss , shorter hospital stay , operating time , fewer complications , and less postoperative discomfort .

endodontic treatment outcome is directly related to thorough mechanical and chemical cleansing of the entire root canal system followed by its complete obturation with an inert filling material . the ability to locate all the canals present in the root canal system is an important factor in determining the eventual success of treatment . undetected root canals are of concern as they are the major reason for endodontic failure . additional hidden second mesiobuccal ( mb2 ) canal may be encountered in up to 93.5% maxillary molars and their presence has been demonstrated in in vitro as well as in vivo studies . optical magnification and use of precision guiding equipment the purpose of this in vivo study was to determine whether use of optical magnification with dental operating microscope ( dom ) and precision guiding by troughing with ultrasonic tips either individually or in combination significantly enhance clinician 's ability to locate the mb2 canal in permanent maxillary first molars . thirty - four male and 26 female healthy subjects of age between 14 and 40 years , requiring root canal therapy for maxillary first molar were randomly included for the study . the study was approved by the institutional ethical committee and informed consent was obtained from all participants . the procedure was carried out under local anesthesia [ xylocaine 2% with adrenaline ( 1:200,000 ) ( astra zeneca pharma , astrazenca - bangalore ) ] and rubber dam isolation . standard access cavities were prepared using endo access bur and cavity access set ( dentsply maillefer , astrazenca - bangalore ) . complete de - roofing of the pulp chamber was achieved by endo z bur ( dentsply maillefer ) . dg-16 endodontic explorer , dom ( carl zeiss , astrazenca - bangalore ) , and ultrasonic tips ( proultra endo tips ; dentsply tulsa dental astrazenca - bangalore ) were used in sequence to locate mb2 [ figure 1 ] . while using ultrasonic tips , a 3-mm - long trough was prepared from the mesiobuccal ( mb ) canal toward the palatal canal and re - examined under dom for location of the canal . once the canal was located , it was negotiated incrementally with k files- mani company japan carl zeiss - bangalore of size no . mesial shift intraoral periapical radiograph was taken with a file in each of the canals of the mb root to confirm the presence of two canals . images of mb2 canal [ figure 2 ] were captured using laptop tv tuner card ( honestech , bangalore ) connected to the dom . 3% sodium hypochlorite was used as endodontic irrigating solution . after the identification procedure , closed dressing statistical comparisons for visualisation of mb2 between the naked eye , dom , and dom with ultrasonic techniques were done by analyzing the receiver operating characteristic ( roc ) curves using spss 16.0 version software . protocol for identification of mb2 canal ( a ) mb2 canal not visible under naked eye . the present study was conducted to determine the presence of mb2 canal in maxillary first molars . with naked eye , the mb2 canal was located in 12 teeth ; with the use of the dom , the mb2 canal was located in 21 additional teeth ; and with the combined use of the ultrasonic tip and dom , the mb2 canal was located in 9 more teeth [ figure 3 ] . mb2 canals were easily identified in younger patients [ table 1 ] . among the 42 canals located , 13 were negotiated up to 16 mm and 29 were partially negotiated , i.e. up to 45 mm from the pulpal floor , and 18 canals were not negotiable . mb2 canal was located within 5 mm distance from the mb canal in majority of cases [ table 2 ] . statistical comparisons between the tested techniques were done by analyzing the roc curves . in this study , a curve for each mb2 canal detection technique was drawn according to its sensitivity ( true - positive performance ) and specificity ( false - positive performance ) [ figure 4 ] . the area under each curve was calculated and compared pair - wise with the others . among the comparisons between naked eye , dom , and ultrasonics dom combination , a statistically significant difference was found only between the naked eye group and the dom group ( p < 0.001 ) [ table 3 ] . results showing mb2 canal location using various techniques age distribution in study and incidence of mb2 canal distance between mb and mb2 canal receiver operating characteristic curves pertaining to mb2 canal detection using various methods roc area of various methods identifying mb2 canals has always been taxing without the use of technological advances like dom and ultrasonic devices . hence , this study was conducted to determine if the efficiency of locating mb2 canal in maxillary molar would increase with the use of dom and ultrasonics . variations in the skill levels and experience of different operators in using dom and ultrasonics can influence the outcomes ; hence , all procedures were performed by a single operator . magnification and variable intensity of light , which is focused down the shaft of the optic piece , parallel to the field of magnification by the dom provides clear view of the pulpal floor , thus leading to easier detection of this small - sized canal orifice . a developmental pattern is suggested for presence of two or more canals in a single root , i.e. a ribbon - shaped isthmus area forms during maturation leaving a larger canal and a smaller canal . multiple canals are frequent in the mb root and these additional canals ( mb2 ) may not terminate in independent foramina . we were able to locate these small canals easily in our younger patients [ table 1 ] . maxillary molars often become pulpally involved due to mesial caries which , in turn , stimulates tertiary dentin formation or other canal calcifications leading to difficulty in locating the canal . to address these obstacles , countersinking is suggested , i.e. variable amount of dentin ( 13 mm ) must be removed by troughing along the mb sub - pulpal groove with a distinct orientation toward the mesial direction to uncover completely the orifice and pursue the mb2 canal deeper into the root . munce discovery burs , round burs , composite finishing burs , and ultrasonics can be used for this purpose . in the present study , we used zirconium nitride coated ultrasonic tips which provide good control while maintaining the cutting efficiency . it has been demonstrated that troughing the chamber floor within 3 mm from the mb canal toward the palatal canal with an ultrasonic tip under dom makes detection of mb2 canal more successful . in our subjects , mb2 orifice openings were usually found mesial to an imaginary line between the mb and palatal canal orifices and commonly about 23 mm palatal to the mb canal orifice . occasionally , mb2 shared orifice with the main mb canal and was oval in shape . in the present study , this may be due to the tortuous pathway of some of these canals that can include one or two abrupt curves in the coronal portion as explained by kulid and peters . leaving the canal untreated may allow microorganisms to colonize the space , leading to infection and treatment failure . the study results suggest that the prevalence of mb2 canal in the maxillary first molar is high , i.e. 70% , and the use of the dom significantly increased the detection of mb2 canals in permanent maxillary first molars and the use of ultrasonics for troughing further enhanced the ability to detect mb2 canal under dom . considering the higher prevalence of mb2 canals in our study as well as other reported studies and additional discoveries of canal orifices by using dom and ultrasonics in our cases , which translated into clinically significant outcome , we suggest the clinical use of dom and ultrasonics to improve treatment prognosis .

objective : the aim of this study was to evaluate the influence of using the dental operating microscope ( dom ) and ultrasonics for the detection of second mesiobuccal ( mb2 ) canal orifice in maxillary first molars.materials and methods : sixty subjects seeking root canal therapy for maxillary first molar were assessed for the presence of mb2 canal using endodontic explorer without magnification . teeth in which the mb2 canal orifice could not be located were examined under magnification using dom . if the mb2 canal orifice could not be found even after using dom , ultrasonic tips were used to prepare 3-mm - long trough from the mesiobuccal canal orifice toward the palatal canal and examined under dom for location of the canal.results:with naked eye , the mb2 canal was located in 12 teeth ; with the use of the dom , the mb2 canal was located in 21 additional teeth ; and with the combined use of ultrasonic tip and dom , the mb2 canal was located in 9 more teeth . statistical comparisons between the tested techniques were done by analyzing the receiver operating characteristic ( roc ) curves ; a statistically significant difference was found ( p < 0.001).conclusion : the results of this study indicate that the dom and ultrasonics provide increased opportunity for the dentist to detect canal orifices .

absolute pitch ( ap ) is the ability to identify the frequency or musical name of a specific tone , or to identify a tone without comparing it with any objective reference tone ( martin and perry , 1999 ; levitin and rogers , 2005 ; vanzella and schnellenberg , 2010 ) . although whether the extraordinary ability of ap is genetically determined or develops dependently on environmental variables still remains a controversial issue ( levitin and zatorre , 2003 ; drayna , 2007 ) , there is a general consensus that musical training in childhood is important for its acquisition ( zatorre , 2003 ) . those investigations have consistently found that while the right hemisphere is in general important for musical processing , increasing musical sophistication causes a shift of musical processing from the right to the left hemisphere ( elbert et al . , 1995 ; schlaug et al . , 1995 ; the notion has been confirmed regarding ap by various structural observations of morphological changes in the cortical regions of the planum temporale ( pt ) in musicians with ap ( keenan et al . , 2001 ; luders et al . , 2004 ; 2008 ) . moreover , more recently , the stronger left pt activation has been recorded in ap possessors than in non - ap possessors ( including professional musicians ; hirata et al . , 1999 ; ohnishi et al . , 2001 ; gaab et al . , 2006 ; wu et al . , 2008 ; oechslin et al . , 2010 ) . the left pt is known as wernicke 's area , and is related to language comprehension . the human pt is a roughly triangular region of the superior temporal plane located posterior to the primary auditory field ( steinmetz et al . , 1991 ) . it is , on the average , larger in the left hemisphere , suggesting that it may play a specialized role in language and language lateralization . why is the left pt involved in music perception in ap possessors , particularly in those of trained musicians ? so far , no behavioral evidence of speech - relevant auditory acuity in any ap possessors has been reported . in order to pursue this issue , here , i have compared the performance of identifying isolated syllables between musicians with ap and musicians without ap . i hypothesized that since ap possessors have special expertise in identifying the tonal label of a specific tone , their sense of identification would be keener than that of non - ap possessors whether the isolated syllables they were asked to identify were the names of musical notes or not . fifteen professional musicians with ap ( referred to below as ap - possessors : 3 males and 12 females ; mean age = 23 years , sd = 3.3 ; mean practice years = 15.4 , sd = 4.3 ; mean age practice began = 4.2 , sd = 3.0 ) , and 14 professional musicians without ap ( referred to below as non - ap possessors : 4 males and 10 females ; mean age = 23 years , sd = 3.8 ; mean practice age = 13.9 , sd = 6.2 , mean age practice began = 9.1 , sd = 5.2 ) participated in the present study . all of them were born in japan , had been educated in the japanese formal education system from the beginning of the entrance into kindergarden at least until the completion of graduation from senior high school , and had acquired japanese as their first language . none of them reported any hearing impairment . both ap and non - ap possessors as participants were selected preliminarily with an in - house test which was conducted prior to the present experiment : they heard 108 pure sine wave tones , presented in pseudorandomized order , which ranged from a3 ( tuning : a4 = 440 hz ) to a5 , with each tone being presented three times . each tone of the ap test had a duration of 1 s , with a 4-s interstimulus interval . during the intervals , the participants heard brown noise . the accuracy was evaluated by counting correct answers and the semitone errors were taken as incorrect to increase the discriminatory power . the participants were not asked to identify the adjacent octaves of the presented tones because for ap it is a most notable prerequisite to identify the correct chroma . in all , ap possessors were those whose accuracy scores were above 80% ( mean = 85.8 ; sd = 6.6 ) whereas non - ap possessors were those whose scores were below 10% ( mean = 7.2 ; sd = 3.9 ) . during the experiment as well as during the preliminary screening test for ap , each participant was seated in an attenuation chamber and wore a headphone . while a pure tone was presented using a notebook computer in a screening test , a syllable was chosen for a presented stimulus from a total of the 111 syllables that constitute the japanese language . a total of 100 isolated syllables , each of which had a duration of 200 ms , were presented to a given participant consecutively with an interstimulus interval being 5 s of silence in a given presentation session , and in all , two such sessions were conducted for each participant . in the first session , the participant was asked to press a key which was located on a table near the participant as quickly as possible when identifying what syllable was the stimulus and to answer it orally ( referred to below as the identification session ) . in the second session , the participant was asked to press the key as quickly as possible when hearing the presented sound ( referred to below as the hearing session ) . prior to the identification session , the participant was also instructed not to press the key before recognizing the presented syllable , and that was actually confirmed in each participant by an interview undertaken after the completion of the entire experiment . as the presented stimuli in each session , the 111 syllables were operationally classified into two categories ; seven were those that are used as japanese tonal labels for seven musical notes constituting an octave , i.e. , do ( c ) , re ( d ) , mi ( e ) , fa ( f ) , so ( g ) , ra ( a ) , si ( b ) ( referred to below as solfege syllables ) , and the remaining 104 were those that are not used as note - names ( referred to below as non - solfege syllables ) . the 100 isolated syllables presented to each participant in a given session comprised 50 solfege syllables and 50 non - solfege syllables . moreover , all of the stimuli were presented randomly to each participant regarding whether they were solfege syllables or non - solfege syllables . as a behavioral measure , in both sessions , the interval between the onset of each stimulus presentation and the onset of the subsequent pressing of the button was used . the mean latency to the answer was computed for each participant in each of the two sessions separately with regard to solfege syllables and to non - solfege syllables . fifteen professional musicians with ap ( referred to below as ap - possessors : 3 males and 12 females ; mean age = 23 years , sd = 3.3 ; mean practice years = 15.4 , sd = 4.3 ; mean age practice began = 4.2 , sd = 3.0 ) , and 14 professional musicians without ap ( referred to below as non - ap possessors : 4 males and 10 females ; mean age = 23 years , sd = 3.8 ; mean practice age = 13.9 , sd = 6.2 , mean age practice began = 9.1 , sd = 5.2 ) participated in the present study . all of them were born in japan , had been educated in the japanese formal education system from the beginning of the entrance into kindergarden at least until the completion of graduation from senior high school , and had acquired japanese as their first language . none of them reported any hearing impairment . both ap and non - ap possessors as participants were selected preliminarily with an in - house test which was conducted prior to the present experiment : they heard 108 pure sine wave tones , presented in pseudorandomized order , which ranged from a3 ( tuning : a4 = 440 hz ) to a5 , with each tone being presented three times . each tone of the ap test had a duration of 1 s , with a 4-s interstimulus interval . during the intervals , the participants heard brown noise . the accuracy was evaluated by counting correct answers and the semitone errors were taken as incorrect to increase the discriminatory power . the participants were not asked to identify the adjacent octaves of the presented tones because for ap it is a most notable prerequisite to identify the correct chroma . in all , ap possessors were those whose accuracy scores were above 80% ( mean = 85.8 ; sd = 6.6 ) whereas non - ap possessors were those whose scores were below 10% ( mean = 7.2 ; sd = 3.9 ) . during the experiment as well as during the preliminary screening test for ap , each participant was seated in an attenuation chamber and wore a headphone . while a pure tone was presented using a notebook computer in a screening test , a syllable was chosen for a presented stimulus from a total of the 111 syllables that constitute the japanese language . a total of 100 isolated syllables , each of which had a duration of 200 ms , were presented to a given participant consecutively with an interstimulus interval being 5 s of silence in a given presentation session , and in all , two such sessions were conducted for each participant . in the first session , the participant was asked to press a key which was located on a table near the participant as quickly as possible when identifying what syllable was the stimulus and to answer it orally ( referred to below as the identification session ) . in the second session , the participant was asked to press the key as quickly as possible when hearing the presented sound ( referred to below as the hearing session ) . prior to the identification session , the participant was also instructed not to press the key before recognizing the presented syllable , and that was actually confirmed in each participant by an interview undertaken after the completion of the entire experiment . as the presented stimuli in each session , the 111 syllables were operationally classified into two categories ; seven were those that are used as japanese tonal labels for seven musical notes constituting an octave , i.e. , do ( c ) , re ( d ) , mi ( e ) , fa ( f ) , so ( g ) , ra ( a ) , si ( b ) ( referred to below as solfege syllables ) , and the remaining 104 were those that are not used as note - names ( referred to below as non - solfege syllables ) . the 100 isolated syllables presented to each participant in a given session comprised 50 solfege syllables and 50 non - solfege syllables . moreover , all of the stimuli were presented randomly to each participant regarding whether they were solfege syllables or non - solfege syllables . as a behavioral measure , in both sessions , the interval between the onset of each stimulus presentation and the onset of the subsequent pressing of the button was used . the mean latency to the answer was computed for each participant in each of the two sessions separately with regard to solfege syllables and to non - solfege syllables . figure 1 shows the mean latency to press the button of ap possessors and of non - ap possessors in the identification session when the presented stimuli were solfege syllables as well as non - solfege syllables . throughout the entire experiment , no identification errors were recorded and all the participants answered all the presented syllables correctly . nonetheless , 2 ( ap possessor versus non - ap possessor ) 2 ( solfege syllable versus non - solfege syllable ) analysis of variance ( anova ) revealed a significant main effect [ f(1,27 ) = 12.176 , p = 0.002 ] , and the mean latency to the presented stimulus was significantly shorter in the ap possessors than in non - ap possessors . the score was not significantly different whether the stimulus was a solfege syllable or a non - solfege syllable [ f(1,27 ) = 0.012 , p = 0.912 ] . interaction between the two main factors was not significant , either [ f(1,27 ) = 0.042 , p = 0.839 ] . mean latency ( error bars : sds ) of ap possessors and of non - ap possessors in the identification session to press the button when the presented stimuli were solfege syllables as well as non - solfege syllables . figure 2 shows the mean latency to press the button of ap possessors and of non - ap possessors in the hearing session when the presented stimuli were solfege syllables as well as non - solfege syllables . anova revealed no significant main effects , and the average latency to the presented stimulus was not different between the ap possessors and the non - ap possessors [ f(1,27 ) = 0.392 , p = 0.536 ] . the score was not different whether the stimulus was a solfege syllable or a non - solfege syllable [ f(1,27 ) = 0.963 , p = 0.334 ] . interaction between the factors was not significant , either [ f(1,27 ) = 1.359 , p = 0.254 ] . mean latency ( error bars : sds ) of ap possessors and of non - ap possessors in the hearing session to press the button when the presented stimuli were solfege syllables as well as non - solfege syllables . previous research demonstrated that the basic auditory capability does not differ between ap possessors and non - ap possessors ( fujisaki and kashino , 2002 ) . the results of the hearing session in the present study are consistent with that conclusion . nonetheless , recent neuroimaging studies have provided suggestive evidence for a strong influence of the pitch - processing expertise of ap possessors on their speech perception ( oechslin et al . , 2010 ) . the plausibility of this notion was tested in the present experiment , which actually presented suggestive evidence for such a link between musical expertise and speech information processing , and the results of the identification session in the present experiment revealed the fact that ap possessors are significantly superior in basic speech processing to non - ap possessors . namely , ap possessors were able to identify a given isolated syllable chosen from their first language significantly more rapidly than non - ap possessors could , whether the syllable was one used as musical note or not . human infants are born with the predispositional capability to distinguish all the sounds in all of the world 's languages ( kuhl , 2000 ) . by the end of their first year , however , they are on their way to perceiving particularly well the sounds that are important for their native languages ( usually around 40 for a given language ) whereas their capability to distinguish foreign speech sounds has decreased ( kuhl , 2003 ) . japanese infants , for example , initially perceive separate sounds for r and l ( as in the words road and load ) but lose the ability to hear this foreign distinction meanwhile , the first recognizable speech infants produce by themselves comprises a single word or what may appear to be a phrase , though at this stage , they are not aware that the words they produce have constituent elements . the question that then arises relates to the segmentation problem : how do children discover the structural components of the fluent speech stream without knowing the identity of the target elements ? in fact , the infant 's task of learning its native language is a daunting one because , unlike written language , spoken language has no obvious markers that indicate the boundaries between words . the commonest answer to the above question is that language learners use supra - segmental cues to locate boundaries in the speech stream ( werker and voutoumantos , 2000 ; falk , 2009 ) . in particular , it is a well - known fact that long before they can speak , infants are sensitive to the frequencies at which combinations of syllables occur and how they differ within and across word boundaries ( wermke and mende , 2006 ) . the study used the example of the phrase pretty baby and noted that among english words , the likelihood of ty following pre was higher than the likelihood that bay would follow ty . thus , with enough repetition , participating 7-month - old infants began to understand that pretty was potentially a word , even before they knew what it meant . prosodic cues embedded in language also help . in conversational english , for example , the majority of words are stressed on their first syllables , as in the words monkey and jungle . this predominantly strong - weak pattern is reversed in some languages . by the time an english - learning infant is seven and half a months old , he spontaneously perceives words that reflect the strong - weak pattern , but not the weak - strong pattern . they perceive taris as a unit because it begins with a stressed syllable . regarding the developmental milestones of vocalizations with segmental features , infants become able to produce them first as canonical babbling around 8 months of age . this period coincides with the period when they become able to segment words from fluent speech ( masataka , 2003 ) . taken together , these facts indicate that producing a limited , sequentially organized phonemic segment entails extracting words from some portion of the speech stream , which in turn is segmented according to prosodic and more global supra - segmental characteristics . extracting words enables infants to code them , which in turn enables the infants to recognize them as familiar words ( masataka , 2007 ) . while this should be the usual process by which one 's sensitivity to word units in fluent speech typically develops , the results of the present experiment indicate that possessing ap provides a person with extraordinarily enhanced acuity to individual syllables per se as fundamental units of a segmented word in the speech stream . presumably this keen sensitivity allows ap possessors to accomplish speech segmentation with less help of supra - segmental information of the speech compared to non - ap possessors . as it were , ap would enable its possessors to discover the structural components of speech stream , and consequently to rely more on identifying purely isolated linguistic elements involved in the stream than on identifying paralinguistic ones . the development of such a capability is closely related to neurological observations on pt changes that should be a consequence of early special musical experiences . admittedly , it is not completely clear how such performance of the isolated syllable identification in terms of reaction time as reported here could be speech segmentation ability because the ap possessors were not examined with regard to their superiority in other non - speech classification processing . also , the fact should be noticeable that all the participants were native speakers of japanese , a language having pitch contrast ( e.g. , tsujimune , 2007 ) . thus , information of the pitch of a word has some importance for the identification of the word as it is . in order to link the current data to speech segmentation , the performance of syllable recognition in a context should be compared between ap and non - ap possessors who are , preferably , native speakers of a language that does not have tone distinction or pitch contrast by presenting meaningful phrases or strings of nonsense syllables in which the target syllable can be in the initial , middle or final position . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

absolute pitch ( ap ) is the ability to identify the frequency or musical name of a specific tone , or to identify a tone without comparing it with any objective reference tone . while ap has recently been shown to be associated with morphological changes and neurophysiological adaptations in the planum temporale , a cortical area in the brain involved in speech perception processes , no behavioral evidence of speech - relevant auditory acuity in any ap possessors has hitherto been reported . in order to seek such evidence , in the present study , 15 professional musicians with ap and 14 without ap , all of whom had acquired japanese as their first language , were asked to identify isolated japanese syllables as quickly as possible after these syllables were presented auditorily . when the mean latency to the syllable identification was compared , it was significantly shorter in ap possessors than in non - ap possessors whether the presented syllables were those used as japanese labels representing the 7 tones constituting an octave or not . the latency to hear the stimuli per se did not differ according to whether the participants were ap possessors or not . the results indicate the possibility that possessing ap provides one with extraordinarily enhanced acuity to individual syllables per se as fundamental units of a segmented word in the speech stream .

prevalence and risk of overweight were assessed in 232 children who were enrolled in the prospective german gdm offspring study between 1989 and 2000 . all children were from singleton births , had a mother with gdm diagnosed according to criteria of the german diabetes association using an oral glucose tolerance test ( ogtt ) with a 75-g glucose load . women were considered to have gdm if two of three capillary blood glucose values exceeded the following limits : > 5 mmol / l ( fasting ) before an oral glucose tolerance test , > 10.0 mmol / l after 60 min , and 8.6 mmol / l after 120 min . prevalence of overweight was compared with that of a cohort of ot1d ( n = 757 ) and offspring of nondiabetic mothers ( ondm ) ( n = 431 ) followed in the babydiab study ( 6 ) . in both the gdm and babydiab studies , mothers and their offspring were recruited between 1989 and 2000 germany - wide through the same network of collaborating obstetric departments and pediatricians and entered into the study before the offspring reached the age of 3 months . all children were followed with similar follow - up visits until the age of 14 years . in both studies , > 98% of the families were caucasian . in families with more than one offspring participating in the study , data on weight and height were collected at birth and at age 2 , 8 , and 11 years by physicians . at birth , length was measured on a measuring board and weight on a calibrated scale by health care professionals . weight and height were obtained for 215 children at age 2 years , 89 children at age 8 years , and 74 children at age 11 years . birth weight was adjusted for sex and gestational age and is expressed as a percentile of the german reference population ( 8) . offspring less than the 10th percentile were defined as small for gestational age , those 90th percentile as large for gestational age and those between > 10th and < 90th percentile as appropriate for gestational age . from age 2 years , height and weight were measured by physicians at regular clinical visits using a standard protocol . weight was measured on a calibrated scale with the subject wearing light clothing , and height was measured with a fixed tape standing barefoot . height , weight , and bmi from age 2 years were expressed as percentiles adjusted for age and sex according to german reference data ( 9 ) . overweight was defined as bmi percentile 90 . maternal height and weight in early pregnancy were collected by physicians at the first pregnancy visit , bmi was calculated and categorized as obese ( 30 kg / m ) , overweight ( 25.029.9 kg / m ) , or normal weight ( < 25 kg / m ) . data on diabetes treatment ( categorized as treated by insulin injections or diet alone ) and maternal smoking behavior during pregnancy ( defined as < or 1 cigarette / day ) were obtained from a questionnaire at birth . since 2003 , fasting blood samples were collected for the determination of insulin resistance in ogdm as well as in offspring from the babydiab study . insulin resistance was measured by homeostasis model assessment of insulin resistance ( homa - ir ) . a total of 751 children ( including 74 ogdm and 425 ot1d ) with data on homa - ir either at age 8 or 11 years were included in this substudy . fasting insulin was determined centrally using an automated immunoassay analyzer ( aia 360 ; tosoh , san francisco , ca ) . the interassay coefficient of variation of the insulin assay is 8.4% at a concentration of 22.6 u / ml and the lower limit of detection is 0.5 u / ml . overweight prevalence in offspring at age 2 , 8 , and 11 years was compared between groups using the test . logistic regression analyses were performed to test the association of maternal bmi at early pregnancy , birth size , maternal smoking during pregnancy , treatment modality during pregnancy , and breast - feeding duration as univariate covariates with overweight risk in ogdm , giving the odds ratio ( or ) and 95% ci for overweight for each of the covariates . factors that were significantly associated with overweight risk in the univariate analysis were included as covariates in the multivariate logistic regression analysis . missing data were not considered in the univariate analyses but were included as a separate categorical value for each of the variables included in the multivariate analysis . linear regression was used to compare homa - ir in ogdm with those in ot1d and in ondm adjusted for age and sex and to analyze factors associated with homa - ir in ogdm . for all analyses , two - tailed p < all statistical analyses were performed using spss 17.0 ( spss , chicago , il ) . data on weight and height were collected at birth and at age 2 , 8 , and 11 years by physicians . at birth , length was measured on a measuring board and weight on a calibrated scale by health care professionals . weight and height were obtained for 215 children at age 2 years , 89 children at age 8 years , and 74 children at age 11 years . birth weight was adjusted for sex and gestational age and is expressed as a percentile of the german reference population ( 8) . offspring less than the 10th percentile were defined as small for gestational age , those 90th percentile as large for gestational age and those between > 10th and < 90th percentile as appropriate for gestational age . from age 2 years , height and weight were measured by physicians at regular clinical visits using a standard protocol . weight was measured on a calibrated scale with the subject wearing light clothing , and height was measured with a fixed tape standing barefoot . height , weight , and bmi from age 2 years were expressed as percentiles adjusted for age and sex according to german reference data ( 9 ) . maternal height and weight in early pregnancy were collected by physicians at the first pregnancy visit , bmi was calculated and categorized as obese ( 30 kg / m ) , overweight ( 25.029.9 kg / m ) , or normal weight ( < 25 kg / m ) . data on diabetes treatment ( categorized as treated by insulin injections or diet alone ) and maternal smoking behavior during pregnancy ( defined as < or 1 cigarette / day ) were obtained from a questionnaire at birth . written informed consent since 2003 , fasting blood samples were collected for the determination of insulin resistance in ogdm as well as in offspring from the babydiab study . insulin resistance was measured by homeostasis model assessment of insulin resistance ( homa - ir ) . a total of 751 children ( including 74 ogdm and 425 ot1d ) with data on homa - ir either at age 8 or 11 years were included in this substudy . fasting insulin was determined centrally using an automated immunoassay analyzer ( aia 360 ; tosoh , san francisco , ca ) . the interassay coefficient of variation of the insulin assay is 8.4% at a concentration of 22.6 u / ml and the lower limit of detection is 0.5 u / ml . overweight prevalence in offspring at age 2 , 8 , and 11 years was compared between groups using the test . logistic regression analyses were performed to test the association of maternal bmi at early pregnancy , birth size , maternal smoking during pregnancy , treatment modality during pregnancy , and breast - feeding duration as univariate covariates with overweight risk in ogdm , giving the odds ratio ( or ) and 95% ci for overweight for each of the covariates . factors that were significantly associated with overweight risk in the univariate analysis were included as covariates in the multivariate logistic regression analysis . missing data were not considered in the univariate analyses but were included as a separate categorical value for each of the variables included in the multivariate analysis . linear regression was used to compare homa - ir in ogdm with those in ot1d and in ondm adjusted for age and sex and to analyze factors associated with homa - ir in ogdm . for all analyses , two - tailed p < all statistical analyses were performed using spss 17.0 ( spss , chicago , il ) . prevalence of overweight in ogdm was 17.2% at age 2 years , 20.2% at age 8 years , and 31.1% at age 11 years . prevalence of overweight was increased in ogdm compared with ot1d ( 15.8 , 11.0 , and 15.8% at age 2 , 8 , and 11 years , respectively ; p < 0.05 , p = 0.03 , and p < 0.01 , respectively ) and with ondm ( 11.4 , 10.3 , and 15.5% ; p = 0.7 , p = 0.02 , and p = 0.005 , respectively ( fig . 1 ) . prevalence of overweight at 2 , 8 , and 11 years of age in ogdm ( ) , ot1d ( ) , and ondm ( ) . in ogdm , maternal obesity was a strong predictor of overweight in children at age 2 , 8 , and 11 years in the univariate and multivariate logistic regression model ( tables 1 and 2 ) . the prevalence of overweight in children at 2 , 8 , and 11 years of age was 24.6 , 36.4 , and 45.8% in children of obese mothers compared with 9.2 , 11.3 , and 11.9% of children born to nonobese mothers ( p = 0.01 , p = 0.02 , and p = 0.003 , respectively ) ( fig . birth size , therapy of gdm during pregnancy , and maternal smoking during pregnancy showed inconsistent associations with overweight risk in the offspring ( tables 1 and 2 ) . associations of maternal bmi in early pregnancy , birth size , therapy modality , and smoking behavior during pregnancy on overweight - risk ( bmi 90th percentile ) in ogdm at 2 , 8 , and 11 years of age : univariate analysis aga , appropriate for gestational age ; lga , large for gestational age ; sga , small for gestational age . associations of maternal obesity in early pregnancy , lga status , and maternal smoking during pregnancy on overweight risk in ogdm at 2 , 8 , and 11 years of age : multivariate analysis variables included in the multivariate analysis : maternal bmi before pregnancy , birth size , and maternal smoking during pregnancy . prevalence of overweight at 2 , 8 , and 11 years of age in ogdm in relation to maternal bmi in early pregnancy : bmi < 25.0 kg / m ( ) , bmi 25.029.9 kg / m ( ) , and bmi 30 kg / m ( ) . in the substudy on insulin resistance , ogdm had increased homa - ir compared with that of children of ondm and of ot1d ( p = 0.04 and p = 0.03 , adjusted for age and sex ) ( fig . homa - ir was associated with the child 's bmi ( p = 0.01 , adjusted for age and sex ) ( supplementary fig . 1a , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0139/dc1 ) but not with maternal obesity in early pregnancy ( supplementary fig . 1b ) . homa - ir ( mean 1 se ) at age 11 years in ogdm compared with ot1d and ondm . prevalence of overweight in ogdm was 17.2% at age 2 years , 20.2% at age 8 years , and 31.1% at age 11 years . prevalence of overweight was increased in ogdm compared with ot1d ( 15.8 , 11.0 , and 15.8% at age 2 , 8 , and 11 years , respectively ; p < 0.05 , p = 0.03 , and p < 0.01 , respectively ) and with ondm ( 11.4 , 10.3 , and 15.5% ; p = 0.7 , p = 0.02 , and p = 0.005 , respectively ( fig . 1 ) . prevalence of overweight at 2 , 8 , and 11 years of age in ogdm ( ) , ot1d ( ) , and ondm ( ) . in ogdm , maternal obesity was a strong predictor of overweight in children at age 2 , 8 , and 11 years in the univariate and multivariate logistic regression model ( tables 1 and 2 ) . the prevalence of overweight in children at 2 , 8 , and 11 years of age was 24.6 , 36.4 , and 45.8% in children of obese mothers compared with 9.2 , 11.3 , and 11.9% of children born to nonobese mothers ( p = 0.01 , p = 0.02 , and p = 0.003 , respectively ) ( fig . birth size , therapy of gdm during pregnancy , and maternal smoking during pregnancy showed inconsistent associations with overweight risk in the offspring ( tables 1 and 2 ) . associations of maternal bmi in early pregnancy , birth size , therapy modality , and smoking behavior during pregnancy on overweight - risk ( bmi 90th percentile ) in ogdm at 2 , 8 , and 11 years of age : univariate analysis aga , appropriate for gestational age ; lga , large for gestational age ; sga , small for gestational age . associations of maternal obesity in early pregnancy , lga status , and maternal smoking during pregnancy on overweight risk in ogdm at 2 , 8 , and 11 years of age : multivariate analysis variables included in the multivariate analysis : maternal bmi before pregnancy , birth size , and maternal smoking during pregnancy . prevalence of overweight at 2 , 8 , and 11 years of age in ogdm in relation to maternal bmi in early pregnancy : bmi < 25.0 kg / m ( ) , bmi 25.029.9 kg / m ( ) , and bmi 30 kg / m ( ) . in the substudy on insulin resistance , ogdm had increased homa - ir compared with that of children of ondm and of ot1d ( p = 0.04 and p = 0.03 , adjusted for age and sex ) ( fig . homa - ir was associated with the child 's bmi ( p = 0.01 , adjusted for age and sex ) ( supplementary fig . 1a , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0139/dc1 ) but not with maternal obesity in early pregnancy ( supplementary fig . homa - ir ( mean 1 se ) at age 11 years in ogdm compared with ot1d and ondm . this study shows an increased prevalence of overweight at 2 , 8 , and 11 years of age in ogdm compared with ot1d and ondm . in ogdm , overweight up to age 11 years was strongly associated with maternal obesity at early pregnancy . birth size of the child , maternal smoking during pregnancy , or treatment modality of gdm were less consistent predictors of overweight in children of mothers with gdm . this gdm offspring study is a prospective study from birth , in which questionnaires addressing in pregnancy - related factors were administered at birth and children 's weight and height were measured regularly by a pediatrician according to standard protocols . recall bias and over- or underestimating of growth that may occur when data are reported by parents are therefore avoided or limited . our results are based on the definition of overweight as bmi percentile 90 , which has been proposed by the german association of obesity during childhood and obesity . findings were consistent when overweight was defined as bmi 85 or 95 percentile ( data not shown ) . although not population based , this is the largest prospective study following caucasian children of mothers with gdm from birth in defined follow - up intervals , enabling a longitudinal analysis of factors affecting overweight risk . nevertheless , the numbers of children of mothers with gdm remains relatively low at ages 8 and 11 years . as a consequence , cis for some of the risk estimates are wide , and some associations may change with a larger number of subjects . a small number of studies have examined the impact of maternal diabetes on overweight risk in offspring , differentiating between ogdm and offspring of mothers with preexisting type 1 diabetes ( 10,11 ) . clausen et al . ( 11 ) reported an increased prevalence of overweight in adults who were from gdm pregnancies ( 40% ) or type 1 diabetes pregnancies ( 41% ) compared with control subjects ( 24% ) . ( 10 ) found only a minor increase in the prevalence of overweight in ogdm children ( 30% ) compared with control children ( 23% ) and ot1d ( 23% ) . the findings of lawlor et al . support our previous report that ot1d are not at higher risk for overweight compared with ondm ( 6 ) . however , in the current study , we saw a pronounced difference between ogdm and ot1d , whereas differences are not significant in the study of lawlor et al . to reconcile this discrepancy , it is noted that our study in germany reported a lower background prevalence of overweight in children compared with the u.s . and that the numbers of ot1d and ogdm are larger in our study . our findings are unlikely to be biased with respect to the comparisons between ogdm and ot1d children because both cohorts were recruited over the same period and from the same region and ethnic background . they are also supported by expected associations of maternal bmi with overweight risk in the ogdm . however , data for potential confounders such as socioeconomic status , dietary habits during childhood , and physical activity were not collected , and we can not exclude the possibility that the associations observed could , in some cases , be due to one or more of these confounding variables . the finding that overweight risk is significantly higher in ogdm compared with ot1d indicates that exposure to hyperglycemia during pregnancy per se can only partly explain the increased prevalence of overweight in these children . maternal obesity in early pregnancy was the strongest predictor of overweight risk at 2 , 8 , and 11 years in ogdm . others have shown that maternal pregravid bmi is the strongest predictor of childhood obesity independent of maternal glucose status or birth weight ( 7 ) . it has also been shown that childhood bmi correlates more closely with maternal bmi than does paternal bmi , indicating that in addition to genetic influences an obese intrauterine environment per se may contribute to the higher overweight risk in children of obese mothers ( 12,13 ) . this finding is strengthened by results from animal studies showing that feeding - induced obesity at conception increases the prevalence of obesity in offspring . programming of obesity in animals was independent of changes in birth weight and was associated with significant changes in metabolic and endocrine parameters and adipose tissue cellularity independent of postnatal caloric intake ( 13 ) . finally , the possibility that the influence of maternal obesity on childhood overweight could also be due to similar adverse dietary and physical activity behaviors in obese mothers and their offspring should be considered . similarly , the observation that maternal smoking during pregnancy is associated with overweight in the child could further indicate an influence via an unhealthy postnatal environment . the relationship between childhood obesity and both maternal obesity and smoking during pregnancy became more pronounced with age , further giving support to this hypothesis . not unexpectedly given the increased risk for overweight in ogdm , our substudy found higher homa - ir in ogdm than in ondm and also in ot1d . ( 14 ) who also reported a relationship between offspring bmi and insulin resistance in a small group of 23 female ogdm . a recently published study found that in offspring of mothers with pregravid obesity fetal insulin resistance strongly correlated with fetal adiposity ( 15 ) . we could not find a significant association between insulin resistance in the offspring and maternal obesity in early pregnancy , but numbers were relatively small . in summary , our results show that prevalence of overweight and , as a consequence , homa - ir during childhood is higher in ogdm than in ot1d , indicating that maternal diabetes type affects overweight risk . the finding that overweight risk was associated with maternal obesity and to a lesser extent with birth size suggests that a combination of genetic predisposition , fetal overnutrition and lifestyle factors are likely to contribute to childhood growth in ogdm .

objectivegestational diabetes mellitus ( gdm ) is associated with high birth weight in the offspring . this may lead to overweight and insulin resistance during childhood . the aim of the study was to assess the impact of gdm on overweight risk and insulin resistance in offspring.research design and methodsbmi measurements were collected at age 2 , 8 , and 11 years from 232 offspring of mothers with gdm ( ogdm ) and compared with those from 757 offspring of mothers with type 1 diabetes ( ot1d ) and 431 offspring of nondiabetic mothers ( ondm ) born between 1989 and 2000 . insulin resistance ( homeostasis model assessment of insulin resistance [ homa - ir ] ) was determined at age 8 and 11 years in 751 children ( 74 ogdm ) . overweight was defined as bmi percentile 90 ; insulin resistance was defined by homa-ir.resultsoverweight prevalence was increased in ogdm compared with ot1d and to ondm throughout childhood ( age 11 years 31.1 , 15.8 , and 15.5% ; p = 0.005 ) . maternal obesity was an important predictor of overweight risk in children ( age 11 years odds ratio 7.0 [ 95% ci 1.827.7 ] ; p = 0.006 ) ; birth size and maternal smoking during pregnancy were inconsistently associated with and treatment of gdm during pregnancy did not affect overweight risk . homa - ir was increased in ogdm compared with offspring of ondm mothers ( p = 0.01 , adjusted for sex and age ) and was associated with the child 's bmi ( p = 0.004).conclusionsoverweight and insulin resistance in children is increased in ogdm compared with ot1d or ondm . the finding that overweight risk is associated mainly with maternal obesity suggests that familial predisposition contributes to childhood growth in these offspring .

recently , a series of epidemiological studies have found that reduced birthweight was associated with impaired glucose tolerance and type 2 diabetes later in life . there are two explanations for it , which are thrifty phenotype hypothesis and fetal insulin hypothesis . fetal insulin hypothesis was proposed that the association between low birth weight and type 2 diabetes is principally genetically mediated . genetic variants affecting pancreatic beta cell function or insulin sensitivity result in low - insulin mediated fetal growth ( that is low birth weight ) and the risk of type 2 diabetes in later life . in recent years , many international studies on the association between birth weight and type 2 diabetes risk gene variants have been reported , which are strong evidence of a fetal insulin hypothesis . as we known , there is a racial difference in gene polymorphisms . to explore the low birth weight risk gene variants in chinese han population , in 2009 , we analyzed the association between type 2 diabetes risk gene variants in or near tcf7l2 , slc30a8 , kcnq1 , and pank4 in chinese han individuals born in peking union medical college hospital ( pumch ) , then found that birth weight was positively associated with kcnq1 rs2074196 ( = 40 g , [ 95% confidence interval [ ci ] : 71 , 0.1 ] , p = 0.04 ) , and did not observe association between birth weight and tcf7l2 , slc30a8 , and pank4 . in this study , we aimed to further explore the relationship between birth weight and other type 2 diabetes risk gene variants in chinese people . we selected variants in or near cdkal1 , hhex , and adcy5 , recently identified the association with reduced birthweight in european , and srr and ptprd , which were recently identified through type 2 diabetes genome - wide association studies in chinese han population and have not been investigated in relation to birth weight . a cohort of 2019 participants born in pumch from 1921 to 1954 were recruited during may 2003 and april 2005 , for the study of the association between low birthweight and later diabetes and impaired glucose regulation ( igr ) , detailed information has been published previously . among them , 1174 subjects with blood samples drawn entered this study , 564 males and 610 females , aged 5085 years ( average age 59 years ) . written informed consent was obtained from each participant , and the scanned document was submitted . standard oral glucose tolerance testing was performed in all the participants except those who had already been diagnosed with diabetes . type 2 diabetes was defined as the presence of one or more of the following : fasting plasma glucose ( fpg ) 7.0 mmol / l , 2-h plasma glucose ( 2hpg ) 11.1 the criteria for igr were fpg 5.6 to < 7.0 mmol / l and/or 2hpg on the oral glucose tolerance test of 7.8 to < 11.1 mmol / l . insulin resistance was determined by homeostasis model assessment ( homa - ir ) : homa - ir = fpg fins/22.5 ; pancreatic cell function was determined by homeostasis model assessment ( homa - b ) : homa - b = 20 fins/(fpg3.5 ) . fpg : ( mmol / l ) , fins : ( iu / ml ) . the loci previously reported to be associated with reduced birthweight at a genome - wide significance level in european were selected , including cdkal1 ( rs7756992 rs10946398 ) , hhex ( rs1111875 ) and adcy5 ( rs 1170806 rs9883204 ) , 5 tagsnps ( rs4678017/rs9881942/rs7641344/rs6777397/rs6226243 ) in adcy5 , rs391300 in srr and rs17584499 in ptprd which have not been investigated in relation to birth weight were also selected . the quality value was set as 95% during data analysis using the sequence detection system version 2.4 software ( applied biosystems , foster city , ca , usa ) . genotyping quality control was performed in 10% of the samples by duplicate checking ( rate of concordance in duplicates 99% ) . data are presented as means standard deviation or median ( interquartile range ) for nonnormally distributed variables . differences among groups were assessed by analysis of variance for normally distributed continuous variables , by nonparametric tests ( kruskall wallis test ) for nonnormally distributed continuous variables ( homa - ir , the number of pregnancies , and parity ) and by a test for frequencies . the effect of the genetic variant on birthweight , length at birth , and ponderal index was assessed by linear regression adjusting for sex , gestational age , parity , and maternal age . consequently for each variant , only participants with complete data were included in the analysis . type 2 diabetes risk was assessed by logistic regression analysis , after adjusting for age , sex , and body mass index ( bmi ) . statistical analyses were performed by spss 13.0 software ( inc . , chicago , il , usa ) . a cohort of 2019 participants born in pumch from 1921 to 1954 were recruited during may 2003 and april 2005 , for the study of the association between low birthweight and later diabetes and impaired glucose regulation ( igr ) , detailed information has been published previously . among them , 1174 subjects with blood samples drawn entered this study , 564 males and 610 females , aged 5085 years ( average age 59 years ) . written informed consent was obtained from each participant , and the scanned document was submitted . standard oral glucose tolerance testing was performed in all the participants except those who had already been diagnosed with diabetes . type 2 diabetes was defined as the presence of one or more of the following : fasting plasma glucose ( fpg ) 7.0 mmol / l , 2-h plasma glucose ( 2hpg ) 11.1 the criteria for igr were fpg 5.6 to < 7.0 mmol / l and/or 2hpg on the oral glucose tolerance test of 7.8 to < 11.1 mmol / l . insulin resistance was determined by homeostasis model assessment ( homa - ir ) : homa - ir = fpg fins/22.5 ; pancreatic cell function was determined by homeostasis model assessment ( homa - b ) : homa - b = 20 fins/(fpg3.5 ) . fpg : ( mmol / l ) , fins : ( iu / ml ) . the loci previously reported to be associated with reduced birthweight at a genome - wide significance level in european were selected , including cdkal1 ( rs7756992 rs10946398 ) , hhex ( rs1111875 ) and adcy5 ( rs 1170806 rs9883204 ) , 5 tagsnps ( rs4678017/rs9881942/rs7641344/rs6777397/rs6226243 ) in adcy5 , rs391300 in srr and rs17584499 in ptprd which have not been investigated in relation to birth weight were also selected . the quality value was set as 95% during data analysis using the sequence detection system version 2.4 software ( applied biosystems , foster city , ca , usa ) . genotyping quality control was performed in 10% of the samples by duplicate checking ( rate of concordance in duplicates 99% ) . data are presented as means standard deviation or median ( interquartile range ) for nonnormally distributed variables . differences among groups were assessed by analysis of variance for normally distributed continuous variables , by nonparametric tests ( kruskall wallis test ) for nonnormally distributed continuous variables ( homa - ir , the number of pregnancies , and parity ) and by a test for frequencies . the effect of the genetic variant on birthweight , length at birth , and ponderal index was assessed by linear regression adjusting for sex , gestational age , parity , and maternal age . consequently for each variant , only participants with complete data were included in the analysis . type 2 diabetes risk was assessed by logistic regression analysis , after adjusting for age , sex , and body mass index ( bmi ) . a total of 1174 participants , 645 ( 50.0% ) had normal glucose tolerance ( ngt ) , 181 ( 15.4% ) had developed type 2 diabetes and 348 ( 29.6% ) igr , respectively . table 1 shows the baseline characteristics of the study subjects and the statistical test of the differences between the impaired glucose metabolism ( igm , including type 2 diabetes and igr ) and ngt groups . however , the birthweight and birth length of igm patients were smaller than ngt groups ( p < 0.001 and p = 0.01 , respectively ) . about adult measurements , there were significant differences in age , waist , bmi , fpg , and 2hpg in the two groups ( p < 0.001 ) . baseline characteristics of the study participants data are shown as mean sd or median ( interquartile range ) . * the statistical tests of the difference between the igm and ngt groups in this table are not adjusted for confounding factors . ponderal index : birth weight / length ; bmi : body mass index ; fpg : fasting plasma glucose ; 2hpg : 2-h plasma glucose ; sd : standard deviation ; ngt : normal glucose tolerance ; igm : impaired glucose metabolism . all genotypes obeyed hardy weinberg equilibrium in nondiabetic participants except ptprd rs17584499 ( p = 0.005 ) . the risk c allele of cdkal1-rs10946398 was associated with reduced birthweight ( per allele = 41 g [ 95% ci : 80 , 3 ] , p = 0.034 ) , after adjusting for sex , gestational weeks , parity and maternal age , there were no significant association ( p = 0.118 ) , while the risk g allele of cdkal1-rs7756992 was associated with reduced birthweight ( per allele = 36 g [ 95% ci : 72 , 0.2 ] , p = 0.048 ) . the risk g allele of srr - rs391300 showed a tendency toward lower birth weight ( = 34 g [ 95% ci : 72 , 5 ] , p = 0.085 ) after adjusting for sex , gestational weeks , parity , and maternal age [ table 2 ] . however , all studied variants were not independently associated with birth length and ponderal index ( data not shown ) . effect of genetic variants on birth weight means sd stratified by t2 dm risk genotypes ; effect in gram ( 95% ci ) ; * linear regression , adjusted for gestational sex , parity , gestational weeks and maternal age . t2 dm : type 2 diabetes mellitus ; sd : standard deviation ; ci : confidence interval . the risk g allele of the hhex - rs1111875 was significantly associated with increased risk of igm ( odds ratio [ or ] : 1.325 [ 95% ci : 1.1091.582 ] , p = 0.002 ) . cc genotype of the cdkal1-rs10946398 was significantly associated with increased igm compared with aa genotype ( or : 1.575 [ 95% ci : 1.0972.261 ] , p = 0.014 ) . we did not analyze the association between adcy5-rs11708067/rs9883204 and the risk of igm , because the frequency of the risk alleles of which were below 1% ( g = 0.4% and t = 0.5% , respectively ) [ tables 3 and 4 ] . genotype distributions and allele frequencies of all the 11 snps * p values were calculated by fisher s exact test ; or : odds ratio for risk allele ; risk allele , allele with higher frequency in cases compared to controls . igm : impaired glucose metabolism ; ngt : normal glucose tolerance ; ci : confidence interval . association between nine genetic variants and the risk of igm * p values were assessed by logistic regression after adjusted by sex , age , and bmi . or : odds ratio ; ci : confidence interval ; bmi : body mass index ; igm : impaired glucose metabolism . the risk alleles ( c and g ) of cdkal1-rs10946398 and adcy5-rs7641344 were associated with impaired beta cell function ( homa - b : p = 0.008 and p = 0.02 ; fins : p = 0.085 and p = 0.05 ) compared with nonrisk - alleles . none of the risk variants was associated with reduced insulin sensitivity [ table 5 ] . effect of genetic variants on insulin secretion and sensitivity data are shown as median ( interquartile range ) , which was stratified by t2 dm risk genotypes . maf : minor allele frequency ; fins : fasting insulin level ; homa - ir : homeostasis model assessment for insulin resistance ; homa - b : homeostasis model assessment for cell function ; t2 dm : type 2 diabetes mellitus . a total of 1174 participants , 645 ( 50.0% ) had normal glucose tolerance ( ngt ) , 181 ( 15.4% ) had developed type 2 diabetes and 348 ( 29.6% ) igr , respectively . table 1 shows the baseline characteristics of the study subjects and the statistical test of the differences between the impaired glucose metabolism ( igm , including type 2 diabetes and igr ) and ngt groups . however , the birthweight and birth length of igm patients were smaller than ngt groups ( p < 0.001 and p = 0.01 , respectively ) . about adult measurements , there were significant differences in age , waist , bmi , fpg , and 2hpg in the two groups ( p < 0.001 ) . baseline characteristics of the study participants data are shown as mean sd or median ( interquartile range ) . * the statistical tests of the difference between the igm and ngt groups in this table are not adjusted for confounding factors . ponderal index : birth weight / length ; bmi : body mass index ; fpg : fasting plasma glucose ; 2hpg : 2-h plasma glucose ; sd : standard deviation ; ngt : normal glucose tolerance ; igm : impaired glucose metabolism . all genotypes obeyed hardy weinberg equilibrium in nondiabetic participants except ptprd rs17584499 ( p = 0.005 ) . the risk c allele of cdkal1-rs10946398 was associated with reduced birthweight ( per allele = 41 g [ 95% ci : 80 , 3 ] , p = 0.034 ) , after adjusting for sex , gestational weeks , parity and maternal age , there were no significant association ( p = 0.118 ) , while the risk g allele of cdkal1-rs7756992 was associated with reduced birthweight ( per allele = 36 g [ 95% ci : 72 , 0.2 ] , p = 0.048 ) . the risk g allele of srr - rs391300 showed a tendency toward lower birth weight ( = 34 g [ 95% ci : 72 , 5 ] , p = 0.085 ) after adjusting for sex , gestational weeks , parity , and maternal age [ table 2 ] . however , all studied variants were not independently associated with birth length and ponderal index ( data not shown ) . effect of genetic variants on birth weight means sd stratified by t2 dm risk genotypes ; effect in gram ( 95% ci ) ; * linear regression , adjusted for gestational sex , parity , gestational weeks and maternal age . t2 dm : type 2 diabetes mellitus ; sd : standard deviation ; ci : confidence interval . the risk g allele of the hhex - rs1111875 was significantly associated with increased risk of igm ( odds ratio [ or ] : 1.325 [ 95% ci : 1.1091.582 ] , p = 0.002 ) . cc genotype of the cdkal1-rs10946398 was significantly associated with increased igm compared with aa genotype ( or : 1.575 [ 95% ci : 1.0972.261 ] , p = 0.014 ) . we did not analyze the association between adcy5-rs11708067/rs9883204 and the risk of igm , because the frequency of the risk alleles of which were below 1% ( g = 0.4% and t = 0.5% , respectively ) [ tables 3 and 4 ] . genotype distributions and allele frequencies of all the 11 snps * p values were calculated by fisher s exact test ; or : odds ratio for risk allele ; risk allele , allele with higher frequency in cases compared to controls . igm : impaired glucose metabolism ; ngt : normal glucose tolerance ; ci : confidence interval . association between nine genetic variants and the risk of igm * p values were assessed by logistic regression after adjusted by sex , age , and bmi . or : odds ratio ; ci : confidence interval ; bmi : body mass index ; igm : impaired glucose metabolism . the risk alleles ( c and g ) of cdkal1-rs10946398 and adcy5-rs7641344 were associated with impaired beta cell function ( homa - b : p = 0.008 and p = 0.02 ; fins : p = 0.085 and p = 0.05 ) compared with nonrisk - alleles . none of the risk variants was associated with reduced insulin sensitivity [ table 5 ] . effect of genetic variants on insulin secretion and sensitivity data are shown as median ( interquartile range ) , which was stratified by t2 dm risk genotypes . maf : minor allele frequency ; fins : fasting insulin level ; homa - ir : homeostasis model assessment for insulin resistance ; homa - b : homeostasis model assessment for cell function ; t2 dm : type 2 diabetes mellitus . this is the first genetic study on the association between birthweight and glucose metabolism in chinese han population . the major finding in this study is that cdkal1 rs10946398/rs7756992 are associated with reduced birthweight . the c allele of single nucleotide polymorphism ( snp ) rs10946398 is associated with a 41 g reduction in birthweight , common variants both associated with increased risk of igm and decreased insulin secretion index . insulin is important for fetal growth and metabolism throughout life , so variants in cdkal1 might affect pancreatic beta cell function resulting in low - insulin mediated fetal growth and the risk of type 2 diabetes in later life , which is a strong evidence for fetal insulin hypothesis . previous studies found that cdkal1 was a member of methyl sulfur transferase family , expressed in human pancreatic islet , might associated with insulin secretion of pancreatic beta cell induced by cyclin - dependent kinase 5 ( cdk5 ) . the down - regulation of cdkal1 expression might increase the activity of cdk5 , resulting in decreased insulin secretion , while the exact mechanisms are not clear . in the future , we need further study to explore the function of cdkal1 on insulin secretion and glucose metabolism . in the present study , srr - rs391300 is nominally associated with reduced birthweight , carrier of g allele had a trend of birthweight reduction . srr was identified as a special risk gene of type 2 diabetes mellitus in the han chinese other than the other race . basic research showed srr affected glutamate metabolism , followed secretion of the pancreatic beta cell . so srr may be the chinese han population characteristic and potential risk gene of type 2 diabetes associated with birth weight . in the future , further study on this association should be carried on in a large sample number and different races . adcy5 was newly identified as a risk gene of type 2 diabetes mellitus in many large meta - analysis studies . freathy et al . reported a meta - analysis of genome - wide association studies in european followed by replication studies showing that the c allele of rs9883204 in adcy5 was associated with lower birthweight ( p = 7 10 ) , andersson et al . showed the inverse association between risk allele of adcy5-rs9883204/rs7756992 and birthweight in danish inter99 population . according to hapmap , the risk allele frequency of these two snps was 21.7% in european while below 1% in this present study . hu et al . reported a meta - analysis of genome - wide association studies in chinese han population showing that there was no association between risk allele of rs11708067 and risk of type 2 diabetes . hence , it is possible that there are other snps of adcy5 associated with risk of type 2 diabetes in chinese han population . adcy5-rs7641344 may be the choice because the risk genotype was associated with reduced insulin secretion of pancreatic beta cell in this study and rs7641344 is a tag snp of adcy5 , which is in linkage disequilibrium with six snps rs9840967 , rs7614840 , rs6794936 , rs9841543 , rs7616545 , rs7641344 ( hapmap ceu phase iii r= 1.0 ) . so rs7641344 may be the special variants of adcy5 associated with type 2 diabetes in chinese han individuals . further study on this association should be carried on in a large sample number and different races . we confirmed the association between risk of igm and variant in rs1111875 of hhex , which was not associated with fasting insulin , homa - b and homa - ir . in conclusion , this study identified the association between type 2 diabetes mellitus risk variants in cdkal1 and reduced birthweight in chinese han individuals for the first time , and the carrier of risk allele within srr had the trend of reduced birth weight . the polymorphism of adcy5 had significant racial difference , variant of rs7641344 in adcy5 was associated with reduced insulin secretion , which may be the special variants of adcy5 associated with type 2 diabetes in chinese han individuals . our study demonstrated that there was a clear overlap between the genetics of type 2 diabetes and fetal growth , supporting the fetal insulin hypothesis , which proposed that lower birth weight and type 2 diabetes could be two phenotypes of one genotype . hence , we can deeply explore the molecular genetic mechanism of type 2 diabetes in the future .

background : fetal insulin hypothesis was proposed that the association between low birth weight and type 2 diabetes is principally genetically mediated . the aim of this study was to investigate whether common variants in genes cdkal1 , hhex , adcy5 , srr , ptprd that predisposed to type 2 diabetes were also associated with reduced birthweight in chinese han population.methods:twelve single nucleotide polymorphisms ( rs7756992/rs10946398 in cdkal1 , rs1111875 in hhex , rs391300 in srr , rs17584499 in ptprd , rs1170806/rs9883204/rs4678017/rs9881942/rs7641344/rs6777397/rs6226243 in adcy5 ) were genotyped in 1174 unrelated individuals born in peking union medical college hospital from 1921 to 1954 by taqman allelic discrimination assays , of which 645 had normal glucose tolerance , 181 had developed type 2 diabetes and 348 impaired glucose regulation . associations of these 12 genetic variants with birthweight and glucose metabolism in later life were analyzed.results:birthweight was inversely associated with cdkal1-rs10946398 ( = 41 g [ 95% confidence interval [ ci ] : 80 , 3 ] , p = 0.034 ) , common variants both associated with increased risk of impaired glucose metabolism and decreased insulin secretion index later in life . after adjusting for sex , gestational weeks , parity and maternal age , the risk allele of cdkal1-rs7756992 was associated with reduced birthweight ( = 36 g [ 95% ci : 72 , 0.2 ] , p = 0.048 ) . the risk allele in srr showed a trend toward a reduction of birthweight ( p = 0.085).conclusions : this study identified the association between type 2 diabetes risk variants in cdkal1 and birthweight in chinese han individuals , and the carrier of risk allele within srr had the trend of reduced birthweight . this demonstrates that there is a clear overlap between the genetics of type 2 diabetes and fetal growth , which proposes that lower birth weight and type 2 diabetes may be two phenotypes of one genotype .

viruses are the most pervasive biological entities in aquatic ecosystems , the majority being bacteriophages ( suttle , 2007 ) . abundances of planktonic viruses are reported to commonly range between 10 ( likely an underestimation due to bad storage conditions ) and 10 ml , being generally higher in freshwater than in marine systems . peak values were found in very productive estuaries and lakes ( peduzzi and luef , 2009 ) . exceptionally high virioplankton abundance ( up to 2 10 ml ) has been documented in the alkaline , hypersaline mono lake , california ( brum et al . , 2005 ) . typically , virus replication rates increase in conjunction with higher host growth rates ( suttle , 2007 ) . beyond the potential to influence and control abundance and diversity of heterotrophic prokaryote hosts , viruses apparently infect a significant proportion of the photoautotrophic plankton community as well ( wommack and colwell , 2000 ; weinbauer , 2004 ; suttle , 2007 ) . particularly cyanophages have been shown to cause mortality in important members of the phytoplankton ( suttle and chan , 1994 ) . the lesser flamingo , phoeniconaias minor , is the dominating and characteristic bird species of alkaline saline lakes and pans of east africa ( krienitz and kotut , 2010 ) and classified as near - threatened ' ( iucn red list , 2012 ) . the dense population of these pinkish waterfowl ( 1.52.5 million in eastern africa , which is around 75% of the worldwide occurrence ) has been rated since decades as one of the most significant wildlife spectacles worldwide ( jenkin , 1929 ) . flamingos are filter feeders of photoautotrophic algal primary producers , putting them on top of a short and direct food chain ( vareschi and jacobs , 1985 ) . the lesser flamingo relies on the very fast - growing alkaliphilic cyanobacterium arthrospira fusiformis as most important food source . fluctuations of this resource are reported as the overwhelming factor influencing lesser flamingo distribution ( vareschi and jacobs , 1985 ; krienitz and kotut , 2010 ) . however , the typically dense ( up to > 700 mg l fresh weight ; krienitz and kotut , 2010 ) population of a. fusiformis collapses unpredictably . this is thought to be the major cause for episodes of disappearance or significant irregular mass movements of flamingos between the lakes ( krienitz and kotut , 2010 ) . causes for the sudden a. fusiformis breakdowns have remained a matter of speculation . at latest eisenberg 's inspiring book the wolf 's tooth ' ( eisenberg , 2010 ) cemented awareness in the scientific community about how trophic cascades can impact food webs . trophic cascades have been documented for all of the world 's major biomes , in terrestrial and aquatic systems , from the poles to the tropics . for example , the anthropogenically mediated loss of apex consumers is discussed as leading to extensive top - down cascading effects in marine , terrestrial and freshwater ecosystems worldwide ( eisenberg , 2010 ; estes et al . , 2011 ) . whereas top - down forcing is well documented ( shurin et al . , 2002 ) this phenomenon occurs if , for example , a primary producer is removed , resulting in diminished population sizes through the community . the effect of trophic cascades is assumed to be stronger in aquatic than in terrestrial food webs ( shurin et al . , 2002 ) . on the one hand , this may be due to the higher mass - specific biomass production in phytoplankton and benthic algae compared with terrestrial vascular plants ( cebrian , 1999 ) ; on the other hand , herbivores consume on average a threefold greater amount of lentic plankton than plant consumption in terrestrial food webs ( cyr and pace , 1993 ) . the consensus is that cascade effects are more severe with decreasing food web complexity ( strong , 1992 ) . we know that nomadic movements or disappearance of the lesser flamingo are linked to changes in food quantity and quality ( tuite , 1979 ; owino et al . , 2001 ; krienitz and kotut , 2010 ; zaccara et al . , 2011 ) . we hypothesized that viruses ( as most abundant biological entities ) are capable of mediating a bottom - up cascade ( sensu hunter and price , 1992 ) , thus being responsible for the frequent breakdowns in a. fusiformis biomass and , as a consequence , for the drastic drop in flamingo numbers at lake nakuru . we document a first example for such a virus - mediated bottom - up cascade in this short food chain from photoautotrophic prokaryotes to birds . lake nakuru is a shallow , alkaline saline water body situated in lake nakuru national park , kenya . its size covers 44 km , the average depth is 2.5 m , which can reach a maximum level of 4.5 m ( http://www.worldlakes.org/ ) . it also occasionally receives inflows from the three seasonal surface streams njoro , makalia and nderit , as well as from the baharini spring at the northern end . . selected limnological characteristics ( temperature , soluble reactive phosphorus , ph , conductivity , salinity , total alkalinity as well as overall bacterial and virus abundance ) were monitored during the investigation period . further and more detailed information on hydrochemistry and lake characteristics was reported earlier ( oduor and schagerl , 2007 ; jirsa et al . , 2013 ) . sampling was done in the central part of the lake at the coordinates 00 21.387 s , 036 05.519 e. surface samples ( 0.51.0 m ) were taken weekly between 7 january and 27 october 2009 . samples for counting heterotrophic bacteria , viruses and phytoplankton were taken with a schindler trap and immediately fixed with glutaraldehyde to a final concentration of 2% . samples for cyanophage screening within a. fusiformis cells by tem ( transmission electron microscopy ) were collected with a plankton net ( mesh size 30 m ) and fixed as above . water temperature was measured by data loggers ( hobo , onset computer corporation , bourne , ma , usa ) with logging intervals of 5 min . salinity , electrical conductivity and ph were measured on site using a multiprobe ( wtw multi 340i wissenschaftlich technische werksttten weilheim , germany ) . soluble reactive phosphorus was analyzed by a modified standard method ( clesceri et al . , 1998 ) , considering the high buffer capacity of the water . for heterotrophic bacteria and total virus counts , 0.51 ml sample water was filtered within 12 h on whatman anodisc filters ( pore size 0.02 m ; 25 mm diameter ) , stained with sybr green 1 and counted with an epifluorescence microscope ( motic ba 4000 , motic , hong kong , china ) ( noble and fuhrman , 1998 ) . to estimate the a. fusiformis biovolume , filaments were classified into three groups based on their morphology and enumerated and measured under an inverted microscope ( nikon diaphot , nikon , japan ) ( utermhl , 1958 ) . filament dimensions of the morphology groups , which included height of coil , coil diameter and number of coils were measured at 400 magnification . each morphological variable was measured on at least 50 filaments . for total biovolume calculation , the number of filaments was multiplied by the respective filament volume . for more details , see kaggwa et al . a conversion factor for density of 1 was used according to wetzel and likens ( 1991 ) . to screen for visible cyanophage infection ( frequency of visibly infected cells ; proctor and fuhrman , 1990 ; binder , 1999 ) in arthrospira cells , the material was concentrated via gentle centrifugation and then preserved in sodium cacodylate buffer after three rinsing steps with buffer solution ( glauert , 1975 ) . the filaments were then transferred into 2 ml eppendorf vials and stored in the dark at 4 c until further analysis . for tem preparation , the samples were rinsed with a buffer solution to get rid of interfering dissolved organic substances . the residue was then deposited in glutaraldehyde ( 2.5% ) for at least 15 h , rinsed five times ( each 10 min ) with double - distilled water and transferred into osmium tetroxide ( 2% ) for 2 h. after post - fixation and three more rinsing steps ( double - distilled water , each step 20 min ) , dehydration was done in 100% acetone ( two times , 15 min each ) . samples were then transferred into agar low viscosity resin ( agar scientific , stansted , essex , uk ) embedding medium , diluted 1 + 1 with acetone and left for 3 h to facilitate infiltration , which was assisted by a specimen rotator . after transfer into pure embedding medium , the samples were left for 4 h and then the supernatant resin was replaced . the sample was then placed for polymerization at 40 c in a cabinet dryer overnight followed by 24 h at 65 c . the slug was trimmed and ultra - thin sections were performed ( 70 nm ; diamond knife , diatome , biel - bienne , switzerland ; ultramicrotome ultracut e , reichert jung , austria ) . to ensure the observation of different filaments , the slug was trimmed again by additional 25 m after every five sections and another series was cut . ultra - thin sections were placed on copper grids and contrasted with uranyl acetate ( 0.5% ) for 30 min followed by lead citrate ( 3% ) for 7 min . 4068 cells were sliced and the ultrathin sections were inspected for visible signs of infection and photographed . on average , 131.2 cells were sliced per each sampling date when a. fusiformis was present . we also considered areas within the host cells exhibiting disintegrated cytoplasm structure ( figures 2a and b ) , which contained only few or no virus particles as visible signs of infection ( these two categories are distinguished in figure 1a ) . this is based on the observation that visible virus particles were always associated with the same disintegrated cytoplasm structure . ultra - thin sections , however , can eventually cut through the fringe of a viruses - infected area , thus resulting in empty or only few ( < 5 ) virus particle - containing sectors . therefore , we decided to include this information to the results , but distinguished between these two categories . the frequency of visible infected cells is expressed as % of the total number of inspected cells ( proctor and fuhrman , 1990 ; binder , 1999 ) . the development of the lesser flamingo abundance at lake nakuru during the investigation period was followed by estimating total numbers , applying ground - based line transects along the lake shoreline ( lloyd et al . , 1998 ) . lake nakuru is a shallow , alkaline saline water body situated in lake nakuru national park , kenya . its size covers 44 km , the average depth is 2.5 m , which can reach a maximum level of 4.5 m ( http://www.worldlakes.org/ ) . it also occasionally receives inflows from the three seasonal surface streams njoro , makalia and nderit , as well as from the baharini spring at the northern end . . selected limnological characteristics ( temperature , soluble reactive phosphorus , ph , conductivity , salinity , total alkalinity as well as overall bacterial and virus abundance ) were monitored during the investigation period . further and more detailed information on hydrochemistry and lake characteristics was reported earlier ( oduor and schagerl , 2007 ; jirsa et al . , 2013 ) . sampling was done in the central part of the lake at the coordinates 00 21.387 s , 036 05.519 e. surface samples ( 0.51.0 m ) were taken weekly between 7 january and 27 october 2009 . samples for counting heterotrophic bacteria , viruses and phytoplankton were taken with a schindler trap and immediately fixed with glutaraldehyde to a final concentration of 2% . samples for cyanophage screening within a. fusiformis cells by tem ( transmission electron microscopy ) were collected with a plankton net ( mesh size 30 m ) and fixed as above . water temperature was measured by data loggers ( hobo , onset computer corporation , bourne , ma , usa ) with logging intervals of 5 min . salinity , electrical conductivity and ph were measured on site using a multiprobe ( wtw multi 340i wissenschaftlich technische werksttten weilheim , germany ) . soluble reactive phosphorus was analyzed by a modified standard method ( clesceri et al . , 1998 ) , considering the high buffer capacity of the water . for heterotrophic bacteria and total virus counts , 0.51 ml sample water was filtered within 12 h on whatman anodisc filters ( pore size 0.02 m ; 25 mm diameter ) , stained with sybr green 1 and counted with an epifluorescence microscope ( motic ba 4000 , motic , hong kong , china ) ( noble and fuhrman , 1998 ) . to estimate the a. fusiformis biovolume , filaments were classified into three groups based on their morphology and enumerated and measured under an inverted microscope ( nikon diaphot , nikon , japan ) ( utermhl , 1958 ) . filament dimensions of the morphology groups , which included height of coil , coil diameter and number of coils were measured at 400 magnification . each morphological variable was measured on at least 50 filaments . for total biovolume calculation , the number of filaments was multiplied by the respective filament volume . for more details , see kaggwa et al . a conversion factor for density of 1 was used according to wetzel and likens ( 1991 ) . to screen for visible cyanophage infection ( frequency of visibly infected cells ; proctor and fuhrman , 1990 ; binder , 1999 ) in arthrospira cells , the material was concentrated via gentle centrifugation and then preserved in sodium cacodylate buffer after three rinsing steps with buffer solution ( glauert , 1975 ) . the filaments were then transferred into 2 ml eppendorf vials and stored in the dark at 4 c until further analysis . for tem preparation , the samples were rinsed with a buffer solution to get rid of interfering dissolved organic substances . the residue was then deposited in glutaraldehyde ( 2.5% ) for at least 15 h , rinsed five times ( each 10 min ) with double - distilled water and transferred into osmium tetroxide ( 2% ) for 2 h. after post - fixation and three more rinsing steps ( double - distilled water , each step 20 min ) , dehydration was done in 100% acetone ( two times , 15 min each ) . samples were then transferred into agar low viscosity resin ( agar scientific , stansted , essex , uk ) embedding medium , diluted 1 + 1 with acetone and left for 3 h to facilitate infiltration , which was assisted by a specimen rotator . after transfer into pure embedding medium , the samples were left for 4 h and then the supernatant resin was replaced . the sample was then placed for polymerization at 40 c in a cabinet dryer overnight followed by 24 h at 65 c . the slug was trimmed and ultra - thin sections were performed ( 70 nm ; diamond knife , diatome , biel - bienne , switzerland ; ultramicrotome ultracut e , reichert jung , austria ) . to ensure the observation of different filaments , the slug was trimmed again by additional 25 m after every five sections and another series was cut . ultra - thin sections were placed on copper grids and contrasted with uranyl acetate ( 0.5% ) for 30 min followed by lead citrate ( 3% ) for 7 min . 4068 cells were sliced and the ultrathin sections were inspected for visible signs of infection and photographed . on average , 131.2 cells were sliced per each sampling date when a. fusiformis was present . we also considered areas within the host cells exhibiting disintegrated cytoplasm structure ( figures 2a and b ) , which contained only few or no virus particles as visible signs of infection ( these two categories are distinguished in figure 1a ) . this is based on the observation that visible virus particles were always associated with the same disintegrated cytoplasm structure . ultra - thin sections , however , can eventually cut through the fringe of a viruses - infected area , thus resulting in empty or only few ( < 5 ) virus particle - containing sectors . therefore , we decided to include this information to the results , but distinguished between these two categories . the frequency of visible infected cells is expressed as % of the total number of inspected cells ( proctor and fuhrman , 1990 ; binder , 1999 ) . the development of the lesser flamingo abundance at lake nakuru during the investigation period was followed by estimating total numbers , applying ground - based line transects along the lake shoreline ( lloyd et al . , 1998 ) . table 1 presents selected limnological characteristics and overall bacterial and virus abundance of this lake during the investigation period . further , the samples revealed very high numbers of viruses ( total counts of the viral community ) of up to 7.0 10 ml . also heterotrophic bacterial abundance was high ; the virus - to - bacteria ratio fluctuated but remained in the same order of magnitude . during the study period , an increase and a subsequent breakdown of both a. fusiformis biomass and lesser flamingo abundance occurred ( figures 1a and b ) . l in june 2009 . here , at peak phytoplankton biomass , almost 100% of the total algal biomass was comprised by a. fusiformis ( kaggwa et al . , 2013 ) . after this maximum , a. fusiformis biomass dropped again ( with a slight recovery in late june and august ) to undetectable levels in october . during the crash of a. fusiformis , visible signs of cyanophage infection the highest frequency of visible infection ( 24.3% of inspected cells ) occurred at peak abundance of a. fusiformis host cells , which clearly marked the onset of this biomass breakdown . from early june until the complete vanishing of a. fusiformis , almost each sampling revealed a high percentage ( on average 5.9% ) of visible infections ( figure 1a ) . low flamingo numbers of around only 100 individuals were detected at the beginning of the year , followed by an increase in april , reaching a dense population of>1 250 000 in june 2009 ( figure 1b ) . together with the breakdown of the algal biomass , a drastic decrease in flamingo abundance was observed , with a lowest number of 1500 individuals in october . the correlation between a. fusiformis biomass and p. minor numbers was significant ( r=0.70 , p=0.001 , n=38 ; spearman , two - tailed ) . along with its specific chemical and biological features , lake nakuru is also extreme in its virioplankton abundance . to our knowledge , the samples revealed the highest numbers of viruses ever reported in any natural aquatic environment . this strongly supports the significant role that viruses may have in such an environment , for example , as important mechanism of mortality in prokaryotes . very recently , a cyanophage was described that infects arthrospira platensis in mass cultures ( jacquet et al . , 2013 ) . in our study , the frequency of visible infected cells between june and september was high ( compare binder , 1999 ) . considering that only a certain fraction is actually in the visible state of infection ( proctor and fuhrman , 1990 ; binder , 1999 ) , most likely an even larger proportion of the population was infected during this period . a virus - mediated impact on the a. fusiformis host population two viral cycles are important in aquatic habitats , lysogeny and lytic infection , the latter being strongly related to host abundance ( weinbauer , 2004 ) . clearly , the host cell density and the linked infection probability were very high , not only at the june biomass peak . furthermore , when considering induction as a significant mechanism , the transition from a lysogenic state to host cell lysis is often linked to various types of inducing agents . some of these might be of particular importance in the investigated environment , such as sunlight and uv radiation , temperature changes , aromatic compounds and changes in ion contents ( jiang and paul , 1996 ; weinbauer , 2004 ) . water temperature was apparently not important because it fluctuated only moderately during the investigation period ( see table 1 ) . salinity , however , almost doubled during the cyanoprokaryote breakdown from june to october , from 32 to 62. we did not collect data on uv exposure , but it could have been important especially during calm periods , when buoyant a. fusiformis filaments develop scums on the lake surface . in addition , other factors can be responsible for dynamic population oscillations in such a virus host system . there is evidence that the presence of bacteriophages is crucial in generating genetic diversity such as strains and ecotypes ( rodriguez - valera et al . , 2009 ) . for a major component of marine picoplankton ( synechococcus ) , genetic diversity has been linked to interactions with co - occurring phages , making phages determinants for the abundance , decline as well as succession of different clones ( mhling et al . , 2005 ) . such results indicate that both the abundance and genetic diversity of cyanophages co - varied with their hosts . this is consistent with cyanophage infection as a major controlling factor in phytoplankton successions ( suttle and chan , 1994 ; wang and chen , 2004 ; mhling et al . , 2005 ) . we assume that the irregular and unpredictable collapses of a. fusiformis may also be the result of dynamic interactions between resistant and non - resistant host strains and co - occurring , genetically diverse cyanophage populations . from our investigation we conclude that , at lake nakuru , a virus - mediated loss of the most important food source triggered the collapse in p. minor abundance . the consumption activity of the flamingos on a. fusiformis is unlikely to be responsible for the stochastic breakdowns of the algal biomass . at a medium phytoplankton biomass at lake nakuru ( 145 mg l ) , a flamingo population of 1 million birds was calculated to extract up to 60 t algal mass per day ( vareschi , 1978 ; krienitz and kotut , 2010 ) . on the basis of the average lake volume , the entire lake then harbors some 13 10 t of a. fusiformis , thus only 0.46% of the standing stock would be consumed per day . further , a. fusiformis tolerates high alkalinity , salinity and elevated temperatures of > 35 c ( vonshak and tomaselli , 2000 ) . during the investigation period , all these parameters remained , together with soluble reactive phosphorus , within a range that does not limit a. fusiformis growth ( see table 1 ) . moreover , top - down control on the a. fusiformis population by zooplankton was recently excluded as a reason for biomass breakdowns in our study environment ( burian et al . , 2013 ) , whereas a virus - mediated control was not considered in any previous study . on the basis of the decrease of a. fusiformis biomass during 8 days after the biomass peak in mid of june , > 60% of the algal biomass has disappeared , which resembles a mortality rate of 16.3 mg l d. however , this mortality rate is likely even an underestimation as it does not consider the ongoing algal production . under continuing infection pressure in the following period the complete a. fusiformis food biomass vanished from the environment , thus being likely the major cause for disappearance also of the flamingos . our results point to a previously undocumented virus attack on the highly specialized algal diet of the lesser flamingo . moreover , this infection is likely responsible for a collapse in flamingo numbers in a bottom - up cascade . the result is low diversity and short food chains , which are apparently highly susceptible to various kinds of episodic disturbance events like virus attack . we suggest more in - depth investigations of the role of viruses in trophic interactions and cascades , and in different types of food webs . we propose that virus - related processes should be more comprehensively incorporated in ecological theory , thus adding complexity to trophic cascade concepts .

trophic cascade effects occur when a food web is disrupted by loss or significant reduction of one or more of its members . in east african rift valley lakes , the lesser flamingo is on top of a short food chain . at irregular intervals , the dominance of their most important food source , the cyanobacterium arthrospira fusiformis , is interrupted . bacteriophages are known as potentially controlling photoautotrophic bacterioplankton . in lake nakuru ( kenya ) , we found the highest abundance of suspended viruses ever recorded in a natural aquatic system . we document that cyanophage infection and the related breakdown of a. fusiformis biomass led to a dramatic reduction in flamingo abundance . this documents that virus infection at the very base of a food chain can affect , in a bottom - up cascade , the distribution of end consumers . we anticipate this as an important example for virus - mediated cascading effects , potentially occurring also in various other aquatic food webs .

lipomyelomeningocele ( lipommc ) known as a close neural tube defect is caused by the process of dysjunction , in which the closed neural tube detaches prematurely from the overlying ectoderm and paraxial mesenchyme tissue finds access to the neural tube and differentiates to adipose tissue . mostly lipommc occurs as a single lesion but there are some reports that describe lipommc associated with other spinal dysraphism like split cord malformation ( scm ) , syringomyelia , dermoid tumor , neurenteric cyst , and tight filum terminale . since 2008 we have operated 115 patients with lipommc in our department and only one of them had two lipommcs at the same time ( about 0.87% ) . here we report the infant who presented with lumbosacral soft tissue mass which his spinal mri and surgical intervention confirmed two separate kind of lipomas ; distal and dorsal types associated with split cord malformation . a three month old male infant presented with a congenital skin covered soft tissue mass in midline lumbosacral area . there were multiple cutaneous hemangiomatous skin changes over the soft tissue mass . thorough urological evaluation confirmed detrusor sphincter dys - synergy with high pressure bladder according to urodynamic study . spinal mri confirmed scm type ii associated with low lying conus and lipomatous stalk coming from spinal canal defect to the spinal cord and attached to the cord dorsally [ figure 1 ] . ( a ) axial view of spinal mri shows lipomyelomeningocele dorsal type and low level conus . ( b ) axial view confirms scm type ii and lipoma attached to the dorsal aspect of spinal cord surgical procedure was done in prone position with an elliptical incision . there were fascial and bony defects at l4 to s1 levels through which lipomatous tissue was invaginating from surface to the dura mater through that defect . laminotomy above the defect was performed and normal dura mater was exposed and incised . there were two dural defects separated from each other with normal dura mater . the dorsal type lipoma was separated carefully from the neural placode in the spinal canal and then pial edges approximated by 6 - 0 non - absorbable sutures . another lipomatous tissue as a distal type lipoma was in continuity with a short right hemicord exiting through a different dural defect in a more caudal and lateral location of the spinal canal . the left hemicord that was separated from lipomyelomeningoceles and right hemicord had a thick fatty filum terminale that was divided from the attachment to distal dural pouch [ figure 2 ] . intraoperative photography after cord untethering shows the interface of lipoma and cord at dorsal ( thick arrow ) and distal type lipommcs ( thin arrow ) . right and left cords are inside the same dural sac , the thick filum was cut at right side disturbances in the sequence of events of secondary neurulation ( between postovulatory day 25 and 48 ) can lead to lumbosacral lipomas . the development of scms as a result of persistent accessory neurenteric canal , during third week of embryogenesis , has been explained so far . further development of various dysraphic entities in conjunction with scms were seen in the literature , but two secondary neurulation errors in a case of previous defective gastrulation is a very rare condition . association of several embryologically different dysraphic lesions in one case as described in our patient is very rare . all previous reports were female and lipomas were located in lumbar or sacral regions [ table 1 ] . two patients reported before had cloacal extrophy and one of them like our patient had scm ii . the probable pathogenesis for this rare association can be genetic basis that has the potential of involving different embryologic phases or being exposed to multiple teratogenic factors at different time . information related to current case and four previous reports ( references 6789 ) good quality spinal mri can discover multiple lesions in one patient but mri in a young infant especially without a high quality mri like this one may miss the multiple lesions and mri findings become inappropriate to the surgical findings . the importance of knowing multiple lesions in one patient is the correction of associated lesions at the same surgery . ignorance of the accompanied malformations and performing the first surgery without correction of all lesions will result in the patient undergoing another surgery . late surgery inside the previous operative adhesions and scars increases the risk of damage to neural tissue and complications . multiple neural tube defect anomalies in one patient are very rare that can be detected with a good quality spinal mri or during precise surgical exploration . awareness of accompanying lesions in a known case of lipomyelomeningocele is important to correct associated lesions at the same operation which prevents from another surgery which is more risky in the presence of previous operative adhesions .

lipomyelomeningocele , a closed neural tube defect , usually occurs in lumbosacral area as a single lesion but can be associated with other spinal dysraphism . we report an infant with a very rare presentation of tandem lumbosacral lipomyelomeningoceles , thick filum terminale and split cord malformation .

neonicotinoids are a relatively new class of insecticides that share a common mode of action that affect the central nervous system of insects , resulting in paralysis and death . studies suggested that neonicotinoids residues can accumulate in pollen and nectar of treated plants and represent a potential risk to pollinators . the honey italian observatory stated that in 2008 more than half of italian hives , and that 600,000 of a total of 1,100,000 have been put out of production for the depopulation of entire apiaries . one result might be expected given that the previous year , the european food safety authority ( efsa ) stated that the bee die - off had hit the 50% bee population , compared to the annual average of 15% . neonicotinoids can also be persistent in the environment and , when used as seed treatments , translocate to residues in pollen and nectar of treated plants . the potential for these residues to affect bees and other pollinators remains uncertain . despite these uncertainties , neonicotinoids are beginning to dominate the market place because of their high systemicity , the broad spectrum of action , and the reduced dose . in light of these findings , the italian ministry of agriculture has asked the ministry of health to suspend action . the ministry of health , after consultation with the pesticides committee , issued the ministerial decree of september 17 , 2008 that stated the precautionary suspension of the authorized use for the seeds tanning of plant protection products containing the active substances clothianidin , thiamethoxam , imidacloprid , and fipronil . on june 25 , 2012 , a decree of the ministry of health extended to january 31 , 2013 stating the neonicotinoids suspension for seeds treatment . similar measures have already been taken by other european states . recently , many researchers detected these insecticides in honey bees , honey , soil , pollen , and treated seeds for agriculture [ 612 ] . measurement of pesticide residues in different matrices involves two basic steps , namely , sample preparation ( extraction and clean up ) and instrumental analysis . ideally , a sample preparation should be rapid , simple , cheap , and environment friendly and provide clean extracts . . quechers ( quick easy cheap effective rugged safe ) technique , which was developed between 2000 and 2002 and first reported in 2003 , is a fast and complete extraction and clean up procedure and also employs the use of dispersive - solid phase extraction ( d - spe ) for sample clean up . in this paper , we report a rapid modified quechers method for multiresidue analysis for 6 neonicotinoids ( acetamiprid , clothianidin , imidacloprid , nitenpyram , thiacloprid , and thiamethoxam ) in honey with good selectivity , sensitivity , and cost effectiveness . in order to demonstrate the suitability of the method for routine regulatory purposes , the certified analytical standards of all the 6 pesticides ( acetamiprid , clothianidin , imidacloprid , nitenpyram , thiacloprid , and thiamethoxam ) and internal standard tris(1-chloro-2-propyl)phosphate ( tcpp ) were purchased from ultra scientific ( bologna , italy ) ( 100.0 0.5 g / ml each ) in acetonitrile . all the solvents and chemicals used in the study were of analytical reagent ( ar ) grade , ethanol was supplied by romil ( milan , italy ) , and formic acid , ammonium formiate , and acetonitrile were by carlo erba ( milan , italy ) . distilled water was purified at 18.2 m with a milliq ultra ( millipore , vimodrone ( mi ) , italy ) purification system . a mixture of dispersive spe citrate extraction tube supelco ( 4 g magnesium sulphate , 1 g sodium chloride , 0.5 g sodium citrate dibasic sesquihydrate , and 1 g sodium citrate tribasic dihydrate ) was used , supplied by sigma - aldrich ( milan , italy ) . ultra high - performance liquid chromatography uhplc - ms / ms ( thermo scientific , tsq quantum access max ) equipped with thermo hypersilgold column ( 50 mm 2.1 mm , 1.9 m ) was used for quantification of neonicotinoids . the flow rate was 400 l / min , the column temperature 30c , and the injection loop volume 5 l . a binary gradient of 0.05% hcooh and hcoonh4 2 mm in water ( a ) and 0.05% hcooh and hcoonh4 2 mm in ch3oh ( b ) was employed . the mobile - phase gradient was programmed as follows : 0 min , 10% b ; 7 min , 95% b ; 8 min , 95% b ; 9 min , 10% b ; and 10 min , 10% b. mass spectral analyses were performed using an lc - tsq quantum access max operating in the positive ion mode using a h - esi interface . the neonicotinoids and the internal standard tcpp were detected in ms / ms conditions , programming the chromatographic run in srm mode ( selected reaction monitoring ) as reported in table 1 . preliminary tunings were carried out with continuous introduction of a dilute solution of certified standards . flow rate of syringe pump infusion of 5 l / min and the voltages on the lenses were optimized in tsq tune master ( excalibur software ) . the standard mix solution at 5 g / ml of standard pesticides was diluted by transferring 500 l ( 100.0 0.5 g / ml ) into a volumetric flask ( 10 ml , class a certified ) . the standard mix solution at 1 g / ml of standard pesticides was diluted by transferring 100 l ( 100.0 0.5 g / ml ) into a volumetric flask ( 10 ml , class a certified ) . the standard mix solution at 0.1 g / ml of standard pesticides was diluted by transferring 200 l of solution at 5 g / ml into a volumetric flask ( 10 ml , class a certified ) . the specificity of the analytical method for neonicotinoids detection was confirmed by obtaining positive results from honey containing the analyte , coupled with negative results from samples which do not contain it ( negative controls ) . the matrix effect was assessed by preparing pesticide standards in blank matrix extracted from untreated honey . the matrix extracts were analyzed before spiking to confirm the absence of the test pesticides in them . the quantification of pesticide was based on a six - point matrix - matched calibration graph by plotting the detector response ( srm area ratio with respect to internal standard tcpp ) against concentration of the calibration standards within the range 150 g / a linear regression of six calibration points for each component was used to determine the relationship with the analyte concentrations calculated for each component on the basis of their occurrence in the reference material . the regression equations with slope , y - intercept , and coefficient of correlation ( r ) were evaluated for acetamiprid , clothianidin , imidacloprid , nitenpyram , thiacloprid , and thiamethoxam . statistical test ( mandel and residual analysis with normal distribution of the calibration points ) loq was estimated by the response of method noise level by approximately ten and lod is , therefore , 3.3-fold lower . method recovery studies were performed at two spiking concentration levels ( 10 g / kg and 40 g / kg ) . the sample matrix was prepared by homogenizing a series of different honeys in order to develop a highly specific method . the samples were prepared by weighing 5.0 0.5 g of honey spiked in 50 ml tube ( meus srl , piove di sacco ( pd ) , italy ) . these sample tubes were vortexed ( velp , usmate ( mb ) , italy ) for 30 seconds after adding 10 ml of water and 10 ml of acetonitrile , in order to homogenize and fluidize the sample , and 50 l of tris(1-chloro-2-propyl)phosphate ( tcpp ) at 50 mg / l . in each tube was added a mixture of salts ( 4 g magnesium sulphate , 1 g sodium chloride , 0.5 g sodium citrate dibasic sesquihydrate , and 1 g sodium citrate tribasic dihydrate ) . the extract was stirred for 1 minute in vortex , in order to maximize the distribution of the analytes in the organic phase . the samples were centrifuged at 3000 rpm for 5 minutes and the supernatant was filtered at 0.45 m ptfe filters ( vwr , milan , italy ) . the extract was analyzed by uhplc - ms / ms , making 6 replicates for each concentration . the average percentage of recovery and the relative standard deviation ( rsd , repeatability ) were evaluated . combined uncertainty in estimation was determined for all the neonicotinoids at the two fortification levels studied ( 10 and 40 g / kg ) as the statistical procedure of the eurachem / citac guide cg 4 . as the uncertainty of standard concentration declared in the supplier 's certificate was given without any confidence level , rectangular distribution was assumed for calculating standard uncertainty ( 1)u1= u(x)/c(x)3 , where u(x ) represents the uncertainty value given in the certificate and c(x ) the concentration of the standard solution . the relative uncertainty due to honey weighing was calculated using normal distribution given by ( 2)u2=(0.00005)wi , where wi is the weight of the sample , and 0.00005 is the value of uncertainty of the balance at 95% confidence level as reported in the certificate . uncertainty associated with the calibration curve , was calculated according to ( 3)u3= ( sb1 ) ( { 1p}+{1n}+ { ( c0c)2sxx})1/2 , where s is the standard deviation of the residuals of the calibration curve , b1 is the slope of the calibration curve , p is the number of measurements of the unknown , n is the number of points used to form the calibration curve , c0 is the calculated concentration of the analyte from the calibration curve , c is the arithmetic mean of the concentrations of the standards used to make the calibration curve , and sxx is calculated as given in ( 4)sxx=(cj c)2 , where j = 1 , 2 , , n. cj is the concentration of each calibration standard used to build up the calibration curve . the random errors of extraction , clean up , and uhplc analyses steps were approximated by standard deviations which were calculated from repeated determinations of analytes expressed as repeatability . the precision was calculated according to ( 5)u4 = s(nx ) , where s is the standard deviation of the results obtained from the recovery study , n is the number of assays and x is the mean value of the concentration recovered . the volume of the solution is subject to 3 sources of uncertainty : calibration , repeatability , and temperature effects . ( a ) calibration : the uncertainty in the certified internal volume of the flask and of the pipettes . for example , the manufacturer gives a volume of 10.00 0.02 ml ( v a ) for the flask , when measured at a temperature of 20c . because the value of the uncertainty is given without a confidence level or distribution information , an assumption is necessary . in this work , the standard uncertainty is calculated by assuming a triangular distribution according to ( 6)u5 = ( a/3)v . in the same way , the volumes of the pipettes used to prepare the solutions at different levels are calculated by assuming a triangular distribution . the contributions due to the dilution operations performed for each concentration level are calculated separately and combined to give the standard uncertainty of the volume . ( b ) repeatability : the uncertainty due to variations in filling is considered in the repeatability experiments . ( c ) temperature : the temperatures of the flask and solution differ from the temperature at which the volume of the flask was calibrated . according to the manufacturer , the flask was calibrated at a temperature of 20c , whereas the laboratory temperature varies by 2c . the uncertainty from this effect can be calculated from the estimate of the temperature range and the coefficient of the volume expansion . in the case of acetonitrile as a solvent , this effect is negligible . the combined uncertainty ( u ) was calculated as = x[(u1 + u2 + u3 + u4 ) ] , where cx is the mean neonicotinoids concentration , and reported as expanded uncertainty ( 2u ) which is twice the value of the combined uncertainty at 95% confidence level . in order to identify the major species produced in collisional experimental fragmentation of ms / ms analysis , a mass characterization study was firstly performed for direct infusion of each investigated neonicotinoids . mass scans in positive ions mode were performed with h - esi source ionization ; all investigated molecules showed a good fragmentation . the collision energy was modulated from 5 to 50 of instrumental maximum to obtain the better fragmentation pattern . the esi spectrum is characterized by the parent ion [ m + h ] for all molecules . the neutral losses of no2 and/or hcl were observed for clothianidin , imidacloprid , nitenpyram , and thiamethoxam . the fragment at m / z 126 , corresponding to [ c6h5-ocl ] was a characteristic for acetamiprid , nitenpyram , and thiacloprid ( table 1 ) . the discussed srm data were in agreement with what reported by sabatino et al . and ferrer et al . the chromatographic method has been developed on the results of preliminary studies carried out on matrix - fortified standards . the best results were obtained using an elution gradient starting with a binary gradient of 0.05% hcooh and hcoonh4 2 mm in water and 0.05% hcooh and hcoonh4 2 mm in ch3oh combined with the thermo hypersil gold 50 2.1 mm ( 1.9 m i.d . ) column . under the described chromatographic conditions , the studied molecules were resolved in less than 5 minutes ( figure 1 ) and well recognizable on the basis of m / z signals , and good sensitivities were obtained ; each analyte showed a typical mass spectrum profile previously identified by direct infusion . the concept of a single extraction and dilution of the extracts was chosen in this study to achieve good results in the shortest time . in 2011 , tanner and czerwenka applied two steps of purification with d - spe applying the quechers methodology to the honey . our protocol eliminated the second purification step , limiting the extraction to the use of d - spe citrate extraction tube and reducing times and costs of analyses . nevertheless , results were satisfactory in terms of statistical parameters , the selectivity for the analytes of interest , and reduction of the matrix effect ( see paragraph below ) . this protocol permitted to analyze a high number of samples per day and is , therefore , suitable for a routine application in control laboratories . the proposed analytical protocol is currently applied in icqrf catania laboratory in the frame of italian ministry quality control investigation . analytical parameters of the proposed method were evaluated according to the criteria given in section 2 . the linearity of each pesticide was established by plotting uhplc response area ratio versus concentration . the analytes showed linear behavior in the studied concentration range of 150 g / l . the correlation coefficient ( r ) was found to be 0.995 for all pesticides . lod and loq were estimated as the lowest concentrations of pesticide injected that yielded a signal / noise ratio of 3 and 10 , respectively . loqs evaluation showed the lowest value 0.10 g / kg for thiacloprid to the higher value of 4.00 g / kg for nitenpyram . the loqs attained in the proposed method fit with maximum residue limits ( mrls ) of 10 g / kg for nonallowed pesticides . the single - step extraction method adopted for honey samples provided satisfactory recovery which ranged from 75% ( nitepyram ) to 114% ( imidacloprid ) for the fortification level of 10 g / kg and from 92 ( thiacloprid ) to 109% ( imidacloprid ) for the fortification level of 40 g / kg . the precision of the method was good , not exceeding a coefficient of variation of 12% , with the exception of nitenpyram at the lowest fortification level . therefore , the method could be considered sufficiently accurate and precise for the purpose . the study of uncertainty was performed at 2 concentration levels ( 10 and 40 g / kg ) , identifying and studying the most important parameters that determined the uncertainty of the analytical method . the parameters selected were point calibration , standard solution , weigh , volume , and precision ; their contributions to method uncertainty were calculated as indicated in the experimental section . the different contributions of uncertainty for each concentration level , together with the relative combined standard uncertainty , are shown in tables 3 and 4 for each neonicotinoid . results showed that the contribution to uncertainty due to the dilution operations and the standard purities was constant for each concentration level and for each analyte . the same value of uncertainty concerning the amount of weighed sample was used for each level and for all pesticides because the quantity of analyzed sample did not change among the experiments ; moreover , this contribution could be considered negligible . the uncertainty associated with repeatability has a moderate contribution to the expanded uncertainties , showing the higher value for nitenpyram , thiamethoxam , and clothianidin . the 10 g / kg level showed the uncertainty of calibration point as the main constituent of total uncertainty , followed by the volume contribution . on the contrary , the volume uncertainty was the major source to total uncertainty at the 40 g / kg level , while the uncertainty of repeatability and calibration point had approximately similar values . when the uncertainty of the result is reported , the combined standard uncertainty is multiplied with a so - called coverage factor , yielding an expanded uncertainty . a factor k = 2 was used because of the resemblance of the expanded uncertainty to a 95% confidence interval . sanco/12495/2011 recommended a default expanded uncertainty of 50% to be used by regulatory authorities in cases of enforcement decisions ( mrl exceedances ) . our results showed a relative uncertainty ( u% ) ranging from 21 ( thiamethoxam ) to 49% ( acetamiprid ) at levels of 10 g / kg . lower values were obtained for the 40 g / kg level . at this level ,

rapid and reliable multiresidue analytical methods were developed and validated for the determination of 6 neonicotinoids pesticides ( acetamiprid , clothianidin , imidacloprid , nitenpyram , thiacloprid , and thiamethoxam ) in honey . a modified quechers method has allowed a very rapid and efficient single - step extraction , while the detection was performed by uhplc / ms - ms . the recovery studies were carried out by spiking the samples at two concentration levels ( 10 and 40 g / kg ) . the methods were subjected to a thorough validation procedure . the mean recovery was in the range of 75 to 114% with repeatability below 20% . the limits of detection were below 2.5 g / kg , while the limits of quantification did not exceed 4.0 g / kg . the total uncertainty was evaluated taking the main independent uncertainty sources under consideration . the expanded uncertainty did not exceed 49% for the 10 g / kg concentration level and was in the range of 1619% for the 40 g / kg fortification level .

thus the detection of sige , in addition to obtaining a clinical history and skin prick testing ( spt ) , is important for allergy workup . later on , the enzyme allergosorbent test and the reversed allergosorbent test have been used for the detection of sige [ 2 , 3 ] . in recent years , rapid assays for sige detection as point - of - care diagnostics have been developed using various strategies [ 46 ] . the objective of our study is the evaluation of a scanner based system for the semiquantitative interpretation of allergy lateral flow assay ( alfa ) results . this includes the comparison of alfa results to results obtained from established laboratory methods ( allerg - o - liq , dr . fooke laboratorien , neuss , germany ; immunocap , phadia , uppsala , sweden ) and spt ( g6 , from allergopharma , germany ) for the detection of allergic sensitizations to timothy grass pollen ( g6 ) using a novel scanning system . participants in this study ( n = 50 ) were tested by spt and obtained sera were assayed for sige to g6 by allerg - o - liq , a reverse type , quantitative , who 75/502 calibrated immunoassay , immunocap , and alfa according to the instructions for use . specific ige values > 100 iu / ml were considered as 100 iu / ml . alfa results were also quantified by a novel scanning software allowing for a barcode - directed recognition of the individual lateral flow cassette in combination with a commercially available desktop scanner ( plustek , cerritos , ca , usa ) . this software measures the intensity of the colour of the test line and evaluates the validity of the test run by measuring the existence and intensity of the control line . interassay variation coefficients were determined by consecutively assaying two sera five times using alfa cassettes . the intensities of the resulting test and control bands were determined by the scanning software . the spt for assessment of individual cutaneous sensitivity to g6 was performed using 1 mm single - peak lancets and g6 extract from allergopharma ( reinbek , germany ) . histamine dihydrochloride at a concentration of 1 mg / ml ( allergopharma ) served as positive control and pure saline solution ( 0.9% ) as negative control . test reactions were read after 15 minutes , surrounded by a marker pen and documented with a strip of tape . all tests with a weal diameter smaller than 3 mm elicited by histamine or with a weal diameter of more than 2 mm by the negative control were considered inconclusive . the mean diameters of allergen - induced weals were calculated from the sum of the largest measurement across the weal and the largest weal measurement perpendicular to this divided by two . the allergen specific reaction in the skin test mean age of the patients was 29.8 years ( 1867 years ) , 22 were female and 28 male . spearman correlation was used to analyse quantitative agreement between methods and cohen 's kappa for qualitative agreement ( analyse - it for ms excel ) . 35/50 ( 70% ) test persons were positive by spt , and the same number and identity of sera showed ige reactivity by allerg - o - liq and immunocap . mean values and standard deviations were found at 21.8 ku / l/21.9 ( immunocap ) , 45.0 iu / ml/38.3 ( allerg - o - liq ) , and 10.2 alfa units/10.9 ( alfa ) . excellent agreement was observed between alfa results , spt , immunocap , and allerg - o - liq . high reproducibility was observed for the alfa system ; interassay coefficients of variation varied between 8.7% and 12.4% , depending on the respective serum ige titer ( data not shown ) . when alfa was analyzed in the context of the spt result , the area under the curve ( auc ) value was found at 1.0 . at a cut - off value of 1.4 alfa units 100% sensitivity and specificity titers of sige to g6 were significantly higher in the group of spt positive patients ( p < 0.0001 ; see table 1 ) . quantitative agreements according to spearman were found at 0.94 ( confidence interval , ci = 0.890.96 ; alfa versus immunocap ) , 0.94 ( ci = 0.900.97 ; alfa versus allerg - o - liq ) and at 0.94 ( ci = 0.900.97 ; allerg - o - liq versus immunocap ) ( see figure 1 ) . in recent years , sige screening and profile tests have been developed using different protocols . in 2004 , comparison between allerg - o - liq and the immunocap system showed good agreement for inhalant allergens and moderate agreement for food allergens . in a recent study , alfa has been evaluated by visual result interpretation and found in good qualitative agreement to spt ( 90.8% ) , to immunocap ( 96.7% ) , and to allerg - o - liq ( 98.3% ) . the diagnostic sensitivity and specificity compared to immunocap was 98.2% and 100% , respectively , when samples > 0.7 ku / l were considered . immunocap rapid ( icr ) , which is also based on lateral flow technology , has been shown to yield results that are concordant with clinical diagnosis . the diagnostic sensitivity of icr for g6 was 52.4% ( ci = 29.8%74.3% ) and the specificity was 99.0% ( ci = 96.3%99.9% ) . no information was provided about the titer distribution of specific ige determined by immunocap in the cohort under investigation and thus a direct comparison of the assay performance was not feasible and requires a comparative study between both rapid tests . technically , the major difference between alfa and icr is that alfa utilizes liquid allergens while icr employs allergens immobilized on membranes . noteworthy , the results obtained with a simple rapid assay ( alfa ) that has a significantly different test architecture and works without a calibration system , are in excellent quantitative agreement to technically advanced laboratory assays such as the immunocap and the allerg - o - liq . although spt is known as a reliable method for allergy diagnosis , it has some drawbacks , including nonspecific reactions in subjects with urticarial dermographism , inconclusive results in case of drug intake with antihistaminic activity , and serious side effects in rare cases [ 9 , 10 ] . therefore , rapid allergy tests may represent a promising alternative to spt , which needs to be verified in further studies . alfa is available as a doctor 's office test and we conclude that the novel scanner based system represents a useful tool for the interpretation of alfa results meeting the growing demand for digital documentation of laboratory results . further studies with more complex allergens ( e.g. , food allergens ) are mandatory to verify the general applicability and reliability of the alfa test system .

type i hypersensitivity is driven by allergen specific immunoglobulin e ( sige ) and thus sige represents a marker for modern allergy diagnosis . recently , a rapid assay for the detection of sige , termed as ( allergy lateral flow assay ) alfa , has been developed . the objective of our study is the evaluation of a scanner - based system for the semiquantitative interpretation of alfa results . agreement to skin prick test ( spt , allergopharma ) , allerg - o - liq system ( dr . fooke ) , and immunocap ( phadia ) was investigated using 50 sera tested for specific ige to timothy grass pollen ( g6 ) . 35/50 sera were positive by spt , allerg - o - liq , and immunocap . excellent agreement was observed between alfa results and spt , immunocap , and allerg - o - liq . area under the curve ( auc ) values were found at 1.0 , and 100% sensitivity and specificity was found versus all other methods . visual- and scanner - based interpretation of the alfa results revealed excellent agreement .

about two to three million cases of non - melanoma cancer and 132,000 melanoma cancers are reported annually worldwide . a staggering mortality rate of 75% is reported in the us alone due to skin cancer melanoma when compared to non - melanoma skin cancers , . an average increase of 2.6% deaths caused due to melanoma has been observed annually in the past decade . cases where early detection of skin cancer melanoma is achieved , the costs incurred towards treatments are fairly low and a five year survival rate of 95% is reported . the cost incurred towards the treatment of advance cases of melanoma is very high and the five year survival rate is only 13% . early detection of skin cancer melanoma is a challenging problem and requires attention . to diagnose and study the skin lesions dermatologists use the dermoscopy technique also referred to as surface skin microscopy , dermatoscopy and epiluminescence microscopy ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ elm ) $ \end{document } . the dermoscopy technique adopted is non - invasive and is generally performed by expert dermatologists . dermoscopy is performed by the application of a gel on the skin lesion , then digital imaging systems like stereomicroscope or dermatoscope are used to obtain magnified images . magnified skin lesion images provide additional color , structure and pattern data not clearly visible to the naked human eye . this additional data enable the dermatologists to identify the type of skin lesion and aid the diagnosis . the use of classical clinical algorithms such as abcd ( asymmetry , border , color and diameter ) , abcde ( asymmetry , border , color , diameter and evolution ) , menzies method and the seven - point checklist is adopted by for the diagnosis of melanoma skin lesions . an improvement of 530% is achieved by using dermoscopy and classical clinical algorithms when compared to the examination carried out by the naked human eye . the skill of the dermatologists is also critical to achieve accurate diagnostic performance considering dermoscopy images , . considering the varied type of melanoma , non - melanoma skin lesions and dependency on the skill level of dermatologist , accurate diagnosis of melanoma is still a problem . availability of advance image processing techniques and decision making mechanisms to build computer aided diagnostic system can provide a wholistic solutions to aid early diagnosis of skin cancer melanoma . the computer aided diagnostic systems are also referred to as computerized dermoscopy . computerized dermoscopy systems primarily constituted of five components a ) dermoscopy image acquisition of skin lesions , b ) region of interest identification or segmentation of skin lesion , c ) feature extraction d ) feature selection and e ) decision making mechanisms achieved through machine learning techniques . numerous studies published lay emphasis on the segmentation or region of interest identification . several computerized dermoscopy systems to have been developed considering all or combinations of shape , texture , color features and incorporating varied decision support mechanisms , . to the best of our knowledge , little or no emphasis is laid so far on depth estimation and 3d reconstruction of skin lesions from 2d dermoscopic images . considering the depth and 3d geometry of the skin lesion is critical to achieve accurate diagnosis . surface illumination based dermoscopy techniques are used to for image acquisition as they are inexpensive and easily available . dermoscopy techniques like nevoscopy , trans - illumination light microscopy , high - frequency ultrasound , acoustic microscopy and 3d high - frequency skin ultrasound images , to name a few are considered by researchers to construct 3d volumes and estimate the depth of skin lesions for accurate diagnosis . the availability and the cost of these special dermoscopy imaging systems is still a problem . a noninvasive computerized dermoscopy system to aid diagnosis of skin lesions is proposed in this paper . an adaptive snake model for segmentation of the 2d dermoscopic skin lesion images . to reconstruct the 3d skin lesion initially a depth map the depth map data is fit to the 2d surface to achieve 3d skin lesion reconstruction . the 3d skin lesion is represented as structure tensors . using the 2d skin lesion data color , texture and 2d shape features the 3d reconstructed skin lesion data is used to obtain the 3d shape features . the 3d shape features encompass the relative depth features estimated . to highlight and study the significance of the features , feature selection methods are considered . for decision making , three different multiclass classifiers have been considered and their performance is compared and studied . the proposed computerized dermoscopy system relies on bag - of - features ( bof ) , adaboost and support vector machines ( svm ) for decision making . good classification results considering melanoma skin lesions ( especially in - situ melanoma ) achieved early in the research motivated authors to further expand the diagnosis on varied skin lesion types . experimental study is conducted using dermoscopic images obtained from atlas of dermoscopy cd , ph2 dataset and isic : melanoma project dataset , . the main contributions of the study is summarized as follows 1.3d reconstruction from 2d dermoscopic images using depth estimation.2.3d shape features considering the 3d lesion constructed.3.considering different algorithms for multiclass decision making.4.comprehensive skin lesion data considered in the study namely melanoma , in - situ melanoma , atypical nevus , common nevus , basal cell carcinoma , blue nevus , dermatofibroma , haemangioma , seborrhoeic keratosis and normal mole lesions . 3d shape features considering the 3d lesion constructed . considering different algorithms for multiclass decision making . comprehensive skin lesion data considered in the study namely melanoma , in - situ melanoma , atypical nevus , common nevus , basal cell carcinoma , blue nevus , dermatofibroma , haemangioma , seborrhoeic keratosis and normal mole lesions . the signifance of depth and the various stages of skin cancer melanoma is discussed in section two . melanoma is typically a type of skin cancer . of all types of skin cancer known , melanoma is the deadliest type and the highest mortality rates are reported from patients suffering from melanoma . melanoma cancer occurrences are predominantly reported in the skin but occurrences in the eyes , nasal passages , throat , brain etc . is considered . to diagnose melanoma of the skin a physical examination by a dermatologist and a biopsy post confirmation , the doctors proceed to identify the stage of the melanoma skin cancer to initiate the relevant treatment . stages of melanoma are described through various scales like clarke scale , breslow scale , tumor node and metastases ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ tnm ) $ \end{document } scales . the clarke and breslow scale basically define the measure of the depth of the tumor i.e. how deep the tumor has gone into the skin . the t stages of melanoma defined by cancer research uk to measure type and size of primary tumor and the is shown in fig . 1 . figure 1.anatomy of the skin ( a ) and t stages of melanoma ( b ) . based on the depth of the primary tumor the stage of melanoma is identified . ( note : depth and dimension of tumor may vary from case to case . figure only intends to highlight the significance of tumor depth in melanoma diagnosis ) ( source : cancer research uk ) . anatomy of the skin ( a ) and t stages of melanoma ( b ) . based on the depth of the primary tumor the stage of melanoma is identified . ( note : depth and dimension of tumor may vary from case to case . figure only intends to highlight the significance of tumor depth in melanoma diagnosis ) ( source : cancer research uk ) . in fig . 1(b ) , tis represents an initial stage of melanoma and the tumor is on the epidermis ( i.e. top layer of the skin ) . the primary tumor is of size t1 if the depth is less than 1 mm and is still in the epidermis . primary tumor is of size of t2 when it has grown into the dermis of the skin and its depth ranges from 1 mm to 2 mm . size of the tumor is t3 if its measured depth is 2 mm to 4 mm thick and is still localized to the dermis . when the growth depth of a primary tumor is greater than 4 mm and is beyond the dermis then it is said to be of t4 size.based on how far the cancer is spread and the size of the tumor melanoma cancer is classified into five stages , . stage 0:it is the initial stage also referred to as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ in situ melanoma $ \end{document}. occurrences of abnormal melanocytes are observed in the top layer of the skin . melanoma detected in this stage is 100% curable.stage 1:the tumor in this stage has spread into the skin but limited to the epidermis layer . the tumor growth depth is between 1 mm to 2 mm and can exhibit ulceration ( i.e. breakage of the skin ) . at this stage through surgical procedures the patients can be cured . two sub classes i.e. 1a and 1b are considered based on the depth of the tumor.stage 2:melanoma tumor is 2 mm to 4 mm in size and can exhibit ulceration . no spread to lymph nodes or other parts of the body . sub classes include 2a , 2b and 2c based on the depth and the ulceration . cure is possible through surgical procedures.stage 3:tumor is more than 4 mm deep and can exhibit ulceration . cancer is spread to the lymph nodes but is still localized . advance surgery and post - surgical care required . sub classes include 3a , 3b and 3c.stage 4:the tumor is more than 4 mm deep and has spread to other organs and lymph nodes . treatment at this stage is expensive and life threatening as the cancer has spread from its primary tumor site . it is the initial stage also referred to as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ in situ melanoma $ \end{document}. occurrences of abnormal melanocytes are observed in the top layer of the skin . the tumor in this stage has spread into the skin but limited to the epidermis layer . the tumor growth depth is between 1 mm to 2 mm and can exhibit ulceration ( i.e. breakage of the skin ) . at this stage through surgical procedures the patients can be cured . two sub classes i.e. 1a and 1b are considered based on the depth of the tumor . melanoma tumor is 2 mm to 4 mm in size and can exhibit ulceration . sub classes include 2a , 2b and 2c based on the depth and the ulceration . sub classes include 3a , 3b and 3c . the tumor is more than 4 mm deep and has spread to other organs and lymph nodes . treatment at this stage is expensive and life threatening as the cancer has spread from its primary tumor site . it is clear that the depth of the tumor is a critical parameter for diagnosis and identification of the cancer stage . early detection of melanoma ( stage 0 and stage 1 ) is the solution to reduce mortality rates amongst patients suffering from melanoma skin cancer . in the research work presented here a computerized dermoscopy system to aid early detection of melanoma identification of the skin lesion or region of interest in dermoscopic images is achieved through segmentation procedures . in type two fuzzy logic illumination correction coupled with texture based segmentation technique is presented in . the mimicking expert dermatologist s segmentation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ meds ) meds technique requires low computation resources and provides accurate segmentation results that concur with the segmentation carried out by dermatologists . a comparison considering gradient vector flow , level set , expectation - maximization level set , adaptive thresholding , fuzzy - based split - and - merge algorithm and adaptive snake segmentation techniques is reported by silveira et al . . of all the techniques considered the adaptive snake and the expectation - maximization level set technique exhibit the best segmentation performance . adaptive snake segmentation technique is considered in the proposed computerized dermoscopy system as low execution time and higher segmentation accuracy is reported ( when compared to the expectation - maximization level set technique ) in . researchers have suggested numerous works to aid early detection of melanoma considering dermoscopic images . a detailed survey can be obtained from . in an inspection system to identify clark nevi and malignant melanoma from pigmented skin lesions is discussed . threshold based segmentation algorithm based on otsu s algorithm is considered . shape color and texture features are extracted and binary classifiers are considered to identify the two classes of dermoscopic images considered . in , using supervised mechanisms , lesion features and maximum a posteriori ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ map ) $ \end{document } technique , a segmentation technique is proposed . hair removal techniques using histograms of oriented gradient ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ hog ) $ \end{document } features is also discussed . a classification mechanism is used to identify the presence of pigment network in skin lesions . the classification is achieved considering gaussian and laplacian of gaussian ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ g - log ) $ \end{document } features . to identify two classes of skin lesions ( malignant and benign ) consider texture , shape and novel boundary features in the spatial and frequency domains . classification is achieved using support svm , hidden naive bayes ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ hnb ) $ \end{document } , random forest tree ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ rf ) $ \end{document } and logistic model tree ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ lmt ) $ \end{document}. a multi parameter extraction and classification system ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ mpecs ) $ \end{document } is proposed to detect early melanoma or in situ melanoma in . a six phase approach is adopted to extract the color , texture and shape features . classification of three skin lesion types , namely advanced melanoma , non - melanoma , early melanoma is achieved through neural networks ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ nn ) $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm $ \end{document } classifiers . highlight the importance of detecting irregular streaks in dermoscopic images to accurately diagnose melanoma . a simple logistic classifier is used to identify the absence / presence ( regular and irregular ) of streaks in dermoscopic images . the significance of color features to classify skin lesions is put forth in . a k - means clustering algorithm the congenital nevi , combined nevi , reed / spitz nevi , melanomas , dysplastic nevi , blue nevi , dermal nevi , seborrheic keratosis and dermatofibroma lesion images are considered for evaluation . using a symbolic regression algorithm the skin lesion are classified into benign or malignant types . the parameters estimated from the model are considered as the features of the skin lesion . classification is performed to identify the globular , reticular and homogeneous patterns in the pigmented cell . saz et al . have obtained the dermoscopic images from interactive atlas of dermoscopy . based on high - level intuitive features ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ hlif ) $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm $ \end{document } classifiers the diagnosis of melanomas and non - melanoma skin lesions is presented in . in addition to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ hilf $ \end{document } features , low - level features and their combinations are also considered . in a novel equation to compute the exposure time for skin to burn is introduced a threshold based segmentation , hair detection and removal techniques is considered as the preprocessing steps in the image analysis module . shape , color and texture features are extracted to define the skin lesion images . a two level \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm $ \end{document } classifier is used to identify the benign , atypical and melanoma moles from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ph2 $ \end{document } dataset . the importance of considering global and local features in computer aided diagnosis methods is discussed in . use of color and texture features ( global and local ) to identify melanoma and non - melanoma images from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ph2 $ \end{document } dataset is presented . the use of , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm $ \end{document } , adaboost and bof classifier is adopted for decision making . based on the literature reviewed it is observed that limited work is carried out considering 3d reconstruction , depth estimation and 3d shape features of skin lesions which is critical to diagnose melanoma skin cancer . the state of art works carried out so far predominantly consider only binary decision making mechanisms . in this paper , authors consider 3d reconstruction of skin lesion images to estimate depth of the tumor and adopt multiclass decision making mechanisms . the main objective of the proposed system is to aid early detection of melanoma , especially in - situ melanoma . additionally , the proposed system can also be adopted to diagnose different skin lesions types . segmentation is performed obtain the region of interest or skin lesion to be diagnosed . depth map is used in constructing a 3d model corresponding to the dermoscopic image . a comprehensive feature set considering the 2d shape , 3d shape , color and texture are extracted per image . a feature selection method to understand the significance of features extracted on decision making the proposed system considers a multiclass classification mechanisms for decision making , enabling its applicability to diagnose a wide variety of skin lesion images . dermoscopic images used for evaluation are obtained from cd - rom of dermoscopy and ph2 dataset . figure 2.overview of the proposed computerized proposed computerized dermoscopy system for skin lesion classification . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { i}= \left \ { { i_{0},i_{1 } , \cdots i_{n } } \right \ } $ \end{document } represent a set of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n $ \end{document } dermoscopic images . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { c}= \left \ { { c_{0},c_{1 } , \cdots c_{m } } \right \ } $ \end{document } represent a set of classes of the dermoscopic images i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \forall i_{x}\in \mathbb { i } : i_{x}\in c_{x } $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c_{x}\in \mathbb { c } $ \end{document}. the set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { i } $ \end{document } consists of images used for training and testing . each image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{n } $ \end{document } is represented by a feature set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{n } $ \end{document}. the training data is represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { r}= \left \ { { \left ( { f_{1},c_{1 } } \right ) , \left ( { f_{2},c_{2 } } \right ) , \cdots \left ( { f_{r},c_{r } } \right ) } \right \ } $ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c_{r}\in \mathbb { c } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{r } $ \end{document } represents the features of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r^{th } $ \end{document } image from the set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { i } $ \end{document}. similarly the testing data vector can be defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { t}= \left \ { { \left ( { f_{1},c_{1 } } \right ) , \left ( { f_{2},c_{2 } } \right ) , \cdots \left ( { f_{t},c_{t } } \right ) } \right \ } $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c_{t } $ \end{document } represent the unknown classes and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{t } $ \end{document } represent a set of features extracted from the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t^{th } $ \end{document } image whose class is to be identified . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d } $ \end{document } represent a decision making mechanism such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d}\left ( { f_{r } } \right ) = c_{r } $ \end{document}. the class identified i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c_{t } $ \end{document } in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { t } $ \end{document } is the diagnostic output of the proposed computerized dermoscopy system . the goal of the proposed computerized dermoscopy system can be defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mathbb { d}\left ( { f_{t } } \right ) = c_{t } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{t } $ \end{document } represents the features of the image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{t}\in \mathbb { i } $ \end{document } that needs to be diagnosed . from ( 1 ) it is clear that a robust feature extraction technique aiding the decision making mechanism \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d } $ \end{document } is to be developed . the features extracted consider the 2d shape , 3d shape , color and texture information of the skin lesions . identification of the skin lesion and 3d construction are also considered as the sub problems that need to be solved . identification of the normal skin and skin lesion is critical to accurately extract features . the proposed system considers an adaptive snake ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as ) $ \end{document } segmentation technique to identify skin lesion regions in a set of images \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { i } $ \end{document}. literature presented in proves the accuracy and speed of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as $ \end{document } segmentation technique . skin texture variations , skin hair and specular reflections present in dermoscopic images tend to induce spurious edges not belonging to the skin lesion . eliminating the spurious edges and accurate segmentation can be achieved using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as $ \end{document } model . based on correlation matching in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ hsv $ \end{document } ( hue saturationvalue ) color of a skin image , intensity variations along radial directions are identified as edges . edges obtained are linked using a continuity criteria to form a contour segment set . subset of the contour segments also known as snakes are approximated using an estimation algorithm to obtain the skin lesion segment . the regions depicting the variations of color ( skin region and lesion region ) are manually selected by the user . let s consider a boolean set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b= \left \ { { b_{1 } , \cdots , b_{k } } \right \ } $ \end{document } associated with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k $ \end{document } number of contour segments identified in an image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{n}\in \mathbb { i } $ \end{document}. the number of contour segment identified are considered as features of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{n } $ \end{document}. the contour segment set is denoted as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a= \left \ { { a_{1 } , \cdots , a_{k } } \right \ } $ \end{document}. the counter model consisting of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } points is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ o= \left \ { { o_{1},\cdots , o_{x } } \right \ } $ \end{document}. the approximation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b $ \end{document } or a subset of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b $ \end{document } , by the model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ o $ \end{document } is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } o^{'}=\arg \max \limits _ { o}\log \left ( { p\left ( { a , b , o } \right ) } \right ) . \end{equation*}\end{document } the approximation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ o^{\mathrm { ' } } $ \end{document } is achieved through the approximate a posteriori criterion . equation ( 2 ) can be solved using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ em$ \end{document } algorithm . according to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ em$ \end{document } algorithm , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { } \log \left ( { p\left ( { a , b , o } \right ) } \right ) $ \end{document } can be substituted with its expected value with respect to the unknown variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b $ \end{document } in ( 2).the substitution is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } s\left ( { o;o^ { ' } } \right ) = e_{b}\left \ { { \log \left ( { p\left ( { a , b , o } \right ) } \right ) a , o^ { ' } } \right \}\ ! , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{b } $ \end{document } represents the energy of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b^{th } $ \end{document } contour and the best estimate of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ o $ \end{document } is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ o^ { ' } $ \end{document}. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p\left ( { o_{x},a } \right ) $ \end{document } represent a potential function defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mathcal { p}\left ( { o_{x},a } \right ) = -\sum \nolimits _ { y } { d^{y}p^{y}\left ( { o_{x } } \right ) } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d^{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p^{y}\left ( { o_{x } } \right ) $ \end{document } represents the confidence degree and potential function of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y^{th } $ \end{document } contour segment . the confidence degree \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d^{y } $ \end{document } is a probability \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { p}$ \end{document } , that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y^{th}$ \end{document } contour segment is valid or true i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d^{y}= \mathbb { p } ( b^{y}=1\vert a^{y},o^ { ' } ) $ \end{document } based on the substitutions presented in and ( 4 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s ( o;o^{\mathrm { ' } } ) $ \end{document } can be simplified and presented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } s ( o;o^ { ' } ) = k+\sum \nolimits _ { x } { \mathcal { p } ( o_{x},a ) + e_{i}(o ) } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k $ \end{document } represents a constant and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e_{i } $ \end{document } is the internal energy . potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { p } $ \end{document } is also referred to as an adaptive potential due to the varying nature its exhibits during estimation . adetailed explanation of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as $ \end{document } segmentation model is found in . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as $ \end{document } segmentation results obtained considering dermoscopic images obtained from the ph2 dataset is shown in fig . the normal skin region and lesion region ( center of the skin lesion ) is selected manually . the major segment ( largest contour segment ) detected adaption of the snake at 10 , 30 , 50 and 70 iterations is shown in column three to six . the ground truth ( provided in the ph2 dataset ) is shown in the last column . results considering skin lesions extending beyond the input image is also shown . in such cases the image boundary is considered as the boundary of the segmented skin lesion . figure 3.the adaptive snake ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as ) $ \end{document } segmentation technique for 6 skin lesion images . the original image is shown in the first column , the major segment identified is shown in the second column . intermediate images obtained at 10 , 30 and 50 iterations are shown in column 3 to 5 . the segmentation result ( at 70 iterations ) and the ground truth obtained the adaptive snake ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ as ) $ \end{document } segmentation technique for 6 skin lesion images . the original image is shown in the first column , the major segment identified is shown in the second column . intermediate images obtained at 10 , 30 and 50 iterations are shown in column 3 to 5 . the segmentation result ( at 70 iterations ) and the ground truth obtained techniques like stereo vision , structure from motion , depth from focus , depth from defocus etc . are used to estimate depth considering multiple images . using constrained image acquisition techniques like active illumination and coded aperture method s , depth the varying or unknown dermoscopic data acquisition parameters / settings used and the non - availability of multiple images render these mechanisms ineffective . in a novel technique to estimate depth , considering a single image obtained from unconstrained image data acquisition techniques is described . the proposed computerized dermoscopy system adopts this technique to estimate the depth in dermoscopic images . depth map obtained is fit to the underling 2d surface to enable 3d surface reconstruction . the 3d surface reconstruction results considering two melanoma and one blue nevus skin lesion images is shown in fig . the structure tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } representing the 3d lesion surface is shown in the last column . ( a ) 3d surface reconstruction results for melanoma image obtained from atlas dataset . ( b ) 3d surface reconstruction results for melanoma image obtained from ph2 dataset .. ( c ) 3d surface reconstruction results for blue nevus image from atlas dataset . the 3d lesion surface reconstruction technique . the original image is shown in column 1 . the edge map used to compute the defocus sparse and the resultant depth map is shown in column 3 - 4 . the structure tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } representing the 3d lesion surface is shown in the last column . ( a ) 3d surface reconstruction results for melanoma image obtained from atlas dataset . ( b ) 3d surface reconstruction results for melanoma image obtained from ph2 dataset .. ( c ) 3d surface reconstruction results for blue nevus image from atlas dataset . the depth of the skin is computed using the estimated defocus occurrence at edges . defocus occurrences at edges ( represented as edge map column 2 of fig.4 ) is obtained , as a ratio between the gradient magnitude of the input skin lesion image and its reblurred version . propagating the blur observed at edges to the whole skin lesion image , enables in computing depth maps . an ideal step edge model considering the edge is located at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x=0 $ \end{document } is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mathcal { e}\left ( { x } \right ) = a \mathcal { s}\left ( { x } \right ) + \mathcal { o } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { o}$ \end{document } represent the amplitude and offset . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { s}\left ( { x } \right ) $ \end{document } represents the step function . defocus blur is obtained by convolving the sharp input skin lesion image with a point spread function . a gaussian function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i.e.~\mathcal { g}(x,\mathcal { s } ) $ \end{document } standard deviation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { s } $ \end{document } , in the gaussian function is directly proportional to the circle of confusion \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( c ) $ \end{document } and it is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { s}= \mathcal { kc } $ \end{document } . the blurred edge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { b}\left ( { x } \right ) $ \end{document } obtained using the edge model and gaussian function is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { b}\left ( { x } \right ) = \mathcal { e}\left ( { x } \right ) \otimes \mathcal { g}\left ( { x,\mathcal { s } } \right ) $ \end{document}. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i $ \end{document } represent an input skin lesion image . reblurring of the input skin lesion image is achieved using a two dimensional isotropic gaussian function and it is represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i^{\mathcal { b } } $ \end{document}. gradient magnitude along the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y $ \end{document } directions of the input image is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \left \| { \nabla i\left ( { x , y } \right ) } \right \|=\sqrt { \nabla i_{x}^{2}+\nabla i_{y}^{2 } } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \nabla } i_{x } , \mathrm { \nabla } i_{y } $ \end{document } represent the gradients along \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y $ \end{document } directions . gradient magnitude of the blurred image is computed in a similar fashion . the gradient magnitude ratio between edge locations ( in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } direction ) of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i^{\mathcal { b } } $ \end{document } is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \left \| { \nabla i\left ( { x } \right ) } \right \|}{\left \| { \nabla i^{\mathcal { b}}\left ( { x } \right ) } \right \| } $ \end{document}. a sparse depth map \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { d\left ( { x } \right ) } $ \end{document } is constructed by estimating the blur scale occurrences at each edge location . inaccurate blur estimates at certain edge locations are eliminated by using a joint bilateral filter and the input skin lesion image as a reference . the resulting sparse depth map ( shown in column 3 of fig.4 . ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { d\left ( { x } \right ) } $ \end{document } is then used to obtain the full depth map \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d\left ( { x } \right ) $ \end{document}. the full depth map \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d\left ( { x } \right ) $ \end{document } is obtained by propagating the defocus blur estimates obtained at edge locations to the entire skin lesion image . image interpolation technique based on matting laplacian technique is used to obtain the full depth map . the optimal depth map \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d $ \end{document } is obtained by solving the following equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \left ( { \mathcal { l}+ \lambda \mathcal { m } } \right ) d=\lambda \mathcal { m}\bar { d } \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { l}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { m}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \lambda $ \end{document } represent the matting laplacian , diagonal matrix and scalar balance factor . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \bar { d } $ \end{document } are the vector representations of the full depth map and sparse depth maps . three dimensional legion surface \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s $ \end{document } is represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s : \mathbb { a}\subset \mathbb { d}^{3}\mapsto \mathbb { d}^{2 } $ \end{document}. where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d}^{3 } $ \end{document } is the three dimensional space in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { a } $ \end{document } lies . a point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a $ \end{document } is represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { x_{a},y_{a},d\left ( { x_{a},y_{a } } \right ) } \right ) $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d\left ( { x_{a},y_{a } } \right ) $ \end{document } represents the depth map . the legion reconstruction is achieved as a gradient descent of the depth map based energy function . nonlinear partial differential equations ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ pde ) $ \end{document } are used to represent the gradient descent . legion reconstruction using the heat equation can be defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \min \limits _ { d : \mathcal { u}\mapsto \mathbb { d}}~{\mathcal { r}\left ( { d } \right ) } = \int _ { \mathcal { u } } { \left ( { \left \| { \mathrm { \nabla } d } \right \|^{2 } } \right ) dxdy } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \nabla } d= \left [ { \frac { \partial d}{\partial x},\frac { \partial d}{\partial y } } \right ] $ \end{document } represents gradient of the depth map and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { u } $ \end{document } is the image domain . perona and malik have shown that ( 9 ) results in smooth surfaces . to preserve lesion edges and attain smooth surfaces , perona and malik introduced nonlinear anisotropic \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ pde $ \end{document } to implement the gradient descent defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \min \limits _ { d : \mathcal { u}\mapsto \mathbb { d}}~{\mathcal { r}\left ( { d } \right ) } = \int \nolimits _ { \mathcal { u } } { \left ( { 1-e^{-\frac { \left \| { \mathrm { \nabla \gamma } d } \right \|^{2}}{\kappa ^{2 } } } } \right ) dxdy } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \kappa } $ \end{document } is the edge preservation constant and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \gamma } $ \end{document } is the diffusion tensor . using gradient decent minimization , ( 10 ) let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } represents a tangential space obtained from the depth map . the 3d lesion reconstructed is considered as a structure tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } t= { \nabla d}^{\rho } . \nabla d. \end{equation*}\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } represents a 3d lesion structure constructed , the corresponding coordinate based eigenvectors represent minimum and maximum gradient directions . the diffusion tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \gamma } $ \end{document } is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \gamma = { f_{a}\left ( { \alpha _ { 1},\alpha _ { 2 } } \right ) \beta _ { 1}\beta _ { 2}}^{\rho } + { f_{b}\left ( { \alpha _ { 1},\alpha _ { 2 } } \right ) \beta _ { 1}\beta _ { 2}}^{\rho } \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { f}_{a}\left ( { x } \right ) , \mathrm { f}_{b}\left ( { x } \right ) $ \end{document } represent functions incorporated to capture the gradient deviations . the 3d skin lesion reconstructed i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } is shown in the last column of fig.4 . characteristics of the skin lesion images are represented as features . in this paper color , texture , 2d shape and 3d shape features accurate and robust feature representation is essential as they directly affect the performance of the skin lesion classification . color characteristics are often used by dermatologists to classify skin lesions . according to dermatologists melanoma skin lesions the variegated coloring induces high variance in the red , green and blue color space . red , green and blue component data of the pixels in the segmented skin lesion is stored as vectors . the mean \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu $ \end{document } and variance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma $ \end{document } of each channel is computed . mean , variance computed is represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { r}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { g}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { b } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { r}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { g}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { b } $ \end{document}.to capture complex non - uniform color distributions within the skin lesion , mean ratios of the mean values is computed i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { r}}{\mu _ { g}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { r}}{\mu _ { b}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { g}}{\mu _ { b } } $ \end{document}. variations in color of the skin lesion with respect to the surrounding skin is also considered as color features . these features are represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { r}}{\bar { \mu } _ { r}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { g}}{\bar { \mu } _ { g}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \frac { \mu _ { b}}{\bar { \mu } _ { b } } $ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \bar { \mu } $ \end{document } represents the mean value of surrounding / normal skin region . to extract the texture features the segmented skin lesion image haralick - features are adopted to obtain the texture characteristics of the skin lesion . considering applicability of the proposed computerized dermoscopy system to classify even low quality skin lesion images , haralick texture features is considered . texture features are computed using gray - tone spatial - dependence matrices i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) $ \end{document}.the angle of the spatial neighborhood \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \theta = 1,2,3,4 $ \end{document}. the matrix denotes the number of the grey tomes of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y $ \end{document } that are spatial neighbors . the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) $ \end{document } is computed at 0 , 45 , 90 and 135 degrees . the energy feature is computed using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } e^{\left [ { \theta } \right ] } = \sum \nolimits _ { x , y } { g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) ^{2 } } \end{equation*}\end{document } the homogeneity texture feature is computed using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } h^{\left [ { \theta } \right ] } = \sum \nolimits _ { x , y } \frac { g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) } { 1+\left | { x - y } \right | } \end{equation*}\end{document } the contrast feature is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } { ct}^{\left [ { \theta } \right ] } = \sum \nolimits _ { x , y } { \left | { x - y } \right |^{2}g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) } \end{equation*}\end{document } mean ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { x } , \mu _ { y } ) $ \end{document } and standard deviations ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \alpha _ { x } , \alpha _ { y } ) $ \end{document } of the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ g_{s}^{\left [ { \theta } \right ] } $ \end{document } considering gray tones of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ y $ \end{document } are computed . using mean and standard deviation the correlation feature is computed as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } { co}^{\left [ { \theta } \right ] } = \sum \nolimits _ { x , y } \frac { \left ( { x-\mu _ { x } } \right ) \left ( { y-\mu _ { y } } \right ) g_{s}^{\left [ { \theta } \right ] } \left ( { x , y } \right ) } { \alpha _ { x } \alpha _ { y } } \end{equation*}\end{document } the mean values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ e^{\left [ { \theta } \right ] } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h^{\left [ { \theta } \right ] } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { ct}^{\left [ { \theta } \right ] } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { co}^{\left [ { \theta } \right ] } $ \end{document } , represented as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { e^{\left [ { \theta } \right ] } } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { h^{\left [ { \theta } \right ] } } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { ct^{\left [ { \theta } \right ] } } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { co^{\left [ { \theta } \right ] } } $ \end{document } are considered as additional texture features . shape , border and asymmetry features are considered as 2d shape features in the proposed computerized dermoscopy system . a total of eleven 2d shape features are extracted from the segmented skin lesion images . area \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { a } $ \end{document } of a skin lesion is defined as the number of pixels present in the lesion . perimeter shape feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { p } $ \end{document } is a count of the number of pixels on the segmented skin lesion boundary . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c$ \end{document } represent the segmented skin lesion centroid . length of a line that connects two furthest boundary points passing through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document } is the greatest diameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { gd } $ \end{document}. length of a line connecting closest lesion boundary points and passing through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document } is considered as the shortest diameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { sd } $ \end{document } shape feature . using area i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { a } $ \end{document } and perimeter i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { p } $ \end{document } of a skin lesion , the circularity index features computed are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ crc=\frac { 4\mathcal { a}\pi } { \mathcal { p}^{2}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ira=\frac { \mathcal { p}}{\mathcal { a}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ir\mathcal { b}=\frac { \mathcal { p}}{\mathcal { gd}}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ irc=\mathcal { p } \left ( { \frac { 1}{\mathcal { sd}}-\frac { 1}{\mathcal { sd } } } \right ) $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ird=\mathcal { gd}-sd $ \end{document}. circularity index features computed quantify irregularity . major , minor axis lengths and asymmetry index features are computed in accordance to . the maximum , minimum and average or relative depth feature is extracted from the 3d skin lesion reconstructed . in addition seven hu invariants and three affine moment invariants are adopted to characterize 3d shape features of the skin lesion . for a skin lesion image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i= f\left ( { x , y } \right ) $ \end{document } of size \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k\times l $ \end{document } , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { k+l } \right ) ^{th } $ \end{document } order geometric moment is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } m_{kl}= \sum \nolimits _ { y=0}^{l-1 } { f\left ( { x , y } \right ) x^{k } y^{l}}. \end{equation*}\end{document } the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { k+l } \right ) ^{th } $ \end{document } order central moment is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mu _ { kl}= \sum \nolimits _ { x=0}^{k-1 } \sum \nolimits _ { y=0}^{l-1 } { f\left ( { x , y } \right ) ( x{-\bar { x})}^{k } ( y{-\bar { y})}^{l } } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \bar { x}=m_{10}/m_{00 } , \bar { y}=m_{01}/m_{00 } $ \end{document } is the center of gravity of an image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m_{00 } $ \end{document}. considering intensity images , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m_{00 } $ \end{document } represents it quality . the moments \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m_{kl } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { kl } $ \end{document } are used to represent the shape of the image . normalization of the central moments of higher orders using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { 0}^{th } $ \end{document } central moment is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \eta _ { kl}=\frac { \mu _ { kl}}{\mu _ { 00}^{r } } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r=\frac { k+l+2}{2 } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k+l=2,3,4\ldots $ \end{document}. using ( 19 ) the hu s moment invariants \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { hm } \right ) $ \end{document } are computed as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } { hm}_{1}=&\eta _ { 20}+\eta _ { 02},\notag \\ { hm}_{2}=&{(\eta _ { 20}-\eta _ { 02})}^{2}+4\eta _ { 11}^{2},\notag \\ { hm}_{3}=&{(\eta _ { 30}-{3\eta } _ { 12})}^{2}+{({3\eta } _ { 21}-\eta _ { 02})}^{2},\notag \\ { hm}_{4}=&{(\eta _ { 30}+\eta _ { 12})}^{2}+{(\eta _ { 21}+\eta _ { 03})}^{2 } , \notag \\ { hm}_{5}=&\biggl ( \left ( { \eta _ { 30}-{3\eta } _ { 12 } } \right ) \left ( { \eta _ { 30}+\eta _ { 12 } } \right ) \biggl [ { { ( \eta _ { 30}+\eta _ { 12})}^{2}}\notag \\&{-\,{2(\eta _ { 21}+\eta _ { 03})}^{2 } } \biggr ] \biggr ) + \biggl ( { \left ( { \eta _ { 30}-{3\eta } _ { 21 } } \right ) \left ( { \eta _ { 03}+\eta _ { 21 } } \right ) } \notag \\&\times { \left [ { { ( \eta _ { 03}+\eta _ { 21})}^{2}-{2(\eta _ { 12}+\eta _ { 30})}^{2 } } \right ] } \biggr ) , \notag \\ { hm}_{6}=&\left ( { \left ( { \eta _ { 20}-\eta _ { 02 } } \right ) \left [ { { ( \eta _ { 30}+\eta _ { 12})}^{2}-{(\eta _ { 21}+\eta _ { 03})}^{2 } } \right ] } \right ) \notag \\&+\left ( { 4\eta _ { 11}\left ( { \eta _ { 30}+\eta _ { 12 } } \right ) \left ( { \eta _ { 21}+\eta _ { 03 } } \right ) } \\ { hm}_{7}=&\big ( \left ( { 3\eta _ { 21}-\eta _ { 03 } } \right ) \biggl ( \left ( { \eta _ { 30}+\eta _ { 12 } } \right ) \biggl [ { \left ( { \eta _ { 30}+\eta _ { 12 } } \right ) ^{2}}\notag \\&{-\,{3\left ( { \eta _ { 21}+\eta _ { 03 } } \right ) } ^{2 } } \biggr ] \biggr ) -\big ( \left ( { 3\eta _ { 12}-\eta _ { 30 } } \right ) \biggl ( \left ( { \eta _ { 03}+\eta _ { 21 } } \right ) \notag \\&\times { \left [ { \left ( { \eta _ { 03}+\eta _ { 21 } } \right ) ^{2}-{3\left ( { \eta _ { 12}+\eta _ { 30 } } \right ) } ^{2 } } \right ] } \biggr ) . \end{align } \end{document } to provide additional 3d shape features , affine moment invariants of the first , second and third order are considered . according to the features affine moment invariants are defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } { am}_{1}=&\left ( { \mu _ { 20}\mu _ { 02}-\mu _ { 11}^{2 } } \right ) / \mu _ { 00}^{4},\notag \\ { am}_{2}=&\left ( { { \begin{array}{l } \mu _ { 30}^{2}\mu _ { 03}^{2}-6\mu _ { 30}\mu _ { 21}\mu _ { 12}\mu _ { 03}+4\mu _ { 30}\mu _ { 12}^{3 } \\ + 4\mu _ { 21}^{3}\mu _ { 03}-3\mu _ { 21}^{2}\mu _ { 12}^{2 } \\ \end{array } } } \right ) \bigg / \mu _ { 00}^{10},\notag \\ { am}_{3}=&\left ( { { \begin{array}{l } \mu _ { 20}\left ( { \mu _ { 21}\mu _ { 03}-\mu _ { 12}^{2 } } \right ) - \mu _ { 11 } ( \mu _ { 30}\mu _ { 03}\\ -\mu _ { 21}\mu _ { 12 } ) + \mu _ { 02}{(\mu } _ { 30}\mu _ { 12}-\mu _ { 21}^{2 } ) \\ \end{array } } } \right ) \!\bigg / \!\mu _ { 00}^{7}\notag \\ { } \end{align } \end{document } feature selection , generally is identifying an optimized subset of features extracted that imparts highest discriminating power to the decision making mechanism adopted . in the proposed computerized dermoscopy system color \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { f^{c } } \right ) $ \end{document } , texture \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { f^{t } } \right ) $ \end{document } , 2d shape \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { f^{s2d } } \right ) $ \end{document } , and 3d shape \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { f^{s3d } } \right ) $ \end{document } features of skin lesion images are extracted . apart from imparting discriminating power , feature selection is adopted to study the impact of color features , texture features , 2d shape features , 3d shape feature and their combinations to classification of skin lesions . the feature set is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{r } = \left \ { { f^{c } , f^{t } , f^{s2d } , f^{s3d } } \right \ } $ \end{document}. a heuristic approach is adopted to obtain the optimized feature set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{r}^{sel } \subseteq f_{r } $ \end{document}. optimized feature set is constructed considering different combinations of the features extracted . resulting performance enable in understanding the significance of features considered on the classification system . experimental study discussed in the subsequent section considers four optimized feature set combining the features extracted . the optimized feature sets considered for evaluation are defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } f_{r}^{sel\_{}1}=&\left \ { { f^{s2d } , f^{s3d } } \right \},\notag \\ f_{r}^{sel\_{}2}=&\left \ { { f^{c } , f^{t } } \right \},\notag \\ f_{r}^{sel\_{}3}=&\left \ { { f^{c } , f^{t } , f^{s2d } } \right \},\notag \\ f_{r}^{sel\_{}4}=&\left \ { { f^{c } , f^{t } , f^{s2d } , f^{s3d } } \right \}. \end{align } \end{document } skin lesion classification is the final step of proposed computerized dermoscopy system . in the research work presented here , three different classes of classifiers i.e. svm , , adaboost and the recently developed bag - of - features ( bof ) , classifiers are adopted . the classifiers adopted are also referred to as decision making mechanisms \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d } $ \end{document}. classification broadly involves two phases namely training and testing . in the training phase the classifiers learn from the training set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { r } $ \end{document}. feature properties with respect to the classes are derived in the training phase . in the testing phase we wish to classify test data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { t } $ \end{document}. based on the feature properties observed in training , the decision making mechanisms \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathbb { d } $ \end{document } classifies a test image \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{t } $ \end{document } represented by feature set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{t } $ \end{document } as the resultant class \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { } c_{t}\mathrm { } $ \end{document}. skin lesion data is complex in nature and can not be considered as a global model . in the bof decision making mechanism , skin lesion data is considered as a combination of individual feature models rather than the complete feature set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{r}^{sel } $ \end{document}. the bof classifier exhibits promising results when adopted for complex image analysis , . therefore , the bof classifier was deemed applicable to solve our skin lesion classification problem . the capability to train a strong classifier from a combination of weak classifiers and appropriate feature selection capabilities exhibited by the adaboost algorithm motivated the authors to consider its inclusion in the proposed system . svm classifiers are robust , simple to implement and provide high degree of classification accuracy . recent works for skin lesion classification , , , prove the applicability of svm classifiers for decision making . a gaussian radial basis function ( rbf ) kernel is considered in the proposed computerized dermoscopy system . the rbf kernel assists in deriving complex relations between the skin lesion classes and complex nonlinear skin lesion data represented as a feature vector space . a linear kernel is a special case of the rbf kernel , hence the authors have considered to adopt a rbf kernel in the svm classifier . in this section experimental studies conducted to evaluate performance of the proposed computerized dermoscopy system is presented . the dermoscopy data used in the experiments , experiment details , performance of the three classifiers proposed , comparisons with existing systems and the experiments based on the 3d reconstruction algorithm proposed for depth estimation is discussed . data to evaluate the performance of the proposed dermoscopy system is acquired from two sources . the datasets used are summarized in table i.table 1dataset details used for experimentsno.dataset nameno of imagesno of classestype of skin lesions1ph2 2004melanomain - situ melanomaatypical nevuscommon nevus2atlas 638melanomain - situ melanomabasal cell carcinomablue nevusdermatofibromahaemangiomaseborrhoeic keratosisnormal mole the ph2 database of 200 dermoscopic images from pedro hispano hospital is considered . four classes i.e. common nevus , atypical nevus , melanoma , in - situ melanoma ( lentigo melanoma ) are considered . the ph2 dataset is also used to evaluate the performance in , , and . the second dataset is obtained from atlas of dermoscopy cd published alongside . comprehensive skin lesion data of varied types with analysis from expert dermatologists is provided in the atlas . theatlas dataset consists of a comprehensive set of skin lesion images rendering it more practical to evaluate the proposed computerized dermoscopy system . the atlas dataset created is complex and 8 type of skin lesions are considered . performance of the proposed system with each classifier individually is evaluated considering the feature selection \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{r}^{sel } $ \end{document } combinations defined in ( 22 ) . training and testing data used in the experiments is obtained in accordance to the procedure described in . experiment details and the notations used to represent them is described in table ii . leave - one - out approach , is adopted for testing due to the limited size of the datasets available . to evaluate performance a cost function overall classification accuracy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { ca } \right ) $ \end{document } considering all skin lesion classes ( i.e. 4 classes for ph2 and 8 classes for atlas dataset ) is computed using the confusion matrix . a tradeoff between specificity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( sp ) $ \end{document } and sensibility \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { se } \right ) $ \end{document } exists hence barata have introduced as cost function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document } for evaluating performance.table 2experiment details using varied feature types and datasetsexp . nodataset usedfeatures selectednotation1ph2 2d shape , 3d shaped1ex12ph2 color , textured1ex23ph2 color , texture , 2d shaped1ex34ph2 color , texture , 2d shape , 3d shaped1ex45atlas 2d shape , 3d shaped2ex16atlas color , textured2ex27atlas color , texture , 2d shaped2ex38atlas color , texture , 2d shape , 3d shaped2ex4 the cost function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document } is defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mathcal { c}= \frac { \mathcal { k}_{fn}\left ( { 1-se } \right ) + \mathcal { k}_{fp}\left ( { 1-sp } \right ) } { \mathcal { k}_{fn}+\mathcal { k}_{fp } } , \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fn},\mathcal { k}_{fp } $ \end{document } are constants and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fn}=1.5 \times \mathcal { k}_{fp } $ \end{document}. constants \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fn } $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fp } $ \end{document } represent the false negative \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { fn } \right ) $ \end{document } and false positive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \left ( { fp } \right ) $ \end{document } costs . in the experimental results presented \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fn}=1.5 $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathcal { k}_{fp}=1.0 $ \end{document } is considered . the bof classifier considers a block size of 50 and the number of histogram bins is set to 25 . classification is achieved using the k - nearest neighbor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( knn ) $ \end{document } classifier . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ knn $ \end{document } employed , considers euclidean distance and the number of neighbors is set to 10 . results obtained in this study is summarized in table iii.table 3experimental results considering bof classifierexp # \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ se $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document}d1ex191%94%0.079d1ex291%94%0.077d1ex393%96%0.062d1ex490%94%0.079d2ex176%95%0.164d2ex267%95%0.216d2ex377%96%0.153d2ex472%96%0.187 considering ph2 dataset best performance is reported considering color , texture and 2d shape features ( d1ex3 ) . inclusion of 2d shape features to texture and color exhibits a 19.4% reduction in the value of cost function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document}. results considering atlas dataset show that color and texture information alone considered in d2ex2 is insufficient to classify skin lesions . considering shape features ( 2d and/or 3d ) improves performance of the bof classifier observed in d2ex1 , d2ex3 and d2ex4 . on the atlas this observation is due to the fact that limited training data and numerous skin lesion types are considered in the atlas dataset enhancing complexity . to overcome this drawback we used two additional classifiers discussed in the latter section of the paper . a noteworthy observation is that classification using the shape descriptors ( i.e. d1ex1 , d2ex1 ) exhibits similar performance to color and texture features ( i.e. d1ex2 , d2ex2 ) . inclusion of shape features to color and texture improve performance considering the bof classifier . this configuration is established based on a number of iterations to obtain best performance . considering ph2 dataset the adaboost classifier exhibits better results when compared to the bof classifier . it must be noted that in d1ex2 we report \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ se=96 $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp=98 $ \end{document } similar to the values observed in that reports \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ se=96 $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp=77 $ \end{document } for color and texture features considering adaboost classifier . in d1ex3 and d2ex4 , adaboost exhibits best classification results considering experiments conducted on the datasets . performance of the adaboost classifier improves on considering the proposed 3d shape features for the atlas dataset . the adaboost classifier exhibits better performance considering ph2 dataset when compared to atlas dataset . adaboost classifier does not achieve acceptable performance on the atlas dataset even if the number of weak classifiers are increased . a similar observation is also reported in . to overcome this drawback and improve performance across varied datasets the authors have considered the svm classifier.table 4experimental results considering adaboost classifierexp # \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ se $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document}d1ex194%97%0.049d1ex296%98%0.029d1ex396%98%0.028d1ex494%97%0.049d2ex140%92%0.394d2ex235%90%0.426d2ex344%92%0.369d2ex447%92%0.349 the bof and adaboost classifier exhibit promising results considering ph2 dataset . on evaluation with the complex atlas dataset these classifiers exhibit low performance authors have adopted a rbf kernel in the svm classifier to overcome this drawback and achieve acceptable performance on both ph2 and atlas datasets . results obtained considering the rbf - svm classifier is shown in table v.table 5experimental results considering svm classifierexp # \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ se $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ sp $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c $ \end{document}d1ex179%86%0.184d1ex229%78%0.518d1ex349%86%0.36d1ex496%97%0.038d2ex180%97%0.136d2ex231%91%0.448d2ex359%94%0.271d2ex498%99%0.013 considering ph2 dataset , the best performance is reported in d1ex4 which outperforms the results presented in and . a marked improvement in performance is reported on the atlas dataset considering the svm classifier . results obtained prove that the svm classifier exhibits better generalization performance on increasing the feature vector when compared to the other classifiers . observe results of d1ex1 , d1ex4 against d1ex2 and d1ex3 in table v. a marked performance improvement considering the proposed 3d shape feature inclusion is reported on ph2 dataset . results of d2ex1 , d2ex4 against d2ex2 and d2ex3 in table v prove the performance improvement . in it is stated that performance of the svm is directly dependent on the features extracted i.e. to project data into separable feature space . based on the results presented it can be concluded that the 3d shape feature extracted improve classification performance ( refer d1ex1 , d1ex4 , d2ex1 and d2ex4 in table v ) . the svm classifier considered exhibits better performance in comparison to the adaboost and bof classifier . a tentative comparison with the other state of art systems is presented here even if we have not considered similar features and datasets . using the asymmetry , border , color , texture features on the dermat dataset in an accuracy of 86% , se = 94% and sp = 68% considering high level , low level features on datasets created from dermquest , dermatology information system the highest accuracy of 83.59% ( se = 91.01% , sp = 73.45% ) is reported by amelard et al . . considerable amount of research work is carried out using the ph2 dataset . using color and texture features in se = 98% , sp = 79% and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c = 0.100 $ \end{document } is reported . using color features alone the best performance of se = 100% , sp = 75% and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c = 0.099 $ \end{document } is reported in . automated skin lesion analysis system developed by abuzaghleh et al . reports a classification accuracy of melanoma , benign , atypical lesions as 97.5% , 95.7% , and 96.3% . report a se = 97.6% , sp = 90.5% and average accuracy of 96.5 in . report performance measures of se = 98% , sp = 90% on ph2 dataset and se = 83% , sp = 76% on edra datasets considering a fusion of features . a recent work , introduces sparse coding of the scale - invariant feature transform ( sift ) features for melanoma classification . reference reports a performance of se = 100% , sp = 90.3% on the ph2 dataset . in comparison the proposed computerized dermoscopy system considering the ph2 dataset ( and svm classifier ) reports performance results se = 96% , sp = 97% and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c = 0.038 $ \end{document}. the classification accuracy of melanoma , common nevus , atypical nevus and in - situ skin lesions is 100% , 93% , 90% , and 100% . the average classification accuracy is 95.75% . using only color features of ph2 dataset and the adaboost classifier a performance of se = 96% , sp = 98% and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c = 0.031 $ \end{document } is observed . the results considering the atlas dataset is se = 98% , sp = 99% and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c = 0.013 $ \end{document}. the classification accuracy is 96.83% . the results obtained prove that the proposed computerized dermoscopy system is efficient and can be adopted to diagnose skin lesions of varied types . use of the proposed computerized dermoscopy system to train or test new dermatologists is also mooted . a major goal of the proposed computerized dermoscopy system is to aid early detection of melanoma i.e. in - situ melanoma . the 3d data / tensor provides useful insight to analyze relative depth of melanoma cancer skin lesions . in the initial experiment we have considered an in - situ melanoma image from chapter 16 ( follow - up of melanocytic skin lesions with digital dermoscopy ) of the atlas crrom . region of interest and estimated depth of the follow - up image is shown in fig . spreading of the melanoma in the region of interest is clearly evident by comparing fig . the marginal increase in relative estimated depth values validate the 3d reconstruction / estimation technique proposed in this paper . figure 5.in-situ melanoma baseline image and 3d depth projections ( a ) baseline image ( top ) and estimated depth ( bottom ) . ( b ) region of interest ( top ) and estimated depth ( bottom ) . figure 6.in-situ melanoma follow - up image and 3d depth projections ( a ) follow - up image ( top ) and estimated depth ( bottom ) . ( b ) region of interest ( top ) and estimated depth ( bottom ) . in - situ melanoma baseline image and 3d depth projections ( a ) baseline image ( top ) and estimated depth ( bottom ) . ( b ) region of interest ( top ) and estimated depth ( bottom ) . in - situ melanoma follow - up image and 3d depth projections ( a ) follow - up image ( top ) and estimated depth ( bottom ) . ( b ) region of interest ( top ) and estimated depth ( bottom ) . dermoscopic images from is consider to further assess performance of the proposed 3d reconstruction technique . an in - situ melanoma image ( top ) and the corresponding relative estimated depth ( bottom ) is shown fig . 7(a ) . a spreading melanoma with a breslow index of 0.5 mm and the relative depth estimated is shown in fig . the relative estimated depth and the spreading melanoma image with a breslow index of 0.9 mm is shown in fig . relative estimated depth of the images computed using our proposed technique is reported as 0.0918 , 0.1388 and 0.2437 . 7(c ) , breslow index difference of 80% is observed . a difference of 75.58% the increase in the relative estimated depth and the difference measure reported validate the relative estimation accuracy . figure 7.melanoma images obtained from and 3d depth projections ( a ) in - situ melanoma image ( top ) and estimated depth ( bottom ) . ( b ) spreading melanoma image with breslow index 0.5 mm ( top ) and estimated depth ( bottom ) . ( c ) spreading melanoma image with breslow index of 0.9 mm ( top ) and estimated depth ( bottom ) . melanoma images obtained from and 3d depth projections ( a ) in - situ melanoma image ( top ) and estimated depth ( bottom ) . ( b ) spreading melanoma image with breslow index 0.5 mm ( top ) and estimated depth ( bottom ) . ( c ) spreading melanoma image with breslow index of 0.9 mm ( top ) and estimated depth ( bottom ) . to evaluate performance of the proposed 3d reconstruction technique on slow growing melanoma , dermoscopic data from is considered . the baseline image and the corresponding relative estimated depth the follow - up image after five years and the relative estimated depth is shown in fig . the follow - up dermoscopic skin lesion was biopsied and the melanoma was found to be 0.15 mm thick . the relative estimated depth for the baseline image and follow - up image is 0.0189 , 0.0436 . the increasing values of the relative estimated depth prove accuracy of the proposed 3d reconstruction technique for slow growing melanoma . figure 8.slow growing melanoma follow - up image from and 3d depth projections ( a ) baseline image ( top ) and estimated depth ( bottom ) . biopsy reveals melanoma is 0.15 mm thick ( top ) and estimated depth ( bottom ) . slow growing melanoma follow - up image from and 3d depth projections ( a ) baseline image ( top ) and estimated depth ( bottom ) . biopsy reveals melanoma is 0.15 mm thick ( top ) and estimated depth ( bottom ) . the international skin imaging collaboration ( isic ) : melanoma project introduced in recent times , is an academia - industry partnership providing dermoscopic data for melanoma diagnosis . a large number of societies have collaborated together in the isic : melanoma project . data provided is by far the most comprehensive set of publicly available melanoma skin lesion images . a total of 4670 skin lesion images collected from various clinical trials are available in the dataset till date . clinical/ diagnosis data corresponding to each skin lesion image is also available . to validate 3d reconstruction technique , a set of 22 images from the isic archive images whose breslow depth is confirmed through biopsies performed are taken for evaluation study . large breslow depth variations , from 0.16 to 0.9 is observed in dataset considered . minor variations ( in order of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { 10}^{-3 } ) $ \end{document } of estimated depth values for images with similar breslow depths is observed . results presented depict that increase in estimated depth is correlated to increase in breslow depth , proving effectiveness of proposed 3d reconstruction technique on large set of dermoscopic images considered.table 6evaluation of 3d skin lesion reconstruction technique using isic data image i d from biopsy confirmed breslow depthrelative estimated depthisic_00114290.160.0448isic_00114300.160.0449isic_00114040.20.0558isic_00114630.220.0614isic_00115140.220.0610isic_00114280.40.1097isic_00115110.40.1100isic_00114380.480.1271isic_00114390.480.1291isic_00114580.490.1336isic_00115260.50.1529isic_00115200.620.1670isic_00115210.620.1693isic_00114050.650.1774isic_00001580.760.2273isic_00114350.80.2276isic_00115070.80.2203isic_00115030.820.2360isic_00115150.90.2544isic_00115170.90.2536isic_00115180.90.2531isic_00115190.90.2513 though actual depth ( currently obtained using invasive biopsy ) can not be computed , accurate estimates can be obtained using the proposed technique . the relative estimated depth is a critical feature for identification of in - situ melanoma . in addition , 3d features extracted using the 3d reconstructed skin lesion improve overall system classification performance as reported in the previous section . amongst all skin cancers known , melanoma accounts for the majority of deaths reported . use of non - invasive computerized dermoscopy techniques to diagnose skin lesions is commonly adopted . identifying depth of the melanoma tumor into the skin is essential to ascertain the stage of cancer . melanoma is skin deep and introduce a computerized dermoscopy system in this paper that incorporates depth estimation . the 3d reconstruction is achieved by fitting the depth map estimated to the underling 2d surface . based on the 3d tensor structure constructed , depth and 3d shape features are extracted . feature selection to study the effects of features and their combinations on decision making is proposed . for decision making bof , adaboost and svm classifiers results considering different feature combinations and bof , adaboost , svm classifiers is presented . in view of the results , it is concluded that inclusion of 3d shape features proposed ( that include the estimated depth features ) enhance performance aiding accurate diagnosis of varied skin lesion types . the svm achieves best classification scores of se = 96% and sp = 97% on ph2 dataset . the svm classification score of se = 98% and sp = 99% on atlas dataset is reported . depth estimation performance is evaluated on isic : melanoma project dataset and data obtained from literature published . though good performance is reported , improvement of the estimation technique is always an open issue . future of the work presented here is to identify depth estimation error using clinical data and devise new techniques to minimize errors . authors welcome dermatologists/ researchers to undertake a collaborative research to develop the proposed system further considering clinical data .

melanoma mortality rates are the highest amongst skin cancer patients . melanoma is life threating when it grows beyond the dermis of the skin . hence , depth is an important factor to diagnose melanoma . this paper introduces a non - invasive computerized dermoscopy system that considers the estimated depth of skin lesions for diagnosis . a 3-d skin lesion reconstruction technique using the estimated depth obtained from regular dermoscopic images is presented . on basis of the 3-d reconstruction , depth and 3-d shape features are extracted . in addition to 3-d features , regular color , texture , and 2-d shape features are also extracted . feature extraction is critical to achieve accurate results . apart from melanoma , in - situ melanoma the proposed system is designed to diagnose basal cell carcinoma , blue nevus , dermatofibroma , haemangioma , seborrhoeic keratosis , and normal mole lesions . for experimental evaluations , the ph2 , isic : melanoma project , and atlas dermoscopy data sets is considered . different feature set combinations is considered and performance is evaluated . significant performance improvement is reported the post inclusion of estimated depth and 3-d features . the good classification scores of sensitivity = 96% , specificity = 97% on ph2 data set and sensitivity = 98% , specificity = 99% on the atlas data set is achieved . experiments conducted to estimate tumor depth from 3-d lesion reconstruction is presented . experimental results achieved prove that the proposed computerized dermoscopy system is efficient and can be used to diagnose varied skin lesion dermoscopy images .

non - steroidal anti - inflammatory drugs ( nsaids ) are one of the most commonly prescribed drugs in the world for their analgesic and anti - inflammatory properties . however , nsaid has limitation in prescribing because of gastrointestinal ( gi ) toxicity . recently , nsaid - induced enteropathy has gained much attention due to the introduction of new emerging diagnostic modalities , capsule endoscopy ( ce ) and device assisted enteroscopy as well as due to the increased use of aspirin and nsaids . recent ce studies have demonstrated nsaid use in healthy volunteers raised the incidence ( 55% to 75% ) of intestinal damage.1 - 6 nsaid - induced enteropathy has been under - examined or even ignored in clinical situation before the times of ce . previous attention or studies have focused primarily on upper gi events but recent researches have shifted to the small bowel and colon during chronic nsaid use . proton pump inhibitor ( ppi ) ca n't protect nsaid - induced intestinal injuries below treitz ligament while ppi is very helpful to reduce nsaid - induced gastroduodenal damages.7 until now , no medications are available yet to prevent or heal nsaid - induced intestinal injuries . it is important to extend the understanding about small bowel and colonic injuries associated with nsaid because all clinician , especially gastroenterologists , should have thorough knowledge of the gi toxicity of nsaids . it has been known that lower gi events accounted for one - third of all clinically relevant gi events . nsaid - induced lower gi complications ( perforation , bleeding , or obstruction ) are increasing while upper gi complications are decreasing.7 in fact , the ratio of upper / lower was 4.1 in 1996 and it has decreased to only 1.4 in 2005.7,8 current evidences suggest that nsaids increase the risk of lower gi bleeding and perforation to a similar extent to that seen in the upper gi tract.9 post - hoc analysis of a large - scale clinical outcome trial showed that lower gi events accounted for 40% of all serious gi events in patients on nsaids.10 there were notable ce studies in short - term nsaid users as well as chronic nsaid users . goldstein et al.5 reported that small bowel mucosal breaks were induced in 55% of healthy volunteers who had taken naproxen for 2 weeks . maiden et al.4 reported that 2-week ingestion of slow release diclofenac resulted in macroscopic injury to the small intestine in 68% to 75% of healthy volunteers . graham et al.3 performed ces in arthritic patients who had been using nsaids for at least 3 months and reported as high as 71% for the incidence of small intestinal mucosal injury after chronic nsaid administration . the japanese study group for double - balloon endoscopy reported that nsaid - induced enteropathy occurred in half of the patients taking nsaids based on the registry of a 2-year period.11 ce and double - balloon enteroscopy ( dbe ) findings suggest that the small intestinal mucosa is very sensitive to nsaid - induced injuries ( table 1 ) . the pathogenesis of nsaid - induced enteropathy is distinct from that of nsaid - induced gastropathy . unlike stomach , nsaid - induced lower gi injuries are not caused by sup - pression of prostaglandin synthesis due to inhibition of cyclo - oxygenase ( cox ) activity12,13 but , most of the time , by gram negative bacteria and bile.4 - 22 bjarnason et al.15 proposed a " three hit " hypothesis . first , nsaids solubilize lipids of phospholipids on the mucosal surface , so the epithelial mitochondria are directly damaged . second , the mitochondrial damage depletes intercellular energy and leads to calcium efflux and to induction of free radicals , a disruption of intercellular junctions occurs , and mucosal permeability increases in the small intestinal mucosa . third , the mucosal barrier becomes weakened , so bile acid , proteolytic enzymes , intestinal bacteria , or toxins can easily penetrate into the epithelial cells , resulting in mucosal injury . nsaid - induced small intestinal injuries augment through the enterohepatic circulation of the nsaid ( fig . nsaids affect the entire gi system and can often cause various abdominal symptoms such as epigastric pain , nausea , indigestion , constipation , and abdominal distension.13 multiple ulcers and erosions are common nsaid - induced small intestinal lesions.14 ulceration often occurs in the gi tract without symptoms because of analgesic effect of nsaids . aspirin itself can also cause stomach and duodenal ulcers , but it is generally believed that aspirin does not usually cause small bowel damage according to intestinal permeability and fecal inflammatory marker studies and aspirin does not have enterohepatic circulation.14 enteric coated aspirin was originally designed to decrease adverse effects on the stomach , but the use of enteric coated aspirin may have shifted the damage to the distal small bowel . low dose aspirin is not safe , and chronic low dose aspirin use induced small bowel enteropathy that has many striking similarities with nsaid - induced enteropathy.23 nsaids were found to induce obscure overt gi bleeding which manifests as hematochezia or melena , or occult obscure gi bleeding , as positive guaiac fecal occult blood test or anemia of unknown etiology.24 nsaid - induced enteropathy is one of the most common causes of obscure gi bleeding.24 nsaid - induced enteropathy might be suspected strongly in patients with obscure gi bleeding and recent history of taking aspirin or nsaids . ce sensitively detects nsaid - induced small intestinal injuries such as red spot , erosion , and ulcer , while nsaid - induced small intestinal injuries are not specific in endoscopic findings and nsaid - induced ulcer is hard to differentiate from other causes of ulcer such as crohn 's disease . nsaid - induced small intestinal bleeding can be effectively controlled by a device assisted enteroscopy ( fig . concentric diaphragmatic stricture is thought to be the pathognomonic symptom of nsaid injury.25 clinical presentation of diaphragm disease is nonspecific and may be presented with obstructive symptoms . it develops from scarring reaction secondary to ulcerative injury during long - term nsaid use . the histological features of the diaphragm - like stricture include fibrosis in the submucosa and thickening of the muscularis mucosa.26 since the muscularis propria layer is intact , the risk of intestinal perforation is low with endoscopic balloon dilation , which is why it is a preferred treatment modality than surgical intervention.27 capsule can be retrieved successfully by enteroscopy in case of the capsule entrapment . in randomized trials in patients with rheumatoid or osteoarthritis , diverticulitis and diverticular bleeding are the most common etiologies of nsaid - induced lower gi injuries , accounting for 30% to 50% of all serious gi events associated with nsaids.28 case control studies have shown a significantly higher prevalence of nsaid use with complications of diverticular diseases ( diverticulitis and bleeding ) compared to controls - their risk estimates varied widely with odds ratios ranging from 1.8 to 16.7 regular use of aspirin or nsaids was associated with increased risks of diverticulitis and diverticular bleeding.7,29 nsaids can induce a variety of abnormalities including ulcerations , perforations , bleeding , and diaphragm - like strictures in the human small intestine.7 difficulties in accessing the entire small intestine prevented comprehensive evaluation of the extent and localization of nsaid - induced small intestinal injury , however . thus , prior to the advent of ce and small bowel enteroscopy , most studies had to rely on surrogate markers such as urinary excretion of chromium-51-labeled ethylenediaminetetraacetic acid , which shows intestinal permeability and fecal level of calprotectin , a proposed marker of intestinal inflammation.30,31 calprotectin is a 36 kda calcium - binding protein constituting up to 60% of the cytosolic proteins in neutrophil granulocytes and plays an important role in inflammatory processes.32 it is excreted in feces and remains stable against bacterial degradation.33 the presence of calprotectin in feces is a consequence of neutrophils ' migration into the gi tissue.33 but now , with ce , we can easily locate the site of injury and evaluate the incidence rates and types of injuries . endoscopic findings of nsaid - induced enteropathy include reddish erosion , multiple sharply demarcated ulcer and concentric stenosis . nsaid - induced small intestinal lesions on ce include denuded areas , erosions and ulcers ( fig . a denuded area is defined as a reddened area without villi.4 maiden et al.4 classified ce findings into five categories : reddened folds , denuded area , red spot , mucosal break , and blood . graham et al.3 divided ce findings into red spots , small erosions , large erosions , and ulcers , and they found mucosal lesions in 13 out of 21 patients ( 62% ) with chronic nsaid use . small lesions such as erosions and red spots are detected more often by ce than dbe . previous efforts have focused primarily on gi events in the upper gi tract , but recently , efforts have been made to reduce nsaid - induced small bowel and colon injuries including mucosal damage , ulceration , overt bleeding , obstruction / perforation , protein - loss and occult blood loss ( with an associated decrease in hemoglobin ) . fujimori et al.34 demonstrated the benefit of treatment with misoprostol in a small pilot study in which small intestinal damage was assessed by ce . misoprostol co - therapy reduced the incidence of small intestinal lesions induced by a 2-week administration of diclofenac sodium in healthy subjects . their subjects were gastric ulcer patients who were orally taking low dose , enteric coated aspirin . they were treated with ppi for 8 weeks , but all patients had redness and erosions in the small intestine on ce at 8 weeks . when misoprostol was administered instead of ppi for additional 8 weeks selective cox-2 inhibitors ( coxibs ) were introduced to the marketplace in the mid-1990s with a promise of improved gi safety . goldstein et al.5 reported that 2-week regimen with celecoxib , a selective coxib , caused less small intestinal injury than a regimen using naproxen . selective coxibs were believed to be less injurious than nonselective nsaids in the small bowel as well as in the stomach , but maiden et al.1 found no difference in the incidence of small intestinal injury between chronic users of nonselective nsaids and selective coxibs . selective coxibs are selective , but not exclusive , and thus have some cox-1 inhibitory activity . further studies with large samples are needed to decide whether or not selective coxibs can entirely control gi toxicity . niwa et al.36 conducted a prospective , double - blind study of ce using a mucosal protective agent , rebamipide , in healthy subjects . it was found that subjects who received placebo had significantly more mucosal injuries in the small intestine compared to those who received rebamipide . mucosal protecting agents such as teprenone , rebamipide , and irsogladine showed protective effect against nsaid - induced small intestinal injuries according to animal experiments using rats ; more specifically , the increase in inos mrna expression and myeloperoxidase activity due to indomethacin was inhibited by pre - treatment with these mucosal protecting agents.24 several strategies have been tried to develop new compounds with improved gi tolerability . several companies are developing drugs that co - release an nsaid ( naproxen , aspirin , or ibuprofen ) and a ppi ( omeprazole , esomeprazole , or lansoprazole ) ; these compounds are already filed with the fda or in very late stages of development.37 these compounds have been shown to reduce the incidence of nsaid - induced gastropathy but not enteropathy . ( houston , tx , usa ) has taken a strategy to reduce the topical irritant property of nsaids and has developed nsaids that are associated with phosphatidylcholine.38 extensive animal works have demonstrated that phosphatidylcholine - nsaids are less likely to damage the gi epithelium in rodents , and produce less severe mucosal injury while retaining the anti - inflammatory effects of the parent nsaid . in a randomized , double - blind , endoscopy study , ibuprofen preassociated with phosphatidylcholine showed superior gi toxicity than an equivalent dose of ibuprofen in the subgroup of patients older than 55 years.39 the discovery that nitric oxide ( no ) is a potent gastroprotective substance that modulates many aspects of gi mucosal defense led to the development of no releasing nsaids , which are also referred to as cox - inhibiting no donors.40 the compound has shown improved gi tolerability in some but not all clinical trials . like no , hydrogen sulfide ( h2s ) has been shown to exert protective effects in the gi tract and to accelerate the healing of preexisting ulcers . h2s - releasing nsaids , derivatives of naproxen , diclofenac , and indomethacin have been reported.41 - 43 phosphatidylcholine associated nsaids as well as no- and h2s - releasing nsaids are under extensive preclinical testing for their influence on nsaid - induced enteropathy . further studies are warranted to develop promising new nsaid with total gi ( upper and lower gi tracts ) tolerability and without cardiovascular toxicity . nsaids are one of the most commonly prescribed drugs in the world for analgesic and anti - inflammatory properties . previous studies have focused primarily on upper gi events ; but recently , nsaid - induced enteropathy has gained much attention due to the introduction of new emerging diagnostic modalities , ce and device assisted enteroscopy . lower gi events accounted for 40% of all serious gi events in patients taking nsaids.14 ce studies have demonstrated nsaid use in healthy volunteers raised the incidence ( 55% to 75% ) of intestinal damage.1 - 6 unfortunately , however , ppi ca n't protect nsaid - induced intestinal injuries below treitz ligament ; selective coxibs improved upper and lower gi safety according to results of clinical trials,23 but coxibs are still capable of triggering gi adverse events and are still concerned to be associated with cardiovascular toxicity issues . thus , there is still a strong clinical need for effective anti - inflammatory drugs with superior safety profiles than the existing nsaids .

non - steroidal anti - inflammatory drugs ( nsaids ) are one of the most commonly prescribed drugs in the world . nsaid - induced lower gastrointestinal ( gi ) complications are increasing while upper gi complications are decreasing . lower gi events accounted for 40% of all serious gi events in patients on nsaids . capsule endoscopy and device assisted enteroscopy are available for detection of small intestinal lesions . capsule endoscopy studies have demonstrated that nsaids use in healthy volunteers raised the incidence ( 55% to 75% ) of intestinal damage . it appears that selective cyclooxygenase-2 inhibitors ( coxibs ) improved upper and lower gi safety based on results of clinical trials . selective coxibs are still capable of triggering gi adverse events and cardiovascular toxicity issues were the main focus of concerns . unfortunately , definite strategies are not available to prevent or heal nsaid - induced intestinal injuries . thus , there is still a strong clinical need for effective drugs with improved safety profiles than the existing nsaids .

the study was reviewed and approved by the institutional review board of the university of pennsylvania . nhc is a company that provided comprehensive wound care management solutions to local wound care centers across the united states . nhc developed clinical pathways to guide treatment based on reviews of best practice and guidelines on proper diagnosis and medical care ( e.g. , debridement , good wound care , and off - loading ) that were taught to the local wound care providers . subjects were included in the study if they were treated at an nhc center between november 2005 and may 2011 , and if the local wound care center had contractually agreed to provide data for research , resulting in 83 centers located in 31 states being available for study . subjects must have had diabetes , adequate lower - extremity arterial flow ( as determined by the clinician ) , and a wound on their plantar foot ( hindfoot [ heel ] , midfoot , or forefoot [ toes ] ) . all subjects must have experienced failure to heal during the first 4 weeks of wound center care and also to have experienced failure of decrease in their wound size by at least 40% . these inclusion criteria are consistent with the inclusion criteria of diabetic foot ulcer randomized clinical trials ( rcts ) registered with the food and drug administration and with the reimbursement guidelines from centers for medicare and medicaid services ( cms ) ( 1012 ) . the cms guidelines for hbo reimbursement suggest that patients should have type i or ii diabetes , adequate lower limb arterial blood flow , a deep skin ulcer penetrating down to ligaments , muscle , or deeper with tissue infection ( e.g. , a wagner wound grade of 3 ) , and a wound that has failed to heal despite a 30-day course of standard wound management . similar criteria recently were approved by the national health insurance program of canada ( 13 ) . to validate our ability to properly access individuals with diabetes who had adequate lower - extremity arterial flow and foot ulcers , we received personal identifier redacted copies of the electronic medical chart with photographs for two groups of patients : one selected for having diabetic nonischemic foot ulcers ( our target population , n = 100 ) and the other selected for having wounds of the lower extremity but not diabetic nonischemic foot ulcers ( n = 100 ) . the nhc codes for an individual having a diabetic foot ulcer were based on a diagnosis of diabetes , diagnosis of arterial and venous disease ( exclusionary codes ) , and the location of the wound ( e.g. , foot ) . however , those in the other lower - extremity wound group could not have the codes listed for diabetic foot ulcers but could have the exclusionary codes . all materials were reviewed by a single investigator ( d.j.m . ) who was blinded to the database classification of the subject . an individual was thought to have a diabetic nonischemic foot ulcer if chart review revealed the following : diagnosis of diabetes ; no mention of lower limb ischemia or venous disease ; and wound location on the plantar aspect of the foot that could be confirmed by a photograph . to compare database ascertainment with actual chart assessment , we calculated the positive and negative predictive values , as well as the sensitivity and specificity of the ascertainment method . the study time began 28 days after an eligible individual was first examined and enrolled in an nhc clinic . follow - up period is part of most clinical trials to ensure that a wound is chronic and not healing . person - time accrued until the subject had left the database , died , healed , had an amputation , or had been followed - up for 20 weeks since enrollment at the nhc center ( i.e. , 16 weeks after they were enrolled in our study population ) . we previously have used this definition of person - time in a similarly designed study ( 14 ) . we studied two outcomes : healed wound ( as determined by the local wound care provider based on a wound being fully epithelized and no longer requiring a bandage ) and lea . all outcomes were assessed 16 weeks after the subject became eligible for our study or 20 weeks after enrollment at an nhc center . covariates included age , sex , wound duration at enrollment , wound size at enrollment , wagner grade , number of wounds on the patient , wound location , history of neuropathy , history of wound recurrence , and history of osteomyelitis or abscess . descriptive statistics were generated for covariates and presented as percentages , means and standard deviations . statistical tests for testing differences between groups or quintiles included tests , t tests , and anova . in this study , hence , selection bias could potentially affect our results if the choice of therapy depended on patient factors that were associated with the outcome ( in this case lea or impaired heal ) . to minimize bias we used a ps approach . this approach allowed us to achieve balance on observed covariates between treatment groups so that the treatment groups were more comparable ( 9 ) . the ps represents a summary value of the potential covariates for each patient and are defined , regardless of the actual treatment choice , as the probability that each patient would receive the treatment of interest given the background covariates of that subject . our ps model was used to balance a number of baseline covariates such as age , sex , wound age , wound size , wagner wound grade 2 , the number of wounds on the patient , history of neuropathy , history of wound recurrence , and history of osteomyelitis or abscess . importantly , wound age , wound size , and wagner wound grade 2 at first visit are highly predictive of the likelihood that a subject will heal and has been used in other ps studies of wound therapies ( 1416 ) . we first used logistic regression to estimate the ps for each individual by regressing treatment assignment on relevant covariates . we used the estimated ps in several models based on matching and stratification ( based on ps quintiles ) as a weighting factor and as a continuous covariate for adjustment in proportional hazards analyses . in all proportional hazard models , the proportional hazards assumption was tested and confirmed using log - log plots and schoenfeld residuals test . we also conducted an instrumental variables analysis , which is an alternative statistical approach used to minimize selection bias from unmeasured and measured confounders ( supplementary materials ) ( 9,17 ) . , an observed association between treatment and outcome may reflect the effects of unknown or unmeasured confounders that might not have been adjusted for using the ps approach . we conducted a sensitivity analyses to assess the effects of an unmeasured binary confounder on the estimated hazard ratio ( hr ) for amputation and healing obtained using the ps model ( 18,19 ) . sensitivity analyses were performed using r version 2.14 ( the r foundation for statistical computing , vienna , austria ) . nhc is a company that provided comprehensive wound care management solutions to local wound care centers across the united states . nhc developed clinical pathways to guide treatment based on reviews of best practice and guidelines on proper diagnosis and medical care ( e.g. , debridement , good wound care , and off - loading ) that were taught to the local wound care providers . subjects were included in the study if they were treated at an nhc center between november 2005 and may 2011 , and if the local wound care center had contractually agreed to provide data for research , resulting in 83 centers located in 31 states being available for study . subjects must have had diabetes , adequate lower - extremity arterial flow ( as determined by the clinician ) , and a wound on their plantar foot ( hindfoot [ heel ] , midfoot , or forefoot [ toes ] ) . all subjects must have experienced failure to heal during the first 4 weeks of wound center care and also to have experienced failure of decrease in their wound size by at least 40% . these inclusion criteria are consistent with the inclusion criteria of diabetic foot ulcer randomized clinical trials ( rcts ) registered with the food and drug administration and with the reimbursement guidelines from centers for medicare and medicaid services ( cms ) ( 1012 ) . the cms guidelines for hbo reimbursement suggest that patients should have type i or ii diabetes , adequate lower limb arterial blood flow , a deep skin ulcer penetrating down to ligaments , muscle , or deeper with tissue infection ( e.g. , a wagner wound grade of 3 ) , and a wound that has failed to heal despite a 30-day course of standard wound management . similar criteria recently were approved by the national health insurance program of canada ( 13 ) . to validate our ability to properly access individuals with diabetes who had adequate lower - extremity arterial flow and foot ulcers , we received personal identifier redacted copies of the electronic medical chart with photographs for two groups of patients : one selected for having diabetic nonischemic foot ulcers ( our target population , n = 100 ) and the other selected for having wounds of the lower extremity but not diabetic nonischemic foot ulcers ( n = 100 ) . the nhc codes for an individual having a diabetic foot ulcer were based on a diagnosis of diabetes , diagnosis of arterial and venous disease ( exclusionary codes ) , and the location of the wound ( e.g. , foot ) . however , those in the other lower - extremity wound group could not have the codes listed for diabetic foot ulcers but could have the exclusionary codes . all materials were reviewed by a single investigator ( d.j.m . ) who was blinded to the database classification of the subject . an individual was thought to have a diabetic nonischemic foot ulcer if chart review revealed the following : diagnosis of diabetes ; no mention of lower limb ischemia or venous disease ; and wound location on the plantar aspect of the foot that could be confirmed by a photograph . to compare database ascertainment with actual chart assessment , we calculated the positive and negative predictive values , as well as the sensitivity and specificity of the ascertainment method . the study time began 28 days after an eligible individual was first examined and enrolled in an nhc clinic . this pretrial follow - up period is part of most clinical trials to ensure that a wound is chronic and not healing . person - time accrued until the subject had left the database , died , healed , had an amputation , or had been followed - up for 20 weeks since enrollment at the nhc center ( i.e. , 16 weeks after they were enrolled in our study population ) . we previously have used this definition of person - time in a similarly designed study ( 14 ) . we studied two outcomes : healed wound ( as determined by the local wound care provider based on a wound being fully epithelized and no longer requiring a bandage ) and lea . all outcomes were assessed 16 weeks after the subject became eligible for our study or 20 weeks after enrollment at an nhc center . covariates included age , sex , wound duration at enrollment , wound size at enrollment , wagner grade , number of wounds on the patient , wound location , history of neuropathy , history of wound recurrence , and history of osteomyelitis or abscess . descriptive statistics were generated for covariates and presented as percentages , means and standard deviations . statistical tests for testing differences between groups or quintiles included tests , t tests , and anova . in this study , hence , selection bias could potentially affect our results if the choice of therapy depended on patient factors that were associated with the outcome ( in this case lea or impaired heal ) . to minimize bias we used a ps approach . this approach allowed us to achieve balance on observed covariates between treatment groups so that the treatment groups were more comparable ( 9 ) . the ps represents a summary value of the potential covariates for each patient and are defined , regardless of the actual treatment choice , as the probability that each patient would receive the treatment of interest given the background covariates of that subject . our ps model was used to balance a number of baseline covariates such as age , sex , wound age , wound size , wagner wound grade 2 , the number of wounds on the patient , history of neuropathy , history of wound recurrence , and history of osteomyelitis or abscess . importantly , wound age , wound size , and wagner wound grade 2 at first visit are highly predictive of the likelihood that a subject will heal and has been used in other ps studies of wound therapies ( 1416 ) . we first used logistic regression to estimate the ps for each individual by regressing treatment assignment on relevant covariates . we used the estimated ps in several models based on matching and stratification ( based on ps quintiles ) as a weighting factor and as a continuous covariate for adjustment in proportional hazards analyses . in all proportional hazard models , the proportional hazards assumption was tested and confirmed using log - log plots and schoenfeld residuals test . we also conducted an instrumental variables analysis , which is an alternative statistical approach used to minimize selection bias from unmeasured and measured confounders ( supplementary materials ) ( 9,17 ) . in nonrandomized studies , an observed association between treatment and outcome may reflect the effects of unknown or unmeasured confounders that might not have been adjusted for using the ps approach . we conducted a sensitivity analyses to assess the effects of an unmeasured binary confounder on the estimated hazard ratio ( hr ) for amputation and healing obtained using the ps model ( 18,19 ) . sensitivity analyses were performed using r version 2.14 ( the r foundation for statistical computing , vienna , austria ) . of the 200 charts reviewed , in those categorized as diabetic nonischemic foot ulcer , the diagnosis was confirmed in 93 of 100 ; in those categorized in the other lower - extremity wound group , 98 of 100 were confirmed . therefore , our database algorithm had a positive predictive value of 97.9% ( 95% ci 92.699.7 ) , a negative predictive value of 93.3% ( 86.797.3 ) , a sensitivity of 93% ( 86.197.1 ) , and a specificity of 98.0% ( 93.099.8 ) . there were 11,301 subjects with 32,021 wounds enrolled at eligible wound care centers with diabetic foot ulcers between november 2005 and may 2011 . per the enrollment criteria , these subjects had adequate lower - extremity arterial flow ( as determined by the clinician ) and a wound on their plantar foot ( hindfoot [ heel ] , midfoot , or forefoot [ toes ] ) . finally , per our inclusion criteria , to more closely mimic food and drug administration approved rcts , our study was limited to 6,259 subjects who did not experience healing or have an lea or who experienced failure to decrease the wound size by at least 40% in the first 28 days of care . the majority ( 83% ) of subjects were excluded because they had healed or had an amputation in the first 28 days of care . the average age of this cohort was 62.8 24.6 years , 43.5% were women , 74.2% were white , and 4.5% had lea ( table 1 ) . the average wound was 1.5 6.7 cm and 1.1 4.0 months old , with 19.3% of the wounds being wagner grade 3 , and 48.6% of the wounds healed by study week 16 ( table 1 ) . in total , we analyzed 767,060 person - days of wound care therapy . hbo was administered to 12.7% of the subjects , most frequently to a depth of 2.0 atm ( 88.5% of treatments ) , 5 days per week ( 88% ) , and for 90-min sessions ( 99.5% ) . basic characteristics of the study cohort within ps strata , with the exception of wound grade , the distributions of all covariates were similar regardless of treatment . as an example , table 2 shows the balance of the three variables most often used to predict that a wound will heal . those in quintile 2 ( a group less likely to receive hbo ) and quintile 5 ( most likely to receive hbo ) were not well - balanced ( p < 0.002 and p = 0.05 , respectively ) with respect to the percentage of individuals with wounds of wagner grade 2 . a sample of the covariate balance based on ps quintiles is illustrated using three variables that are highly predictive of wound healing survival analyses with adjustment for the ps showed that individuals receiving hbo therapy were more likely to have a lower limb amputation ( hr 2.37 [ 95% ci 1.843.04 ] ) and were less likely to heal ( 0.68 [ 0.630.73 ] ) when compared with those who did not receive this therapy ( table 3 ) . these estimates were nearly identical for all analytic methods ( ps adjustment , stratification , or nearest neighbor matching ) . however , because of our failure to fully balance wagner wound grade and also to be consistent with cms criteria , we separately evaluated individuals with wagner wound grade 3 ( table 3 ) . these subjects also were less likely to heal and more likely to have an amputation with hbo therapy ( table 3 ) . the wound center had no effect on the likelihood that a wound might heal after controlling for wound duration , wound size , and wound grade . hrs for the effectiveness of hbo versus conventional care using ps approach among those who received hbo , a median of 29 ( 2575% ; 1548 ) treatments were received . it is important to note that those who received hbo received their leas 3 weeks later than those who did not ( p = 0.02 ) . on average , amputations occurred at 88.6 90.1 days for those who did not receive hbo versus 106.1 days 113.2 for those who received hbo . to assure treatment acceptance , hbo exposure was altered to require at least eight treatments ; hbo therapy still was associated with increased amputation , ( hr 2.03 [ 95% ci 1.492.77 ] ) and fewer healed wounds ( 0.73 [ 0.660.81 ] ) . we conducted a regression - based sensitivity analysis to assess the effects of an unmeasured confounder on the estimated treatment effect hr resulting from the ps analysis . analyses were conducted for both the estimated hrs for amputation ( hr 2.37 [ 95% ci 1.843.04 ] ) and healing ( 0.66 [ 0.630.73 ] ) . for the amputation outcome , the effect estimate was quite robust in the presence of an unmeasured confounder . for example , if there was an unmeasured confounder that was prevalent in 80% of those who received hbo and in only 10% of those in the comparison group , and if the hr for this unmeasured confounder was quite extreme with respect to amputation ( i.e. , hr 2.5 ) , then after adjustment for the hypothetical unmeasured confounder we would have an hbo - based hr of 1.24 with 95% ci of 0.961.59 , meaning that those who received hbo had a similar outcome as those who did not . for the healing outcome , similarly , the estimated hr after adjustment for measured confounders via the ps was 0.66 ( 0.630.70 ) . in this case , if the unmeasured confounder was present only in 10% of the treated group and in 80% of the comparison group , and if the hazard associated with the unmeasured binary confounder was 2.0 , then the new adjusted hr would be 1.08 with 95% ci of 1.031.19 . in this situation , our hr would be < 1 and no longer statistically significantly . however , it is important to realize that our a priori assumption was that hbo prevented amputations and healed more wounds . for this assumption to be true , the direction of the effect estimates would have to be reversed ( e.g. , the 2.37 we measured changed to 0.5 to show fewer amputations ) and would have to be statistically significant . for this to occur , the magnitude of the unmeasured confounder presented would need to be even more extreme and therefore is even more unlikely to exist . finally , we also conducted analyses using an instrumental variable that yielded similar results ( supplementary data ) . of the 200 charts reviewed , in those categorized as diabetic nonischemic foot ulcer , the diagnosis was confirmed in 93 of 100 ; in those categorized in the other lower - extremity wound group , 98 of 100 were confirmed . therefore , our database algorithm had a positive predictive value of 97.9% ( 95% ci 92.699.7 ) , a negative predictive value of 93.3% ( 86.797.3 ) , a sensitivity of 93% ( 86.197.1 ) , and a specificity of 98.0% ( 93.099.8 ) . there were 11,301 subjects with 32,021 wounds enrolled at eligible wound care centers with diabetic foot ulcers between november 2005 and may 2011 . per the enrollment criteria , these subjects had adequate lower - extremity arterial flow ( as determined by the clinician ) and a wound on their plantar foot ( hindfoot [ heel ] , midfoot , or forefoot [ toes ] ) . finally , per our inclusion criteria , to more closely mimic food and drug administration approved rcts , our study was limited to 6,259 subjects who did not experience healing or have an lea or who experienced failure to decrease the wound size by at least 40% in the first 28 days of care . the majority ( 83% ) of subjects were excluded because they had healed or had an amputation in the first 28 days of care . the average age of this cohort was 62.8 24.6 years , 43.5% were women , 74.2% were white , and 4.5% had lea ( table 1 ) . the average wound was 1.5 6.7 cm and 1.1 4.0 months old , with 19.3% of the wounds being wagner grade 3 , and 48.6% of the wounds healed by study week 16 ( table 1 ) . in total hbo was administered to 12.7% of the subjects , most frequently to a depth of 2.0 atm ( 88.5% of treatments ) , 5 days per week ( 88% ) , and for 90-min sessions ( 99.5% ) . basic characteristics of the study cohort within ps strata , with the exception of wound grade , the distributions of all covariates were similar regardless of treatment . as an example , table 2 shows the balance of the three variables most often used to predict that a wound will heal . those in quintile 2 ( a group less likely to receive hbo ) and quintile 5 ( most likely to receive hbo ) were not well - balanced ( p < 0.002 and p = 0.05 , respectively ) with respect to the percentage of individuals with wounds of wagner grade 2 . a sample of the covariate balance based on ps quintiles is illustrated using three variables that are highly predictive of wound healing survival analyses with adjustment for the ps showed that individuals receiving hbo therapy were more likely to have a lower limb amputation ( hr 2.37 [ 95% ci 1.843.04 ] ) and were less likely to heal ( 0.68 [ 0.630.73 ] ) when compared with those who did not receive this therapy ( table 3 ) . these estimates were nearly identical for all analytic methods ( ps adjustment , stratification , or nearest neighbor matching ) . however , because of our failure to fully balance wagner wound grade and also to be consistent with cms criteria , we separately evaluated individuals with wagner wound grade 3 ( table 3 ) . these subjects also were less likely to heal and more likely to have an amputation with hbo therapy ( table 3 ) . the wound center had no effect on the likelihood that a wound might heal after controlling for wound duration , wound size , and wound grade . hrs for the effectiveness of hbo versus conventional care using ps approach among those who received hbo , a median of 29 ( 2575% ; 1548 ) treatments were received . it is important to note that those who received hbo received their leas 3 weeks later than those who did not ( p = 0.02 ) . on average , amputations occurred at 88.6 90.1 days for those who did not receive hbo versus 106.1 days 113.2 for those who received hbo . to assure treatment acceptance , hbo exposure was altered to require at least eight treatments ; hbo therapy still was associated with increased amputation , ( hr 2.03 [ 95% ci 1.492.77 ] ) and fewer healed wounds ( 0.73 [ 0.660.81 ] ) . we conducted a regression - based sensitivity analysis to assess the effects of an unmeasured confounder on the estimated treatment effect hr resulting from the ps analysis . analyses were conducted for both the estimated hrs for amputation ( hr 2.37 [ 95% ci 1.843.04 ] ) and healing ( 0.66 [ 0.630.73 ] ) . for the amputation outcome , the effect estimate was quite robust in the presence of an unmeasured confounder . for example , if there was an unmeasured confounder that was prevalent in 80% of those who received hbo and in only 10% of those in the comparison group , and if the hr for this unmeasured confounder was quite extreme with respect to amputation ( i.e. , hr 2.5 ) , then after adjustment for the hypothetical unmeasured confounder we would have an hbo - based hr of 1.24 with 95% ci of 0.961.59 , meaning that those who received hbo had a similar outcome as those who did not . for the healing outcome , similarly , the estimated hr after adjustment for measured confounders via the ps was 0.66 ( 0.630.70 ) . in this case , if the unmeasured confounder was present only in 10% of the treated group and in 80% of the comparison group , and if the hazard associated with the unmeasured binary confounder was 2.0 , then the new adjusted hr would be 1.08 with 95% ci of 1.031.19 . in this situation , our hr would be < 1 and no longer statistically significantly . however , it is important to realize that our a priori assumption was that hbo prevented amputations and healed more wounds . for this assumption to be true , the direction of the effect estimates would have to be reversed ( e.g. , the 2.37 we measured changed to 0.5 to show fewer amputations ) and would have to be statistically significant . for this to occur , the magnitude of the unmeasured confounder presented would need to be even more extreme and therefore is even more unlikely to exist . finally , we also conducted analyses using an instrumental variable that yielded similar results ( supplementary data ) . unfortunately , the literature on the efficacy of hbo therapy is not clear . perhaps more importantly , the importance of effectiveness studies on therapies for the treatment of diabetic foot ulcer is critical , as evidenced by the poor utilization of efficacious therapies like skin substitutes , recombinant human platelet - derived growth factor , and total contact casting ( 2022 ) . these are therapies that have been extensively investigated with high - quality efficacy studies , although not all are consistently reimbursed and therefore are not used extensively in clinical practice . in our study , we were not able to show that hbo prevented amputation or improved the likelihood that a wound would heal . in fact , using multiple analytic approaches , it appeared that those who received hbo were 1.5 to 3 times more likely to have an amputation than those who did not receive hbo , and they were also 1.2 to 3 times less likely to heal their foot ulcer . hbo has the potential to have many differing effects on a chronic wound and it may not be reasonable to assume that this therapy should be used to fully heal a wound . in fact , those who study wound care have been concerned that the requirement by regulators that a wound care product must heal a wound to receive approval may be nave and not consistent with the biology of wound repair . hbo therapy may have a beneficial effect on microbial balance , soft tissue infection , and angiogenesis . hbo simply may be a part of the answer and not a therapy that should be used until a wound fully heals . approximately one - third of the subjects in our study who received hbo received more than the recommended maximum number of treatments ( e.g. , > 40 ) ( 23 ) . most wound care guidelines explicitly mention the need to try other therapies if a therapy is not successful ( 24 ) . it is conceivable that more attention needs to be given to the coordination between hbo usage and debridement or the use of other adjuvants like skin substitutes , recombinant human platelet - derived growth factor , total contact cast , and others ( 24 ) . further , this potentially improper usage may explain the conflicting reports in the literature about the efficacy of hbo ( 8) . osteoradionecrosis of the jaws is another condition treated with hbo and the timing of surgical intervention is an important variable with respect to success ( 25 ) . a standard protocol shown to be effective is 20 preoperative hbo treatments before oral surgery . when hbo is used without surgical intervention it has no lasting benefit ( 26 ) . in 2012 , an rct - based meta - analysis was published on the efficacy of hbo ( 8) . the authors found seven rcts of 369 subjects that evaluated the efficacy of hbo as compared with conventional therapy ( 8) . hbo therapy varied from 2 to 3 atm for 45 to 120 min , administered once or twice per day , 4 to 5 times per week ( 8) . the case definition for diabetic foot ulcer varied across studies but mostly included wounds below the ankle in individuals with diabetes . the wound grade varied from wagner grade 0 to 4 , with most studies including individuals with wounds of grade 2 and allowing individuals with poor lower - extremity arterial blood flow ( 8) . the conventional therapy arm also varied by trial but usually involved off - loading the foot . their meta - estimates did not show an advantage of hbo therapy as compared with standard therapy with respect to a healed wound at 6 months ( two trials , n = 112 ) or 1 year ( three trials , n = 212 ) ( 8) . they also were not able to show fewer amputations , fewer minor amputations ( four trials , n = 242 ) , or fewer major amputations ( five trials , n = 309 ) in those who received hbo as compared with standard therapy ( 8) . although they could not show a statistically significant advantage , in most circumstances hbo appeared to be superior to conventional therapy . pooled data from three of the trials ( n = 140 ) at 6 weeks did show that individuals who received hbo were more likely to heal ( 8) . interestingly , a recent randomized trial had demonstrated that another device was approximately two times more efficacious than hbo , but this same device was later shown not to be more efficacious than standard off - loading ( 27 ) . it is important to realize that many of the subjects studied in the trials described would not have met cms eligibility criteria for treatment . our goal was to compare those who received hbo with those who did not receive hbo . the nhc algorithm suggests that individuals with diabetic foot ulcers receive debridement , off - loading , good wound care , and the consideration of other therapies like recombinant human platelet - derived growth factor and skin substitutes . we did not investigate which other therapies were the most successful , but our results indicate that as a group they were more successful than hbo and these other strategies need to be considered . even though we used statistical methods such as ps or instrumental variable , to try to understand and adjust for treatment selection bias , it is still possible that our results are biased . however , we did include in our ps variables that have been shown to correctly predict whether a wound will heal and to explain > 90% of variability between predicted and actual outcomes ( 16,28,29 ) . the covariates that are most highly predictive of wound healing were either well - balanced ( i.e. , wound duration and wound size ) or evaluated by exclusion ( i.e. , wound grade ) . these variables have been used in other ps assessments of the effectiveness of wound therapies ( 14,28 ) . finally , we conducted a sensitivity analysis to evaluate the effect of unmeasured confounders on our results . the results of the sensitivity analysis demonstrate the robustness of our results with any potential unknown confounders . however , until multiple , well - designed , large rcts evaluate outcomes other than a fully healed wound , it may not be possible to truly understand if hbo provides a benefit . also , because we conducted our study in wound care centers , our results might not be generalizable to all wound care providers . however , all wound care providers do not have hbo facilities ; however , many wound care centers like nhc do have hbo on site , thereby improving the likelihood that our study generalizes to those that have hbo on site . in conclusion , hbo did not appear to be useful for the prevention of amputation and did not improve the likelihood that a wound would heal in a cohort of patients defined by cms eligibility criteria . to date , this is the largest study of hbo . it is important to note that our findings may not be consistent with the food and drug administration clearance for this device , which was based on the undersea and hyperbaric medical society approved indications . per these recommendations , it is entirely likely that hbo therapy enhances a specific aspect of wound repair and should not be used as a single agent to completely heal a wound . this also was likely true for several scientifically valid compounds , which when tested in vitro and in animal studies had shown promise with respect to wound healing but when tested in humans were not shown to heal chronic wounds ( 11,30 ) . the usefulness of hbo in the treatment of diabetic foot ulcers needs to be better - clarified , preferably using well - designed rcts and perhaps using other healing - based end points other than a healed wound .

objectivehyperbaric oxygen ( hbo ) is a device that is used to treat foot ulcers . the study goal was to compare the effectiveness of hbo with other conventional therapies administered in a wound care network for the treatment of a diabetic foot ulcer and prevention of lower - extremity amputation.research design and methodsthis was a longitudinal observational cohort study . to address treatment selection bias , we used propensity scores to determine the propensity that an individual was selected to receive hbo.resultswe studied 6,259 individuals with diabetes , adequate lower limb arterial perfusion , and foot ulcer extending through the dermis , representing 767,060 person - days of wound care . in the propensity score adjusted models , individuals receiving hbo were less likely to have healing of their foot ulcer ( hazard ratio 0.68 [ 95% ci 0.630.73 ] ) and more likely to have an amputation ( 2.37 [ 1.843.04 ] ) . additional analyses , including the use of an instrumental variable , were conducted to assess the robustness of our results to unmeasured confounding . hbo was not found to improve the likelihood that a wound might heal or to decrease the likelihood of amputation in any of these analyses.conclusionsuse of hbo neither improved the likelihood that a wound would heal nor prevented amputation in a cohort of patients defined by centers for medicare and medicaid services eligibility criteria . the usefulness of hbo in the treatment of diabetic foot ulcers needs to be reevaluated .

benign prostate hyperplasia ( bph ) is a very common disease among aging men and prevalence rate increase in men over the age of fifty ( mcvary , 2006 ) and it has been estimated that actively managed cost for bph is 12.2 million annually in united states ( vuichoud and loughlin , 2015 ) . in korea , according to recent statistics from the health insurance review and assessment service , 1,021,222 people were registered as patients with bph in 2014 . it is the fourth most common diagnosis in older men who has lower urinary tract symptoms ( luts ) that consist of bothersome , impairment of psychological and functional well - being , and interference with daily life activities ( coyne et al . , 2009 ) . 5-alpha reductase converts testosterone to dihydrotestosterone ( dht ) that is most potent androgen and affected in prostate growth and could bind to intracytoplasmic androgen receptor . and then complex of ar with dht enhances activation of androgen response elements ( andriole et al . , 2004 ; steers , 2001 ) . recently , type iii 5-reductase was reported and was overexpressed in prostate cancer ( uemura et al . , 2008 ) . type i 5-reductase ( srd5a1 ) and type ii 5-reductase ( srd5a2 ) were well known and those inhibitors were clinically used as medication for alopecia and bph ( cindolo et al . , 2013 ; sudduth and koronkowski , 1993 ) . however , the roles of type iii 5-alpha reductase ( srd5a3 ) in bph patients are unknown . we evaluated whether srd5a3 polymorphism was associated with susceptibility of bph or not and the combined effects in bph risk between the type of short tandem repeat ( str ) in srd5a3 and the length of cag repeats in ar gene in patients with bph . from july 2014 to june 2016 , bph patients who had a histological confirmed diagnosis after transurethral resection of prostate at chungnam national university hospital were involved in this study . after approval from the institutional review board , informed consent was obtained from the study participants . genomic dna was obtained from 188 patients with bph ( mean age , 69.78.1 years ) and from 98 healthy controls ( mean age , 63.78.1 years ) . the healthy controls were selected by age over 50 years old with less than 10 points of international prostate symptoms scores and without evidences of prostate cancer using a prostate - specific antigen and digital rectal examinations . for the analysis of str , genomic dnas were isolated from 1-ml blood sample using by genomic dna prep kit ( cat . sgd61-s120 , solgent co. , daejeon , korea ) according to the manufacturer s instruction . the qualities of isolated genomic dna samples were tested using 1% agarose gel electrophoresis and their quantities were measured by nanodrop ( thermo fisher scientific , waltham , ma usa ) . the polymerase chain reaction ( pcr ) products for the loci containing the str region ( srd5a3 gene , ncbi reference sequence : nm_024592.4 ) were obtained using a mixture containing 10 pmol from each primer : 5-gta gat gag act tct cca agc tg-3 ( forward ) and 5-caa caa aca gtt att gag cac -3 ( reverse ) , 10x h - taq buffer , 10 mm dntp(t ) , h - taq dna polymerase ( solgent co. ) , and 200-ng genomic dna isolated from blood sample . and we also obtained the pcr products for the loci containing the cag repeats ( ar gene , national center for biotechnology information [ ncbi ] reference sequence : bc132975.1 ) using each primer : 5-tcc aag acc tac cga gga gct-3 ( forward ) and 5-tgt gaa ggt tgc tgt tcc tca tc-3 ( reverse ) . after the initial denaturation of the reaction mixture at 95c for 3 min , amplification was achieved by 35 cycles at 95c for 20 sec , 58c for 40 sec and 72c for 60 sec , and a final extension at 72c for 5 min . after purifying the pcr product using pcr purification kit ( solgent co. ) , sequences of the str region were confirmed by the direct sequencing analysis . for the genescan analysis of str , ac repeat loci were amplified by the fluorescent dye - tagged forward primer and reverse primer . after the purification of amplified pcr product , 2-ng pcr products were mixed with hi - di tm formamide ( applied biosystems , foster city , ca , usa ) and pop-4 tm polymer ( applied biosystems ) size marker . the reaction mix were incubated at 96c , 2 min and 4c , 3 min , and then analyzed by abi 3100 genetic analyzer ( applied biosystems ) . the obtained data of samples were analyzed by genemapper 4.0 ( applied biosystems ) program . according to the values analyzed by genemapper 4.0 and nucleotide sequence , the repeat numbers of str for each sample were estimated as previously reported ( park et al . , 2013 ) . statistical analysis was performed to identify an association between the bph status and the length of str of srd5a3 , the length of cag repeats in the ar gene , and in combination . the odds ratios ( ors ) and 95% confidence intervals ( 95% cis ) were calculated using a chi - square test and binary logistic regression . from july 2014 to june 2016 , bph patients who had a histological confirmed diagnosis after transurethral resection of prostate at chungnam national university hospital were involved in this study . after approval from the institutional review board , informed consent was obtained from the study participants . genomic dna was obtained from 188 patients with bph ( mean age , 69.78.1 years ) and from 98 healthy controls ( mean age , 63.78.1 years ) . the healthy controls were selected by age over 50 years old with less than 10 points of international prostate symptoms scores and without evidences of prostate cancer using a prostate - specific antigen and digital rectal examinations . for the analysis of str , genomic dnas were isolated from 1-ml blood sample using by genomic dna prep kit ( cat . sgd61-s120 , solgent co. , daejeon , korea ) according to the manufacturer s instruction . the qualities of isolated genomic dna samples were tested using 1% agarose gel electrophoresis and their quantities were measured by nanodrop ( thermo fisher scientific , waltham , ma usa ) . the polymerase chain reaction ( pcr ) products for the loci containing the str region ( srd5a3 gene , ncbi reference sequence : nm_024592.4 ) were obtained using a mixture containing 10 pmol from each primer : 5-gta gat gag act tct cca agc tg-3 ( forward ) and 5-caa caa aca gtt att gag cac -3 ( reverse ) , 10x h - taq buffer , 10 mm dntp(t ) , h - taq dna polymerase ( solgent co. ) , and 200-ng genomic dna isolated from blood sample . and we also obtained the pcr products for the loci containing the cag repeats ( ar gene , national center for biotechnology information [ ncbi ] reference sequence : bc132975.1 ) using each primer : 5-tcc aag acc tac cga gga gct-3 ( forward ) and 5-tgt gaa ggt tgc tgt tcc tca tc-3 ( reverse ) . after the initial denaturation of the reaction mixture at 95c for 3 min , amplification was achieved by 35 cycles at 95c for 20 sec , 58c for 40 sec and 72c for 60 sec , and a final extension at 72c for 5 min . after purifying the pcr product using pcr purification kit ( solgent co. ) , sequences of the str region were confirmed by the direct sequencing analysis . for the genescan analysis of str , ac repeat loci were amplified by the fluorescent dye - tagged forward primer and reverse primer . after the purification of amplified pcr product , 2-ng pcr products were mixed with hi - di tm formamide ( applied biosystems , foster city , ca , usa ) and pop-4 tm polymer ( applied biosystems ) size marker . the reaction mix were incubated at 96c , 2 min and 4c , 3 min , and then analyzed by abi 3100 genetic analyzer ( applied biosystems ) . the obtained data of samples were analyzed by genemapper 4.0 ( applied biosystems ) program . according to the values analyzed by genemapper 4.0 and nucleotide sequence , the repeat numbers of str for each sample were estimated as previously reported ( park et al . , 2013 ) . statistical analysis was performed to identify an association between the bph status and the length of str of srd5a3 , the length of cag repeats in the ar gene , and in combination . the odds ratios ( ors ) and 95% confidence intervals ( 95% cis ) were calculated using a chi - square test and binary logistic regression . all the tests in this article were implemented using ibm spss statistics ver . 20.0 ( ibm co. , armonk , ny , usa ) . we defined that a short type of allele had less than 21 copies of ac repeats in the str region of srd5a3 gene . short and short type of str in srd5a3 gene was 1.89 times more likely to occur in bph patients than in controls ( or , 1.89 ; 95% ci , 0.983.64 ; p=0.058 ) . the men who had at least one of short type have a 3.1 times of bph risk compared with controls ( or , 3.10 ; 95% ci , 1.875.16 ; p=0.000 ) ( table 1 ) . we divided into two groups as the cutoff value was 22 copies of the length of cag repeats in ar gene . the men who had a less than 22 copies cag repeats of ar gene had a 1.5 times of bph risk but there was no statistical significance . however , if cutoff value was 23 copies of the length of cag repeats in ar gene , the men who had a less than 23 copies cag repeats of ar gene had a 2.35 times of bph risk compared with controls ( or , 2.35 ; 95% ci , 1.182.36 ; p=0.016 ) ( table 2 ) . we calculated age - adjusted p - value to be compared ss and sl type with ll type of str in srd5a3 gene and cutoff value was 23 copies of the length of cag repeats in ar gene according to strong statistical evidences from tables 1 and 2 . the age adjustment did not affect the statistical significance of two variables in the multivariate logistic regression model ( table 3 ) . in table 4 , we examined the combined effects of the type of str and the length of cag repeats . the men who have the large type of str and 23 copies of cag repeats have 5.3 times bph risk compared to the reference group of men who have the at least one of the short type of str and < 23 copies of cag repeats ( p<0.000 ) . similarly , the men who have the large type of str and < 23 copies of cag repeats have 2.2 times bph risk compared to the reference group ( p=0.027 ) . the upward trend of bph risk along the risk groups is observed ( p<0.000 ) . testosterone is irreversibly converted to dht by 5 alpha - steroid reductase ( srd5a ) that have a major roles to make various secretory proteins to associate with prostate development and growth ( zhu and imperato - mcginley , 2009 ) . 5-alpha reductase has three isoenzymes to be identified as srd5a1 , srd5a2 , and srd5a3 ( thomas et al . , 2005 ; uemura et al . , 2008 ) srd5a1 , srd5a2 are well known and developed inhibitors to decreased prostate volume and prevent alopecia and widely used therapeutic agents for bph ( sudduth and koronkowski , 1993 ) . srd5a3 had been called srd5a2l , srd5a2l1 , 3-oxo-5-alpha - steroid 4-dehydrogenase 3 , and cdg1p , but srd5a3 was approved as the symbol in the hugo gene nomenclature committee database . 2008 ) reported that srd5a3 is overexpressed in hormone - refractory prostate cancer tissues and regulates growth and viability in a prostate cancer cell line . however the roles of srd5a3 in bph condition have been unknown , there were a few literatures in association study between polymorphisms in srd5a and bph . ( 2004 ) reported that polymorphisms in srd5a2 were not associated with severity of bph but srd5a1 . this study was evaluated cag repeat of ar gene , single nucleotide polymorphism of srd5a2 gene and two silent polymorphisms in srd5a1 however not assessed combined effects each genes . our results in association between polymorphism in srd5a3 and bph risk suggested that the men who had at least one of short type have a 3.1 times of bph risk compared with the men who had both of large type ( or , 3.10 ; 95% ci , 1.875.16 ; p=0.000 ) ( table 1 ) . riley and krieger ( 2003 ) reported that longer strs provided larger and more accessible loops for processing resulting in transcripts with shorter half - lives and induced abnormal function of protein . and we already suggest possible mechanism that longer ac repeats of srd5a3 gene may induce the dysfunction of srd5a3 to be converted testosterone to dht ( park et al . , 2013 ) . it is involved prostate proliferation and may affect with risk of bph . ar gene is located in x - chromosome q12 and has major roles in proliferation and developments of prostate cells . in brazilian study , there was no evidence for an association between ar cag repeat length in bph risk in a population - based sample ( biolchi et al . , 2012 ) 2008 ) reported that there was an association between short ar gene cag repeat length and a small prostate volume , which confirms a previous finding in the finnish population . our findings in polymorphism in ar gene was similar result with finnish population study that if cutoff value was 23 copies of the length of cag repeats , the men who had a less than 23 copies cag repeats of ar gene had a 2.4 times of bph risk compared with the men who had an equal and greater than 23 copies cag repeats ( or , 2.35 ; 95% ci , 1.182.36 ; p=0.016 ) ( table 2 ) . the association of the length of cag repeats in ar with bph is still controversial ; we suggest that the longer repeats of cag would have stronger susceptibility with bph . we also evaluated combined effect polymorphism in srd5a3 and ar gene because srd5a and ar gene play a key roles in prostate proliferation by binding ar with dht converted from testosterone which is more powerful form of androgen . we assumed that if it may occur aberrant translation in srd5a3 and ar genes , the functions of srd5a3 and ar would be impaired and caused less bph incidence , however , statistically significant results were not proven meanwhile statistical trend was shown . the main limitation of this study is the lack of age matched controls . because most of the middle aged patients visiting the urology department had previous urinary tract infection , systemic diseases , or luts , it was difficult for us to enroll the age match controls for comparison in this pilot study . in conclusion , we investigated polymorphisms of srd5a3 gene in bph risk and evaluated association between the length of cag repeat of ar and polymorphisms of srd5a3 gene . we observed that short ac repeats of srd5a3 and a less than 23 copies of cag repeats of ar gene were associated with an increased risk for bph . however , the interaction between the length of cag repeats in ar gene and the length of ac repeats in srd5a3 was not affected in risk of bph .

we evaluated whether type iii 5-alpha reductase ( srd5a3 ; steroid reductase 5-alpha 3 ) polymorphism was associated with susceptibility of benign prostate hyperplasia ( bph ) and the combined effects in bph risk between the type of short tandem repeat ( str ) in srd5a3 and the length of trinucleotide ( cag ) repeats in androgen receptor ( ar ) gene . we compared the length of ac repeats in str region of srd5a3 gene and a cag repeat in ar in 188 bph patients who underwent transurethral resection of prostate ( turp ) and 98 controls by polymerase chain reaction - based methods . we defined short type was less than 21 copies of ac repeats . the odds ratio for bph between the men with at least one of short type and with both large types of str in srd5a3 gene was 3.10 ( 95% confidence interval [ ci ] , 1.875.16 ; p=0.000 ) . and bph was 2.35 times more likely to occur in with less than 23 copies of cag repeats than men equal or greater than 23 copies in ar gene ( 95% ci , 1.182.36 ; p=0.016 ) . the men with the large type of str and 23 copies of cag repeats have 5.3 times bph risk compared to the reference group with the at least one of the short type of str and < 23 copies ( p<0.000 ) . in conclusion , these results suggest that shorter ac repeats of srd5a3 gene and shorter cag repeats of ar gene were associated with an increased risk for bph . however , the interaction between above two factors was not affected in risk of bph .

traumatic asphyxia is probably much more common than the surgical literature shows and should always be kept in mind as a possible complication of injuries of the chest and abdomen . traumatic asphyxia or perte s syndrome results from a severe crush injury causing sudden compression of the thorax . during a 3-year period , we treated five cases of traumatic asphyxia , which we report in this manuscript . they suffered different types of crushing injuries : industrial accidents in two patients , run over by motor vehicles in two patients , and a farm accident in one patient . most of the patients suffered some associated injuries , including fracture of the sternum in one patient , fracture of the right clavicle in one patient , and bilateral hemopneumothoraces in one patient . the treatment included bilateral chest tube thoracostomy in one patient , and the others required supportive treatment . treatment for traumatic asphyxia is supportive , and patient recovery is related to the generally associated injuries . traumatic asphyxia should always be kept in mind as a possible complication of injuries of the chest and abdomen . perte s syndrome or traumatic asphyxia is a clinical syndrome associated with craniocervical cyanosis , subconjunctival hemorrhage , multiple petechiae , and neurological symptoms . this syndrome occurs as a result of sudden or severe compression of the thorax or upper abdomen , or both . prompt treatment with attention to the reestablishment of oxygenation and perfusion may result in a good outcome . the patients ' recoveries were related to the severity of the injuries and the associated injuries . a total of five patients with diagnoses of traumatic asphyxia in our department were reported during the 3-year period . they suffered different types of crush injuries : industrial accidents , two patients ; being run over by a motor vehicle , two patients ; farm accident , one patient . we quickly evaluated all patients with computed tomography of the brain , thorax , and abdomen , and if needed with ultrasonography . laboratory studies showed elevated levels of creatine phosphokinase ( ck ) , lactic dehydrogenase ( ldh ) , aspartate aminotransferase ( ast ) , and alanine aminotransferase ( alt ) . most of the patients suffered some associated injuries including fracture of the sternum in one patient , fracture of the right clavicle in one patient , and bilateral hemopneumothoraces in one patient . their head , neck , and upper chest were strikingly cyanotic and edematous , with multiple petechiae ; they also had bilateral subconjunctival hemorrhages ( figs . 1 , 2 ) . the treatment included bilateral chest tube thoracostomy in one patient and measurement of arterial blood gases , oxygen , and fluid supplementation as conservative management in all patients . the hospital stay ranged from 4 to 7 days ( mean 5.6 days ) . the head , neck , and upper chest were strikingly cyanotic and edematous , with multiple petechiae ; he also had bilateral subconjunctival hemorrhages and bilateral hemopneumothoraxfig . the head , neck , and upper chest were strikingly cyanotic and edematous , with multiple petechiae ; he also had bilateral subconjunctival hemorrhages and bilateral hemopneumothorax photograph showing subconjunctival hemorrhages with traumatic asphyxia the first explanation for the development of the syndrome was offered by tardieu in 1866 , when he stated punctiform ecchymosis of the face , neck and chest are caused by the effort in which resistance to suffocation manifested itself . it manifests with facial and upper chest petechiae , subconjunctival hemorrhages , cervical cyanosis , and occasionally neurological symptoms . factors implicated in the development of these striking physical characteristics include thoracoabdominal compression after deep inspiration against a closed glottis , which results in venous hypertension in the valveless cervicofacial venous system . the typical pathological features of traumatic asphyxia consist of craniofacial purple congestion with petechial hemorrhages of the face , neck , upper chest , and conjunctivae . all of these findings ( conjunctival / facial petechiae , craniofacial congestion / swelling ) were present in all of our patients . the reason cyanosis , petechia , and edema are confined to the upper part of the body may be because the lower part of the body is protected from the elevated venous pressure by a series of valves . alternatively , increased airway pressure may compress or obliterate the inferior vena cava to protect the lower part of the body . the outcome in traumatic asphyxia is improved by rapid restoration of ventilation and blood circulation by thoracic decompression and fluid replacement . management of these cases included measurement of arterial blood gases , oxgen supplementation , and intubation with mechanical ventilation if needed . the prognosis is good , but a prolonged thoracic compression could lead to cerebral anoxia and neurological sequelae . the patient s recovery is related to the severity of the injury , the duration of the injury , and the associated injuries . traumatic asphyxia should always be kept in mind as a possible complication of injuries of the chest and abdomen .

backgroundtraumatic asphyxia is probably much more common than the surgical literature shows and should always be kept in mind as a possible complication of injuries of the chest and abdomen . aimstraumatic asphyxia or perte s syndrome results from a severe crush injury causing sudden compression of the thorax . during a 3-year period , we treated five cases of traumatic asphyxia , which we report in this manuscript.methodsthe patients were all male , ranging in age from 26 to 64 . they suffered different types of crushing injuries : industrial accidents in two patients , run over by motor vehicles in two patients , and a farm accident in one patient . most of the patients suffered some associated injuries , including fracture of the sternum in one patient , fracture of the right clavicle in one patient , and bilateral hemopneumothoraces in one patient . resultsthe treatment included bilateral chest tube thoracostomy in one patient , and the others required supportive treatment . there was no mortality.conclusiontreatment for traumatic asphyxia is supportive , and patient recovery is related to the generally associated injuries . traumatic asphyxia should always be kept in mind as a possible complication of injuries of the chest and abdomen .

obstructive sleep apnea - hypopnea syndrome ( osahs ) is a common disease characterized by repetitive episodes of partial or complete upper - airway obstruction during sleep . although estimates of disease prevalence are in the range of 37% for adult men and 25% for adult women in the general population , with prevalence rates reaching up to 33% in certain populations , osahs tends to be underdiagnosed in clinical practice . osahs is associated with various neurobehavioral and cardiovascular sequelae , among which sexual dysfunction remains the least studied . however , erectile dysfunction ( ed ) is a highly prevalent condition in patients with osahs [ 4 , 5 ] , and its frequency and severity appear to correlate with the severity of osahs . in addition , up to 91% of patients with erectile dysfunction may be diagnosed with osahs . ed has been defined as the persistent inability to attain and maintain an erection sufficient to permit satisfactory sexual performance . the prevalence of ed is also high , varying in different countries between 3% and 71% according to age . in greece , doumas and colleagues reported an ed prevalence of 14.1% in normotensive patients compared to 35.2% in patients with essential hypertension . there is a growing body of evidence suggesting that ed is predominantly a disease of vascular origin , with endothelial cell dysfunction as the unifying link . circulating markers of endothelial cell damage that have previously been reported include cytokines and chemokines , soluble adhesion molecules , and acute - phase reactants . several inflammatory cytokines , such as interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , tumor necrosis factor - a ( tnf - a ) , and high - sensitivity c - reactive protein ( hscrp ) , have been investigated and could be proposed as laboratory markers for potential use in ed . moreover , in vitro studies demonstrate that the above inflammatory factors suppress the production of adiponectin , which is released from adipose cells and exerts a variety of anti - inflammatory effects . several mechanisms have been proposed , including nerve involvement caused by hypoxemia , low levels of testosterone , and vascular endothelial dysfunction through diverse pathways such as hypoxemia , oxidative stress , and sympathetic activation . there is convincing evidence for endothelial dysfunction in osahs ; however , there are contradictory findings if administration of continuous positive airway pressure ( cpap ) reverse endothelial dysfunction associated with osahs [ 15 , 16 ] . endothelial dysfunction , as already mentioned , is also a key finding in the patient with ed , suggesting a mechanistic paradigm whereby osahs may lead to ed . however , limited information exists regarding the pathophysiologic basis of ed in osahs , in particular , regarding how intermittent nocturnal hypoxemia is likely to contribute to impaired penile tumescence by promoting endothelial dysfunction , which might be mediated by elevated levels of inflammatory markers as well as decreased levels of anti - inflammatory markers . therefore , the aim of this study was to assess whether osahs is associated with activation of the inflammatory cytokine system in patients with ed compared to patients with osahs and normal sexual function , evaluating a wide range of inflammatory ( hscrp , il-6 , il-8 , and tnf - a ) and anti - inflammatory ( adiponectin ) markers . from june 2011 to june 2012 , patients were recruited from a prospective study cohort comprising consecutive patients , between 18 and 65 years old , who visited the sleep disorders center , department of thoracic medicine , medical school university of crete , for evaluation of suspected sleep - disordered breathing . the inclusion criteria were the following : newly diagnosed osahs by polysomnography ( psg ) according to standard criteria , severe osahs , and an above - elementary school education . the exclusion criteria were the following : refusal to participate , previous cpap treatment , hypertension , coronary artery disease , congestive heart failure , history of life - threatening arrhythmias , cardiomyopathy , history of stroke , chronic renal disease , endocrine dysfunction , such as diabetes mellitus , cushing 's syndrome , and abnormal pituitary function , chronic obstructive pulmonary disease , morbid obesity ( bmi > 40 kg / m ) , dyslipidemias , undertreatment with cardiovascular medication , or pharmacologically treated depression , with severe cognitive impairment , with a family or personal history of mental illness , with drug or alcohol abuse , with concurrent oncologic diseases , metabolic or neurological disorders known to induce peripheral neuropathy or ed , deep vein thrombosis , peripheral vascular disease , connective tissue disorders , and history of narcolepsy . moreover , if patients were diagnosed with ed before the enrolment of this study or had already undergone therapy with medications that affect erectile function , such as b - blockers and h2 blockers , they were also eliminated from the study . all subjects provided written informed consent , and ethical approval was provided by the university hospital ethics committee . the study subjects underwent a detailed evaluation that included age ( years ) , body mass index ( bmi ) ( kg / m ) , clinical history focused on sleep - related symptoms , associated conditions and comorbidities , medication use , smoking history , and alcohol intake . furthermore , a urology specialist assessed sexual history and analyzed the use of medications that may interfere with erection function . each patient was asked to complete the greek version of the validated international index of erectile function ( iief ) questionnaire as a comprehensive tool for assessing erectile function . in addition , the epworth sleepiness scale ( ess ) was used to assess the degree of diurnal somnolence . after signing the informed consent form , the patients arrived at the sleep disorders center at least 2 hours before their normal bedtime and went to sleep at their habitual bedtimes , and blood samples were collected the following morning for biochemical and haematological analyses . a sleep physician coordinated any necessary clinical followup and made the results of the exams available for each patient . during these appointments , they received one - on - one counselling by a sleep physician regarding the results of their psg studies , basic information on osahs , its known effects on comorbid conditions , proper sleep hygiene , adjunctive / conservative methods to improve sleep , and the importance of treatment adherence . the iief is divided into five domains ( erectile function , intercourse satisfaction , orgasm , sex drive , and overall satisfaction ) . the erectile function domain consists of six questions ( questions 1 to 5 and question 15 ) and has a maximum score of 30 and a minimum score of 6 . each question is scored on a 5-point likert scale , with 5 representing the best score . the ess is currently the most used subjective test of daytime sleepiness in clinical practice . it is a simple , self - administered , and eight - item questionnaire that measures the risk of falling asleep in eight situations that are commonly met . the higher the score ( from 10 to 24 ) , the greater the reported subjective daytime sleepiness . all patients underwent a single - night full diagnostic psg ( alice 4 , 5 , diagnostics system , respironics , usa ) according to standard techniques , with monitoring of the electroencephalogram ( eeg ) using frontal , central , and occipital leads , electrooculogram ( eog ) , electromyogram ( emg ) , flow ( by oronasal thermistor and nasal air pressure transducer ) , thoracic and abdominal respiratory effort ( induction plethysmography ) , oximetry , and body position . polysomnographic recordings were manually interpreted over 30-second periods , in accordance with the 2007 guidelines of the american academy of sleep medicine ( aasm ) . the scorer was always the same person , blinded to the clinical condition of the patients and the previous results of the questionnaires . the determination of sleep stages and arousals was performed according to the aasm 2007 criteria using eeg montages , including frontal , central , and occipital leads . apnea was defined as a cessation of airflow ( 90% ) for at least 10 seconds , and hypopnea was defined as a 30% reduction of airflow ( from the nasal pressure transducer signal ) lasting at least 10 seconds with 4% desaturation . the apnea - hypopnea index ( ahi ) shortly after the conclusion of the overnight sleep recordings , venous blood was collected from all subjects between 8:00 a.m. and 9:00 a.m. , following an overnight fast , for measurement of hscrp , tnf - a , il-6 , il-8 , and adiponectin . all venous samples were centrifuged within 30 minutes at 3000 rpm for 15 min , and serum was separated into multiple aliquots and stored at 80c until analysis . hscrp levels , expressed in mg / dl , were measured by particle - enhanced immunonephelometry using bn systems ( dade behring inc . ; newark , usa ) . quantitative measurements of tnf - a , il-6 , and il-8 in serum were made using an automated chemiluminescence analyzer ( immulite 1000 , dpc ) with reagents from the same manufacturer . for these three markers , the results were expressed as pg / ml . serum levels of adiponectin were determined using a quantitative sandwich enzyme immunoassay technique ( quantikine human adiponectin immunoassay , r&d systems ) , which measures total human adiponectin ( low , middle , and high molecular weight forms ) . the results were expressed as ng / ml . values are expressed as mean ( sd ) or median value with interquartile range ( iqr ) , depending on the data distribution . the degree of osahs , assessed by the ahi , in the whole group and separately in patients with osahs - ed and controls was correlated with the levels of crp and other cytokines using pearson 's correlation analysis . all statistics were calculated using the statistical package for social sciences software , version 17.0.0 ( spss , chicago , illinois , usa ) . during the study period , 1034 patients underwent diagnostic psg for a suspected sleep disorder . of them , 404 patients volunteered to participate in this study and completed the iief questionnaire . most of the participants were middle aged ( 42.6 9.3 years old ) and moderately obese ( bmi : 33.3 5.05 kg / m ) . the overall ed prevalence , based on iief score , was 40.9% ( 165 patients ) of the study population . the patients with severe osahs had higher incidence of ed compared with those moderate or mild osahs patients ( 53.5% versus 27.1% ; p = 0.005 ) . the mean iief score was significantly lower in patients with severe osahs ( 23.84 6.38 versus 26.1 4.9 , p = 0.001 ) . of the 404 patients , most of them were not eligible for further evaluation because of the presence of at least one of the exclusion criteria . finally , 31 patients with severe osahs who suffered from ed ( iief < 26 ) and met the inclusion criteria agreed to participate in the study and were available for analysis . fifteen patients with normal erectile function , who had similar age , bmi , and ahi distributions to the study group , served as controls . table 1 shows the baseline demographic and psg characteristics of the ed patients and controls . apart from their iief scores , there were no statistically significant differences between the two groups . patients with ed had significantly higher median ( interquartile range ( iqr ) ) plasma levels of hscrp [ 0.32 ( 0.38 ) versus 0.1 ( 0.17 ) mg / dl , p < 0.001 ] than controls ( figure 1 ) . tnf - a was significantly elevated in ed patients compared to controls [ 13.8 ( 5.8 ) versus 11.55 ( 2.2 ) pg / ml , p = 0.01 ] ( figure 2 ) . furthermore , significantly higher levels of il-6 [ 4.38 ( 2.74 ) versus 2 ( 0.39 ) pg / ml , p < 0.001 ] ( figure 3 ) and il-8 [ 8.29 ( 5.2 ) versus 4.98 ( 7.0 ) pg / ml , p = 0.034 ] were observed in ed patients ( figure 4 ) . adiponectin levels were lower in osahs - ed patients , but the difference did not reach statistical significance [ 4680.6 ( 3154 ) versus 4864.6 ( 8418 ) ng / ml , p = 0.5 ] ( figure 5 ) . the degree of osahs in the whole group assessed by the ahi showed a statistical significant correlation only with crp values ( p = 0.013 and r = 0.52 ) and not with the other measured cytokines . it is notable that the significant association between osahs severity and crp was observed only in osahs patients with ed ( p < 0.001 , r = 0.896 and p = 0.013 , r = 0.52 , osahs patients with and without ed , resp . ) . sexual dysfunction represents a significant health problem and may have a strong negative impact on the quality of life . therefore , the identification of potentially modifiable risk factors , such as osahs , may be important for disease prevention and treatment . although ed is a frequent occurrence in male patients with osahs , the precise mechanisms mediating this morbidity are currently unknown . the present study is the first to investigate low - grade inflammation and an altered endothelial state in patients with ed and severe osahs by evaluating a wide spectrum of circulating markers and mediators . with a view to explaining the association between ed and osahs , patient selection was strict enough to exclude the presence of other comorbidities that are also known as risk factors for ed . our results showed that the combination of ed and osahs was associated with higher levels of inflammatory markers compared to osahs alone . previous studies have shown that ed could be a disease of low - grade inflammation [ 20 , 21 ] . vlachopoulos et al . demonstrated an increase in the levels of inflammatory markers such as hscrp , il-6 , il-1 , and tnf - a in patients with ed , suggesting that low - grade systemic inflammation is present in these subjects , similar to that seen in insulin resistance , obesity , type 2 diabetes mellitus , hypertension , hyperlipidaemia , and metabolic syndrome x. moreover , other investigators reported that ed was associated with increased levels of tnf - a and crp , which increased progressively with the severity of penile vascular disease , supporting the role of these markers in the pathophysiology of ed [ 22 , 23 ] . furthermore , recently , matos et al . showed that there was an association between tnf - a levels and ed complaints in men independent of osahs . it is worth noting that , in a recent study , reduced levels of adiponectin , an anti - inflammatory cytokine that attenuates endothelial cell adhesion molecules , and the levels of inflammatory cytokines , such as tnf - a , il-8 , and il-6 , were observed in patients with ed . there are several explanations regarding why this link between ed and osahs exists , implicating hormonal , neural , and endothelial mechanisms . an important link connecting ed and patients with osahs tend to have lower levels of nitric oxide ( no ) , which is responsible for vasodilatation and erection . similarly , oxidative stress , one of the promoters of endothelial dysfunction , is enhanced in osahs , promoting ed . the combination of repetitive hypoxemia and sleep deprivation in osahs patients may be associated with no deficiency , resulting in increased levels of well - known inflammatory cytokines , such as il-6 , il-8 , adhesion molecules , and hscrp [ 27 , 28 ] . increased expression of these inflammatory cytokines may contribute to endothelial dysfunction , which may cause ed . indeed , a number of studies have demonstrated a causal relationship between osahs and endothelial dysfunction , which was improved or not by cpap treatment [ 16 , 29 , 30 ] . a chronic hypoxic condition also contributes to low levels of adiponectin ; however , there are conflicting opinions regarding adiponectin levels in patients with osahs . although a few studies reported that adiponectin was more strongly correlated with ahi in patients who had osahs , compared with various other factors such as age and obesity [ 3134 ] , other studies have reported that adiponectin levels were largely unaffected by the syndrome [ 3537 ] . the elevated markers of endothelial damage and decreased anti - inflammatory adiponectin found in our study underline the involvement of endothelial dysfunction in the pathogenesis of ed , which also comprises a pathophysiological link between this entity and osahs . considering osahs as a chronic disease state , it must play a role in the development of ed , leading to subsequent overall dissatisfaction with sexual function . however , as the two groups of patients were matched for severity of osahs , one could argue why one group would experience the elevated cytokine levels and ed while the other group was spared . possibly , the disparity in responses among the two groups is based on the heterogeneity of the magnitude of end - organ morbidity in sleep apnea among patients and shows that not everyone will be affected to the same extent . furthermore , the observation that treatment for sleep apnea restores erectile function argues for a direct role of osahs in the pathogenesis of ed [ 38 , 39 ] . these novel findings clearly warrant further research aimed at defining the roles of inflammatory markers and associated factors , such as testosterone levels and hormonal disorders , in subjects who are at high risk of ed in the early detection of low - grade systemic inflammation and in the prevention , prediction , and prognosis of ed . subsequently , larger scale studies should be conducted and performed including subjects with comorbidities and various age groups . screening and multidisciplinary approach must be adopted in patients at risk of ed , and there is a need not only for clinical , but also for public health interventions . firstly , the analysis was conducted on a small population and not based on a power calculation , depriving the ability to conduct more robust statistical methods . this was due to the difficulty of including only middle - aged patients with newly diagnosed osahs who had no comorbidities and were not under treatment with cardiovascular medication . in addition , a high refusal rate limited the patients ' participation and may have been related to embarrassment at reporting such symptoms . secondly , ed was assessed based exclusively on the iief questionnaire , a self - reported , subjective estimation of erectile function in men , and not on objective measures , such as nocturnal penile tumescence , testosterone measurements . however , this questionnaire has been validated by multiple studies for evaluating erectile function in various patient populations and has been shown to be clinically appropriate for the evaluation of erectile function ; therefore , it can serve as an indirect indicator of ed . thirdly , we included only patients with severe osahs , so further studies are necessary in order to investigate possible relations between ed and cytokine profile in osa patients with moderate or even mild disease . finally , patients with other additional risk factors for ed were excluded , which does not represent well the situation found in clinical practice . however , as per huang et al . , subclinical endothelial dysfunction may underlie organic ed in young patients without well - known related risk factors . in conclusion , our results showed that the presence of ed in patients with severe osahs is associated with higher levels of inflammatory markers and lower levels of an anti - inflammatory marker , adiponectin , compared to patients with osahs of the same severity but without ed . the increased markers of endothelial damage underline a possible involvement of endothelial dysfunction in the pathogenesis of ed .

patients with obstructive sleep apnea - hypopnea syndrome ( osahs ) show a high prevalence of erectile dysfunction ( ed ) . although the underlying pathogenesis is still unknown , endothelial dysfunction , induced by inflammatory cytokines , chemokines , and adhesion molecules , has been proposed as a possible mechanism . the aim of this study was to assess whether osahs is associated with activation of the inflammatory cytokine system in patients with ed compared to the matched osahs patients with normal sexual function . thirty - one patients with severe osahs and ed were included . fifteen patients with severe osahs and without ed served as controls . serum concentrations of high - sensitivity c - reactive protein ( hscrp ) , tumor necrosis factor- ( tnf - a ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and adiponectin were measured after the diagnostic polysomnography . we found that hscrp levels were significantly elevated in osahs patients with ed compared to controls . similarly , tnf - a levels , il-6 , and il-8 were elevated in osahs patients with ed compared to controls . serum adiponectin levels were lower in osahs - ed patients , but the difference did not reach statistical significance . the presence of ed in patients with severe osahs is associated with elevated levels of inflammatory markers , underlining a possible involvement of endothelial dysfunction in the pathogenesis of ed .

mesothelioma is a primary neoplasm originating from the lining of serous cavities of the human body . the pleural cavity is the most frequent localization of the disease , whereas approximately only one fifth of the lesions occur in the peritoneal cavity . mesothelioma develops more commonly in males and the median age of diagnosis is 50 to 60 years . particularly , the peritoneal form of mesothelioma is associated with prolonged exposure to airborne asbestos fibres . mesothelioma occurs in the form of multifocal tumours and infiltrates of various size and shape affecting the parietal peritoneum and the visceral layer . those lesions coalesce into larger nodules and tumours over time and may cover the entire peritoneal surface eventually . pain , increasing abdominal girth , anorexia and weight loss , and recurrent ascites are the most frequent presenting symptoms . in most cases , this insidious disease is recognized at an advanced stage under the clinical mask of carcinomatous peritonitis . most often , the final diagnosis is made after postoperative histopathological assessment supplemented with immunohistochemical tests . malignant mesothelioma cells are characterized by positive staining for epithelial membrane antigen ( ema ) , wilms tumour 1 ( wt1 ) , cytokeratins ( 5/6 , 7 ) and calretinin , whereas there is absence of antigens such as the carcinoembryonic one . the treatment of mesothelioma is palliative in most patients . before 2000 , a combination of cytoreductive surgery and systemic chemotherapy was applied in most centres and the median survival rate ranged from 9 to 15 months . afterwards , more aggressive methods of treatment were implemented , consisting of surgery , followed by intraperitoneal , hyperthermic , multidrug chemotherapy and whole abdominal radiation , and the rate of 5-year survival increased to 50% . however , such comprehensive management is undertaken only in patients after complete surgery , whereas the patients with incomplete cytoreduction , unresectable tumours like in the presented patient and those with extra abdominal lesions are candidates for palliative systemic chemotherapy [ 6 , 7 ] . the article presents a 50-year - old male patient suffering from an incurable form of advanced malignant peritoneal mesothelioma under the clinical mask of fever of unknown origin . the patient , r.o . , age 50 , tall and of heavy build , fell ill at the end of may 2009 , being treated first in an out - patient department of internal medicine . he presented with symptoms of generalized viral infection such as fever , sore throat and myalgia , whereas physical examination was negative . transient alleviation occurred after introduction of nsaids and clarithromycin , followed however by recurrent rises in temperature , paroxysmal and persistent cough , and malaise after a few days . additional studies were negative except for an increase in erythrocyte sedimentation rate ( esr ) to 95 mm / h , a rise in c reactive protein ( crp ) to 46 mg / l and a single gallstone in the gallbladder found on ultrasonography . anti - mycoplasma and anti - epstein - barr virus ( ebv ) antibody levels were assessed due to suspected atypical tracheitis . whereas antibody titres were non - specific , there was still an increase in esr to 120 mm / h . because of persistent symptoms such as fever , weight loss ( 4 kg in a fortnight ) , hacking cough and rising esr , the patient was suspected to have developed sarcoidosis . then , he was admitted to the private clinic certus on 23.06.2009 . there were no abnormalities on physical examination . all the laboratory tests such as complete blood count ( cbc ) , blood smear , liver and kidney function tests , cardiac enzyme studies , and tumour antigens were normal except for a further increase in crp to 103.5 mg / l and non - specific changes on urinalysis . however , urine culture was negative . there were non - specific changes on the chest ct scan and the mediastinal lymph nodes were not enlarged . the bacteriological tests such as mycobacterium tuberculosis detection ( mb / bac t ) , cytological and mycological examination of the bronchial mucus secretion were negative . due to persistent cough and recognized chronic bronchitis , the patient was given methylprednisolone orally , inhalatory steroids and bronchodilatory medications . looking for other sources of infection , abdominal computed tomography ( ct ) was performed on 25.06.2009 . this examination showed inflammatory infiltration along the wall of the hepatic flexure of the colon , a small amount of fluid under the right lobe of the liver , small lymph nodes in the liver hilus and along the aorta and a gallstone 4 cm in diameter in the gallbladder ( fig . diverticula were located in the transverse , descending and sigmoid colon according to subsequent colonoscopy . the patient responded to the treatment , the temperature dropped and the cough went away . he was released home on 2.07.2009 in good general condition on intravenous amikacin for 5 days , to be followed by oral ciprofloxacin , ceftibuten , fluconazole , methylprednisolone and inhalatory ciclesonide and tiotropium . due to recurrent fever up to 39c , malaise and further weight loss at the end of july he consulted a surgeon , who suspected the patient to have developed either perforation of the diverticulum or gallbladder empyema . pyrexia , malaise , mild abdominal pain in the right epigastrium and weight loss ca . physical examination revealed an extensive , immovable mass with blurred borders in the right epigastrium with mild peritoneal signs . there was a small amount of clear fluid in the peritoneal cavity , whereas the entire abdomen was filled with an unresectable omental mass which was encasing the colon , duodenum and small bowel loops , and reached the lateral walls of the peritoneal cavity and the minor pelvis . the parietal peritoneum was covered with multiple nodules similar to cancerous metastases from 1 to a few mm in diameter . there were enlarged lymph nodes in the liver hilus and in the vicinity of the stomach . biopsy specimens were obtained from the omentum and peritoneal nodules and the abdomen was closed . negative immunohistochemical staining for cd20 excluded lymphatic origin of the neoplasm , whereas positive staining for wt1 , cytokeratin 5/6 ( ck5/6 ) , ck7 and calretinin confirmed the diagnosis of the epithelial type of malignant mesothelioma . the patient was then admitted to the department of oncology on 2.09.2009 for chemotherapy . despite intensive treatment after death the patient 's wife reported that he had lived in the vicinity of an asbestos factory until the age of 14 . computed tomography , venous portal phase . selected transverse sections at the level of the lower margin of the right lobe of the liver ( a ) , liver hilus ( b ) , and the antral part of the stomach ( c ) . extensive pathological mass infiltrating the visceral surface of the right liver lobe , the liver hilus , the hepatic flexure of the colon , the antral part of the stomach , and anterior wall of the abdomen in the middle and right epigastric region ( white arrowheads ) pain , increasing abdominal girth , anorexia and weight loss , and recurrent ascites are the most frequent and non - specific presenting symptoms of peritoneal mesothelioma . less common signs , such as persistent fever , night sweats and mechanical bowel obstruction , are regarded as poor prognostic factors . mesothelioma can be also associated with such paraneoplastic signs as thrombocytosis , hypoglycaemia , thrombophlebitis and liver failure . malignant peritoneal mesothelioma manifested clinically under the mask of fever of unknown origin in the presented 50-year - old male patient . pyrexia of more than three weeks duration , with temperatures over 38c and a failure to identify the cause of the fever , is defined as fever of unknown origin . infections and collagen - vascular diseases are the most common causes of the systemic inflammatory response syndrome with elevated temperatures . the presented patient was suspected to have developed sarcoidosis due to the following symptoms : persistent fever , weight loss , non - productive cough and elevated esr . however , persistent fever can also be a presenting sign of more frequently reported malignant diseases such as hodgkin 's lymphoma , leukaemia and renal cancer . indeed , it has been observed that any other malignancy , either a localized tumour or already disseminated disease , can cause neoplastic fever . no clinical features reliably differentiate neoplastic fever from fever due to other causes . therefore , neoplastic fever is a diagnosis of exclusion , confirmed after careful evaluation of other causes of pyrexia . awaiting results of additional studies , an empirical treatment with standard broad - spectrum antibiotic therapy is followed for at least 7 days . if no clinical response to antibiotics occurs and there is no evident cause of infection , the naproxen test can be applied . although the pathophysiology of neoplastic fever and its differences from other causes of fever remain uncertain , it has been shown that significantly more patients with neoplastic fever respond to naproxen compared to others . nevertheless , patients during naproxen treatment require further examination to rule out fever due to other non - neoplastic aetiology . ct facilitates the diagnosis and evaluation of the clinical staging and resectability of the lesions , and it assists the percutaneous needle biopsy . treatment is monitored with ct and it is also a prognostic factor of the neoplasm . a large tumour in the epigastric or hypogastric region , irregular or nodular peritoneal thickening limited to a single quadrant of the abdominal cavity and omental mass are the ct features of malignant mesothelioma . additional findings that could be helpful in the differential diagnosis from other malignancies , including peritoneal carcinomatosis , are the lack of a primary lesion , encasement rather than infiltration of the adjacent organs , absence of lymph nodes or distant metastases and small amount of ascites . a tumour exceeding 5 cm in diameter , nodular thickening of the peritoneum within the small bowel and its mesentery and segmental obstruction of the bowel indicate rather incomplete surgical debulking . ct scans of the presented patient from 25.06.2009 were sent for reassessment to an independent radiologist who was not aware of the final diagnosis . 16 cm in diameter , localized in the liver hilus and under the right lobe of the liver , spreading to the level of the lower pole of the right kidney and encasing the hepatic flexure of the colon , the antrum of the stomach and small bowel loops , infiltrating the anterior wall of the abdomen in the medial and right epigastric region ( fig . a discrepancy between both ct results indicates difficulties in radiological evaluation of lesions originating from the peritoneum such as peritoneal studding , mesenteric thickening and omental mass . however , magnetic resonance imaging ( mri ) , according to some authors , is superior to ct , both in the differentiation of malignant from benign lesions and because of better resolution in visualization of soft tissues . perfusion and diffusion mri are promising new techniques for the assessment of tumour cellularity and microvasculature and can be used for assessment of treatment response . in turn , positron emission tomography ( pet ) is also useful for the differentiation of benign from malignant lesions , and for staging , as well as monitoring response to treatment .

the authors present a patient suffering from malignant peritoneal mesothelioma . differential diagnosis has become the major concern in the fatally ill patient . pain , increasing abdominal girth , anorexia and weight loss , and recurrent ascites are the most frequent presenting symptoms . in this patient , fever of unknown origin was a clinical mask of mesothelioma . the diagnostic process was focused on infections and collagen - vascular diseases since they are the most common causes of the systemic inflammatory response syndrome . however , persistent pyrexia can also occur , less frequently , in the course of any malignant disease .

ventilator - associated pneumonia ( vap ) is thought to develop from microorganisms entering the sterile lower respiratory tract by aspiration of oropharyngeal secretions containing bacteria endemic to the digestive tract or exogenous pathogens acquired from contaminated equipment or health care workers . less commonly , the lower respiratory tract may be inoculated by direct inhalation of pathogens , hematogenous spread from a remote infection , or direct extension of a contiguous infection . the most common etiologic agents are pseudomonas aeruginosa , klebsiella pneumoniae , escherichia coli , acinetobacter , and staphylococcus aureus . in healthy hosts , mucociliary clearance and innate immunity protect against pneumonia . however , placement of an endotracheal tube impairs mucociliary clearance and provides a direct pathway for inoculation of the lower respiratory tract while critical illness weakens the immune system , putting critically ill , ventilated patients at high risk for developing pneumonia . prevention strategies focus on decreasing bacterial colonization of the oropharynx , reducing the frequency of aspiration , maintaining the immune system , and liberating patients from the ventilator as early as possible . vap previously occurred in 9 - 18% of mechanically ventilated patients and was associated with a 20 - 50% mortality rate and a 7- to 9-day increase in hospitalization . the cost of diagnosing and treating vap is us $ 5,000 to $ 40,000 per incident . routine prevention strategies are summarized in table 1 . emerging prevention strategies to consider in selected patient populations in 2005 , the infectious diseases society of america and the american thoracic society published a comprehensive guideline for vap prevention focusing on modifiable risk factors . in 2008 , the canadian critical care trials group published a similar guideline . to decrease bacterial colonization of the oropharynx and endotracheal tube , these guidelines advocate using orotracheal rather than nasotracheal intubation , continuous subglottic secretion drainage , and standard infection control measures , including frequent hand washing , sterile central venous catheter placement , and isolation of resistant organisms . while continuous subglottic secretion drainage requires a special endotracheal tube that costs about us $ 12 more than a standard tube , several studies have shown a significant reduction of vap incidence with this intervention , as summarized in a recent review . decontamination of the oropharynx and digestive tract with systemic antibiotics , selective digestive decontamination , and selective oropharyngeal decontamination have all been shown to decrease bacterial colonization and vap incidence , but the practice remains controversial . while many studies have demonstrated decreased vap incidence in patients treated with prophylactic antibiotics , the guidelines recommend against their use until more data on the effect on mortality and the risk of developing resistant organisms emerge . in 2009 , de smet et al . published the largest randomized trial to date on selective gut decontamination . after baseline differences between the treatment arms were adjusted for , there was a significant decrease in mortality for those treated with selective gut decontamination . however , concerns about the external validity of the study remain because of the low incidence of resistant organisms encountered in the study population . oral cleansing with topical antiseptics such as chlorhexidine also reduces bacterial colonization with less potential for selecting resistant organisms but is similarly controversial . many subsequent trials on chlorhexidine have been published , but the results are conflicting so the controversy persists . several meta - analyses and clinical trials suggest a benefit of chlorehexidine in decreasing vap , whereas others have yielded negative results [ 16 - 18 ] . since the 2008 guidelines were published , a new silver - coated endotracheal tube designed to decrease bacterial colonization and biofilm formation was introduced . the nascent trial , a prospective , randomized , multi - center study comparing standard and coated endotracheal tubes , showed a significant reduction in vap ( 4.8% versus 7.5% , p = 0.03 ) in patients treated with this coated tube . however , mechanical ventilation duration , hospital length of stay , and intensive care unit ( icu ) length of stay were unchanged between the control and intervention groups . a numeric increase in mortality among patients assigned to the coated tubes ( 30.4% versus 26.6% for standard tubes , p = 0.11 ) needs to be evaluated further . furthermore , the cost of a coated tube is us $ 90 compared with $ 2 for a routine tube , but a recent cost - effectiveness analysis concluded that silver - coated tubes would likely save money because of their ability to prevent vap . enteral nutrition predisposes patients to aspiration of gastric contents and subsequent vap but is still considered preferable to parenteral nutrition because of the many complications associated with parenteral nutrition . the 2005 guidelines recommend post - pyloric feeding tubes and semi - recumbent positioning with a head of bed angle of greater than 45 degrees to decrease the frequency of aspiration associated with enteral feeding . unfortunately , it is difficult to maintain patients at 45 degrees , and 30 degree elevation may not be as effective . a 2002 single - center study evaluating initial trophic feeding followed by delayed full - calorie nutrition found a significant reduction in vap with no change in mortality compared with the control group . stress ulcer prophylaxis with acid suppression predisposes patients to developing vap by raising the gastric ph levels and allowing bacterial overgrowth . sucralfate is an appealing option because it does not affect gastric ph , but it has been associated with increased bleeding and vap incidence . citing conflicting evidence , more recently , acid suppression therapy with more potent proton pump inhibitors has become widespread , but it was associated with a greater incidence of vap than h2 blockers in a retrospective study . to help maintain natural immunity , the guidelines also recommend a conservative transfusion policy and intensive insulin therapy since blood transfusion and hyperglycemia have been associated with increased infectious complications . however , intensive insulin regimens have been increasingly scrutinized as new data suggesting their potential for harm emerge [ 28 - 30 ] . the 2005 guidelines recommend avoiding intubation , and particularly reintubation whenever possible , as well as installing protocols to reduce sedation and accelerate ventilator weaning . in 2008 , the awakening and breathing controlled trial confirmed the importance of combining sedation and ventilator weaning protocols by showing a shorter duration of mechanical ventilation and significant mortality improvement in the intervention group . for patients who are unlikely to be liberated from the ventilator quickly , early tracheostomy has been used to mitigate the risks associated with endotracheal intubation , but its role is controversial . a 2004 randomized clinical trial showed a significant reduction in vap , duration of mechanical ventilation , and mortality in patients who received tracheostomy on ventilator day 2 rather than prolonged endotracheal intubation with tracheostomy on ventilator day 14 . a 2006 meta - analysis showed decreased ventilation duration and icu length of stay but no difference in mortality or vap in those treated with early tracheostomy . in 2008 , a small randomized trial also showed no difference between tracheostomy within the first 4 days of intubation and prolonged intubation with tracheostomy allowed only after ventilator day 14 , other than improved patient comfort , but the study was too underpowered to be conclusive . in 2005 , the infectious diseases society of america and the american thoracic society published a comprehensive guideline for vap prevention focusing on modifiable risk factors . in 2008 , the canadian critical care trials group published a similar guideline . to decrease bacterial colonization of the oropharynx and endotracheal tube , these guidelines advocate using orotracheal rather than nasotracheal intubation , continuous subglottic secretion drainage , and standard infection control measures , including frequent hand washing , sterile central venous catheter placement , and isolation of resistant organisms . while continuous subglottic secretion drainage requires a special endotracheal tube that costs about us $ 12 more than a standard tube , several studies have shown a significant reduction of vap incidence with this intervention , as summarized in a recent review . decontamination of the oropharynx and digestive tract with systemic antibiotics , selective digestive decontamination , and selective oropharyngeal decontamination have all been shown to decrease bacterial colonization and vap incidence , but the practice remains controversial . while many studies have demonstrated decreased vap incidence in patients treated with prophylactic antibiotics , the guidelines recommend against their use until more data on the effect on mortality and the risk of developing resistant organisms emerge . in 2009 , de smet et al . published the largest randomized trial to date on selective gut decontamination . after baseline differences between the treatment arms were adjusted for , there was a significant decrease in mortality for those treated with selective gut decontamination . however , concerns about the external validity of the study remain because of the low incidence of resistant organisms encountered in the study population . oral cleansing with topical antiseptics such as chlorhexidine also reduces bacterial colonization with less potential for selecting resistant organisms but is similarly controversial . many subsequent trials on chlorhexidine have been published , but the results are conflicting so the controversy persists . several meta - analyses and clinical trials suggest a benefit of chlorehexidine in decreasing vap , whereas others have yielded negative results [ 16 - 18 ] . since the 2008 guidelines were published , a new silver - coated endotracheal tube designed to decrease bacterial colonization and biofilm formation was introduced . the nascent trial , a prospective , randomized , multi - center study comparing standard and coated endotracheal tubes , showed a significant reduction in vap ( 4.8% versus 7.5% , p = 0.03 ) in patients treated with this coated tube . however , mechanical ventilation duration , hospital length of stay , and intensive care unit ( icu ) length of stay were unchanged between the control and intervention groups . a numeric increase in mortality among patients assigned to the coated tubes ( 30.4% versus 26.6% for standard tubes , p = 0.11 ) needs to be evaluated further . furthermore , the cost of a coated tube is us $ 90 compared with $ 2 for a routine tube , but a recent cost - effectiveness analysis concluded that silver - coated tubes would likely save money because of their ability to prevent vap . enteral nutrition predisposes patients to aspiration of gastric contents and subsequent vap but is still considered preferable to parenteral nutrition because of the many complications associated with parenteral nutrition . the 2005 guidelines recommend post - pyloric feeding tubes and semi - recumbent positioning with a head of bed angle of greater than 45 degrees to decrease the frequency of aspiration associated with enteral feeding . unfortunately , it is difficult to maintain patients at 45 degrees , and 30 degree elevation may not be as effective . a 2002 single - center study evaluating initial trophic feeding followed by delayed full - calorie nutrition found a significant reduction in vap with no change in mortality compared with the control group . stress ulcer prophylaxis with acid suppression predisposes patients to developing vap by raising the gastric ph levels and allowing bacterial overgrowth . sucralfate is an appealing option because it does not affect gastric ph , but it has been associated with increased bleeding and vap incidence . citing conflicting evidence , the 2005 guidelines recommend using either sucralfate or h2 blockers for stress ulcer prophylaxis . more recently , acid suppression therapy with more potent proton pump inhibitors has become widespread , but it was associated with a greater incidence of vap than h2 blockers in a retrospective study . to help maintain natural immunity , the guidelines also recommend a conservative transfusion policy and intensive insulin therapy since blood transfusion and hyperglycemia have been associated with increased infectious complications . however , intensive insulin regimens have been increasingly scrutinized as new data suggesting their potential for harm emerge [ 28 - 30 ] . the 2005 guidelines recommend avoiding intubation , and particularly reintubation whenever possible , as well as installing protocols to reduce sedation and accelerate ventilator weaning . in 2008 , the awakening and breathing controlled trial confirmed the importance of combining sedation and ventilator weaning protocols by showing a shorter duration of mechanical ventilation and significant mortality improvement in the intervention group . for patients who are unlikely to be liberated from the ventilator quickly , early tracheostomy has been used to mitigate the risks associated with endotracheal intubation , but its role is controversial . a 2004 randomized clinical trial showed a significant reduction in vap , duration of mechanical ventilation , and mortality in patients who received tracheostomy on ventilator day 2 rather than prolonged endotracheal intubation with tracheostomy on ventilator day 14 . a 2006 meta - analysis showed decreased ventilation duration and icu length of stay but no difference in mortality or vap in those treated with early tracheostomy . in 2008 , a small randomized trial also showed no difference between tracheostomy within the first 4 days of intubation and prolonged intubation with tracheostomy allowed only after ventilator day 14 , other than improved patient comfort , but the study was too underpowered to be conclusive . while many prevention strategies have proven successful in decreasing the incidence of vap by a small amount , significant reduction requires a multimodal approach encompassing positioning , equipment , nutritional support , infection control , sedation minimization , and ventilator weaning . adjunctive therapies such as stress ulcer prophylaxis , glycemic control , and blood transfusion also play a role because of their impact on the immune system . vigilant attention to all of these details by means of a ventilator bundle has been shown to significantly reduce the incidence of vap . many other promising therapies , such as selective gut decontamination , early tracheostomy , and coated endotracheal tubes , remain controversial because of their mixed or limited data , so we await further trials to determine their role . vap prevention remains an area of active research , with 28 clinical trials currently listed on www.clinicaltrials.gov .

ventilator - associated pneumonia , broadly defined as pneumonia that develops after 48 hours of intubation , is a common mechanical ventilation complication that causes significant morbidity and mortality in critically ill patients . prevention strategies are continually evolving to decrease the impact of this serious and costly disease .

allenes readily undergo thermal pericyclic reactions , including diels alder , 1,3-dipolar , and ( 2 + 2 ) cycloadditions . there is some evidence that these reactions are stepwise , although few systematic investigations are available . we report multiconfigurational complete active space ( cas ) computational studies of the reactions of allene with butadiene and with benzene , aliphatic and aromatic dienes in diels alder reactions ( figure 1 ) . for the butadiene allene reaction , we have discovered that a single ambimodal transition state leads to a path bifurcation to either the ( 4 + 2 ) cycloadduct , via a concerted reaction , or to a diradical intermediate that can subsequently give either diels alder or ( 2 + 2 ) adduct . in contrast , benzene and allene react through a transition state that leads only to a concerted pathway , forming both c diels alder and ( 2 + 2 ) cycloaddition reactions of allene with butadiene and benzene . pericyclic reactions involving allenes are known and have been used extensively in the syntheses of natural products . the relative reactivity of allenes , alkynes , and alkenes in these processes have been the subject of some interest . for instance , the cope rearrangement was found to proceed through similar transition structures , independent of the identity and degree of unsaturation of the -components . allenes also participate in ( 4 + 2 ) cycloadditions , 1,3-dipolar cycloadditions , and ( 2 + 2 ) cycloadditions ; examples of each of these studied experimentally are shown in figure 2 . maier utilized both cyclopentadiene and boc - protected pyrrole with monosubstituted allenes to generate bridged bicyclic compounds through the diels alder reaction . the 1,3-dipolar cycloaddition of c - phenyl - n - methylnitrone with electron - deficient allenes produces methyleneisoxazolidines at 40 c . allene dimerization has been known for decades , and dolbier investigated the preference for formation of 1,2-dimethylenecyclobutane over the 1,3-regioisomer . ( 4 + 2 ) , 1,3-dipolar , and ( 2 + 2 ) cycloadditions of allenes . computational mechanistic studies of allenes as reaction partners in 1,3-dipolar and ( 2 + 2 ) cycloadditions have been reported . there are , however , limited theoretical investigations of allenes as dienophiles in ( 4 + 2 ) reactions . venuvanalingam studied the concerted diels alder cycloadditions of dienes with allenes and fluoroallenes as dienophiles with semiempirical am1 and pm3 methods . gandolfi studied concerted diels alder cycloadditions of allene and fluoroallene with cyclopentadiene and furan with the ab initio hartree houk and co - workers conducted a dft study of the concerted and stepwise pathways of the parent butadiene allene cycloaddition as well as some furan cycloadditions with allene but were unable to locate a number of important stationary points . in light of the numerous studies contrasting the diels alder reactions of alkene and alkyne dienophiles , diels alder reactions of dimethyl 1,3-allenedicarboxylate 2 with danishefsky dienes 1 . a variety of substituted dienes undergo diels alder reactions with allenes . as shown in figure 3 , danishefsky dienes 1 react with unsymmetrically 1,3-disubstituted allenes 2 to give aromatic products 4 and 5 . however , jung and co - workers have shown for similar cases that ( 2 + 2 ) adducts may precede diels alder adduct formation . reactions of dienes 6 with allenoic ester 7 give exo - methylenevinylcyclobutane intermediates 8 , formal ( 2 + 2 ) adducts , when the reaction time is 5 h ( figure 4 ) . these adducts undergo formal cope rearrangements to give the diels alder products 9 and 10 after extended reaction times . the cope rearrangement of the unsubstituted exo - methylenevinylcyclobutane was found in previous computational studies by houk and co - workers to rearrange to the diels alder adduct in a stepwise fashion through a bis - allylic diradical . based on previous studies and experimental results in the literature , it is proposed that ( 4 + 2 ) reactions of this nature are stepwise and proceed first through a formal ( 2 + 2 ) cycloaddition , followed by a formal 1,3- or 3,3-shift to afford the diels alder adduct . formation of exo - methylenevinylcyclobutane intermediate prior to rearrangement to diels alder adducts . himbert and henn have shown that intramolecular ( 4 + 2 ) cycloadditions between allenyl amides and tethered aryl groups occur efficiently at elevated temperatures , despite the required disruption of aromaticity ( figure 5a ) . the polar stepwise mechanism was ruled out by the insensitivity of the kinetics of the reaction to varying electron - donating and electron - withdrawing groups on the benzene and allene moieties . however , although a concerted mechanism was initially proposed , a stepwise diradical mechanism could not be ruled out . vanderwal has recently explored this dearomatizing intramolecular diels alder reaction and has incorporated a subsequent ring - rearranging metathesis to form complex polycyclic scaffolds ( figure 5b ) . together , our groups uncovered important mechanistic insights into these intramolecular cycloadditions of allene to benzene derivatives . in order to understand the energetics of concerted and stepwise pathways in benzene allene cycloadditions and to make direct comparisons with nonaromatic diene reactions , we have undertaken a systematic investigation of the benzene - allene and butadiene - allene reactions with multiconfigurational casscf and caspt2 methods . we have studied these reactions with complete active space ( cas ) multiconfigurational methods . stationary point structures were optimized using the casscf(8,8)/6 - 31g(d ) and casscf(10,10)/6 - 31g(d ) methods in gaussian 09 for the butadiene / allene and benzene / allene systems , respectively . single - point calculations with second - order perturbation theory caspt2/6 - 31g(d ) were carried out on the optimized structures , using the program molcas version 7.4 , to account for dynamic electron correlation . casscf thermal corrections and zero - point energies are included in the caspt2 electronic energies . vibrational frequencies were computed for all optimized structures in order to verify that they are minima or transition states . intrinsic reaction coordinate ( irc ) calculations were also performed on several transition structures to verify that these transition structures originated from the correct reactants and led to the expected intermediates or products . casscf and caspt2 has been found by houk and co - workers to provide reasonable energetics for various diradical and pericyclic reactions . dft methods were also employed for optimizations , but we had difficulty locating relevant stationary points . furthermore , several unrestricted dft methods gave unrealistically high energy diradicals for the benzene consequently , we have used more robust multiconfigurational methods for the entirety of the investigation . the reaction of butadiene 16 with allene 17 can occur by either a concerted or stepwise radical mechanism ( figure 6 ) . the concerted pathway has previously been studied using semiempirical as well as ub3lyp methods . alternatively , the reaction can give diradical 18 that can subsequently cyclize to diels alder adduct 19 or to the ( 2 + 2 ) adduct 3-methylenevinylcyclobutane 20 . the ( 2 + 2 ) adduct can reopen to 18 and then cyclize to yield 19 . this cope rearrangement to the diels alder adduct of the unsubstituted 3-methylenevinylcyclobutane was found in previous computational studies by houk and co - workers to occur in stepwise fashion through a bis - allylic diradical intermediate . reaction of the diene in the s - cis conformation is necessary to permit cyclization to the diels alder adduct ; the transoid diradical 18(trans ) could be formed and undergo bond rotation around the partial double bond to furnish the cisoid diradical 18(cis ) , which can then cyclize to 19 , but this would require rotation around the partial double bond of the allyl radical . the active space was chosen to include the electrons involved in the formation of new bonds , namely the eight -electrons of butadiene and allene . a schematic of the energy surface was generated from the quantum - chemically calculated values and is shown in figure 7 . reported energies are relative to the lowest energy conformations of separated allene and s - trans butadiene . at the left of the diagram , the s - cis and s - trans butadiene reactants are shown . the s - cis butadiene is 3.0 kcal / mol higher in energy , consistent with the 2.64.0 kcal / mol values for the gauche conformation of s - cis butadiene found in prior calculations and experiments . the barrier to interconversion is approximately 6 kcal / mol to switch from s - trans butadiene to s - cis butadiene . to the right of the diagram in figure 7 free energies calculated at room temperature ( 25 c ) have also been included , since reaction rates are determined from free energies through transition state theory . because of the entropic penalty ( ts term in free energy ) of bringing two molecules together , g values are uniformly 1114 kcal / mol higher than the corresponding e values for all stationary points other than the separated reactants . consequently , the reaction surfaces generated from both electronic and free energies have similar topologies , and we will proceed by referring to electronic energies for consistency . schematic of the potential energy surface for the reaction between butadiene and allene . caspt2//casscf(8,8)/6 - 31 g * gas - phase energies are shown in kcal / mol . red arrows refer to the stepwise pathways , the blue arrow is the concerted pathway , and black arrows are for cis / trans and s - cis / s - trans interconversions . along the lower border , the concerted diels 19 is the concerted transition state at 27.7 kcal / mol but is described in detail in the next section ; this is also the transition state leading to the cis - diradical 18(cis ) . singleton has also studied a bifurcation that occurs in the diels alder reactions of ketenes with cyclopentadiene which leads to an intermediate or a cycloadduct , as found here . at 28.1 kcal / mol , the transition state leading to the trans - diradical , 18(trans ) both the trans and cis diradicals can give the 3-vinylmethylenecyclobutane 20 through transition states of only 1011 kcal / mol . the transition state for formation of diels alder product , 19(closure ) , is 7.7 kcal / mol with respect to the reactant and only 0.9 kcal / mol higher in energy than the diradical intermediate 18(cis ) . alder adduct 19 and 3-vinylmethylenecyclobutane 20 should both be formed thermally , with the former being the thermodynamically and kinetically favored major product . in order to understand the region around 19 , a detailed potential energy surface was generated ( figure 8) . the energies were calculated by fixing the distance between the internal carbon of the allene and a terminal carbon of butadiene ( bond 1 ) and varying the distance corresponding to the second forming -bond ( bond 2 ) . casscf(8,8 ) single - point calculations were conducted on each structure , and the same protocol was applied to increasing lengths of bond 1 . examination of the surface shows that only one saddle point exists , corresponding to ambimodal transition state 19 . an irc calculation shows that the steepest downhill trajectory leads to formation of diradical 18(cis ) . from this diradical , there is only a small barrier 19(closure ) to radical recombination to form 4-methylenecyclohexene 19 ( red arrows ) . however , an alternative trajectory can lead directly to 19 which , although not the steepest trajectory , bypasses 18 and 19(closure ) ( blue arrow ) . in a study of the allenic cope rearrangement of 1,2,6-heptatriene , borden observed a similar phenomenon where both a concerted and a stepwise pathway can emerge after traversing a common transition state . despite the large preference for reaction at the central carbon of allenes , the allylic stabilization found in the diradical intermediates is not substantial in the transition structures , suggesting the possibility for direct formation of product without passage through an intermediate . this result is in line with the discovery that cope rearrangements involving alkenes , allenes , and alkynes are all mechanistically and kinetically similar . left : potential energy surface ( pes ) region of the possible transition states of initial bond formation , generated with casscf(8,8)/6 - 31g*. energy levels are designated by the following color spectrum : red = high energy , violet = low energy . the red arrows outline the stepwise pathway from ambimodal transition state 19 , while the blue arrow outlines the concerted pathway . right : side view of the pes , demonstrating the saddle point for 19 . the lengths of the forming -bonds in 19 differ by 1.5 , suggesting significant diradical character . the occupations of the homo and lumo natural orbitals are 1.65 and 0.36 , respectively ; occupations of 2 and 0 are expected for ideal closed - shell species , while 1 and 1 would represent a pure diradical . to further probe the existence of a distinct concerted transition state , pseudo-19(conc ) was optimized with bond distance restraints of 2.17 and 2.36 , established from successful location of the concerted stationary point using the 3 - 21 g basis set ; the greater synchronicity of the transition state may be an artifact of the smaller basis set . the potential energy surface connecting 19 and pseudo-19(conc ) is very flat , requiring only a minor geometric change to interconvert the two structures . hence , when butadiene is in the cis conformation , only a single transition state 19 leads to diradical 18(cis ) and to diels although both the blue and red downhill trajectories in figure 8 are barrierless on the potential energy surface , singleton has shown that inclusion of entropic factors can reveal hidden dynamical bottlenecks . from 19 , formation of a single c c bond resulting in diradical 18(cis ) will have a lower entropic penalty than simultaneously establishing the two new bonds of 19 . also , examination of the transition - state region shows that the location of the highly asynchronous transition structure 19 is skewed toward 18(cis ) . this may cause an entropic bottleneck between 19 and 19 , establishing a barrier for the blue concerted pathway in figure 8 and leading to exclusive formation of intermediate 18(cis ) prior to forming the diels alder adduct 19 . thus , despite the fact that the potential energy surface contains only one initial bond - forming transition state that can seemingly form either a cycloadduct or a diradical , accounting for entropy would likely lead to preferential diradical formation . molecular dynamics simulations may be a valuable tool in validating this notion and further probing the surface around the transition state . a similar situation where an irc predicts a concerted pathway while dynamics suggests a stepwise route has been uncovered in the intramolecular heterolysis of pinacolyl alcohol . formation of the bis - allyl diradical can result in either the cisoid ( 18(cis ) ) or the transoid ( 18(trans ) ) intermediate , depending on the orientation of the butadiene prior to bond formation . the intermediates are essentially isoenergetic , but transition state 19 lies 0.4 kcal / mol lower than 18(trans ) . the cisoid and transoid intermediates can interconvert only by traversing a 13 kcal / mol barrier due to rotation around the partial double bond of the allyl radical . optimized structures of the stationary points for the cycloaddition of butadiene 16 and allene 17 . from the cisoid diradical intermediate 18(cis ) , both 3-methylenevinylcyclobutane 20 and 4-methylenecyclohexene 19 can be formed by radical combination through 20(cis ) and 19(closure ) , respectively , while 18(trans ) can only form cyclobutane product 20 . the transition states 20(cis ) and 20(trans ) have the same energy ; the structures are identical except for the conformation of the distal double bond . the formation of 20 is exoergic by 29.7 kcal / mol ; longer reaction times or higher temperatures result in radical ring - opening back to either stereoisomer of bis - allyl diradical 18 . although the barrier for the ring - opening of 20 is high ( 40 kcal / mol ) for the unsubstituted system , substituents stabilizing the diradical intermediate will result in a lower barrier for the ring - opening of 20 . the cisoid intermediate can then irreversibly produce the diels alder product 19 through transition state 19(closure ) , which is lower in energy than 20(cis ) and 20(trans ) by 3 kcal / mol . these results parallel the experimental results reported by jung on substituted substrates ( figure 4 ) . after a few hours of heating mixtures containing substituted butadienes and allenyl ester 7 , heating the cyclobutanes over a period of days resulted in rearrangement to the formal diels allene dimerizes readily , and the mechanism has been studied theoretically at a coupled - cluster level of theory . johnson calculated the dimerization to occur with an energetic barrier e = 32.9 kcal / mol for initial diradical formation , approximately 5 kcal / mol higher than our calculated barrier for reaction with butadiene . previous successes in ( 4 + 2 ) cycloadditions with substituted butadienes illustrate this preference of diels the ( 4 + 2 ) reaction of benzene 21 and allene 17 was also explored ( figure 10 ) . this cycloaddition does not occur in the parent cases because allenes dimerize and oligomerize more rapidly than they react with benzene . as mentioned previously , himbert , orahovats , and more recently vanderwal have demonstrated that substituted benzenes and allenes can form intramolecular cycloadducts . the intramolecular cycloadditions of n - arylallenylamides are known ( figure 5 ) and prompted our study of the benzene allene reaction . the cycloaddition can occur through a concerted ( 23(conc ) ) mechanism or through the stabilized pentadienyl radical 22 . either route can lead to ( 4 + 2 ) cycloadduct 23 , with the latter proceeding through 23(closure ) . optimizations were carried out with casscf(10,10 ) involving an active space of the six -electrons of benzene and the four -electrons of allene . a schematic of the reaction profile and energy values are shown in figure 11 . the concerted transition state 23(conc ) lies 5.0 kcal / mol lower than the stepwise 22(step ) , in contrast to the union of these into a single transition state found with s - cis butadiene and ethylene . formation of the first c c bond gives intermediate 22 , containing allyl and pentadienyl radicals . although these radicals are stabilized , loss of aromaticity offsets the favorable conjugation so that 22 is 32.2 kcal / mol higher than the reactants . the allyl radical resulting from the allene does not initially benefit from delocalization ; rotations about the c conversely , the concerted 23(conc ) better offsets the loss of aromaticity and maintains most of the benzene stabilization by providing an aromatic transition state . schematic of the potential energy surface for the reaction between benzene 21 and allene 17 . caspt2//casscf(10,10)/6 - 31 g * gas - phase energies are shown in kcal / mol . red arrows refer to the stepwise pathways , blue arrow for the concerted pathway . ring closure of the diradical to form the ( 4 + 2 ) adduct 23 is favored over formation of the ( 2 + 2 ) adduct 24 by 5.9 kcal / mol . the formation of 24 is endoergic by 5.0 kcal / mol and is reversible . the methylenecyclobutane 24 can ring - open to 22 and ultimately form the thermodynamically favorable product 23 . optimized structures of the stationary points for the cycloaddition of benzene 21 and allene 17 . the 37.1 kcal / mol required for the ( 4 + 2 ) cycloaddition of benzene and allene is greater than the 32.9 kcal / mol barrier for dimerization , as calculated by johnson ( figure 13 ) . furthermore , 1,2-dimethylenecyclobutane formation is exoergic by 45.0 kcal / mol , compared to only 8.9 kcal / mol for 23 . the dimerization of allene is thermodynamically and kinetically favored relative to diels alder reaction with benzene , consistent with the lack of formation of 23 . energetics of the diels alder reaction of benzene and allene ( left ) and the dimerization of allene ( right ) . dft optimizations using both ub3lyp/6 - 31g(d ) and um06 - 2x/6 - 31g(d ) methods were also utilized for the butadiene allene and benzene allene systems ; energetics and optimized structures can be found in supporting information . for the butadiene allene system , um06 - 2x predicts energies for all stationary points to within 5 kcal / mol of caspt2 . however , dft calculations on the benzene allene system resulted in largely overestimated energies for the open - shell diradical species . the spin - contamination observed with dft methods , which changes over the course of the reaction pathways , may be a large contribution . aside from the unexpectedly high energies for the intermediate in benzene allene system , unrestricted m06 - 2x computations predict values that are comparable to the casscf and caspt2 methods . having established the energetics and mechanism of reactions of allene with butadiene and benzene , we conclude by comparing these results to previously reported studies of the dienes with ethylene and acetylene ( figure 14 ) . the diels alder reactions of allenes , with both butadiene and benzene , have higher activation barriers than their diatomic counterparts . the reactions of ethylene and acetylene with butadiene have a barrier of 22.4 kcal / mol for the concerted cycloaddition , 5.3 kcal / mol lower than that for allene . with benzene , reactions with ethylene and acetylene have reported barriers of 31.9 and 35 kcal / mol , respectively . an allene dienophile raises the activation barrier to 37.1 kcal / mol . despite the destabilizing cumulated double bonds of allene , computations suggest diminished reactivity toward dienes relative to the [ 4 + 2 ] reaction of ethylene and acetylene . table of activation energies ( kcal / mol ) for the concerted diels alder reaction of butadiene and benzene with unsaturated dienophiles . ( b ) experimentally derived ( c ) calculated using mp2/6 - 31g(d ) . the cycloaddition reactions of allene with butadiene and with benzene have been elucidated using multiconfigurational caspt2 calculations . although the reactions investigated here are not explicitly observed experimentally due to the presence of more favorable processes ( allene oligomerization ) or decomposition under the required reaction conditions ( high temperatures ) , many substituted analogues have resulted in successful diels alder cycloadditions . reaction with butadiene occurs through a single ambimodal transition state that can proceed to product along both concerted and stepwise pathways , although inclusion of entropy may ultimately favor the latter . if a diradical intermediate is formed , either the ( 2 + 2 ) or ( 4 + 2 ) cycloadduct can result ; the ( 2 + 2 ) adduct can reversibly ring - open to yield the diradical and proceed to the more thermodynamically stable ( 4 + 2 ) product . conversely , the loss of aromaticity largely affects the reaction profile of benzene and allene cycloaddition ; the propensity of benzene to retain aromaticity prompts the cycloaddition of allene and benzene to occur through a concerted yet asynchronous mechanism , forming both -bonds simultaneously through a pericyclic transition state . the resulting cycloadduct also suffers from the disruption of aromaticity , causing a large decrease in reaction exothermicity relative to the butadiene allene system . in lieu of computationally intensive casscf optimizations , unrestricted dft methods can be also used to model such systems , but care must be taken when applying them to cycloadditions of aromatic compounds with allenes . additionally , molecular dynamics simulations on the butadiene - allene diels alder reaction may increase our understanding of possible ambimodal transition states and subsequent bifurcations in allene chemistry .

multiconfigurational complete active space methods ( casscf and caspt2 ) have been used to investigate the ( 4 + 2 ) cycloadditions of allene with butadiene and with benzene . both concerted and stepwise radical pathways were examined to determine the mechanism of the diels alder reactions with an allene dienophile . reaction with butadiene occurs via a single ambimodal transition state that can lead to either the concerted or stepwise trajectories along the potential energy surface , while reaction with benzene involves two separate transition states and favors the concerted mechanism relative to the stepwise mechanism via a diradical intermediate .

cardiac resynchronization therapy ( crt ) has been validated as an effective therapeutic approach for patients with drug - refractory heart failure associated with left ventricular ( lv ) dyssynchrony . in this population , crt not only improves heart failure symptoms and quality of life [ 1 , 2 ] but also leads to reverse remodeling and reduces the risk of death . despite this unquestionable efficacy , 30% of patients do not appear to benefit from crt , and substantial effort has been made to better identify potential responders [ 5 , 6 ] . several reports have indicated that lv lead placement at the site of latest mechanical contraction is a critical determinant of crt outcome [ 7 , 8 ] . the identification of these sites of greater dyssynchrony by echocardiography has been suggested by several authors to be associated with acute or long - term success of crt . however , echo - guided lead positioning requires sophisticated techniques for assessment of lv dyssynchrony and selection of the site of latest mechanical activation [ 7 , 9 , 10 ] . the use of these techniques , during crt , is challenging and may significantly prolong the procedure duration . furthermore , data from the prospect trial illustrated the limited intra- and interobserver reproducibility of these measurements . another method of identifying sites of latest activation is the use of epicardial electrogram ( egm ) . pacing at the site of maximal electrical delay ( ed ) determined electrophysiologically or by electroanatomical mapping [ 13 , 14 ] has been reported to result in greater acute hemodynamic response . however , data on the long - term value of this technique are very limited . the goal of our study was to assess the value of the ed for the prediction of the long - term response and to determine the degree of conduction delay that was more likely to be associated with positive outcome . we conducted a retrospective single - centre study on patients with a crt device and in whom local epicardial egm was available at the time of the procedure . patients were included in our study if they had successful implantation of a crt device for drug - refractory congestive heart failure : nyha functional class iii or iv , due to severe systolic lv dysfunction ( lv ejection fraction ( lvef ) 35% and long qrs duration ( 120 ms ) ) and if epicardial egms were obtained during the procedure . patients with severe ischemic heart disease and extensive myocardial scar ( involving more than 4 lv segments ) or history of lateral or posterolateral myocardial infarction who had a low likelihood of response were not considered for crt and , therefore , were not included in our study . technical aspects of lead and device implantation were described in detail in previous publications [ 16 , 17 ] . efforts were made to place the lv lead in a lateral tributary of the coronary sinus . at the end of the procedure and before the lv lead was connected to the crt device , simultaneous surface 12-lead ecg and epicardial egms were continuously acquired with a filter bandwidth of 0.05 to 40 hz and 30 to 500 hz , respectively , and displayed on a high - resolution video monitor at 100 mm / second paper speed for inspection and subsequent review ( prucka engineering ) . the ed was measured from the onset of qrs to the peak of sharpest deflection of the egm ( figure 1 ) . we also determined the ratio of the ed to baseline qrs duration ( ed / qrs duration ) . during the study period , the lv lead position was not modified on the basis of the ed . after the implant , each patient had a chest x - ray in the anteroposterior and left anterior oblique ( lao ) views , and the final lv lead position was recorded in the latter view . after implantation of the crt device , patients were followed prospectively in our institution at 1 , 6 , and 12 months and every year thereafter . the following parameters were collected at baseline and each visit : functional status defined by nyha class , 6-minute walked distance , and lv volumes by echocardiography : lv end - systolic volume ( lvesv ) and lv end - diastolic volume ( lvedv ) . lvef was determined by echocardiography or nuclear angiography . when lvef was evaluated at baseline by one of the 2 techniques , some parameters of lv dyssynchrony were also assessed at baseline and followup , but results of these parameters will not be reported in the present study , since their value in selection of patients for crt has not been validated by the prospect trial . response to crt was defined by either improvement of functional status by at least 2 nyha classes alone or by one nyha class associated with increased lvef by at least 5% . categorical data are expressed as incidence , and noncategorical data are expressed as mean standard deviation . a comparison of categorical data was performed using the chi - square test , and noncategorical data were compared by student 's t - test . logistic regression analysis was used for identification of independent predictors of long - term response to crt . our study population comprised 72 patients ( 47 men ) who were implanted with a crt device and in whom epicardial egm was available at the time of the procedure . almost all patients had left bundle branch block ( lbbb , n = 69 ) . mean ed was 132 36 ms , and the mean ratio of ed / qrs duration was 0.75 0.17 . after a mean followup of 30 20 months , 47 patients were classified as responders and 25 as nonresponders . no significant difference was observed between the 2 groups in baseline characteristics including age , nature of underlying heart disease , prevalence of sr at baseline , qrs duration , nyha functional class , 6-minute walked distance , followup duration , and medications at baseline and followup . baseline lvef was higher in responders ( 25 8% versus 20 7% , p = .01 ) . as expected , responders had significant improvement of their nyha functional class and lvef compared to baseline ( nyha class = 1.6 0.7 , p < .0001 , lvef = + 19 12% , p < .0001 ) , whereas in nonresponders , there was no significant change of these same parameters at long - term followup ( nyha class = 0.2 0.9 , p = .63 , lvef = 0.6 8% , p = .60 ) . nonresponders had significantly larger lvedv and lvesv at baseline compared to responders ( table 2 ) . at followup , responders exhibited significant reduction of their lv volumes ( lvedv = 55 72 ml , p < .001 , lvesv = 68 66 ml , p < .001 ) . in nonresponders , there was no significant change of lv volumes compared to baseline ( lvedv = + 5 62 ml , p = .73 , lvesv = + 5 58 ml , p = .69 ) . again , the difference between the 2 groups was highly significant ( p = .002 for lvedv and p = .001 for lvesv ) . ed was significantly longer in responders ( 139 35 ms versus 119 37 ms , p = .03 ) . the ratio of ed / qrs duration was also significantly greater in responders ( 0.79 0.16 versus 0.67 0.18 , p = .005 ) . linear regression analysis showed a weak but significant positive correlation between ed and difference of lvef from baseline to followup ( lvef ) ( r = + 0.307 , p = .009 ) ( figure 2(a ) ) and also a weak but significant negative correlation with change of nyha class from baseline to followup ( nyha ) ( figure 2(b ) ) ( r = 0.310 , p = .008 ) . we also found a significant positive correlation between the ratio of ed / qrs duration and lvef ( r = + 0.232 , p = .05 ) ( figure 3(a ) ) and a significant negative correlation with nyha ( r = 0.283 , p = .016 ) ( figure 3(b ) ) . receiver operating characteristic analysis showed that an ed 150 ms predicted a response to crt with 80% specificity and 47% sensitivity ( odds ratio ( or ) : 3.5 , confidence interval ( ci ) : 1.111 , p = .025 ) . a ratio of ed / qrs duration 0.83 was associated with a response to crt with 89% specificity and 53% sensitivity ( or : 8.3 , ci : 2.231.7 , p = .001 ) . logistic regression analysis ( table 3 ) showed that , after adjustment for baseline rhythm and underlying heart disease , independent predictors of positive outcome were baseline lvef and the ratio of ed / qrs duration . the value of ed to predict response to crt in patients with ischemic and nonischemic cardiomyopathy was analyzed separately ( table 4 ) . we did not find a stronger correlation between ed and change of lvef or nyha class during long - term followup in patients with nonischemic dilated cardiomyopathy versus those with ischemic heart disease . the same result was observed when the correlation was examined with the ratio of ed / qrs duration . figure 4 shows the distribution of ed ( figure 4(a ) ) and the ratio of ed / qrs duration ( figure 4(b ) ) based on the lv lead location in the lao view . in 1 patient the lv lead was left in the great cardiac vein and in 2 patients , the final lv lead position was at 12:30 . as shown in figures 4(a ) and 4(b ) , longer eds and greater ratios ed / qrs duration were more likely to be located between 2:30 and 5:00 o'clock , but short eds and small ratios were also observed in the same locations indicating that not all lateral sites were equal . the present study demonstrates the value of intraprocedural epicardial egm recording to direct placement of the lv lead in regions of long ed in order to increase the likelihood of long - term response to crt . although mechanical rather than electrical resynchronization has been suggested to be the primary determinant of hemodynamic benefit , we hypothesized that these two components are closely linked . controversial data from the literature raise the question whether positioning the lv lead should be guided by echocardiography to determine the site of latest mechanical contraction . some of these reports indicate an enhanced response rate in patients in whom there is concordance between the position of the lv lead tip and the latest area of contraction [ 7 , 9 , 10 , 19 ] , whereas other investigators suggest that pacing at these sites is not always associated with acute or long - term response [ 20 , 21 ] . on the other hand , adequate assessment of lv dyssynchrony and , more precisely , the site of latest mechanical activation requires sophisticated techniques that may prolong the implantation time , and their use is limited by high intra- and interobserver variability as illustrated by the results of the prospect trial , whereas intraoperative assessment of ed is straightforward and can be used as a surrogate method for selection of optimal lv pacing sites . another method that can be used intraoperatively to select optimal sites of lv pacing is intracardiac mapping . in a human study conducted on 14 candidates for crt , tse et al . showed the greater hemodynamic improvement by lv pacing in patients presenting with larger amount of lv area with late endocardial activation time and preserved lv myocardium measured by electroanatomical mapping . previously , singh et al . reported the value of the ed to predict response to crt defined by one - year mortality and hospitalizations for heart failure . they found that a reduced lv electrical delay less than 50% of the qrs duration was associated with worse clinical outcome within the entire patients ' population as well as when stratified into ischemic and nonischemic subgroups . our results confirm these data at longer follow - up durations using a different endpoint for response to crt . as in the latter study , we purposely expressed , in our multivariate analysis , the ed as the percentage of the baseline qrs duration and not the absolute value , in order to eliminate a potential impact of the qrs duration on the predictive value of the local ed . despite this adjustment indeed , the presence of scar does not preclude electrical capture of the myocardium , but this may not translate into mechanical contraction and , therefore , may not result in effective correction of lv dyssynchrony in the presence of extensive myocardial infarction . this hypothesis has been verified by several studies using different imaging techniques of scar quantification : bleeker et al . defined lv scar burden using contrast - enhanced mri and reported that patients who failed to respond to crt were more likely to have transmural scar in the posterolateral region of the lv ( an important target for lead placement ) . assessed the importance of transmural scar quantified by gated spect in the lv pacing target region and showed that pacing at these sites was negatively correlated to subsequent lv reverse remodeling . these observations confirm some study reports where crt results in greater improvement of lvef and reduction of lv end - diastolic volume in patients with nonischemic dilated cardiomyopathy compared to those with ischemic heart disease and extensive transmural scar . in our series , we took every precaution not to implant crt in patients with severe ischemic heart disease and extensive myocardial scar ( involving more than 4 lv segments ) or history of lateral or posterolateral myocardial infarction who had a low likelihood of response . that may explain the lack of significant difference in the prevalence of nonischemic cardiomyopathy between responders and nonresponders . multivariate analysis of our results did not identify the nonischemic nature of the cardiomyopathy as an independent predictor of positive outcome . furthermore , correlation between ed and long - term response was analyzed separately in patients with nonischemic cardiomyopathy and was not superior in this subgroup of patients . on the other hand , fibrosis might also be present in patients with nonischemic dilated cardiomyopathy and may also decrease the efficacy of lv pacing . mri studies are required to quantify fibrosis in patients with nonischemic heart disease at the sites of latest mechanical contraction or greatest ed . our study results indicate that there is no correlation between ed and the location of the lv lead documented in the lao view . a wide range of values of ed or ed / qrs duration are observed in the postero - lateral or lateral locations which are known as important targets for lv lead placement . this finding implies that not all lbbbs are created equal : the ventricular conduction abnormality in patients with lbbb and lv dysfunction is not a uniform conduction system lesion . both endocardial and epicardial electroanatomical mapping of lv activation disclose significant variations during intrinsic conduction in both ischemic and nonischemic cardiomyopathy [ 2628 ] . in some patients , wavefronts from multiple directions contribute to overall lv activation . in others , activation spreads from the anterior to the inferior wall , or the reverse . alternatively , lv epicardial activation starts from the septoapical region , spreading laterally and ending at the lateral or posterolateral base . wavefront propagation is sometimes influenced by areas of slow conduction or lines of conduction block , some of which are fixed and correlate with areas of scar and others shift to other locations during pacing maneuvers due to their functional character . these variations , which occur with similar qrs configurations on surface ecg , may result from any combination of conduction tissue lesion , scar and fibrosis , and slow cell - to - cell conduction . therefore , electroanatomical mapping or , more simply , local electrical delay may refine lv lead placement to achieve the best effect . since the presumed mechanism for the efficacy of crt is the correction of conduction delay , response is more likely when pacing is delivered at an area of greater lv conduction delay , as suggested in our study . although we found a significant relationship between ed and long - term response to crt , the correlation was weak . based on our results , long ed recorded during spontaneous lbbb predicts reverse remodeling and positive clinical outcome , with good specificity but low sensitivity , which means that in the presence of ed 150 ms or ed / qrs duration 0.83 , the likelihood of positive outcome is very high , but shorter eds or lower ed / qrs durations do not necessarily preclude long - term improvement following crt . the mechanism of this finding is unclear and may result from our recording technique that was performed from the tip of the final lv lead position in a tributary of the cs indicating activation of the epicardial and not endocardial side of the left ventricle . mapping might have elucidated the complex mechanism of positive response in some patients with short eds measured from the epicardial egm recording . it is also unknown if ed can vary at the same lv site with the level of patient 's activity . on the other hand , in our study , we did not map the whole lv to determine the site of maximal electrical delay . with the use of electroanatomical mapping , we could have identified areas of latest electrical activation where lv pacing could have resulted in better outcome . the definition of responders in our study was a pure clinical endpoint when improvement of functional status was important and a composite clinical and echocardiographic endpoint when improvement of functional status was more modest . the reason for this selection is that patients were followed at regular intervals in our institution and improvement of functional status by one nyha class with no improvement of lvef might have resulted from the optimization of medical therapy at each visit and not from crt . a plethora of endpoints for response to crt have emerged in the literature : some are merely clinical , defining response as improvement of functional status by at least one nyha class [ 19 , 29 , 30 ] or including composite factors such as peak vo2 [ 31 , 32 ] , quality of life score [ 33 , 34 ] , and 6-minute walked distance [ 19 , 31 , 33 , 35 ] , others are based on echocardiographic parameters including reduction of lv end - systolic volume by at least 15% [ 3537 ] or improvement of lvef by at least 5% [ 38 , 39 ] . even though our population was highly selected ( mostly lbbb , nonischemic heart disease , long qrs duration ) , the proportion of responders in our series was in the range of that previously reported in the literature [ 5 , 6 ] . with softer clinical endpoints ( improvement of function status by one nyha class ) another explanation for our result is our longer follow - up duration . on the other hand , in large crt trials , although the cut - off value for qrs duration was usually 120130 ms , the actual average mean qrs duration of included patients was in the range of values reported in our study ( > 150 ms ) . the small number of our patients might have been another limitation of the study . with larger number of patients selecting the lv lead position at the site of the delayed electrical activation may provide an important criterion for appropriate pacing site in patients with both ischemic and nonischemic cardiomyopathy , with high specificity but low sensitivity . gross anatomic lateral location of the lv lead is not always correlated with electrical delay and by itself is not enough to predict chronic response to crt .

cardiac resynchronization therapy ( crt ) has been demonstrated to improve symptoms and survival in patients with left ventricular ( lv ) systolic dysfunction and dyssynchrony . to achieve this goal , the lv lead should be positioned in a region of delayed contraction . we hypothesized that pacing at the site of late electrical activation was also associated with long - term response to crt . we conducted a retrospective study on 72 crt patients . for each patient , we determined the electrical delay ( ed ) from the onset of qrs to the epicardial egm and the ratio of ed to qrs duration ( ed / qrs duration ) . after a followup of 30 20 months , 47 patients responded to crt . responders had a significantly longer ed and greater ratio of ed / qrs duration than nonresponders . an ed / qrs duration 0.38 predicted a response to crt with 89% specificity and 53% sensitivity .

condylar hyperplasia ( ch ) is a rare disorder characterized by excessive bone growth that usually presents unilaterally , resulting in facial asymmetry.1 its etiology and pathogenesis are poorly understood . facial asymmetry is often the reason that patients present for treatment of the disorder . in many cases , occlusal discrepancies and temporomandibular joint disorder an attempt has been made to separate and classify different types of ch based on clinical characteristics and etiology.2 in 1986 , obwegeser and makek3 specifically detailed two hemimandibular anomalies , hemimandibular hyperplasia and hemimandibular elongation . many diagnostic tools and criteria have been used to aid in the correct diagnosis of ch , which in turn is critical to determining the appropriate treatments and timing . with proper diagnosis , timing , and treatment , ch can occur at any age and can continue past the growth period.4 previous literature has suggested that ch may predominantly affect women.4 a meta - analysis was conducted by raijmakers et al.5 in 2012 to assess this hypothesis , which revealed that women develop ch significantly more frequently , with 64% ( 95% ci , 58 - 70% ; n = 275 patients ) of ch patients being women . ch was also proposed to demonstrate sex - based laterality , with women and men affected more frequently on the right and left side , respectively.4 however , the meta - analysis by raijmakers et al.5 did not find any evidence that the side affected by ch was linked to sex . possible etiologies include endocrine distortions ( e.g. , insulin - like growth factors [ igfs ] ) , metabolic hyperactivity , trauma , arthrosis , and genetics . typical mandibular condyle soft tissue histology includes four layers : fibrous articular layer , undifferentiated mesenchymal layer , transitional layer , and hypertrophic cartilage layer.6 active ch has been found to display a broader mesenchymal layer than that in the normal condyle.7 igf-1 and igf-1 receptor ( igf-1r ) expression was found to significantly increase in chondrocytes affected by ch.7 igf-1 is an essential growth modulator that has been described as a pathological factor when produced in excess8 and ' has been shown to reach higher - than - normal levels in ch'.79 in an in vitro model , chen et al.7 demonstrated that igf-1 was able to significantly increase the proliferation of chondrocytes from the cartilage of normal condyles and , conversely , that nvp - aew541 ( an inhibitor of igf-1r kinase ) was able to significantly decrease the proliferation capacity of chondrocytes cultured from ch . gene expression of collagen type ii a1 ( col2a1 ) was found to significantly increase in ch compared to that in normal condylar cartilage , and when chondrocytes from normal condylar cartilage were treated with igf-1 , gene expression of col2a1 also significantly increased . however , when ch chondrocytes were treated with nvp - aew541 , no significant reduction in col2a1 expression occurred.7 these results indicate that igf-1 has an impact on chondrogenesis of the mandibular condyle ; however , the extent and full underlying mechanism of this effect is not known . a study by saridin et al.10 analyzed metabolism and blood flow in seven patients ( mean age 25.5 years ) with unilateral ch using f and h2o positron emission tomography ( pet ) imaging . four patients had hemimandibular elongation , one had hemimandibular hyperplasia , and two had a combination of these anomalies . their results highlighted the possibility that ch can also be caused by a decrease in growth activity of the contralateral condyle with normal growth in the affected condyle . in patients with condylar hyperactivity , f uptake , used to assess bone formation , was significantly higher in the affected condyle than in the contralateral condyle.10 however , the f uptake of the hyperactive condyles was similar to that of control condyles , while the contralateral condyles had significantly less f uptake than control condyles.10 these results suggest that in this patient sample , the affected condyle grows at a normal rate , while a cessation of growth occurs in the contralateral condyle . in contrast to previous literature,11 the study by saridin et al.10 did not find any relationship between bone formation and blood flow . correct diagnosis of ch is essential when deciding how to treat the condition . to prevent post - surgical reversion , accurate diagnosis of ch activity is also of upmost importance.12 diagnostic methods such as clinical examination , radiographs , and nuclear imaging ( figure 1 ) can be used to determine the type of ch as well as its activity . clinical diagnosis has been described as the diagnostic gold standard.13 nuclear imaging is capable of providing physiological details of ch using radionuclide - labeled tracers.14 examples of different types of nuclear imaging include planar scintigraphy , single - photon emission computed tomography ( spect ) and pet . spect and planar scintigraphy utilize the radionucleotide technetium-99-labelled methylene diphosphonate ( tc - mdp ) , while pet utilizes the radionucleotide [ f]-fluoride.15 prior to the development of tc - mdp , [ f]-fluoride was the standard radionucleotide tracer for spect.15 planar scintigraphy produces a two - dimensional image , as opposed to spect and pet , which produce three - dimensional images . bone scintigraphy has high sensitivity and low specificity for bone metabolism , meaning that it can identify when a change in bone metabolism is present but is limited in its ability to differentiate among various conditions ( e.g. , bone healing , growth , infection , arthritic changes , or tumors).14 generally when condyles are being evaluated with bone scintigraphy , a difference in uptake levels of less than 10% indicates either normal condyles or individuals without progressive asymmetry.13 a meta - analysis by saridin et al.13 found that the spect technique of bone scintigraphy had a significantly higher sensitivity ( 0.90 ) in detecting unilateral ch than the planar technique ( 0.71 ) ( p = 0.04 ) . pet has been described as having better spatial resolution than spect.15 further research is needed to establish a more formalized method for scintigraphy analysis . the current literature provides various methods such as comparing right and left condylar activity in the form of a percentage or ratio and comparing condylar activity to a different bony landmark such as the lumbar vertebrae.13 an attempt was made to relate spect findings to histopathological differences in ch , in which spect was found insufficiently sensitive to detect histopathological differences.16 temporomandibular joint ch has been described as a rare unilateral growth of the mandibular condyle . this growth causes both functional and esthetic problems , which often manifest as facial asymmetry , occlusal interferences ( figure 2 ) , and joint dysfunction that can lead to pain.1 excessive growth can occur in several different locations in the mandible . the growth can be the result of an enlarged condyle , an elongated condylar neck , or outward bowing or downward growth of the body and ramus.17 due to the variations in locations of excessive growth , multiple classification systems have been developed to better characterize the pathology . obwegeser and makek3 developed a classification system based on the asymmetry and predominant growth vector ( table 1 ) . in their article , they classified ch into 3 different categories . they defined type 1 as hemimandibular elongation , with excessive growth displayed in the horizontal vector . type 1 ch is associated with chin deviation toward the unaffected side , with no corresponding vertical asymmetry . due to the overgrowth , , the contralateral mandibular molars often deviate lingually in order to remain in proper occlusion with the maxillary molars . if the contralateral molars are unable to adapt to the growth , a crossbite may develop . normally , the condyle is unaffected , but the neck is often misshapen and slender . the ramus is elongated , which is the basis for referring to type 1 as hemimandibular elongation . type 2 ch was defined as hemimandibular hyperplasia , which is associated with excessive growth in the vertical vector . it is often characterized by a sloping rima oris with minimal chin deviation . due to the excessive downward growth of the mandible , the maxillary molars on the affected side compensate by following the mandible 's downward growth . the maxillary alveolar bone on the ipsilateral side grows excessively to maintain occlusion . if the maxillary molars are not able to follow the excessive downward growth , an open bite on the affected side results . in type 2 ch , the condyle often appears enlarged , and the head is usually irregular or deformed . the neck of the condyle has also been reported as thickened and/or elongated.18 type 3 ch is a combination of types 1 and 2 . wolford et al.2 developed an updated classification system that they considered more inclusive of pathologies causing ch . their report classifies ch into four different categories based on clinical , imaging , growth , and histological characteristics . this system was developed to classify ch into more specific types in order to provide optimal treatment to patients based on their specific disease characteristics ( table 2 ) . in this system , type 1 and 2 ch are similar to the classification system developed by obwegeser and makek3 with the following exceptions . type 1 is characterized by an accelerated and prolonged growth that causes condylar and mandibular elongation and split into 1a and 1b . ch type 1a is defined as mandibular elongation that occurs bilaterally , while ch type 1b occurs unilaterally . ch type 2 consists of unilateral overgrowth of the condyle caused by an osteochondroma and results in vertical overgrowth of the mandible . wolford et al.19 further classified ch type 2a and b. type 2a results from vertical elongation of the condylar head and neck . type 2b involves horizontal exophytic tumor growth of the condyle in addition to vertical elongation of the head and neck . ch type 3 consists of other benign tumors that cause ch , including but not limited to osteomas , neurofibromas , and fibrous dysplasia , and results in unilateral facial enlargement . type 4 ch is caused by malignant tumors that originate in the condyle and cause enlargement and facial asymmetry . some malignant tumors attributed to type 4 ch include chondrosarcoma , multiple myeloma , osteosarcoma , and ewing sarcoma . slootweg and mller20 were among the first to create a histological classification system based on a study they conducted in 1986 , in which they classified 22 patients into four categories based on histological findings in various layers of hyperplastic condyles . specifically , they analyzed the fibrous articular layer , the undifferentiated mesenchymal layer , the transitional layer , and the hypertrophic cartilage layer and characterized each layer based on histological findings . ch type 1 displayed a broad proliferation zone with an underlying thick layer of hyaline growth cartilage and bone that contained numerous cartilage islands . ch type 2 demonstrated a patchier distribution of proliferation zones with a smaller number of cartilage islands . ch type 3 was characterized by irregular - shaped masses of cartilage found in the bone of the condylar neck or encroaching onto the superficial articular layer . type 4 ch was commonly characterized by a burned - out appearance of the condyle due to a very cell - poor fibrocartilaginous layer covering the subchondral bone plate . slootweg and mller20 also noted that type 4 ch did not demonstrate a proliferation layer of the hyaline growth cartilage like that seen in the other types . one of the first steps in managing ch cases is to determine if the mandible is actively growing . this determination can be made with many methods , but bone spect is an essential diagnostic tool to assess active growth.21 in this quantitative method , tc - mdp is injected and absorbed into hydroxyapatite crystals and calcium in the bone.22 the bone is then scanned using the spect technique , and the hyperplastic condyle is quantitatively compared to the contralateral side.23 often only a 0 - 5% difference in positive area is observed between normal condyles . differences greater than 10% between two condyles are considered to indicate active growth due to ch.12 therefore , a relative 55% uptake in the affected hyperplastic condyle is considered to be abnormal . some other tests used to diagnose ch include three - dimensional tomography , standard radiology and cephalometry , and pet scans.24 once a detailed diagnosis of ch has been obtained , a treatment plan must be established . treatment plans must consider the degree of asymmetry , resulting malocclusion , and condylar growth activity . usually , the selected strategy is dependent on growth activity and the patient 's age . a study by naini et al.25 found that the patient 's desire for surgical intervention increases as the degree of asymmetry increases . interestingly , they also found that asymmetries as small as 5 mm can be noticed by the laypersons . ch treatment options are detailed from the simplest , least invasive to most complex procedures . one of the simplest procedures that can be performed is a mandibular ramus osteotomy of affected condyles . seven of the patients were treated with bilateral osteotomies alone , six were treated with unilateral osteotomies , and two of those procedures were combined with le fort i procedures . the study concluded that patients with unilateral ch can effectively be treated with unilateral ramus osteotomies on the affected side . bilateral osteotomies did not show any advantages over unilateral procedures ; however , they may be indicated for patients with severely prognathic profiles and patients in whom unilateral osteotomies could possibly lead to excessive rotation of the unaffected condyle . combining the osteotomies with le fort an analysis of 22 patients treated with high condylectomies who completed a 4-year follow - up concluded that this method is a viable treatment option for patients , which often has a good and predictable surgical outcome.20 lippold et al.27 also found that high condylectomy was an appropriate treatment for unilateral ch . in their study , 4 - 5 mm was removed from the upper pole of the affected condyle , which appeared to effectively limit growth in ch . the most complex surgical treatment for ch involves both a condylectomy and orthognathic surgery . in a classic study conducted by wolford et al.19 in 2002 , a group of patients was treated with both a high condylectomy and orthognathic surgery , and this treatment was found to be very effective for correcting both functional and esthetic problems resulting from ch . another study supporting this claim found that combined condylectomy and orthognathic surgery was successful both functionally and esthetically in 30 of 36 patients.28 however , it also indicated that orthognathic surgery without any condylar intervention could possibly lead to future issues , because condylar growth may not be complete at the time of surgery . thus , without treating the hyperplastic condyle , growth could possibly continue following orthognathic surgery . by contrast , orthognathic surgery alone may be an acceptable treatment if the excessive condylar growth has halted . two studies treated a combined 44 ch patients after the excessive condylar growth was determined to have ceased.29 the cessation of condylar growth was confirmed by taking multiple spect images over a determined time period to compare the amount of active growth in each condyle . for all surgical options , timing and the patient 's wishes are critical in determining the correct treatment plan for ch . the literature pertaining to orthodontic treatment in patients with ch is very limited and generally consists of case reports . in a report of five cases by rajkumar et al.30 , the authors ' final assessment was that mild to severe ch can be treated with surgery alone . other case reports have described the use of orthodontic treatment after surgical treatment to finalize occlusion . in a case involving two - stage surgery , xavier et al.31 described their procedure as consisting of condylectomy followed by orthodontic treatment and orthognathic surgery . each of these reports only describes the treatment used and what worked in those particular circumstances . if orthodontic treatment is chosen , it is vital to confirm that growth has halted before the orthodontic treatment begins . ch can occur at any age and can continue past the growth period.4 previous literature has suggested that ch may predominantly affect women.4 a meta - analysis was conducted by raijmakers et al.5 in 2012 to assess this hypothesis , which revealed that women develop ch significantly more frequently , with 64% ( 95% ci , 58 - 70% ; n = 275 patients ) of ch patients being women . ch was also proposed to demonstrate sex - based laterality , with women and men affected more frequently on the right and left side , respectively.4 however , the meta - analysis by raijmakers et al.5 did not find any evidence that the side affected by ch was linked to sex . possible etiologies include endocrine distortions ( e.g. , insulin - like growth factors [ igfs ] ) , metabolic hyperactivity , trauma , arthrosis , and genetics . typical mandibular condyle soft tissue histology includes four layers : fibrous articular layer , undifferentiated mesenchymal layer , transitional layer , and hypertrophic cartilage layer.6 active ch has been found to display a broader mesenchymal layer than that in the normal condyle.7 igf-1 and igf-1 receptor ( igf-1r ) expression was found to significantly increase in chondrocytes affected by ch.7 igf-1 is an essential growth modulator that has been described as a pathological factor when produced in excess8 and ' has been shown to reach higher - than - normal levels in ch'.79 in an in vitro model , chen et al.7 demonstrated that igf-1 was able to significantly increase the proliferation of chondrocytes from the cartilage of normal condyles and , conversely , that nvp - aew541 ( an inhibitor of igf-1r kinase ) was able to significantly decrease the proliferation capacity of chondrocytes cultured from ch . gene expression of collagen type ii a1 ( col2a1 ) was found to significantly increase in ch compared to that in normal condylar cartilage , and when chondrocytes from normal condylar cartilage were treated with igf-1 , gene expression of col2a1 also significantly increased . however , when ch chondrocytes were treated with nvp - aew541 , no significant reduction in col2a1 expression occurred.7 these results indicate that igf-1 has an impact on chondrogenesis of the mandibular condyle ; however , the extent and full underlying mechanism of this effect is not known . a study by saridin et al.10 analyzed metabolism and blood flow in seven patients ( mean age 25.5 years ) with unilateral ch using f and h2o positron emission tomography ( pet ) imaging . four patients had hemimandibular elongation , one had hemimandibular hyperplasia , and two had a combination of these anomalies . their results highlighted the possibility that ch can also be caused by a decrease in growth activity of the contralateral condyle with normal growth in the affected condyle . in patients with condylar hyperactivity , f uptake , used to assess bone formation , was significantly higher in the affected condyle than in the contralateral condyle.10 however , the f uptake of the hyperactive condyles was similar to that of control condyles , while the contralateral condyles had significantly less f uptake than control condyles.10 these results suggest that in this patient sample , the affected condyle grows at a normal rate , while a cessation of growth occurs in the contralateral condyle . in contrast to previous literature,11 the study by saridin et al.10 did not find any relationship between bone formation and blood flow . correct diagnosis of ch is essential when deciding how to treat the condition . to prevent post - surgical reversion , accurate diagnosis of ch activity is also of upmost importance.12 diagnostic methods such as clinical examination , radiographs , and nuclear imaging ( figure 1 ) can be used to determine the type of ch as well as its activity . clinical diagnosis has been described as the diagnostic gold standard.13 nuclear imaging is capable of providing physiological details of ch using radionuclide - labeled tracers.14 examples of different types of nuclear imaging include planar scintigraphy , single - photon emission computed tomography ( spect ) and pet . spect and planar scintigraphy utilize the radionucleotide technetium-99-labelled methylene diphosphonate ( tc - mdp ) , while pet utilizes the radionucleotide [ f]-fluoride.15 prior to the development of tc - mdp , [ f]-fluoride was the standard radionucleotide tracer for spect.15 planar scintigraphy produces a two - dimensional image , as opposed to spect and pet , which produce three - dimensional images . bone scintigraphy has high sensitivity and low specificity for bone metabolism , meaning that it can identify when a change in bone metabolism is present but is limited in its ability to differentiate among various conditions ( e.g. , bone healing , growth , infection , arthritic changes , or tumors).14 generally when condyles are being evaluated with bone scintigraphy , a difference in uptake levels of less than 10% indicates either normal condyles or individuals without progressive asymmetry.13 a meta - analysis by saridin et al.13 found that the spect technique of bone scintigraphy had a significantly higher sensitivity ( 0.90 ) in detecting unilateral ch than the planar technique ( 0.71 ) ( p = 0.04 ) . pet has been described as having better spatial resolution than spect.15 further research is needed to establish a more formalized method for scintigraphy analysis . the current literature provides various methods such as comparing right and left condylar activity in the form of a percentage or ratio and comparing condylar activity to a different bony landmark such as the lumbar vertebrae.13 an attempt was made to relate spect findings to histopathological differences in ch , in which spect was found insufficiently sensitive to detect histopathological differences.16 temporomandibular joint ch has been described as a rare unilateral growth of the mandibular condyle . this growth causes both functional and esthetic problems , which often manifest as facial asymmetry , occlusal interferences ( figure 2 ) , and joint dysfunction that can lead to pain.1 excessive growth can occur in several different locations in the mandible . the growth can be the result of an enlarged condyle , an elongated condylar neck , or outward bowing or downward growth of the body and ramus.17 due to the variations in locations of excessive growth , multiple classification systems have been developed to better characterize the pathology . obwegeser and makek3 developed a classification system based on the asymmetry and predominant growth vector ( table 1 ) . in their article , they classified ch into 3 different categories . they defined type 1 as hemimandibular elongation , with excessive growth displayed in the horizontal vector . type 1 ch is associated with chin deviation toward the unaffected side , with no corresponding vertical asymmetry . due to the overgrowth , , the contralateral mandibular molars often deviate lingually in order to remain in proper occlusion with the maxillary molars . if the contralateral molars are unable to adapt to the growth , a crossbite may develop . normally , the condyle is unaffected , but the neck is often misshapen and slender . the ramus is elongated , which is the basis for referring to type 1 as hemimandibular elongation . type 2 ch was defined as hemimandibular hyperplasia , which is associated with excessive growth in the vertical vector . it is often characterized by a sloping rima oris with minimal chin deviation . due to the excessive downward growth of the mandible , the maxillary molars on the affected side compensate by following the mandible 's downward growth . the maxillary alveolar bone on the ipsilateral side grows excessively to maintain occlusion . if the maxillary molars are not able to follow the excessive downward growth , an open bite on the affected side results . in type 2 ch , the condyle often appears enlarged , and the head is usually irregular or deformed . the neck of the condyle has also been reported as thickened and/or elongated.18 type 3 ch is a combination of types 1 and 2 . wolford et al.2 developed an updated classification system that they considered more inclusive of pathologies causing ch . their report classifies ch into four different categories based on clinical , imaging , growth , and histological characteristics . this system was developed to classify ch into more specific types in order to provide optimal treatment to patients based on their specific disease characteristics ( table 2 ) . in this system , type 1 and 2 ch are similar to the classification system developed by obwegeser and makek3 with the following exceptions . type 1 is characterized by an accelerated and prolonged growth that causes condylar and mandibular elongation and split into 1a and 1b . ch type 1a is defined as mandibular elongation that occurs bilaterally , while ch type 1b occurs unilaterally . ch type 2 consists of unilateral overgrowth of the condyle caused by an osteochondroma and results in vertical overgrowth of the mandible . wolford et al.19 further classified ch type 2a and b. type 2a results from vertical elongation of the condylar head and neck . type 2b involves horizontal exophytic tumor growth of the condyle in addition to vertical elongation of the head and neck . ch type 3 consists of other benign tumors that cause ch , including but not limited to osteomas , neurofibromas , and fibrous dysplasia , and results in unilateral facial enlargement . type 4 ch is caused by malignant tumors that originate in the condyle and cause enlargement and facial asymmetry . some malignant tumors attributed to type 4 ch include chondrosarcoma , multiple myeloma , osteosarcoma , and ewing sarcoma . slootweg and mller20 were among the first to create a histological classification system based on a study they conducted in 1986 , in which they classified 22 patients into four categories based on histological findings in various layers of hyperplastic condyles . specifically , they analyzed the fibrous articular layer , the undifferentiated mesenchymal layer , the transitional layer , and the hypertrophic cartilage layer and characterized each layer based on histological findings . ch type 1 displayed a broad proliferation zone with an underlying thick layer of hyaline growth cartilage and bone that contained numerous cartilage islands . ch type 2 demonstrated a patchier distribution of proliferation zones with a smaller number of cartilage islands . ch type 3 was characterized by irregular - shaped masses of cartilage found in the bone of the condylar neck or encroaching onto the superficial articular layer . type 4 ch was commonly characterized by a burned - out appearance of the condyle due to a very cell - poor fibrocartilaginous layer covering the subchondral bone plate . slootweg and mller20 also noted that type 4 ch did not demonstrate a proliferation layer of the hyaline growth cartilage like that seen in the other types . one of the first steps in managing ch cases is to determine if the mandible is actively growing . this determination can be made with many methods , but bone spect is an essential diagnostic tool to assess active growth.21 in this quantitative method , tc - mdp is injected and absorbed into hydroxyapatite crystals and calcium in the bone.22 the bone is then scanned using the spect technique , and the hyperplastic condyle is quantitatively compared to the contralateral side.23 often only a 0 - 5% difference in positive area is observed between normal condyles . differences greater than 10% between two condyles are considered to indicate active growth due to ch.12 therefore , a relative 55% uptake in the affected hyperplastic condyle is considered to be abnormal . some other tests used to diagnose ch include three - dimensional tomography , standard radiology and cephalometry , and pet scans.24 once a detailed diagnosis of ch has been obtained , a treatment plan must be established . treatment plans must consider the degree of asymmetry , resulting malocclusion , and condylar growth activity . , the selected strategy is dependent on growth activity and the patient 's age . as always , the patient 's demands and expectations are other important considerations . a study by naini et al.25 found that the patient 's desire for surgical intervention increases as the degree of asymmetry increases . interestingly , they also found that asymmetries as small as 5 mm can be noticed by the laypersons . ch treatment options are detailed from the simplest , least invasive to most complex procedures . one of the simplest procedures that can be performed is a mandibular ramus osteotomy of affected condyles . seven of the patients were treated with bilateral osteotomies alone , six were treated with unilateral osteotomies , and two of those procedures were combined with le fort i procedures . the study concluded that patients with unilateral ch can effectively be treated with unilateral ramus osteotomies on the affected side . bilateral osteotomies did not show any advantages over unilateral procedures ; however , they may be indicated for patients with severely prognathic profiles and patients in whom unilateral osteotomies could possibly lead to excessive rotation of the unaffected condyle . many studies and case reports advocate high condylectomies to treat ch . an analysis of 22 patients treated with high condylectomies who completed a 4-year follow - up concluded that this method is a viable treatment option for patients , which often has a good and predictable surgical outcome.20 lippold et al.27 also found that high condylectomy was an appropriate treatment for unilateral ch . in their study , 4 - 5 mm was removed from the upper pole of the affected condyle , which appeared to effectively limit growth in ch . the most complex surgical treatment for ch involves both a condylectomy and orthognathic surgery . in a classic study conducted by wolford et al.19 in 2002 , a group of patients was treated with both a high condylectomy and orthognathic surgery , and this treatment was found to be very effective for correcting both functional and esthetic problems resulting from ch . another study supporting this claim found that combined condylectomy and orthognathic surgery was successful both functionally and esthetically in 30 of 36 patients.28 however , it also indicated that orthognathic surgery without any condylar intervention could possibly lead to future issues , because condylar growth may not be complete at the time of surgery . thus , without treating the hyperplastic condyle , growth could possibly continue following orthognathic surgery . by contrast , orthognathic surgery alone may be an acceptable treatment if the excessive condylar growth has halted . two studies treated a combined 44 ch patients after the excessive condylar growth was determined to have ceased.29 the cessation of condylar growth was confirmed by taking multiple spect images over a determined time period to compare the amount of active growth in each condyle . for all surgical options , timing and the patient 's wishes are critical in determining the correct treatment plan for ch . the literature pertaining to orthodontic treatment in patients with ch is very limited and generally consists of case reports . in a report of five cases by rajkumar et al.30 , the authors ' final assessment was that mild to severe ch can be treated with surgery alone . other case reports have described the use of orthodontic treatment after surgical treatment to finalize occlusion . in a case involving two - stage surgery , xavier et al.31 described their procedure as consisting of condylectomy followed by orthodontic treatment and orthognathic surgery . each of these reports only describes the treatment used and what worked in those particular circumstances . if orthodontic treatment is chosen , it is vital to confirm that growth has halted before the orthodontic treatment begins . additional research is needed to establish a more standardized approach for diagnosing the activity of ch . clinical examination , radiography , planar scintigraphy , spect , and pet are all diagnostic methods that can be utilized by clinicians when planning surgery . longer follow - up studies must be completed to determine which treatment options are the most successful . additionally , more studies are needed to understand the underlying causes of ch for earlier diagnosis and better treatment options .

condylar hyperplasia ( ch ) is a rare disorder characterized by excessive bone growth that almost always presents unilaterally , resulting in facial asymmetry . classification of the different types of ch can differ depending on the authors . correct diagnosis is critical in determining the proper treatments and timing . this paper is a review of the recent literature on the epidemiology , etiology , diagnosis , classification , and surgical treatments of ch .

stroke is the third most common cause of death and the most common cause of disability in developed countries . due to the aging population , it has been reported by the world health organization ( who ) that about 15 million people suffer from ischemic stroke ( is ) every year , of which 5 million were dead and 5 million became permanently disabled . , a variety of risk factors have been identified to contribute to is , including hypertension , smoking , diabetes , obesity , advanced age , and so on . twins , families , and animal - based studies provide substantial evidence that genetic factors are important in the pathogenesis of is . angiotensinogen ( agt ) is a component of the renin - angiotensin system , which is converted to angiotensin i by renin , and subsequently forms angiotensin ii via angiotensin i - converting enzyme . angiotensin ii is responsible for the regulation of systemic blood pressure , salt and water homeostasis , and the maintenance of vascular tone , as well as increasing the levels of vasopressin and adrenocorticotropic hormone in the central nervous system . the agt gene is located at lq42 - 43 and consists of five exons , and express pre agt or agt precursor protein in the liver . a threonine to methionine substitution at amino acid 174 is a common polymorphism called t174 m ( rs699 ) , designating the t and m alleles , respectively . previous meta - analyses demonstrated that agt t174 m polymorphism was associated with susceptibility to coronary heart disease and hypertension . however , little is known regarding the association between t174 m polymorphism and susceptibility to is . over the past decade , several case - control studies have focused on the association between t174 m polymorphism and is risk . however , the results remain controversial . in the present study , we investigated whether the agt t174 m polymorphism is associated with is risk by performing a meta - analysis . in this study , the terms is or cerebral infarction or cerebrovascular disease or stroke in combination with angiotensinogen or t174 m and polymorphism or variant or gene were used to search the electronic databases ( pubmed , embase and china national knowledge infrastructure databases ) for all studies on agt t174 m polymorphism and is ( last search was updated on october 2014 ) . all the searched studies were retrieved , and their references were checked for other relevant publications . if sequential or multiple publications from the same data occurred , the publication that reported data from the largest or most recent study was included . human studies were included if they met the following criteria : case - control studies that addressed is cases and healthy controls;studies that evaluated the association between agt t174 m polymorphism and is risk;all patients with clinically diagnosed is;studies that included sufficient genotype data for extraction;the studies contained at least two comparison groups ( case group vs. control group);the studies included detailed genotyping data . case - control studies that addressed is cases and healthy controls ; studies that evaluated the association between agt t174 m polymorphism and is risk ; all patients with clinically diagnosed is ; studies that included sufficient genotype data for extraction ; the studies contained at least two comparison groups ( case group vs. control group ) ; the studies included detailed genotyping data . in addition , the following exclusion criteria were also used : not case - control studies that evaluated the association between agt t174 m polymorphism and is risk;animal studies;studies that were based on incomplete raw data or no usable data reported;duplicated publications . not case - control studies that evaluated the association between agt t174 m polymorphism and is risk ; studies that were based on incomplete raw data or no usable data reported ; duplicated publications . the extraction of data from all eligible publications was performed by two investigators independently , according to the inclusion and exclusion criteria listed above . if there was a discrepancy between them , it was settled by discussion until a consensus was reached . the following data were extracted : the first author 's name , year of publication , country of the study , ethnicity , numbers of genotyped cases and controls and deviation from hardy - weinberg equilibrium ( hwe ) of the control group . we assessed hwe in the controls for each study using test and a p < 0.05 was considered as significant disequilibrium . the odd ratios ( ors ) together with the 95% confidence intervals ( cis ) were used to assess the strength of association . the combined ors and 95% cis were calculated respectively for a homozygote comparison ( mm vs. tt ) , a heterozygote comparison ( mt vs. tt ) , a dominant model ( mm + mt vs. tt ) , and a recessive model ( tt + mt vs. mm ) between groups . between - i values of 25 , 50 and 75% were defined as low , moderate and high estimates , respectively . when i > 50% indicated heterogeneity across studies , the random effects model was used for meta - analysis , or else the fixed effects model was used . subgroup analyses were based on ethnicity to identify possible causes of heterogeneity and assess the robustness of the relationships . only one study was performed in african patients ; therefore , the result of subgroup analysis by ethnicity could not be reliable for africans . sensitivity analysis was performed to assess the stability of the results by excluding one study with genotype distributions not in hwe . and publication bias was examined by plotting a begg 's funnel plot ( p < 0.05 was considered representative of statistically significant publication bias ) . all analyses were performed by stata 12.0 ( stata college station , tx , usa ) . in this study , the terms is or cerebral infarction or cerebrovascular disease or stroke in combination with angiotensinogen or t174 m and polymorphism or variant or gene were used to search the electronic databases ( pubmed , embase and china national knowledge infrastructure databases ) for all studies on agt t174 m polymorphism and is ( last search was updated on october 2014 ) . all the searched studies were retrieved , and their references were checked for other relevant publications . if sequential or multiple publications from the same data occurred , the publication that reported data from the largest or most recent study was included . human studies were included if they met the following criteria : case - control studies that addressed is cases and healthy controls;studies that evaluated the association between agt t174 m polymorphism and is risk;all patients with clinically diagnosed is;studies that included sufficient genotype data for extraction;the studies contained at least two comparison groups ( case group vs. control group);the studies included detailed genotyping data . case - control studies that addressed is cases and healthy controls ; studies that evaluated the association between agt t174 m polymorphism and is risk ; all patients with clinically diagnosed is ; studies that included sufficient genotype data for extraction ; the studies contained at least two comparison groups ( case group vs. control group ) ; the studies included detailed genotyping data . in addition , the following exclusion criteria were also used : not case - control studies that evaluated the association between agt t174 m polymorphism and is risk;animal studies;studies that were based on incomplete raw data or no usable data reported;duplicated publications . not case - control studies that evaluated the association between agt t174 m polymorphism and is risk ; studies that were based on incomplete raw data or no usable data reported ; duplicated publications . the extraction of data from all eligible publications was performed by two investigators independently , according to the inclusion and exclusion criteria listed above . if there was a discrepancy between them , it was settled by discussion until a consensus was reached . the following data were extracted : the first author 's name , year of publication , country of the study , ethnicity , numbers of genotyped cases and controls and deviation from hardy - weinberg equilibrium ( hwe ) of the control group . we assessed hwe in the controls for each study using test and a p < 0.05 was considered as significant disequilibrium . the odd ratios ( ors ) together with the 95% confidence intervals ( cis ) were used to assess the strength of association . the combined ors and 95% cis were calculated respectively for a homozygote comparison ( mm vs. tt ) , a heterozygote comparison ( mt vs. tt ) , a dominant model ( mm + mt vs. tt ) , and a recessive model ( tt + mt vs. mm ) between groups . between - i values of 25 , 50 and 75% were defined as low , moderate and high estimates , respectively . when i > 50% indicated heterogeneity across studies , the random effects model was used for meta - analysis , or else the fixed effects model was used . subgroup analyses were based on ethnicity to identify possible causes of heterogeneity and assess the robustness of the relationships . only one study was performed in african patients ; therefore , the result of subgroup analysis by ethnicity could not be reliable for africans . sensitivity analysis was performed to assess the stability of the results by excluding one study with genotype distributions not in hwe . and publication bias was examined by plotting a begg 's funnel plot ( p < 0.05 was considered representative of statistically significant publication bias ) . all analyses were performed by stata 12.0 ( stata college station , tx , usa ) . with our search criterion , 25 individual records were found , and 12 13 full - text publications were preliminarily identified for further detailed evaluation . according to the exclusion criteria , six publications were excluded , including one duplicate study , one meta - analysis , and four without sufficient data for extraction . finally , as shown in figure 1 , six studies with 1290 cases and 1125 healthy controls were included into this meta - analysis . the ethnicity of eligible subjects involved populations ranging from african ( n = 1 ) and asian ( n = 5 ) . only one of the six studies was deviated from hwe . in addition , the controls in two of these eligible studies were based on common populations , while the others were based on hospital populations . furthermore , all of the genotyping methods applied in the including studies are pcr - based . the flow chart of the included studies in the meta - analysis distribution of agt t174 m polymorphism genotype between cases and controls and genotyping methods the main results of meta - analysis and heterogeneity are listed in table 2 . four models suggested remarkable between - study heterogeneity , thus both fixed effects model and random effects model were performed to calculate the pooled estimates . overall , no significant association between agt t174 m polymorphism and is was observed under all genetic models . ( mm vs. tt : or = 1.64 , 95% ci = 0.51 - 5.28 , p = 0.01 ; mt vs. tt : or = 0.93 , 95% ci = 0.66 - 1.31 , p = 0.07 ; dominant model : or = 1.08 , 95% ci = 0.69 - 1.72 , p = 0.00 ; recessive model : or = 0.61 , 95% ci = 0.20 - 1.91 , p = 0.02 ) . in the subgroup analysis based on ethnicity , results of subgroup analysis confirmed that there was significant associations between agt t174 m polymorphism and is risk in asian populations ( figure 2 , mm vs. tt : or = 3.28 , 95% ci = 1.79 - 6.02 , p = 0.32 ; mt vs. tt : or = 0.96 , 95% ci = 0.61 - 1.53 , p = 0.04 ; dominant model : or = 1.17 , 95% ci = 0.65 - 2.09 , p = 0.00 ; recessive model : or = 0.31 , 95% ci = 0.17 - 0.57 , p = 0.35 ) . summary of different comparative results forest plots for the association of angiotensinogen t174 m polymorphism with risk of ischemic stroke in asians . for each study , the estimates of odds ratio ( or ) and its 95% confidence interval ( ci ) were plotted with a box and a horizontal line . the symbol filled diamond indicates pooled or and its 95% ci in the subgroup analysis based on hwe , we detected no significant association between agt t174 m polymorphism and is risk ( mm vs. tt : or = 1.08 , 95% ci = 0.53 - 2.21 , p = 0.11 ; mt vs. tt : or = 0.86 , 95% ci = 0.68 - 1.09 , p = 0.09 ; dominant model : or = 0.90 , 95% ci = 0.62 - 1.32 , p = 0.05 ; recessive model : or = 0.91 , 95% ci = 0.44 - 1.85 , p = 0.12 ) the results implied that the publication bias was low in the present meta - analysis . funnel plot of angiotensinogen gene t174 m polymorphism and susceptibility of ischemic stroke ( mm vs. tt ) with our search criterion , 25 individual records were found , and 12 13 full - text publications were preliminarily identified for further detailed evaluation . according to the exclusion criteria , six publications were excluded , including one duplicate study , one meta - analysis , and four without sufficient data for extraction . finally , as shown in figure 1 , six studies with 1290 cases and 1125 healthy controls were included into this meta - analysis . the ethnicity of eligible subjects involved populations ranging from african ( n = 1 ) and asian ( n = 5 ) . only one of the six studies was deviated from hwe . in addition , the controls in two of these eligible studies were based on common populations , while the others were based on hospital populations . furthermore , all of the genotyping methods applied in the including studies are pcr - based . the flow chart of the included studies in the meta - analysis distribution of agt t174 m polymorphism genotype between cases and controls and genotyping methods four models suggested remarkable between - study heterogeneity , thus both fixed effects model and random effects model were performed to calculate the pooled estimates . overall , no significant association between agt t174 m polymorphism and is was observed under all genetic models . ( mm vs. tt : or = 1.64 , 95% ci = 0.51 - 5.28 , p = 0.01 ; mt vs. tt : or = 0.93 , 95% ci = 0.66 - 1.31 , p = 0.07 ; dominant model : or = 1.08 , 95% ci = 0.69 - 1.72 , p = 0.00 ; recessive model : or = 0.61 , 95% ci = 0.20 - 1.91 , p = 0.02 ) . in the subgroup analysis based on ethnicity , results of subgroup analysis confirmed that there was significant associations between agt t174 m polymorphism and is risk in asian populations ( figure 2 , mm vs. tt : or = 3.28 , 95% ci = 1.79 - 6.02 , p = 0.32 ; mt vs. tt : or = 0.96 , 95% ci = 0.61 - 1.53 , p = 0.04 ; dominant model : or = 1.17 , 95% ci = 0.65 - 2.09 , p = 0.00 ; recessive model : or = 0.31 , 95% ci = 0.17 - 0.57 , p = 0.35 ) . summary of different comparative results forest plots for the association of angiotensinogen t174 m polymorphism with risk of ischemic stroke in asians . for each study , the estimates of odds ratio ( or ) and its 95% confidence interval ( ci ) were plotted with a box and a horizontal line . in the subgroup analysis based on hwe , we detected no significant association between agt t174 m polymorphism and is risk ( mm vs. tt : or = 1.08 , 95% ci = 0.53 - 2.21 , p = 0.11 ; mt vs. tt : or = 0.86 , 95% ci = 0.68 - 1.09 , p = 0.09 ; dominant model : or = 0.90 , 95% ci = 0.62 - 1.32 , p = 0.05 ; recessive model : or = 0.91 , 95% ci = 0.44 - 1.85 , p = 0.12 ) . no material alteration was detected , indicating that our results were statistically robust . the results implied that the publication bias was low in the present meta - analysis . funnel plot of angiotensinogen gene t174 m polymorphism and susceptibility of ischemic stroke ( mm vs. tt ) accumulated evidence indicate incontestably that is is determined by a complex interaction of environmental and genetic factors . agt is the component of ras that is released from the liver and is cleaved by rennin , which has been shown to play a vital role in affecting the cardiovascular system . recently , a variety of studies have focused on the association between the agt t174 m polymorphism and is . however , the observed associations of these studies were inconclusive . the most likely reason for the inconsistencies among these studies is that they are single case - control studies with small sample sizes . the aim of meta - analysis is to combine the same kind of studies to increase the sample size and statistical power , and thereby get a more authentic result . to our knowledge , this is the first meta - analysis examining the association between t174 m polymorphism and is risk . and a total of six studies with 1290 cases and 1125 controls were included to systematically explore the association between the agt t174 m polymorphism and the risk of is in this meta - analysis . from the combined statistical results , the meta - analysis did not show any significant association between t174 m polymorphism and is risk in the overall populations . because of the difference in genetic backgrounds and the environment in which they lived , we perform an ethnicity - specific subgroup analysis , and we found a significant association between agt t174 m polymorphism and is risk in asians . for only one study was performed in africans and no study aiming at caucasians , these results still need further investigation in africans and caucasians . moreover , if the distribution of genotypes in the control groups were not in hwe , the results of genetic association studies might be spurious . when limiting the analysis to the studies within hwe , no significant relationship was detected , suggesting that this factor probably had little effect in the present meta - analysis . the mechanism of the agt gene t174 m relates to is risk is still unclear . the serum agt level was shown to be higher in subjects carrying the t allele . and agt interacts with renin to produce angiotensin ii , which activates vascular cell apoptosis , contributing to vascular remodeling , and cardiomyocyte loss in ischemia - reperfusion . in addition , the m235 t polymorphism was in strong linkage disequilibrium with the t174 m polymorphism . a recent meta - analysis showed that the agt m235 t polymorphism was a risk factor for is among asians , the linkage disequilibrium of m235 t and t174 m in exon 2 of the agt gene may synergistically increase the risk of is . first , heterogeneity was observed in some models , so all cases and controls should be matched for age , gender , ethnicity , and type of is , but these issues could not be analyzed precisely because of inadequate clinical information for individual person . second , the number of published studies was not sufficiently large for a comprehensive analysis , and some included studies of small size might not have had enough statistical power to explore the real association between the t174 m polymorphism and susceptibility to is . finally , the inclusion or exclusion of confounding variables ( hypertension , hyperlipidemia , coronary artery disease , diabetes ) in the original studies is inconsistent . this meta - analysis suggests that the agt gene t174 m polymorphism might be associated with is risk in asians . large - scale case - control and population - based association studies are warranted to validate the risk identified in the current meta - analysis and investigate the potential gene - gene and gene - environment interactions on is risk . zlo contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . hbch contributed in the conception of the work , approval of the final version of the manuscript , and agreed for all aspects of the work . gl contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . cl contributed in revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . syl contributed in revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . jwl contributed in the conception and design of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

background : numerous studies have evaluated the association between the angiotensinogen ( agt ) t174 m polymorphism and ischemic stroke(is ) risk . however , the specific association is still controversial.materials and methods : in order to explore this association more deeply , we performed a meta - analysis . all of the relevant studies were identified from pubmed , embase , and chinese national knowledge infrastructure database up to october 2014 . statistical analyses were conducted with stata 12.0 software . odds ratio ( or ) with 95% confidence interval ( ci ) values were applied to evaluate the strength of the association.results:six studies with 1290 cases and 1125 controls were included . no significant variation in is risk was detected in any of the genetic models in the overall ( mm vs. tt : or = 1.64 , 95% ci = 0.51 - 5.28 ; mt vs. tt : or = 0.93 , 95% ci = 0.66 - 1.31 ; dominant model : or = 1.08 , 95% ci = 0.69 - 1.72 ; recessive model : or = 0.61,95% ci = 0.20 - 1.91 ) . taking into account the effect of ethnicity , further stratified analyses were performed . the results showed that agt gene t174 m polymorphism might be associated with is risk in asians ( mm vs. tt : or = 3.28 , 95% ci = 1.79 - 6.02 ; recessive model : or = 0.31 , 95% ci = 0.17 - 0.57).conclusion : in conclusion , the agt t174 m polymorphism may be a susceptible predictor of the risk of is in asians . further , large and well - designed studies are needed to confirm this conclusion .

dental caries constitutes a multifactorial disease with a complex origin where the acidogenicity of dental plaque as a consequence may affect dental hard tissues [ 13 ] . a reduction in plaque ph occurs following the release of organic acids , primarily lactate , acetate , and propionate , by oral microorganisms as fermentation products . over the years , different approaches have been designed intended to prevent this disease from occurring . apart from strengthening the tooth mineral using fluoride , pronounced changes in environmental factors such as diet , oral hygiene measures , and the use of antimicrobials have been suggested in order to induce ecological shifts in biofilm composition [ 3 , 4 ] . the latter are designed to inhibit the fermentation activity of cariogenic microorganisms , particularly those harboured in the oral biofilm , which will in turn determine a shift from a diseased to a healthy state . several possible mechanisms by agents of this kind have been suggested . they include the prevention of bacterial adhesion , a reduction in plaque formation , and interference with the bacterial metabolism . the most commonly used antimicrobial agent is chlorhexidine , a bisbiguanid , with known strong antimicrobial activity , but xylitol , fluoride , and essential oils are also known to possess similar , albeit weaker , activity [ 7 , 8 ] . chemical components able to produce one or more of the actions on different kinds of biological activity have also been shown to be present in a number of foods [ 9 , 10 ] . the presence of compounds with antibacterial activity on different pathogens , as well as antiadhesive activity and inhibitory activity on matrix formation , has been demonstrated by different food products . when it comes to different food products and their constituents , interest has recently focused on an edible mushroom , shiitake ( lentinus edodes ) . the extract from l. edodes has been studied in rats and an inhibitory effect on one of the virulence factors of streptococcus mutans has been demonstrated . there are few reports related to the general antimicrobial effects of different compounds obtained from shiitake . an aqueous extract from l. edodes displayed high antimicrobial activity on food - borne pathogenic bacterial strains . a diet containing 5% of dried l. edodes has been found to reduce the viable counts of the total number of microorganisms , streptococci , escherichia coli , and lactic acid bacteria in the intestinal flora of piglets . in a series of in vitro studies , a number of biological activities relevant to caries prevention have been identified , the most prominent of which are the induction of the detachment of cariogenic microorganisms from hydroxyapatite , changes in cell surface hydrophobicity , bactericidal activity against cariogenic microorganisms , the prevention of the coaggregation of microorganisms , and the disruption of signal transduction in streptococcus mutans [ 15 , 16 ] . the hypothesis was that frequent mouth rinses with low - molecular - weight fraction of edible mushroom shiitake extract may reduce plaque metabolic activity , change plaque cariogenic microflora towards a healthier oral flora , and reduce plaque amount . thus , the aim of the present study was to conduct a short - term clinical trial to determine the in vivo potential of rinsing with a low molecular weight extract ( < 5.000 da ) of shiitake ( lentinula edodes ) on the acidogenicity of dental plaque , microbial composition , and plaque index score . this study was carried out as a double - blind , randomized , placebo - controlled , three - leg , cross - over clinical trial . one at the department of cariology , university of gothenburg ( got ) and one at the department of preventive dentistry in collaboration with the department of periodontology , academic centre for dentistry amsterdam ( acta ) . despite being very similar , the two substudies were not identical in study design and the data that were obtained and , for this reason , the protocol can not be regarded as a multicentre approach . the study was performed within an international eu sixth framework programme consortium project ( nutrident , food - ct-2006 - 036210 ) , which was granted in order to identify beverage / food constituents that are able to reduce the risk of dental caries and gingivitis . this study focused on the opportunity to prevent dental caries and inhibit dental plaque formation . the two series were approved by the ethical committee at the university of gothenburg ( dnr 10209 ) and by the institutional review board at acta ( metc vumc , protocol number bl21480.029 - 08 ) , respectively . all volunteers made eight visits to the laboratory ( got ) respective clinic ( acta ) , for a first visit , when a clinical examination was carried out and information about the study was given , and for a total of seven subsequent test visits . in all , there were four washout periods . the study started with a two ( got ) and a three - week ( acta ) preexperimental washout period followed by three two - week periods with daily mouth rinsing with the assigned product intermitted with two - week washout periods . the total duration of the study was , therefore , 14 ( got ) or 15 ( acta ) weeks . at the screening ( got ) or at the first visit ( acta ) , a medical questionnaire was completed and the oral health status of the participants professional oral hygiene for got was performed at the start of each test period as well as directly after each test period ( prior to washout ) and for acta before the start of the first washout ( before the baseline samples ) . at each of the following visits , the subjects underwent the following data collection in the order mentioned : ( 1 ) collection of resting and fermented plaque for protein / acid analyses , ( 2 ) plaque acidogenicity ( only got ) , ( 3 ) collection of plaque for microbiological analyses , and ( 4 ) assessment of plaque score . at the end of each two - week test period , the subjects were asked to fill in a questionnaire with questions related to the usage and experience of the product used . the investigators involved in plaque sampling and ph measurements were blinded with respect to the treatment allocation of the subjects . the research population at got was made up of students and staff at the institute of odontology , as well as individuals in the nearby vicinity recruited via advertisements on bulletin boards . , recruitment was performed using the existing database ( approximately 600 entries being not - dental students ) at the department of periodontology . the inclusion criteria were healthy adults , possessing at least three premolars / molars in each quadrant , who were able to reduce their plaque ph by at least one ph unit after a mouth rinse with 10% sucrose solution for 1 min ( only got ) , no metal fillings in the premolar / molar region ( only got ) , and a stimulated saliva secretion rate of > 0.7 ml / min ( only got ) . the exclusion criteria were subjects with untreated caries or periodontal disease , wearing partial dentures , wearing orthodontic bands , and the use of antibiotics less than three months prior to the start of the study . all the subjects were given verbal and written information about the study and signed an informed consent form prior to the start of the study . sample size calculations were made by using the results of plaque acidogenicity relating to the effect of meridol mouthwash ( amf - snf2 ) on the amount of lactate in sucrose - fermenting plaque where an effect size of 0.759 had been found . since no data were available on the effect of shiitake extract rinse on lactate production , a more conservative effect ( 75% ) an a priori two - tailed analysis of the required sample size with an alpha - error probability of 0.05 , a power of 0.8 , and an effect size of 0.569 was performed . in got , 30 subjects who fulfilled the inclusion criteria were enrolled , while at acta 35 subjects were enrolled compensating for potential dropouts in order to complete the study with at least 30 individuals . the subjects were randomised using a computer - generated allocation schedule , and the subjects were not informed of their allocation . apart from the specific instructions given to participants in got / acta for each test period , the volunteers were asked to refrain from any oral hygiene procedures during the last 72 hours ( got ) and 48 hours ( acta ) , respectively , prior to each visit , as well as eating / drinking during the last two hours prior to the test . a toothpaste containing 1450 ppm f as naf was distributed to all subjects to be used twice daily throughout the entire study : pepsodent super fluor , unilever sverige ab , stockholm , sweden ( got ) and prodent , sara lee , the netherlands ( acta ) , respectively . the following three products were tested : ( 1 ) shiitake ( low - molecular - weight fraction of shiitake mushroom ( lentinula edodes ) extract ) , ( 2 ) placebo ( negative vehicle control without active ingredients ) , and ( 3 ) meridol ( amf - snf2 , positive control ) . the active product was produced and shipped by the subcontractor micropharm ltd ( uk ) in 20 ml aliquots . the product was prepared at micropharm ltd according to the gmp guidelines at the company . in addition , the placebo formulation ( negative control ) was distributed by micropharm ltd in identical vials containing 20 ml aliquots . the positive control ( meridol , gaba international ab , mnchenstein , switzerland ) contained 125 ppm amf + 125 ppm snf2 . prior to the start of the study , the solution was aseptically distributed in 20 ml aliquots into empty vials identical to those used for the active and placebo solutions . at the start of each test period , the subjects received a total of 30 vials ( 28 vials + 2 extra ) to be used during the 14-day test period . the volunteers were asked to rinse with the assigned solution twice daily . on each rinsing occasion , they were instructed to rinse vigorously with 10 ml ( 1/2 of the volume of the vial ) for 30 sec , after which they expectorated the solution . a second identical rinsing procedure with the remaining 10 ml was repeated directly after the first one . the total daily exposure was , therefore , 40 ml for 120 sec . no food or drink intake was allowed for at least one hour after the rinse . to standardise the sampling procedure after two weeks ' use of the mouthwash , all the volunteers were asked to rinse exactly three hours before the visit on day 14 . no food or drink intake was allowed for at least one hour after the rinse . in got , changes in plaque acidogenicity were measured before and after a mouth rinse with 10% sucrose using the microtouch method . an iridium microelectrode ( beetrode meph-1 , wpi instruments , new haven , conn , usa ) was inserted into the plaque in an interproximal area in the left and right upper premolar / molar region . the electrode was connected to an orion sa720 ph / ise meter ( orion research , boston , mass , usa ) to which a reference electrode was also connected . the reference electrode was placed in a solution of 3 m kcl into which a finger of the volunteer was also inserted in order to create a salt bridge . prior to and during each test session , the electrode was calibrated against a standard buffer at ph 7 . after baseline registration ( 0 min ) , the subjects rinsed with the sucrose solution for 1 min , after which ph was measured at seven different time points up to 45 min . two plaque samples were collected for the protein and acid anion profile , before ( resting ) and 10 min after the start of rinsing ( fermented ) . the collection of resting plaque was carried out on the buccal surface of the right upper second molar using a sterile carver ( got ) and teflon spatula ( acta ) , respectively . the volunteers then rinsed for 2 min ( acta ) or 1 min ( got ) with 10 ml of 10% sucrose ( w / v ) solution . fermented plaque , collected 10 min after the start of the sucrose rinse , was collected from the contralateral buccal surface ( left second upper molar ) . for got , the fermented plaque sample was collected at the same time point as the ph measurements . the plaque was transferred to a precooled eppendorf tube containing 50 l of milliq water . the samples were immediately spun down by centrifuging the tube for 30 sec at 16.100 g and put on ice until they were further processed within one hour . the samples from got were sent on dry ice to acta for further processing and analyses . the vials with plaque were centrifuged at 16.100 g for 15 min at 4c . the supernatants were transferred into vials with a microspin filter ( ultrafree - mc 0.22 m , millipore , bedford , mass , usa ) and centrifuged at 13.684 g for 5 min at 4c . organic acids in resting and fermenting plaque were determined as their anions by capillary electrophoresis on a beckman p / ace mdq system . sodium salts of formic , acetic , propionic , butyric , succinic , and lactic acid were used to prepare mixture standard solutions in milliq water . formic , butyric , succinic , propionic , acetic , and lactic acid were determined in duplicate samples . a stimulated saliva sample was collected by chewing on a piece of paraffin for 5 min . the samples were dispersed on a whirlimixer , diluted in 10-fold stages in a potassium phosphate buffer and plated in duplicate on msb agar ( mutans streptococci ) , ms agar ( total streptococci ) , rogosa sl agar ( lactobacilli ) , blood agar ( total viable count ) . after being incubated in its respective atmosphere , the number of mutans streptococci was identified by their characteristic colony morphology on the msb agar . at acta , all visible plaque was collected from a buccal surface of the upper first molar using a teflon spatula . plaque was put into sterile eppendorf tubes and kept on ice until stored at 80c . samples were sent on dry ice to the department of microbial diseases ( ucl eastman dental institute , university college , london , uk ) for analyses of microbiological composition . the numbers of streptococcus sanguinis , streptococcus mutans , lactobacillus casei , veillonella dispar , neisseria subflava , actinomyces naeslundii , prevotella intermedia , fusobacterium nucleatum , and total bacterial 16srdna were determined by using multiplex quantitative pcr ( qpcr ) . in brief , dna was extracted from plaque biofilms using a phenol : chloroform : isoamyl alcohol ( 25 : 24 : 1 ) bead - beating extraction method , which involves physical cell lysis , protein removal , and finally dna precipitation using polyethylene glycol . three triplex qpcr assays were then carried out using 2 l of extracted dna to enumerate eight oral taxa as well as the total number of organisms . the assays were performed using the rotor - gene 6500 ( qiagen ) instrument and sensimix probe ( bioline ) qpcr mix according to the manufacturers instructions , using previously published oligonucleotide sequences . the plaque score was in got calculated using the turesky modification of the quigley - hein index ( tqhpi - index ) . the toothsurface coverage with plaque was for each tooth scored on six surfaces ( mesiobuccal , buccal , distobuccal , distolingual , lingual , and mesiolingual ) on a scale of 05 . at acta all buccal and lingual areas in the lower jaw were assessed for each tooth at six sites ( mesiobuccal , buccal , distobuccal , distolingual , lingual , and mesiolingual ) on a scale of 03 . at the end of each test period , the volunteers were requested to complete a questionnaire with a visual analogue scale ( vas ) with a total of nine questions related to their experience of using the assigned mouth rinse solution . they marked their answer on a 100 mm line with the negative extreme on the left and the positive extreme on the right . ph , the mean of the values for the left and right side was collected . from each ph curve , the area under the curve ( auc5.7 and auc6.2 ) , minimum - ph , and maximum - ph decrease was calculated . for the plaque score , the mean score for each tooth was first calculated , after which the mean score for the whole dentition was calculated . protein content was expressed in g and the amount of organic acids as mol / mg protein . for acta , the total number of the different microorganisms was calculated . for got , the distribution of mutans streptococci and total streptococci in comparison to the total streptococcal flora and total oral flora ( % ) , respectively , was also calculated . for acta , the log10 cfu was calculated . for the answers on the vas , the distance ( in mm ) from the left side was measured for each question and a mean score was calculated . in got , two - way analysis of variance , anova , was used to test the significance of differences between the seven test occasions ( after each test period and the washout periods ) . when anova rejected the multisample hypothesis of equal means , multiple comparison testing was performed using fisher 's plsd . p < 0.05 was regarded as statistically significant . at acta , a paired t - test was used to compare the amounts of different organic acids in resting and fermented plaque from the same visit . the general linear model repeated measures test and the bonferroni post - hoc test were used to compare the output parameters ( amount of each acid , relative abundance of oral microorganisms , protein amount ) after each of the three treatment periods and separately from the test periods , that is , between pre - experimental baseline and each consecutive washout period . the difference between the mean plaque score at the start of each test period and upon completion of each test period was calculated and used as an input variable in glm - rm test . all 30 and 35 individuals , respectively , completed the study , apart from the final washout period for one subject in got . the mean age of the volunteers was 31 13 years ( mean sd ) at got , including 19 females/11 males , and 23 3 years ( mean sd ) with 32 females/3 males at acta . the most pronounced metabolic activity for the sucrose rinse at the end of the three test periods was found after rinsing with the placebo , and the least attenuated ph fall was found for the positive control ( amf - snf2 ) , while the active compound ( shiitake ) resulted in an intermediate position ( figure 1 ) . a statistically significant difference when comparing the ph values at the different time points was found at 2 min between shiitake and placebo ( p < 0.05 ) . in the case of minimum ph , there was also a numerical difference between the three products , with a difference of 0.2 ph units between shiitake and placebo and the positive control , respectively ( ns ) . minor differences in plaque acidogenicity were found when evaluating the maximum ph decrease , as well as auc5.7 and auc6.2 . only minor numerical differences in plaque acidogenicity were found when comparing the results for the four washout periods ( baseline and posttreatment ; ns ) . there was no difference between shiitake and placebo in plaque protein mass , while the positive control ( amf - snf2 ) resulted in significantly less plaque protein than the test or placebo rinse ( p < 0.001 ) . no significant differences in plaque protein were found between the four washout periods . in the case of acta , there were no differences in the amount of protein from resting versus fermented plaque , except for the positive control washout period , where less protein was found in the resting plaque ( p < 0.01 ) . for got , there was a tendency towards a generally somewhat lower protein content for the resting plaque ( ns ) . the protein plaque content varied for the seven test periods and two conditions ( resting and fermented ) for got between 14.7 0.1 and 40.3 31.4 g and for acta between 30.3 23.5 and 81.2 65.3 g . significantly less protein was found for the positive control compared with the other two test products ( p < 0.001 ) ( acta ) . the profiles of acetate , lactate , and minor acids ( propionate , formate , succinate , and butyrate ) for the resting and fermented plaque gave similar values for got and acta ( figure 2 ) . the highest values for all the test sessions were found for lactate for fermented plaque , with a larger variation for got compared with acta . the rinse period of the positive control ( amf - snf2 ) resulted in significantly less lactate and acetate for fermented plaque compared with shiitake and placebo for got ( p < 0.01 ) . the corresponding data for acta showed that the positive control period resulted in significantly less lactate and minor acids in fermented plaque compared with shiitake and placebo ( p < 0.001 ) . a higher amount of minor acids was found in resting plaque after the shiitake rinse compared with the positive control for acta ( p < 0.01 ) , while no such effect was seen for got . all the microbial data are presented in table 1 . for salivary microorganisms in got , no statistically significant differences were found for lactobacilli or mutans streptococci in saliva between the three test periods . rinsing with the positive control ( amf - snf2 ) resulted in a significantly lower number of oral streptococci ( p < 0.05 ) and total number of microorganisms ( p < 0.001 ) when compared with the shiitake rinse . the lowest proportion of mutans streptococci in comparison to the total number of streptococci was found for shiitake ( ns ) . no significant differences were found for any of the groups of oral microorganisms or the proportion of bacteria when comparing baseline and posttreatment washout periods . for plaque samples from acta analysed by pcr assays , neisseria subflava was the most predominant microorganism of all the tested organisms , followed by veillonella dispar and fusobacterium nucleatum . there were significantly fewer microbial cells and individual organisms in the panel , apart from s. mutans counts in plaque samples collected after positive control than after shiitake or placebo periods ( p < 0.001 ) . the shiitake mouth rinse reduced the proportion of n. subflava significantly compared with the placebo rinse ( p < 0.05 ) , while n. subflava and s. sanguinis were significantly reduced by the positive control compared with shiitake ( p < 0.01 ) . for got , the three test periods resulted in the numerically lowest plaque scores ( ns ) , while the positive control resulted in significantly less plaque than shiitake and placebo ( p < significantly less plaque was found for got after the shiitake washout period compared with placebo ( p < 0.05 ) . for acta , significantly less plaque was found at the overall preexperimental baseline compared with the other three washout periods ( p < 0.001 ) . taste experience was , when marked from very poor to very good , described as significantly worse by the volunteers after the shiitake test period ( got 47.0 32.7 , acta 3.0 4.6 ; mean sd ) compared to both the placebo ( got 62.4 22.4 , acta 59.2 21.5 ) and positive control ( got 70.6 18.9 , acta 54.9 25.6 ) ; rinses for both got and acta ( p < 0.01 or p < 0.001 ) . similar significant differences were found for duration of the taste , taste perception , and experienced rinsing time ( data not shown ) . a significantly higher perception of sensitivity of the teeth after shiitake ( 36.2 27.2 ) compared to the positive control ( 19.6 27.7 ) and higher perception of staining after shiitake ( 24.8 27.2 ) in comparison to the placebo ( 12.1 18.8 ) rinse ( p < 0.05 ) was reported for acta . the burning sensation of the mouth was also significantly higher after the shiitake ( got 27.4 28.6 , acta , 43.2 30.8 ) and placebo ( got 29.1 31.5 , acta 38.2 29.1 ) rinses compared to the positive control ( got 12.6 18.8 , acta 15.0 23.4 ) for both test series ( got p < 0.01 , acta p < 0.002 ) . the scientific approach and study design of this paper are based on the results of previous studies performed within the nutrident project . the study was planned as a consequence of the initial chemical characterization of the shiitake mushroom , evaluation of the fractions and subfractions of the shiitake mushroom , and further evaluation in different in vitro settings [ 16 , 25 ] . due to different technical limitations at each of the two centres , however , the fact that the conclusions are based on the results from 30 and 35 volunteers from two international centres strengthens its scientific value . however , the additional inclusion criteria in got , where the subjects are known to have reduced their plaque ph by at least one ph unit after a sugar rinse , indicates that these subjects may have higher caries activity . the possibility that the numerical variation seen between the two substudies may be a consequence of the selection of subjects can not , therefore , be ruled out . due to the above - mentioned factors , the main finding in this study is that rinsing twice daily with a natural food extract may reduce the metabolic activity of the dental biofilm . although not evaluated in the present study , a reduction of this kind may result in the long term in a lower degree of demineralisation . this is supported by recent data where a subfraction of shiitake showed a strong inhibiting effect on dentine demineralisation when evaluated in an environment using saliva - derived microcosms . there may be multiple explanations for the present findings of a change in the acidogenic potential of the biofilm . previous work focusing on shiitake mushroom extract has demonstrated biological activity relevant to caries prevention [ 16 , 25 ] . this includes mechanisms such as bactericidal activity against cariogenic microorganisms , the prevention of the coaggregation of cariogenic microorganisms , the induction of the detachment of cariogenic microorganisms from hydroxyapaite , and changes in cell surface hydrophobicity . the antimicrobial , antiadhesive , and antiplaque properties of polyphenol - rich beverages have previously been demonstrated [ 26 , 27 ] . recent studies have focused on the oral health variables of tea in particular , both when consumed naturally or when evaluating tea and cranberry in an in vitro or in vivo design [ 2831 ] . similar findings relating to the plaque - lowering potential have been found both after using both different sweeteners and essential oils . the interpretation of the acid anion profiles of the resting and fermented plaque is complicated . although a corresponding pattern when comparing the data with the results from the plaque - ph measurements would have seemed logical , it was difficult to obtain a clear and consistent picture from the current data . activity of the food compound that was tested , a poor cooperation of the subjects , or a weak experimental design . when evaluating the same low - molecular - weight fraction of the shiitake mushroom in an in vitro caries model , a stronger inhibitory effect on acid production potential was observed by one of the subfractions ( sf4 ) in comparison to the whole low - molecular - weight fraction . microbiological analyses included both the total cell count and bacteria related to periodontal diseases , dental caries , and oral health . only minor differences in both the salivary levels ( got ) and the plaque levels ( acta ) of oral microorganisms were found between the different visits . the numerically lowest salivary number of mutans streptococci in comparison to the total number of streptococci was found for shiitake . for plaque microflora , significantly reduced proportions of microorganisms were only found for the gram - negative organism n. subflava when comparing the shiitake mouth rinse with placebo . these findings are supported by a recent study in which 11 days of frequent mouth rinses with the same mushroom extract resulted in a reduced amount of plaque but a weaker effect on the decrease in total bacterial counts as well as some specific oral pathogens when compared with a placebo test period . while got found a significant reduction in plaque score when comparing shiitake with placebo , no such difference was found for acta . however , a reduction in dental plaque deposition has also been found when evaluating the active compound against gingivitis- and periodontitis - related variables . this finding is furthermore supported by previous studies in which inhibited plaque formation was found when using mouth rinses of oolong tea and pomegranate . neither the mushroom extract nor the placebo was capable of reducing plaque formation to the same degree as chlorhexidine , an antimicrobial compound known to inhibit biofilm development and maturation . the subjects reported a less favourable outcome for the different questions related to taste for the shiitake extract mouth rinse . however , following the reported negative reaction to the taste of the shiitake mouth rinse by a large number of the volunteers , one can not exclude that this may have had a negative impact on compliance . as a consequence , some of the subjects may not have rinsed with the active compound according to instructions and that they may have rinsed their mouth with water shortly after using the active substance can not be excluded . in order to secure the regular use of potential future products , it is important that this aspect is also considered seriously , as this factor alone may determine whether or not an oral health product is used . functional foods have not been introduced in order to replace traditional caries - prevention strategies but instead to add another tool to offer patients at higher risk . the positive finding of reduced plaque fermentation activity indicates that there is an opportunity to add one more strategy to the palette of preventive methods . it is not surprising that a stronger effect was found for this variable and that only a limited effect was found for several of the other variables . the metabolic activity of the dental biofilm is the end result of a large number of biological and biochemical caries - related factors . as shown by previous laboratory work [ 16 , 25 ] , the active compounds of shiitake mushroom may exert multiple actions on different caries - related variables . even if the effect of each of them may appear weak , they may interfere in a positive way in the complex and diverse microbial community constituted by the biofilm . the limited effect on several dental biofilm properties seen in the present study may indicate that frequent exposure for a longer period is needed . one important factor is believed to be the contact time between the active compound and the different oral properties . the repeated rinsing with 10 + 10 ml for 30 + 30 sec was used to diminish the dilution effect of saliva and to prolong the contact time of active compounds with the oral cavity . for this reason , both further laboratory and clinical studies are needed in order to evaluate not least the effect of a longer exposure period or variations in the concentration of these naturally derived biologically active compounds . the main finding of this study is that frequent mouth rinses with a natural food extract ( shiitake mushroom ) may reduce the metabolic activity of the dental biofilm . only a limited effect on other dental plaque properties related to the caries disease was found and not to the same extent as the positive control .

the main objective was to investigate whether low - molecular - weight fraction of edible mushroom shiitake extract ( lentinus edodes ) possesses caries - preventive properties . the study was designed as a double - blind , three - leg , cross - over , randomized , controlled clinical trial carried out on two series of volunteers at the university of gothenburg , and the academic centre for dentistry amsterdam . volunteers rinsed twice daily with a solution containing low - molecular - weight fraction of edible mushroom , placebo ( negative control without active ingredients ) , or meridol ( positive control , amf - snf2 ) for two weeks , with a two - week washout period between each rinsing period . changes in the acidogenicity of dental plaque before and after a sucrose challenge , shifts in microbial composition , and plaque scores were determined . frequent rinses with shiitake reduced the metabolic activity of dental plaque . no reduction of plaque scores and no inhibition of the production of organic acids in plaque was found . minor differences in microbial composition between test sessions were found . to conclude , the results indicate that shiitake extract has anticariogenic potential , but not to the same extent as the positive control .

ocular trauma , although relatively rare , is a proven etiology of focal corneal stromal edema . not many cases have been reported since it was described in 1978 by cibis et al.1 ocular manifestations include lesions of transient , gray - white , endothelial ring - shaped opacities with focal corneal stromal edema and most cases show remission within a few days with conservative management.2 here we report a case of traumatic corneal stromal edema discovered by anterior segment optical coherence tomography ( as - oct ) . a 31-year - old man presented to our clinic for an ocular trauma to his left eye . the patient had a blast injury which occurred on the previous day by a plastic bottle cap when trying to open a beverage bottle . his best - corrected visual acuity ( bcva ) was 20/200 os ( left eye ) . anterior segment examination showed an epithelial defect , which implies a direct injury to the cornea . a marked focal stromal edema accompanied by descemet s membrane folds were located at 10 oclock in the paraxial area , about 4 mm apart from the corneal center ( figure 1a ) . grade 1 gross hyphema was observed . as - oct ( as - oct ; carl zeiss meditec ag , jena , germany ) revealed marked corneal swelling of the lesion ( figure 1b ) . the stromal edema resolved , showing no signs of epithelial defect , although small opacity remained ( figure 1d ) . as - oct revealed complete resolution of corneal swelling ( figure 1e ) . despite the resolution of corneal edema , however , specular microscopy ( sp-9000 ; konan medical , tokyo , japan ) showed substantially decreased endothelial cell density in the lesion compared to the fellow eye ( 2,062 versus 2,959 mm ) ( figure 1f ) . one month later , the patient s 2959 cells / mm bcva improved to 20/25 os . there were no signs of corneal edema and no further decrease in endothelial cell density . a diffuse corneal edema and its recovery is often observed in patients with eye injury caused by ocular trauma . the main etiologies of transient diffuse stromal edema include post - traumatic inflammation inside the anterior chamber and persistently increased intraocular pressure . however , a local corneal edema accompanied by substantially decreased endothelial cell is a rare and notable case . this case differs from traumatic corneal endothelial rings in that there is no distinct endothelial ring lesion on slit lamp examination . furthermore , despite the difference in corneal morphology , this case shares a similarity with the traumatic corneal endothelial rings : the corneal lesion was caused by traumatic endothelial cell damage in this case . to our knowledge , only one case of traumatic corneal endothelial rings with imaging of as - oct has been reported,3 in which the as - oct revealed a ruffled disruption to the endothelial cells directly posterior to the stromal thickening . endothelial cells around the traumatic lesion undergo the greatest torsion and energy absorption as the cornea moves axially posterior and then relaxes to its original position.4 this process could have induced the damage and inflammation of the corneal endothelial cells . another possible mechanism that caused the injury would be a direct contact between the corneal endothelium and a lens or iris . the proper functioning of the corneal endothelium is regarded to be crucial to the maintenance of corneal clarity . once damaged , the endothelium is not able to recover from the injury . therefore , significant endothelial damage may lead to severe complications such as persistent corneal edema and bullous keratopathy at a later stage . in conclusion , permanent corneal endothelial cell loss could occurs by traumatic mechanism , which manifests as corneal stromal edema in the early phase of trauma . corneal endothelial layer evaluation using specular microscopy and as - oct can be useful for diagnosing and monitoring the damage to cornea .

a 31-year - old man presented to our clinic for an ocular trauma to his left eye . his best - corrected visual acuity was 20/200 os ( left eye ) . anterior segment examination showed an epithelial defect , which implies a direct injury to the cornea . a marked focal stromal edema accompanied by descemet s membrane folds were located at 10 oclock in the paraxial area , about 4 mm apart from the corneal center . anterior segment optical coherence tomography ( as - oct ) revealed marked corneal swelling of the lesion . five days later , the patient s best - corrected visual acuity improved to 20/50 os . the stromal edema resolved showing no signs of epithelial defect , although small opacity remained . as - oct revealed complete resolution of corneal swelling . despite the resolution of corneal edema , however , specular microscopy showed substantially decreased endothelial cell density in the lesion compared to the fellow eye . here we report a case of traumatic corneal stromal edema discovered by as - oct .

the interaction of stem cell factor with kit , and its receptor , is critical for the survival , proliferation , differentiation and migration of melanocytes ( 1 ) . however , the regulation of kit pathway is complex and depends on other multiple cellular factors . kit is a trans - membrane receptor tyrosine kinase encoded by the proto - oncogene kit at 4q11 - 12 ( 1 ) . kit activation mutations are associated with a variety of malignant human tumors as well as malignant melanoma ( 1,2 ) . it was speculated that melanoma cells should lose kit expression to acquire proliferative activity and escape from the epidermal boundaries , ( 2 ) . this hypothesis was supported by previous observations in which kit expression in melanoma was strong in the in situ and junctional components of invasive lesions , while kit expression was lost once the melanoma became invasive and metastatic ( 3,4 ) . it was observed that more than a third of the melanomas without detectable kit mutation , or increased copy number showed overexpression of kit by ihc . this observation led the authors to consider other mechanisms than gene mutation or amplification to explain the kit overexpression ( 5 ) . in literature the role of ihc in the assessment of kit in cutaneous and mucosal melanomas and their metastases , and its relation to the mutational status of the kit gene is not well established , and a few articles are found on this subject . according to the previous studies the kit mutations prevalence in acrallentiginous/ mucosal melanomas is relatively low ( not more than 1520% ) but they can have profound therapeutic implications for localized high risk or metastatic disease ( 6 ) . in this study , kit protein expression by ihc in a large series of melanomas with emphasis on cutaneous and mucosal melanomas and their metastasis was evaluated . we designed a cross - sectional study and selected 50 cases of malignant melanoma including primary cutaneous , mucosal and their metastasis that were referred to affiliated hospitals of shiraz university of medical sciences ( 20082012 ) . according to multistage sampling method , we targeted a confidence interval of 95% ( z=1.96 ) , range of variation of 0 to 100 ( s= 16.67 ) , and critical difference of 4.5% ( d=4.5 ) . the analyzed melanomas , totally 50 cases , consisted of the 10 primary acrallentiginous , 10 primary mucosal , 14 primary nodular , 14 metastatic , and two primary uveal melanomas . h&e ( hematoxylin and eosin stain ) slides and paraffin tissue blocks were retrieved from the archives . immunohistochemical analysis for kit was performed using an anti - cd117 polyclonal rabbit antihuman antibody ( dilution 1:1000 , dakocytomation , carpinteria , ca , usa ) on tissue sections . negative controls were prepared by substituting the primary antibody with non - immune rabbit serum . percentage of positive cells was recorded as following : 0 ( negative ) , < 5% of cells staining , 550% of cells staining , 5195% of cells staining , and > 95% of cells staining . intensity was scored as 0 ( negative ) , 1 ( weak ) , 2 ( moderate ) , and 3 ( strong ) ( 7 ) . the ihc data were collected along clinical and morphological findings including age , sex , site , breslow thickness , stage and other specifications . a total of 50 cases of cutaneous and mucosal ( primary and metastatic ) melanoma including 31 ( 62% ) male and 19 ( 38% ) female with ratio of 1.77 were enrolled in the study . the age range was wide ( 1 - 87 ) with meansd age of 5317.5 years . for the mucosal malignant melanoma the head and neck primaries were the most frequent site , followed by anorectal area and colon mucosa . lymph nodes were the sites most frequently involved by metastatic tumors in our series ( 7 cases ) followed by skin subcutaneous ( 6 cases ) and bone marrow metastases ( 1 case ) . table 1 summarizes patientsdemographics and clinical features of the analyzed tumors . the cases were also evaluated for neurotropism 4 out of 50 ( 8% ) , tumor lymphocytic infiltration 4 out of 50 ( 8% ) , vascular invasion 5 out of 50 ( 10% ) , microsatellites 0 out of 50 , regressive changes 1 out of 50 ( 2% ) . we also investigated the chi - square test between sex and site of the lesions and stage of the tumors . results revealed that male sex correlated with higher stage ( p<0.05 ) but there was no correlation between site and stage of the tumors . overall , 19 ( 38% ) cases showed 0 to less than 5% of positive cells , 9 ( 18% ) cases 550% , 8 ( 16% ) cases 51 -95% , and 14 ( 28% ) cases showed greater than 95% of cells expressing kit ( fig . c - kit positive immunohistochemical staining in melanoma.a : 3 + positivity b : 2 + positivity , c : 1 + positivity , some melanin pigment for comparison is seen at upper outer quadrant of the lesion ( x 200 ) . a high percentage of cutaneous and mucosal melanomas , both primary and metastatic , showed at least 5% of kit - positive cells , 30 of 48 cases ( 62.5% ) . one of the two uveal melanoma cases showed more than 95% of tumor cells positive for kit , whereas another case was negative for kit staining ( fig . tumor cell positivity for kit ihc staining as well as its intensity did not correlate with type of melanoma . ( p>0.05 ) intensity of staining and percentage of positive cells were positively correlated ( p<0.001 , table 2 ) . acrallentiginous and nodular types of cutaneous melanoma and mucosal primary melanomas showed a comparable level of kit ihc expression . while cases showing at least 5% of positive cells were outstanding ( 8 out of 10 primary acrallentiginous , 8 out of 10 primary nodular melanomas , and 8 out of 10 primary mucosal melanomas ) . though cases are not quite sufficient for statistical analysis , metastatic melanomas seems to be less likely to be kit - positive than non - metastatic melanomas ( 6 out of 14 cases , 43% v / s 24 out of 36 cases , 66% , respectively ) . tumor stage was also positively correlated with tumor cell immunoreactivity and intensity ( p<0.05 ) . * p - value from chi- square test overall , 19 ( 38% ) cases showed 0 to less than 5% of positive cells , 9 ( 18% ) cases 550% , 8 ( 16% ) cases 51 -95% , and 14 ( 28% ) cases showed greater than 95% of cells expressing kit ( fig . c - kit positive immunohistochemical staining in melanoma.a : 3 + positivity b : 2 + positivity , c : 1 + positivity , some melanin pigment for comparison is seen at upper outer quadrant of the lesion ( x 200 ) . a high percentage of cutaneous and mucosal melanomas , both primary and metastatic , showed at least 5% of kit - positive cells , 30 of 48 cases ( 62.5% ) . one of the two uveal melanoma cases showed more than 95% of tumor cells positive for kit , whereas another case was negative for kit staining ( fig . tumor cell positivity for kit ihc staining as well as its intensity did not correlate with type of melanoma . ( p>0.05 ) intensity of staining and percentage of positive cells were positively correlated ( p<0.001 , table 2 ) . acrallentiginous and nodular types of cutaneous melanoma and mucosal primary melanomas showed a comparable level of kit ihc expression . while cases showing at least 5% of positive cells were outstanding ( 8 out of 10 primary acrallentiginous , 8 out of 10 primary nodular melanomas , and 8 out of 10 primary mucosal melanomas ) . though cases are not quite sufficient for statistical analysis , metastatic melanomas seems to be less likely to be kit - positive than non - metastatic melanomas ( 6 out of 14 cases , 43% v / s 24 out of 36 cases , 66% , respectively ) . tumor stage was also positively correlated with tumor cell immunoreactivity and intensity ( p<0.05 ) . * p - value from chi- square test the role of ihc in the assessment of kit status in melanomas is not well established yet . although the reported prevalence of kit mutations in acrallentiginous / mucosal melanomas is relatively low , the detection of that mutation can have profound therapeutic implications ( 7 ) . a study reported kit expression in 96% of primary melanomas , while its expression was 55% in metastatic melanomas ( 8) . another study showed a possible role of kit in some types of melanoma , such as mucosal melanomas ( 21% kit mutations , and 61% kit overexpression ) , acral cutaneous melanomas ( 11% kit mutations , and 75% c - kit overexpression ) and cutaneous melanomas on skin with chronic sun damage ( 17% kit mutations , and 100% c - kit overexpression ) . in another study kit mutation was detected in 14 out of 39 ( 35% ) ( 10 ) . in contrast , kit mutations are rarely found in the major subtype of cutaneous melanoma originating from skin without chronic sun damage ( 5 ) ; it is not common in unselected cutaneous melanomas ( 2 out of 100 ) ( 9 ) . this suggests that c - kit may have pathogenetic relevance and used as a therapeutic target in these subtypes of melanoma . cytoplasmic c - kit staining was significantly correlated with poor survival in patients with acral melanoma . there is significant difference between c - kit immunoreactivities and the mortality risks of melanomas on acral and non - acral sites . it may change site - specific targeted therapeutic concepts in melanoma in future ( 11 ) . among patients with advanced melanoma harboring kit alterations , treatment with the aim of this study was to further clarify c - kit alterations in patients with cutaneous and mucosal melanomas and their metastasis . our results are in keeping with those recent studies showing that some types of melanomahassignificant expression of kit which can be detected by ihc . we used a highly sensitive detection method employing a rabbit polyclonal antibody , widely tested in previous trials ( 7 , 9 ) . we set acutoff point of 5% for kit expression positivity.in our study , c - kit expression was detected in 66.6% of cutaneous melanomas , while other authors have reported the expression of c - kit from 22.8% ( 12 ) up to 84% ( 13 ) . in cases of mucosal melanomas we achieved a positive rate of 80% which is similar to previous studies , including primary mucosal melanomas of the anal / rectal mucosa 12 out of 16(75% ) ( 13 ) , oral cavity 16 out of 18 ( 88% ) ( 14 ) , and primary mucosal melanoma 35 out of 39 ( 90% ) ( 15 ) . however , one study reported lower number , 6 out of 26 ( 23% ) of c - kit positivity in mucosal melanomas of the anal / rectal tract ( 15 ) . the high range of differences between these studies could be explained with the different qualities of ihc and different cutoff point for tumor cell positivity ( e.g. 20% ) . c - kit expression appears to be in a similar range in mucosal melanomas and cutaneous melanomas in our study . in contrast to other categories , metastatic melanoma revealed the lowest percentage of positivity in c - kit expression which is similar to the results of metastaic cutaneous melanomas in other studies ( 4,7 ) . our results in cases of metastatic melanomas were in contrast to another study in which the expression was found in 9 of 9 metastases of primary mucosal melanoma . that study included only mucosal melanomas ( 39 patients ) of different locations ( 15 ) . other study also detected c - kit reactivity in 6 out of 6 metastases from 20 cases of anal melanomas ( 16 ) . one of the sources of these discrepancies may be due to evaluating the metastatic cutaneous and mucosal melanomas in a single group.the other reason was studying only mucosal melanomas in large groups.immunohistochemistry may be not sufficient to detect tumors with mutations susceptible for kit blockade , as overexpression can also occur in tumors without mutation ( 14 ) . moreover , therapeutic studies with the kit blocker imatinib in unselected melanoma patients withoutknown mutation status were disappointing ( 17 - 19 ) . in summary , according to our findings , primary melanomas including acrallentiginous , nodular , mucosal , and uveal melanomas shows high kit expression . therefore , ihc evaluation may be a useful tool for screening patients that are subjected to kit mutation and can be used as a patient selection method for targeted therapy . we have also encountered some limitations including melanin interference with kit expression within ihc study . we recommend further study to evaluate mutational status of the kit gene to assess the efficacy of immunohistochemistry in order to predict mutations in kit .

background : melanoma causes the greatest morbidity and mortality of all skin cancers . mucosal melanoma is a rare but highly aggressive neoplasm . according to previous studies the prevalence of kit mutations in acral lentiginous and mucosal melanomas is relatively low ( less than 1520% ) , but it can have profound therapeutic implications for localized high risk or metastatic diseases . our goal was to evaluate c - kit expression in different types of primary and metastatic melanoma to discriminate potential candidates for targeted therapy . methods : we designed a cross - sectional study and selected 50 cases of malignant melanoma ( primary , metastatic cutaneous , and mucosal ) from the affiliated hospitals of shiraz university of medical sciences in the period of 2008 to 2012 . immunohistochemistry for kit expression was performed . multistage sampling method was selected for sampling and chi - square test was used for statistical analysis . results : in our study , male to female ratio was 1.77 . the male sex was correlated with higher tumor stage ( p < 0.05 ) . 62% ( n= 31 ) of cases showed at least 5% of kit - positive cells , consist of 18% ( n= 9 ) with 550% , 16% ( n= 8) with 5195% , and 28% ( n= 14 ) of cases showed more than 95% of cells expressing kit . but in 38% ( n= 19 ) of cases kit expression was less than 5% of positive cells . tumor stage was positively correlated with tumor cell immunoreactivity and intensity ( p < 0.05 ) . metastatic melanoma showed lower percentage ( 43% ) of positivity . intensity of staining and percentage of positive cells were positively correlated ( p < 0.001 ) . conclusion : in primary melanomas , significant kit expression was found by immunohistochemistry , which may be useful to screen the patients for advising to kit mutation analysis and targeted therapy .

copd is characterized by a progressive decline in lung function and mental and physical comorbidities ( eg , depression , dystrophy , and heart failure).1 an exacerbation of copd is an acute event caused by several factors . to those patients who need ventilator support because of lung infection the clinician s concern is the optimum timing regarding the condition of the patient to wean mechanical ventilation . unfortunately , there is no consensus or guideline that can give us a distinct conclusion . the aim of this paper is to summarize the evidence - based optimum timing to wean invasive ventilation ( iv ) in patients with acute exacerbations of copd ( aecopd ) or copd with pulmonary infection . copd is currently a significant burden in the people s republic of china because of greater risk exposure and uneven medical resource allocation between urban and remote areas . a population - based , cross - sectional survey conducted between 2002 and 2004 suggested a copd prevalence of 8.2% among chinese population over 40 years old.2 other studies reported varied prevalence ranging from 5% to 13%.36 according to the 2004 global burden of disease study , an annual sum of 3 million people die of copd worldwide . in the people s republic of china alone , copd - related mortality was 27.3 in males and 21.3 in females per 100,000 heads according to a national , prospective cohort study conducted between 1990 and 2000 . acute chronic respiratory failure , heart failure , pulmonary infection , pulmonary embolism , cardiac arrhythmia , and lung cancer are the major causes of death in patients with copd according to a cross - sectional study involving ten european centers.7 aecopd are a common cause of comorbidities and copd - related mortality.811 they are characterized by an increase in the symptoms of dyspnea , sputum volume , and sputum purulence with or without symptoms of upper respiratory infection.12 they may also involve worsening of existing symptoms , which require alterations in treatment ranging from antibiotic administration , short courses of oral corticosteroids , and increased bronchodilator usage.1314 reported aecopd incidence varied between 2.5 and 3 episodes per patient and year.15 being its major cause , infection accounted for 50%70% aecopd occurrences worldwide and 80%90% aecopd occurrences in the people s republic of china alone.16,17 other predisposing factors include environmental pollution , low temperatures , and concomitant heart failure.18,19 subsequent onset of acute respiratory failure ( arf ) may result if aecopd are accompanied by bronchial infections , bronchospasm , left ventricular failure , pneumonia , pneumothorax , or thromboembolism . once arf occurs , in - patient mortality ( 4%30% ) substantially rise up to 50% among elderly patients and 11%26% among intensive care unit ( icu ) patients.10,2023 lung infection other than acute exacerbation is quite common in patients with copd . bronchoscopic studies have shown that at least 50% of patients have bacteria existence in their lower airway during exacerbations of copd.1 viruses are also the common etiology of copd exacerbation next to bacteria . many patients with copd who have comorbidities such as hypertension , diabetes , cardiovascular disease are susceptible to lung infection . only the patients who meet the criteria for hospital admission or icu admission need to be treated in a timely manner . hospital admission is warranted in an increasing number of patients with aecopd to prevent arf onset.24 an abc approach involving antibiotics , bronchodilators , and corticosteroids is generally extended to maximize lung function and to reverse the predisposing causes of exacerbations.25 mechanical ventilation is also suggested in 26%74% of patients with copd so that the respiratory muscle load may be alleviated to reduce dyspnea and respiratory rate and improve arterial oxygenation , partial pressure of carbon dioxide in arterial blood ( paco2 ) , and ph.2628 the criteria to start ventilatory support vary but commonly involve the following : 1 ) moderate to severe dyspnea where accessory muscles are recruited and abdominal breathing prevails ; 2 ) hypercapnic acidosis ( ph < 7.35 ) ; 3 ) tachypnea ( > 25 rpm).29 non - invasive ventilation ( niv ) is validated in patients with early aecopd because their tolerable coughing ability suggests a stronger need for respiratory muscle fatigue relief rather than airway clearance . it is thus initially provided for patients with severe aecopd with respiratory acidosis to reduce intubation rate , shorten icu stay , and decrease patient mortality . nonetheless , iv is indicated if niv measures fail to improve clinical manifestation and blood gas parameters 1 hour after implementation.29 any patient with ineffective airway clearance ( eg , post - surgical patients or patients with copd with hypercapnic arf and pneumonia ) needs iv support to improve sputum discharge and ventilation . table 1 summarizes the indications for both ventilation types.30 in spite of its importance , iv should be discarded whenever appropriate to avoid time - dependent complications associated with intubation , tracheotomy , or ventilation ( table 2).3133 to derive optimum advantages from mechanical ventilation , one should identify the optimum timing for withdrawal so that complications may be prevented while respiratory function is restored . niv has been suggested to serve the purpose because it was identically capable in unloading respiratory muscles.34 its application after iv also significantly reduced weaning time , alleviated ventilator - associated complications , and improved survival.3537 however , no consensus has yet been reached regarding the optimal time when niv should replace iv . according to the chinese guideline for mechanical ventilation in patients with acute exacerbation of chronic obstructive pulmonary disease 2007 , weaning with t - tube is advised in copd patients without obvious bronchial - pulmonary infection while replacement by niv at pulmonary infection control ( pic ) window is advised in copd patients with obvious bronchial - pulmonary infection . tobin summarized six stages in mechanical ventilatory support : 1 ) treatment of arf ; 2 ) suspicion that weaning may be possible ; 3 ) assessment of readiness to wean ; 4 ) spontaneous breathing trial ( sbt ) ; 5 ) extubation ; and 6 ) reintubation when required . one is only liberated from iv when he successfully passes the first five steps and avoids the last . weaning accounts for almost half of the time in mechanical ventilation and allows resumption of spontaneous breathing after gradual reduction of mechanical support.3842 randomized and non - randomized historical cohort studies verified a more significant reduction in the duration of mechanical ventilation when a weaning protocol , involving weaning parameter evaluation and subsequent breathing trials , was used instead of mere clinical judgment.4346 in view of general delays in weaning and the associated increase in mortality,41,47,48 assessment should be performed every day to allow prompt initiation of weaning . variations exist between protocols but weaning parameters essentially stem from observations in respiratory mechanics , gas exchange , and breathing patterns . table 3 is a list of parameters to be considered before weaning.47 daily screen of weaning parameters was found to have predicted a successful extubation with 82% accuracy , nearly 90% sensitivity and positive predictive values . significance of passing the screen could also be extended to hospital survival prediction during the 1st week and a half of mechanical ventilation . its prognostic significance seemed to be limited though , since up to 29% of the patients failed the screen but withstood extubation.49 thus , a successful weaning attempt does not necessarily require fulfillment of all the mentioned criteria . sbt failure is defined by 1 ) objective indices such as tachypnea , tachycardia , hypertension , hypotension , hypoxemia or acidosis , and arrhythmia ; 2 ) subjective indices such as agitation or distress , depressed mental status , diaphoresis , and evidence of increasing effort.47 subsequently , weaning failure refers to sbt failure or the need for reintubation within 48 hours of extubation.50,51 according to the number of attempts or days prior to successful weaning , patients may be classified into three groups:47 1 ) simple weaning ( patients who proceeded from initiation of weaning to extubation on the first attempt without difficulty ) ; 2 ) difficult weaning ( patients failing the first attempt who took up to three attempts or as long as 7 days from the first sbt to achieve successful weaning ) ; and 3 ) prolonged weaning ( patients who failed at least three weaning attempts or required > 7 days of weaning after the first sbt ) . independent factors suggestive of prolonged weaning include copd occurrence,47 higher paco2 and heart rate during the first sbt.52 sellares et al also found a higher paco2 and heart rate among prolonged weaning patients before their first sbt , implying their worse condition when subjected to the initial trial.52 sbt is commonly delivered via pressure support ventilation ( psv ) at 7 cm h2o , continuous positive airway pressure , or t - piece . a conventional protocol - directed sbt takes 120 minutes but a 30 minutes trial performed via either t - tubes or psv was found to be equally effective in identifying successful extubations.44,53 comparing the delivery frequency , once - daily and multiple - daily t - piece trials were found to be equally effective.50 consensus has also been established over the identical validity of pressure support and t - tube in sbt.54 a similar conclusion was valid among infants and children when pressure support was 10 cm h2o.55 further to these findings , pressure support was found to overrun t - tube in difficult - to - wean patients since success of the former and failure of the latter regardlessly indicated successful extubation with unchanged reintubation rate.56 indeed , pressure support might have surpassed t - tube because it compensated for the extra breathing workload caused by an endotracheal tube.5761 in addition , cabello et al observed more successful pressure support trials in difficult - to - wean patients when positive end - expiratory pressure ( peep ; 5 cm h2o ) was incorporated to psv.62 this could be explained by the abilities of peep to 1 ) reduce respiratory muscle energy expenditure;63 2 ) attenuate intrinsic peep so that the work of breathing required to trigger the ventilator reduces;64,65 and 3 ) decrease pulmonary artery occlusion pressure.62 for the past few years , computer - driven automated weaning was introduced to perform sbt automatically in intubated patients but its value remained questionable from results of different studies.66 generally , the technology failed to facilitate weaning in surgical patients but prevailed in difficult - to - wean patients,67 such as those with copd , ischemic heart disease , and immunosuppresion.68,69 niv has been proposed as an alternative weaning tool in copd patients who failed sbt . according to a number of randomized controlled studies and meta - analysis , such application was associated with reduced mechanical ventilation , shortened icu and hospital stay , decreased incidence of septic shock , and pneumonia and improved survival.35,37,70 theoretically speaking , extubated patients administered with niv should not be declared as weaning success unless they ultimately get rid of the ventilatory support . increased use of niv as a weaning tool thus leads to a new weaning category called weaning in progress wherein extubated patients continue to be supported by niv . in spite of its benefits among copd patients , niv should not be indicated in all patients failing sbt because they may be exposed to extubation failure due to substantial comorbidities.47 to summarize , the amount of time needed for iv liberation depends on a sequence of events including suspicion for weaning possibility , performance of weaning assessment , and weaning itself . in order to promptly discard ventilatory support , weaning assessment when sbt fails in selected copd patients , niv may be recruited to shorten the duration of iv . in the people s republic of china , 80%90% aecopd cases occur as a result of bronchial - pulmonary infection . a significant proportion of them further develop into hypercapnic respiratory failure which requires invasive ventilatory support . in order to restore respiratory function and avoid time - dependent complications , pic window has been defined as a prompt stage of controlled pulmonary infection following artificial airway establishment , sputum drainage , and antibiotic administration . it was marked by thinning and decrease of sputum ; clearing of sputum cloudiness ; decreases in body temperature , radiographic infiltrations , and leukocytes ( table 4).17 at this stage , copd patients with severe hypercapnic respiratory failure tend to be more stable and respiratory muscle fatigue becomes relatively more significant in the development of respiratory failure.17 wang et al17 proposed this stage as an optimum timing to replace iv with niv so that ventilatory insufficiency and respiratory muscle fatigue may be resolved while lower airway infection and ventilator - associated pneumonia can be avoided . such a hypothesis was verified through a prospective cohort study and a prospective , multi - centered , randomized controlled trial among copd patients with severe hypercapnic respiratory failure.7173 in both research plans , the study groups were liberated from iv in exchange for niv at pic window while the control groups proceeded with iv throughout . by the end of the trials , the study groups were found to possess lower ventilator - associated pneumonia risks and mortality rate while requiring shorter durations of iv , ventilatory support , and icu stay.17,74 another study with nine patients yielded similar results except in two patients who presented with unstable hemodynamic condition and consciousness disturbance correspondingly.75 while the significance of pic window has been verified , one should be reminded of how proper training , skills , and observation ascertain the identification of such a stage . zhang failed to recognize pic window in several clinical cases thus led to delay in response and subsequently compromised prognosis and increased medical costs.75 an increased intubation rate has also been associated previously with inexperienced labor among hypercapnic arf patients.76,77 such results suggested how proper caring and observation hold the key to the betterment of all ventilated patients above other clinical advancements . copd is currently a significant burden in the people s republic of china because of greater risk exposure and uneven medical resource allocation between urban and remote areas . a population - based , cross - sectional survey conducted between 2002 and 2004 suggested a copd prevalence of 8.2% among chinese population over 40 years old.2 other studies reported varied prevalence ranging from 5% to 13%.36 according to the 2004 global burden of disease study , an annual sum of 3 million people die of copd worldwide . in the people s republic of china alone , copd - related mortality was 27.3 in males and 21.3 in females per 100,000 heads according to a national , prospective cohort study conducted between 1990 and 2000 . acute chronic respiratory failure , heart failure , pulmonary infection , pulmonary embolism , cardiac arrhythmia , and lung cancer are the major causes of death in patients with copd according to a cross - sectional study involving ten european centers.7 aecopd are a common cause of comorbidities and copd - related mortality.811 they are characterized by an increase in the symptoms of dyspnea , sputum volume , and sputum purulence with or without symptoms of upper respiratory infection.12 they may also involve worsening of existing symptoms , which require alterations in treatment ranging from antibiotic administration , short courses of oral corticosteroids , and increased bronchodilator usage.1314 reported aecopd incidence varied between 2.5 and 3 episodes per patient and year.15 being its major cause , infection accounted for 50%70% aecopd occurrences worldwide and 80%90% aecopd occurrences in the people s republic of china alone.16,17 other predisposing factors include environmental pollution , low temperatures , and concomitant heart failure.18,19 subsequent onset of acute respiratory failure ( arf ) may result if aecopd are accompanied by bronchial infections , bronchospasm , left ventricular failure , pneumonia , pneumothorax , or thromboembolism . once arf occurs , in - patient mortality ( 4%30% ) substantially rise up to 50% among elderly patients and 11%26% among intensive care unit ( icu ) patients.10,2023 lung infection other than acute exacerbation is quite common in patients with copd . bronchoscopic studies have shown that at least 50% of patients have bacteria existence in their lower airway during exacerbations of copd.1 viruses are also the common etiology of copd exacerbation next to bacteria . many patients with copd who have comorbidities such as hypertension , diabetes , cardiovascular disease are susceptible to lung infection . as stated earlier , lung infection usually accounts for 50%70% of aecopd . not all lung infections in copd need intensive care . only the patients who meet the criteria for hospital admission or icu admission need to be treated in a timely manner . hospital admission is warranted in an increasing number of patients with aecopd to prevent arf onset.24 an abc approach involving antibiotics , bronchodilators , and corticosteroids is generally extended to maximize lung function and to reverse the predisposing causes of exacerbations.25 mechanical ventilation is also suggested in 26%74% of patients with copd so that the respiratory muscle load may be alleviated to reduce dyspnea and respiratory rate and improve arterial oxygenation , partial pressure of carbon dioxide in arterial blood ( paco2 ) , and ph.2628 the criteria to start ventilatory support vary but commonly involve the following : 1 ) moderate to severe dyspnea where accessory muscles are recruited and abdominal breathing prevails ; 2 ) hypercapnic acidosis ( ph < 7.35 ) ; 3 ) tachypnea ( > 25 rpm).29 non - invasive ventilation ( niv ) is validated in patients with early aecopd because their tolerable coughing ability suggests a stronger need for respiratory muscle fatigue relief rather than airway clearance . it is thus initially provided for patients with severe aecopd with respiratory acidosis to reduce intubation rate , shorten icu stay , and decrease patient mortality . nonetheless , iv is indicated if niv measures fail to improve clinical manifestation and blood gas parameters 1 hour after implementation.29 any patient with ineffective airway clearance ( eg , post - surgical patients or patients with copd with hypercapnic arf and pneumonia ) needs iv support to improve sputum discharge and ventilation . table 1 summarizes the indications for both ventilation types.30 in spite of its importance , iv should be discarded whenever appropriate to avoid time - dependent complications associated with intubation , tracheotomy , or ventilation ( table 2).3133 to derive optimum advantages from mechanical ventilation , one should identify the optimum timing for withdrawal so that complications may be prevented while respiratory function is restored . niv has been suggested to serve the purpose because it was identically capable in unloading respiratory muscles.34 its application after iv also significantly reduced weaning time , alleviated ventilator - associated complications , and improved survival.3537 however , no consensus has yet been reached regarding the optimal time when niv should replace iv . according to the chinese guideline for mechanical ventilation in patients with acute exacerbation of chronic obstructive pulmonary disease 2007 , weaning with t - tube is advised in copd patients without obvious bronchial - pulmonary infection while replacement by niv at pulmonary infection control ( pic ) window is advised in copd patients with obvious bronchial - pulmonary infection . tobin summarized six stages in mechanical ventilatory support : 1 ) treatment of arf ; 2 ) suspicion that weaning may be possible ; 3 ) assessment of readiness to wean ; 4 ) spontaneous breathing trial ( sbt ) ; 5 ) extubation ; and 6 ) reintubation when required . one is only liberated from iv when he successfully passes the first five steps and avoids the last . weaning accounts for almost half of the time in mechanical ventilation and allows resumption of spontaneous breathing after gradual reduction of mechanical support.3842 randomized and non - randomized historical cohort studies verified a more significant reduction in the duration of mechanical ventilation when a weaning protocol , involving weaning parameter evaluation and subsequent breathing trials , was used instead of mere clinical judgment.4346 in view of general delays in weaning and the associated increase in mortality,41,47,48 assessment should be performed every day to allow prompt initiation of weaning . variations exist between protocols but weaning parameters essentially stem from observations in respiratory mechanics , gas exchange , and breathing patterns . table 3 is a list of parameters to be considered before weaning.47 daily screen of weaning parameters was found to have predicted a successful extubation with 82% accuracy , nearly 90% sensitivity and positive predictive values . significance of passing the screen could also be extended to hospital survival prediction during the 1st week and a half of mechanical ventilation . its prognostic significance seemed to be limited though , since up to 29% of the patients failed the screen but withstood extubation.49 thus , a successful weaning attempt does not necessarily require fulfillment of all the mentioned criteria . sbt failure is defined by 1 ) objective indices such as tachypnea , tachycardia , hypertension , hypotension , hypoxemia or acidosis , and arrhythmia ; 2 ) subjective indices such as agitation or distress , depressed mental status , diaphoresis , and evidence of increasing effort.47 subsequently , weaning failure refers to sbt failure or the need for reintubation within 48 hours of extubation.50,51 according to the number of attempts or days prior to successful weaning , patients may be classified into three groups:47 1 ) simple weaning ( patients who proceeded from initiation of weaning to extubation on the first attempt without difficulty ) ; 2 ) difficult weaning ( patients failing the first attempt who took up to three attempts or as long as 7 days from the first sbt to achieve successful weaning ) ; and 3 ) prolonged weaning ( patients who failed at least three weaning attempts or required > 7 days of weaning after the first sbt ) . independent factors suggestive of prolonged weaning include copd occurrence,47 higher paco2 and heart rate during the first sbt.52 sellares et al also found a higher paco2 and heart rate among prolonged weaning patients before their first sbt , implying their worse condition when subjected to the initial trial.52 sbt is commonly delivered via pressure support ventilation ( psv ) at 7 cm h2o , continuous positive airway pressure , or t - piece . a conventional protocol - directed sbt takes 120 minutes but a 30 minutes trial performed via either t - tubes or psv was found to be equally effective in identifying successful extubations.44,53 comparing the delivery frequency , once - daily and multiple - daily t - piece trials were found to be equally effective.50 consensus has also been established over the identical validity of pressure support and t - tube in sbt.54 a similar conclusion was valid among infants and children when pressure support was 10 cm h2o.55 further to these findings , pressure support was found to overrun t - tube in difficult - to - wean patients since success of the former and failure of the latter regardlessly indicated successful extubation with unchanged reintubation rate.56 indeed , pressure support might have surpassed t - tube because it compensated for the extra breathing workload caused by an endotracheal tube.5761 in addition , cabello et al observed more successful pressure support trials in difficult - to - wean patients when positive end - expiratory pressure ( peep ; 5 cm h2o ) was incorporated to psv.62 this could be explained by the abilities of peep to 1 ) reduce respiratory muscle energy expenditure;63 2 ) attenuate intrinsic peep so that the work of breathing required to trigger the ventilator reduces;64,65 and 3 ) decrease pulmonary artery occlusion pressure.62 for the past few years , computer - driven automated weaning was introduced to perform sbt automatically in intubated patients but its value remained questionable from results of different studies.66 generally , the technology failed to facilitate weaning in surgical patients but prevailed in difficult - to - wean patients,67 such as those with copd , ischemic heart disease , and immunosuppresion.68,69 niv has been proposed as an alternative weaning tool in copd patients who failed sbt . according to a number of randomized controlled studies and meta - analysis , such application was associated with reduced mechanical ventilation , shortened icu and hospital stay , decreased incidence of septic shock , and pneumonia and improved survival.35,37,70 theoretically speaking , extubated patients administered with niv should not be declared as weaning success unless they ultimately get rid of the ventilatory support . increased use of niv as a weaning tool thus leads to a new weaning category called weaning in progress wherein extubated patients continue to be supported by niv . in spite of its benefits among copd patients , niv should not be indicated in all patients failing sbt because they may be exposed to extubation failure due to substantial comorbidities.47 to summarize , the amount of time needed for iv liberation depends on a sequence of events including suspicion for weaning possibility , performance of weaning assessment , and weaning itself . in order to promptly discard ventilatory support , weaning assessment when sbt fails in selected copd patients , niv may be recruited to shorten the duration of iv . in the people s republic of china , 80%90% aecopd cases occur as a result of bronchial - pulmonary infection . a significant proportion of them further develop into hypercapnic respiratory failure which requires invasive ventilatory support . in order to restore respiratory function and avoid time - dependent complications , pic window has been defined as a prompt stage of controlled pulmonary infection following artificial airway establishment , sputum drainage , and antibiotic administration . it was marked by thinning and decrease of sputum ; clearing of sputum cloudiness ; decreases in body temperature , radiographic infiltrations , and leukocytes ( table 4).17 at this stage , copd patients with severe hypercapnic respiratory failure tend to be more stable and respiratory muscle fatigue becomes relatively more significant in the development of respiratory failure.17 wang et al17 proposed this stage as an optimum timing to replace iv with niv so that ventilatory insufficiency and respiratory muscle fatigue may be resolved while lower airway infection and ventilator - associated pneumonia can be avoided . such a hypothesis was verified through a prospective cohort study and a prospective , multi - centered , randomized controlled trial among copd patients with severe hypercapnic respiratory failure.7173 in both research plans , the study groups were liberated from iv in exchange for niv at pic window while the control groups proceeded with iv throughout . by the end of the trials , the study groups were found to possess lower ventilator - associated pneumonia risks and mortality rate while requiring shorter durations of iv , ventilatory support , and icu stay.17,74 another study with nine patients yielded similar results except in two patients who presented with unstable hemodynamic condition and consciousness disturbance correspondingly.75 while the significance of pic window has been verified , one should be reminded of how proper training , skills , and observation ascertain the identification of such a stage . zhang failed to recognize pic window in several clinical cases thus led to delay in response and subsequently compromised prognosis and increased medical costs.75 an increased intubation rate has also been associated previously with inexperienced labor among hypercapnic arf patients.76,77 such results suggested how proper caring and observation hold the key to the betterment of all ventilated patients above other clinical advancements . while niv is increasingly suggested as a primary option , iv may not be avoided under certain conditions . in such cases , strategies should be implemented to discard iv as soon as possible so that time - dependent complications may not arise as a result of prolonged iv . in aecopd patients , weaning by sbt ( assisted with niv ) is suggested under insignificant bronchial - pulmonary infection while replacement by niv at pic window is encouraged under significant bronchial - pulmonary infection .

copd is characterized by a progressive decline in lung function and mental and physical comorbidities . it is a significant burden worldwide due to its growing prevalence , comorbidities , and mortality . complication by bronchial - pulmonary infection causes 50%90% of acute exacerbations of copd ( aecopd ) , which may lead to the aggregation of copd symptoms and the development of acute respiratory failure . non - invasive or invasive ventilation ( iv ) is usually implemented to treat acute respiratory failure . however , ventilatory support ( mainly iv ) should be discarded as soon as possible to prevent the onset of time - dependent complications . to withdraw iv , an optimum timing has to be selected based on weaning assessment and spontaneous breathing trial or replacement of iv by non - iv at pulmonary infection control window . the former method is more suitable for patients with aecopd without significant bronchial - pulmonary infection while the latter method is more suitable for patients with aecopd with acute significant bronchial - pulmonary infection .

hospital information systems ( his ) have evolved from closed and proprietary - linked systems into open and standards - based . this change has encouraged medical equipment vendors and software developers to create uniform and interoperable systems . standardized interfaces let developers link different internal department information systems together : for example , connecting the laboratory information system ( lis ) of a clinical chemistry or a pathology department with the radiology information system in a radiology department . linked data repositories make all patient - related material ( e.g. , clinical history and images from different modalities ) available to all institution personnel . for instance , pathologists and radiologists can view breast ultrasound and x - ray images simultaneously with corresponding histological specimens . the most widely used medical imaging standard is the digital imaging and communications in medicine ( dicom)1 , which is routinely used in several medical specialties , especially radiology . in pathology , digitization of whole microscope specimens has only recently become possible with high - throughput slide scanners2 . the digitized versions of microscope glass slides are called virtual slides or whole - slide images ( wsis ) . acquiring , handling , and displaying wsis is commonly called virtual microscopy ( or whole - slide imaging)3,4 . wsis can be used for local viewing or , more practically , for remote viewing by transmitting them over networks5 . within the internal network of a hospital or pathology department , personnel can use wsis in case meetings , slide seminars , and instructional live - audience presentations6 . by allowing access over the internet , wsis can also be used more widely in second - opinion consultations , national and international conferences , and inter - laboratory quality assurance programs7 . since microscope specimens are often up to 20 30 mm in size , a wsi can contain up to 40 gb of uncompressed image data ( with a scanning resolution 0.20.5 m per pixel)2 . the amount of data increases further if scanning is done at a higher optical magnification and/or if several focus layers ( along z - axis ) are scanned ( e.g. , in cytopathology)8 . due to the large size of wsis , all viewing systems described to date apply the on - demand principle : that is , only a user - requested area ( with a desired resolution ) of the wsi is decoded and displayed . moreover , the large image size necessitates the use of lossy image compression . lossy compression can yield a 10- to 30-fold compression ratio compared to lossless compression , without affecting the diagnostic properties of a wsi9 . thus , a suitable image format for virtual microscopy needs to be based on an effective image compression algorithm , as well as to provide a sophisticated random access technique . there are several proprietary image formats that are tied to specific scanner vendors , such as svs ( by aperio technologies , usa ) , ndp ( by hamamatsu photonics , japan ) , and mirax ( by carl zeiss microimaging , usa ) . we have previously shown that the open jpeg2000 standard is a suitable format for wsis , allowing fast random slide access and efficient lossy compression10 . although jpeg2000 compression is computationally intensive , the process can be matched with current slide scanner speeds by utilizing multi - core processor environments10 . jpeg2000 is a family of standards supervised by the joint photographic experts group standardization committee11,12 . the standard family currently consists of 13 parts , three of which are essential for virtual microscopy . part 1 ( core coding system)13 specifies the code - stream syntax and the jp2 file format , which uses part 2 ( extensions)14 provides extensions for the first part . part 9 ( interactivity tools , apis , and protocols)15 introduces the jpeg2000 interactive protocol ( jpip ) for remote serving and viewing of jpeg2000 images . we have previously developed and released a free jpeg2000 software package ( called jvs , for jpeg2000 virtual slide ) comprising wsi compression , viewing , and network server applications10 . della mea et al.16 have presented a survey of currently available jpeg2000 viewing software . for clinical diagnostic use , wsis must be compatible with existing imaging standards , such as dicom17 . although a dicom object definition for visible light microscopy exists18 , wsis are too large ( both pixel dimensions and byte size ) to be used directly as these objects . a base standard correction item ( cp 896)19 to overcome the image dimension limitation was recently rejected by the dicom standards committee20 . nevertheless , the committee will continue to consider the possibility of introducing this change to the standard as a supplement . in addition to image size , the access characteristics of wsis differ from conventional dicom images . panning and zooming within a huge wsi require fast random access with on - demand decoding . currently , the dicom standard includes the basic parts of the jpeg2000 standard in supplements 6121 and 10522 . supplement 106 ( jpeg 2000 interactive protocol)23 describes two jpip - based transfer syntaxes as methods of delivering image pixel data apart from patient data : the noncompressed jpip referenced transfer syntax and the deflate - compressed24 jpip referenced deflate transfer syntax . thus , the dicom standard specification already contains necessary elements for transmitting wsis over jpip . when using the jpip transfer syntaxes in a dicom - based picture archiving and communication system ( pacs ) , a dicom server sends its client a uniform resource locator ( url ) string that refers to the wsi pixel data provider ( i.e. , a jpip server ) , together with the image name , which can be arbitrary and unrelated to patient data ( as shown in fig . 1 ) . upon receiving the pixel data provider reference , the client dicom workstation can either use a built - in jpip viewer or invoke an external one for retrieving the wsi from the specified jpip server . all network messaging between the pacs and the client end is done according to the dicom protocol , except the jpip transmission , which is by default performed on top of the hypertext transfer protocol ( http/1.1)25 for compatibility with existing web infrastructure , but it can also be done using a lower - level transport protocol ( such as transmission control protocol , tcp)26 . image serving performance of jpip has been demonstrated to be excellent and upwards scalable in multi - client systems10,27 . fig 1the principle of transmitting whole - slide images ( wsis ) within a dicom - based pacs by using jpeg2000 interactive protocol ( jpip ) . the principle of transmitting whole - slide images ( wsis ) within a dicom - based pacs by using jpeg2000 interactive protocol ( jpip ) . although jpip is described in the dicom standard specification and a brief description of a commercial application exists28 , we are not aware of any open dicom software solutions or libraries supporting it . in this study , we explore the feasibility of linking jpeg2000 wsis with a dicom - based pacs by using jpip . first , we modified an open - source dicom library by adding support for jpip as described in the supplement 106 . second , the modified library was used as a basis for a software package ( jvsdicom ) , which provides a proof - of - concept for a dicom client - server system that can transmit patient data , conventional dicom imagery , and jpeg2000 wsis over jpip . the software package consists of a compression application ( jvsdicom compressor ) for producing dicom - compatible jpeg2000 wsis , a dicom pacs server application ( jvsdicom server ) , and a dicom pacs client application ( jvsdicom workstation ) . finally , we present a dicom - compatible whole - slide imaging system based on the software developed . an open - source dicom library ( offis dcmtk dicom toolkit , version 3.5.4)29 was modified by adding support for jpip , as described in the dicom supplement 106 . the modified library was used to embed dicom functionality to the developed jvsdicom software package . jvsdicom compressor is based on our previously described jpeg2000 compression application ( jvscomp , version 2.1)10 . jvsdicom server and workstation utilize the qt open - source software framework ( version 4.3)30 for core application functionality and the tango icon library31 for graphical user interface elements . the software package was written in c++ programming language and built for 32-bit windows platforms but can be run under a 64-bit windows platform as well . reference material representing typical diagnostic imagery of breast cancer was obtained from tampere university hospital . the material comprises radiology imagery ( ultrasound , mammography , bone scan , and magnetic resonance imaging ) and histological specimen slides ( routine h&e stains and immunohistochemistry ) . the standard - sized slides ( 75 25 mm ) were scanned with aperio scanscope xt ( aperio technologies , usa ) using uncompressed bigtiff32 as the primary output format . whole - mount section slides ( 75 50 mm ) were acquired with a zeiss axioskop40 microscope ( carl zeiss microimaging , usa ) as described equipped with a charge - coupled device color camera ( qicam fast ; qimaging , canada ) and a motorized specimen stage ( mrzhuser wetzlar gmbh , germany ) . the automated image acquisition was controlled by the surveyor imaging system ( objective imaging , uk ) using uncompressed bitmap as the primary output format . the developed jvsdicom compressor application was used to convert the histological reference material into the dicom - compatible jpeg2000 wsi format . the jvsdicom software package was developed as a proof - of - concept for a dicom client server system that can transmit patient data , conventional dicom imagery ( e.g. , radiological ) , and jpeg2000 wsis using the jpip referenced transfer syntax . the software package consists of a compression application ( jvsdicom compressor ) , a dicom pacs server application ( jvsdicom server ) , and a dicom pacs client application ( jvsdicom workstation ) . jvsdicom compressor is a free , command line - based image compression application capable of converting multiple image formats ( e.g. , bmp , ppm , and bigtiff ) into the dicom - compatible jpeg2000 wsi format . the output jpeg2000 file follows the optimized code - stream parameterization we have previously described10 . in addition to the jpeg2000 wsi file , the application generates a supplementary dicom file containing medical information , image properties , and a jpip server reference to the wsi file location , which is used by the dicom client to access the wsi . the resulting dicom file uses the general microscopy modality18 and visible light microscopic image information object definition ( iod)18 with a minimal set of required attributes . jvsdicom server is an open - source dicom pacs server application that acts as a storage service class provider ( scp ) and as a query / retrieve scp ( table 1 ) . server can be configured to contain several filesystem - based storage areas with different application entity ( ae ) titles , as well as to limit access to these areas from a predefined ae network ( fig . 2 ) . alternatively , the server features a public mode , which can be used to grant open access to the server . for open access , new dicom entries can be added into the server storage with pixel data either coming from existing image files or replaced with a jpip reference ( i.e. , in case of jpeg2000 wsis ) . dicom image objects can be added to the storage by importing existing image files or by creating a new jpip - referenced object.table 1main features of jvsdicom serverlist of featuresfully dicom - compliant pacs serversupport for several storage sop classessupport for jpeg2000 wsis with jpip referenced transfer syntaxa public mode with open access to servercan be used as a server for jvsdicom workstationopen - source a screenshot of jvsdicom server storage management view . dicom image objects can be added to the storage by importing existing image files or by creating a new jpip - referenced object . main features of jvsdicom server jvsdicom workstation is an open - source dicom pacs client application that acts as a query / retrieve service class user ( scu ) and a storage scu ( table 2 ) . with it , users can query and retrieve images from a dicom - compatible pacs server ( fig . 3 ) . users can view the images with several image enhancement options , as well as view a summary of patient- and treatment - related information . jvsdicom workstation interacts with a dicom server as a conventional dicom client , but upon receiving a jpip reference to a jpeg2000 wsi , it invokes an external jpeg2000 viewing application . the external viewing application displays the image pixel data , while jvsdicom displays the associated dicom medical information . thus , by having an external viewer for wsis , users can view conventional dicom imagery and corresponding histopathologic specimens side by side ( fig . regions of interests from the dicom images and wsis can both be opened in a public domain image analysis software , imagej33 , which features a multitude of analysis tools for medical imaging . jvsdicom workstation is compatible with commercial - grade dicom servers , supporting several storage sop classes ( a comprehensive conformance statement appears in the software documentation ) . conventional dicom imagery is displayed in the thumbnail window , while jpeg2000 wsis are opened in an external jpip viewer.fig 4viewing of a breast cancer specimen x - ray and corresponding histological wsi ( whole - mount section ) side by side with jvsdicom workstation and an external jpip viewer . within jvsdicom , users can rotate the image , adjust width and center values , and measure distances by using the ruler tool . the jpip viewer displays the wsi using an overview and a main navigation window.table 2main features of jvsdicom workstationlist of featuresfully dicom - compliant pacs clientsupport for jpeg2000 wsis with jpip referenced transfer syntaxviewing of radiological images and corresponding histological wsis side by sidea functional link with public domain image analysis software ( imagej)can be used as a client for jvsdicom serveropen - source a screenshot of jvsdicom workstation showing the pacs query view . conventional dicom imagery is displayed in the thumbnail window , while jpeg2000 wsis are opened in an external jpip viewer . viewing of a breast cancer specimen x - ray and corresponding histological wsi ( whole - mount section ) side by side with jvsdicom workstation and an external jpip viewer . within jvsdicom , users can rotate the image , adjust width and center values , and measure distances by using the ruler tool . main features of jvsdicom workstation the jvsdicom software package is fully dicom - compliant and designed to run on windows xp but is also windows vista compatible . the binaries and source code , as well as a comprehensive conformance statement , are available on our web site ( http://jvsmicroscope.uta.fi/ ) . the web site also features a public jvsdicom server , containing example images produced in the diagnostics of breast cancer . we developed the jvsdicom software package to study the feasibility of linking jpeg2000 wsis with a dicom - based pacs by using jpip . jvsdicom compressor is used for creating dicom - compatible jpeg2000 wsis , which consist of the actual jpeg2000 image file and a supplementary dicom file containing patient - related information . the supplementary dicom file can be served using the jvsdicom server , while the jpeg2000 image files are served using a separate jpip server . querying and retrieving images from the jvsdicom server can be done using the jvsdicom workstation , which handles conventional dicom imagery directly but uses an external jpip client application for jpeg2000 wsi viewing . a complete , dicom - compatible whole - slide imaging system can be constructed by combining jvsdicom with our previously described jpeg2000 viewing application ( jvsview ) and jpip network serving application ( jvsserv ; fig . 5 ) . in this model system , jvsdicom compressor produces a jpeg2000 file containing the actual wsi image data and a dicom file containing the associated medical data ( i.e. , patient information ) as well as some mandatory image properties , such as width and height . by default , the produced dicom file contains anonymized dicom entries , but it could be linked with a lis or a his for retrieving patient information . a straightforward way to name the jpeg2000 wsi file is to use the microscope slide label identification string , which can be read automatically if bar - coded labels are used . the jpeg2000 wsi file is then moved into a jvsserv server , and the dicom file is moved into a jvsdicom server , which both are parts of the same pacs . both files can be stored separately inside a server - specific storage area within the pacs . jvsdicom can also receive imagery from other imaging modalities , which are in turn linked with the lis or his . end - users ( e.g. , pathologists or physicians ) query the jvsdicom server with a jvsdicom workstation and retrieve patient - linked image objects . they can view and analyze conventional dicom imagery within jvsdicom workstation , while wsis are opened with jvsview in another viewing window . the dicom data is transmitted using the jpip referenced transfer syntax , and the jpeg2000 wsi data is transmitted via an auxiliary channel over jpip . the system architecture makes it possible to use jvsserv separately outside the pacs , since wsis do not contain any dicom references . fig 5a model system for linking whole - slide images ( wsis ) with dicom by using jpeg2000 , jpip , and the jvs software . the resulting dicom and jpeg2000 wsi files are moved into the pacs to jvsdicom server and jvsserv , from which they are queried with jvsdicom workstation and viewed with jvsview . a model system for linking whole - slide images ( wsis ) with dicom by using jpeg2000 , jpip , and the jvs software . the resulting dicom and jpeg2000 wsi files are moved into the pacs to jvsdicom server and jvsserv , from which they are queried with jvsdicom workstation and viewed with jvsview . an open issue with using jpeg2000 wsis and jpip is the image dimension restriction in the dicom standard specification . since image width and height tags are currently described using 16-bit values , oversized ( width or height > 65,535 pixels ) wsis will show false information with regards to these tags . however , since the definitive image width and height information are always also stored in the jpeg2000 code - stream header , the dicom counterpart values can be bypassed . this requires the jpip client software to use the code - stream values instead of the dicom - specific values . if a dicom supplement equivalent to the cp 896 ( to eliminate 16-bit image dimension restrictions ) is introduced to the standard , the jpip viewing software can also operate using the dicom - specific image dimension tags . current limitations for dicom image object size due to 32-bit addressing ( 4 gb ) do not affect jpeg2000 wsis as long as they are stored in a separate jpip server , such as the one described in this study . as such , the jpeg2000 standard does not limit individual file size and image dimensions , even in the largest future wsis11,13 . the dicom working group for pathology imaging ( wg-26 ) is currently making an effort to consolidate pathology concepts and whole - slide imaging with dicom34 . wg-26 has recently finalized supplement 122 ( specimen module and revised pathology sop classes ) , introducing a new mechanism for pathology specimen identification in dicom35 . the scope of our study was to exemplify a dicom wsi solution based on jpip . wg-26 has also recently started preparing a draft for a new dicom supplement for whole - slide imaging containing an iod and sop classes for wsis ( as discussed in the wg-26 meetings)37 . at its current state approach in which the original resolution wsi is split into small image tiles ( usually thousands of them ) . in addition , a number of successively lower resolution versions of the original tiles may be precomputed , creating a pyramid - shaped resolution structure . all the image tiles are stored as a conventional dicom series and accessed with the dicom wsi object , which provides a mapping between the series and the conceptual image pyramid . the pyramidal approach is similar to what various wsi scanner vendors have used in their web viewing solutions for several years . prior to its finalization , the supplement draft is subject to changes . both approaches for whole - slide imaging in dicom , the pyramidal and the jpip - based , as described in this study , can be implemented with minor modifications to the existing standard specification . an advantage of the pyramidal approach is that wsis are treated equal to other dicom imagery residing within the same server . in the jpip - based approach however , a disadvantage of the pyramidal approach is that because wsis are stored in a dicom - specific data structure using lossy compression , as is required in virtual microscopy , relocating wsis from the pacs ( e.g. , for teaching purposes ) requires lossy recompression . on the other hand , the jpeg2000 wsis are not stored using a dicom - specific data structure , making them directly interchangeable with non - dicom systems . moreover , since patient - related dicom data is not embedded in the jpeg2000 wsi files , the same jpip server can be readily used inside and outside of a pacs without the need for anonymization . since the dicom standard specification already includes support for jpip , the pyramidal approach and the jpip - based approach for whole - slide imaging are not mutually exclusive and can be used simultaneously in the same pacs . regardless of the approach , changes in existing pacs workstation software are also required because of the wsi - specific image viewing characteristics . at the time of preparation of this article , the detailed contents of the upcoming wg-26 wsi supplement , as well as its expected release date , are open . in the future , when the supplement is finalized and implemented in practice , comparisons between the two wsi approaches might turn out useful . to our knowledge , the software package described in this study is the first practical solution to overcome the limitations of dicom in virtual microscopy . compared to other approaches , such as the pyramidal approach of the dicom wg-26 , jpeg2000 with jpip is a good alternative , enabling use of wsi archives either with or without a linked dicom system . further , since jpeg2000 has also many other advantageous features over those of existing wsi image formats , we anticipate that jpeg2000 will become a widely - accepted standard wsi format in virtual microscopy .

the use of digitized histopathologic specimens ( also known as whole - slide images ( wsis ) ) in clinical medicine requires compatibility with the digital imaging and communications in medicine ( dicom ) standard . unfortunately , wsis usually exceed dicom image object size limit , making it impossible to store and exchange them in a straightforward way . moreover , transmitting the entire dicom image for viewing is ineffective for wsis . with the jpeg2000 interactive protocol ( jpip ) , wsis can be linked with dicom by transmitting image data over an auxiliary connection , apart from patient data . in this study , we explored the feasibility of using jpip to link jpeg2000 wsis with a dicom - based picture archiving and communications system ( pacs ) . we first modified an open - source dicom library by adding support for jpip as described in the existing dicom supplement 106 . second , the modified library was used as a basis for a software package ( jvsdicom ) , which provides a proof - of - concept for a dicom client server system that can transmit patient data , conventional dicom imagery ( e.g. , radiological ) , and jpip - linked jpeg2000 wsis . the software package consists of a compression application ( jvsdicom compressor ) for producing dicom - compatible jpeg2000 wsis , a dicom pacs server application ( jvsdicom server ) , and a dicom pacs client application ( jvsdicom workstation ) . jvsdicom is available for free from our web site ( http://jvsmicroscope.uta.fi/ ) , which also features a public jvsdicom server , containing example x - ray images and histopathology wsis of breast cancer cases . the software developed indicates that jpeg2000 and jpip provide a well - working solution for linking wsis with dicom , requiring only minor modifications to current dicom standard specification .

study on photosensitizers has received more and more attention . however , the effect of the photosensitization on dna , protein , cell , or even an organ should be given more attention , no matter whether the effect is harmful or helpful for the potential clinical application . it can be extracted from h. perforatum , a popular medicine for the treatment of depression [ 18 ] . it has been used as a conventional medication for the treatment of depression and wound healing for a long time . recently , more and more interest has been given to some other important pharmaceutical potentials of this species , such as antivirus activity , anti - hiv , antibacterial activity and antitumor activity [ 1012 ] . most of the clinic functions are proposed to be related to the phototoxicity of hy . for instance , the cytotoxic activity can be greatly enhanced after light activation [ 9 , 1316 ] . the mechanism how it works is still to be explored , however , it is proposed that after being exposed in visible light at the wavelength of 540600 nm , hy will transfer light energy to oxygen to generate reactive oxygen species ( ros ) , which may further induce the apoptosis of cells . some other studies report that hy preferentially accumulates in the cell membranes , especially the mitochondria membrane , which might be the target in the photodynamic therapy . both the type i and type ii photodynamic reactions can take place in the photoactivation of hy , resulting in the formation of radicals , such as singlet oxygen and superoxide radical . in the paper , we report our studies of the photodynamic effect of hy on the structure and function of a protein by employing uv - vis spectroscopy and electrochemical methods . hemoglobin ( hb ) , a kind of heme protein , is used as the target protein for this work . on one hand , the structure and function of hb are relatively very clear , which is easier to get to know the information of the conformational and functional changes . on the other hand , it is relatively easier to design the experimental protocols , since its electrochemistry has been somewhat largely studied in our lab [ 19 , 20 ] . besides , we have found that the reactive molecules , such as hydroxyl radical , superoxide anions , may influence the peroxidase activity of the protein , which may be ascribed to the conformational transformation of the protein . based on the data obtained in this study , we found that visible light irradiation of the protein in the presence of hy would cause the change of its conformation . and , the peroxidase activity of the protein towards hydrogen peroxide ( h2o2 ) can be obviously enhanced . since the structural and formational changes can take place after the treatment of the protein with visible light , the process of which is so common to occur , this study should be very interesting to lots of scientists in different research areas . uv - vis spectroscopy is a very helpful technique to study the conformational changes of heme proteins , since the soret band of the heme which is located at 407 nm can provide very useful information on the secondary structure of heme proteins [ 2326 ] . the position of the soret band will shift or the absorption will decrease if the structure of a heme protein is transformed . figure 1 shows that the absorption of the soret band remains almost unchanged even after 3h irradiation by visible light in the absence of hy , however , the peak will decrease evidently only after 1 hour treatment if this medicinal species is in the presence . therefore , the heme moiety of the protein can be hardly influenced only by visible light . nevertheless , irradiation treatment on hb together with hy , the structure of the protein , especially the microenvironment of the heme ring , will be changed or even be badly damaged . we propose that the ros , generated by the irradiation of hy , may react with the protein at the heme position , and change the microenvironment of the heme ring . it can be also observed from fig.1 that the decrease of the absorption is much less significant for the case of lower hy concentration . so , the photodynamic effect of hy on hb is not only a time dependent but also a concentration dependent process . we have further employed electrochemical method to check whether the dynamic activity of hy may have any effect on the peroxidase activity of the protein . as is well known , , some other activities of this protein have also been revealed . especially in recent years , the peroxidase activity of hb has been largely studied , and these findings have been employed for the development of third - generation biosensors . based on our previous work of the preparation of protein - film modified electrodes and the fabrication of hb - based h2o2 biosensors with this protein [ 1921 ] , we have first prepared an hb - hy film modified electrode to study the catalytic activity of hb towards h2o2 . as is shown in figure 2 , the reduction peak of hb will increase gradually with the addition of h2o2 in the test solution . further studies reveal that a linear relationship between the increase of the reduction peak and the h2o2 concentration can be obtained from 110mol / l to 510mol / l , and the linear regression equation is : y = 2.15459 + 0.27599x , r = 0.999 . in order to obtain an obvious contrast , and to more clearly show the change of the peroxidase activity of the protein , the value of the reduction peak with the addition of 110mol / l h2o2 is chosen as the baseline , and is set to be 0 . as is shown in figure 3 , the slope of the catalytic linearity is enhanced obviously after the irradiation of visible light on hb together with hy . , the slope increases with the irradiation time , which suggests that a longer time treatment on the samples will result in a higher catalytic ability of the protein . meanwhile , concentration of hy is found to be another key factor . as is shown in figure 4 , although the slopes will also increase for the case of low hy concentration , the changes are indistinct in the first two hours . and the increase is much smaller than that for the case with high hy concentration . these experimental results obtained with electrochemical method coincide very well with the results obtained by uv - vis spectroscopy , which further reveals that the photodynamic effect of hy is not only a time - dependent , but also a concentration - dependent process . from our further studies , this effect has also been known to be an oxygen - dependent process , although o2 is not required for the antivirus activity of hy . as is shown in fig . 5 , if the samples are prepared and irradiated in an anaerobic situation , the catalytic slope will keep unchanged even if a 3h treatment has been made . this is reasonable , since o2 is required in a photodynamic process to generate ros , which is crucial in photoactivating hy . so , no influence of this medicinal species on the peroxidase activity of hb can be obtained without o2 being involved . these results have also confirmed that the phtosensitization of hy would be achieved only in the presence of oxygen . after absorbing the light energy , hy will be transformed to be an excited state , which may further transfer the energy to oxygen directly or indirectly , both of which induce the formation of the ros , especially the singlet oxygen . ros then react with hb , which may induce the change of the microenvironment of the heme position . as a result , the catalytic ability of the protein towards h2o2 is enhanced . in summary , with uv - vis spectroscopic and electrochemical techniques , we have found that irradiation on the mixture of hb and hy by the very common visible light can activate hy to generate ros , which will make the change of the structure of hb and enhance the catalytic activity of the protein towards the h2o2 reduction . this process depends not only on the irradiation time but also on the concentration of hy . meanwhile , o2 , which is required for the generation of ros , is also found to be a vital element in the photoactive effect . this work has not only confirmed the photosensitization of hy and revealed the enhanced peroxidase activity of hb , but also be helpful to the development of more sensitive h2o2 biosensor and the usage of this medicinal herb molecule . stock solutions were prepared by dissolving known amounts of hy in 1 ml of dimethyl sulphoxide ( dmso ) . stock solutions were prepared by dissolving known amounts of hb in 1 ml of double - distilled water . dmso and h2o2 ( 30% ( w / v ) solution ) were provided from shanghai jinshan tingxin chemical reagent co. and nanjing chemical reagent co. , respectively . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18 m cm , was used in all the experiments , and all the solutions were stored in the refrigerator at 4 c . sample i preparation : 1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample ii preparation : 0.5l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample iii preparation:1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . this sample was thoroughly deoxygenated by blowing high - purity nitrogen for at least 1 min , and then sealed by parafilm . the samples were exposed in the visible light emitted by a 200w filament lamp , which was filtered by an orange filer to get a wide band illumination above 580 nm , for 1h , 2h , 3h , respectively , at a distance of 20 cm . uv - vis spectroscopy was performed using a uv-2550 spectrophotometer ( shimadzu , japan ) . after the treatment with visible light irradiation , the samples were then twentyfold diluted and analyzed immediately with the spectrophotometer . the substrate pyrolytic graphite ( pg ) electrode ( a = 6.28 mm ) was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood 's alloy ( a fusible bismuth - based alloy ) . before the modification of the substrate electrode with hy and the protein , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . 10l of hy and hb mixture with their concentration being 110mol / l and 6.210mol / l , respectively , was mixed with 10l dmso ( 20% ) and then spread evenly on the surface of the substrate pg electrode . the hy and hb mixture had been previously treated with visible light irradiation before its immobilization onto pg electrode surface . after that , this electrode was thoroughly rinsed with double - distilled water and could be ready for use . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25 0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all the test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiments . stock solutions were prepared by dissolving known amounts of hy in 1 ml of dimethyl sulphoxide ( dmso ) . stock solutions were prepared by dissolving known amounts of hb in 1 ml of double - distilled water . dmso and h2o2 ( 30% ( w / v ) solution ) were provided from shanghai jinshan tingxin chemical reagent co. and nanjing chemical reagent co. , respectively . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18 m cm , was used in all the experiments , and all the solutions were stored in the refrigerator at 4 c . sample i preparation : 1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample ii preparation : 0.5l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . sample iii preparation:1l hy solution ( 0.1 mol / l ) , 5l hb ( 8mg / ml , ph 7.0 ) and 4l tris - hcl buffer ( ph 7.0 ) were mixed in a microcentrifuge tube . this sample was thoroughly deoxygenated by blowing high - purity nitrogen for at least 1 min , and then sealed by parafilm . the samples were exposed in the visible light emitted by a 200w filament lamp , which was filtered by an orange filer to get a wide band illumination above 580 nm , for 1h , 2h , 3h , respectively , at a distance of 20 cm . uv - vis spectroscopy was performed using a uv-2550 spectrophotometer ( shimadzu , japan ) . after the treatment with visible light irradiation , the samples were then twentyfold diluted and analyzed immediately with the spectrophotometer . the substrate pyrolytic graphite ( pg ) electrode ( a = 6.28 mm ) was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood 's alloy ( a fusible bismuth - based alloy ) . before the modification of the substrate electrode with hy and the protein , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . 10l of hy and hb mixture with their concentration being 110mol / l and 6.210mol / l , respectively , was mixed with 10l dmso ( 20% ) and then spread evenly on the surface of the substrate pg electrode . the hy and hb mixture had been previously treated with visible light irradiation before its immobilization onto pg electrode surface . the modified electrode was dried overnight at room temperature in the dark . after that , this electrode was thoroughly rinsed with double - distilled water and could be ready for use . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25 0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all the test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiments .

hypericin , extracted from h. perforatum , can induce the generation of reactive oxygen species by visible light irradiation , which may consequently induce the conformational change of hemoglobin . we have not only employed uv - vis spectroscopy to observe the changes of uv - vis spectra of the protein , which reveals the conformational changes of the protein , but also employed electrochemical method to obtain its enhanced peroxidase activity . the photodynamic effect of hypericin on the conformation and catalytic activity of the protein has also been proven to be strongly dependent on the irradiation time , the hypericin concentration and the presence of oxygen . this work is beneficial not only to the fabrication of more sensitive hydrogen peroxide biosensor , but also to the guidance of the usage of this medicinal herb molecule , since the conformational change of the protein and the enhanced peroxidase can be easily obtained only by visible light irradiation on hypericin , the process of which is so common to happen .

malnutrition from loss of appetite , indigestion , malabsorption , and metabolic problems is a common condition in cancer patients undergoing treatment . side effects of chemotherapy include anemia , appetite loss , dysgeusia , changes in sensitivity to food temperature , constipation , diarrhea , dysphagia , xerostomia , fatigue , and early satiation that can decrease food intake and lead to extreme weight loss . the goal of nutritional intervention for such patients is to improve their nutritional condition by mitigating treatment side effects , offering individualized patient care , and improving food intake while respecting patients ' food habit with regard to administering more progressive interventions . additionally , as the main method of nutritional intervention , patients are recommended to eat small and frequent meals 5 - 6 times a day to increase their overall intake since most patients experience appetite loss resulting from metabolic problems and chemotherapy treatment . if the amount of food does not exceed 60% of the daily recommended intake , tube feeding or intravenous alimentation is performed . if diets are appropriated to each patient 's condition , oral food intake is recommended whenever possible . hot or cold foods and drinks are offered depending on patient 's receptivity and variations are made according to patient 's condition . when a low bacteria diet is needed , appropriate foods are given and the patient is educated about the dietary practices . the purpose of the current case study is to record the process of nutritional diagnosis and intervention conducted on malnourished cancer patients with chemotherapy for treatment at a hospital . in this case , after admission to the hospital for general weakness , the patient was identified as being at risk of malnutrition according to an early nutritional assessment program by the nutrition team at hospital . approval of the institutional review board at ( khnmc irb 2013 - 116 ) the kyung hee university hospital at gangdong was obtained . on october 21 , 2013 , a 61-year - old man was admitted to the gastroenterology ward . to measure the patient 's nutritional condition , medical history , physical measurements , biochemical data , medical examinations and treatments , nutritional physical examination data , and food / nutrition - related diet history the patient was diagnosed with diabetes mellitus 10 years ago , hypertension ( 10 years ago ) , and rectal cancer 3 years ago , for which the patient underwent an ileostomy on november 14 , 2011 and received leucovorin plus fluorouracil ( lv5fu2 ) as preop # 1 and postop # 8 chemotherapy . afterward , the patient underwent ileostomy repair on december 3 , 2012 , and then a liver wedge resection on july 18 , 2013 for liver s4 metastasis during the follow - up ( f / u ) . afterward the patient received chemotherapy # 4 from september 2 through october 14 , 2013 using oxaliplatin , folinic acid , and 5-fluorouracil ( flofox4 ) . the patient 's current drug prescriptions include lantus 10 u / day , apidra 6 u # 3/day , and nutriflex lipid peri [ + humulin - r ( regular insulin ) 15 u ] every other day ( eod ) , and chemotherapy was paused due to general weakness . the first screening visit was conducted on october 22 , 2013 . at the time of hospital admission ( october 21 , 2013 ) , the physical measurements were : height , 167 cm ; body weight , 53.2 kg ; ideal body weight ( ibw ) , 61.4 kg ; % ibw , 86.6% ; body mass index ( bmi ) , 19.1 kg / m ; usual weight , 64.8 kg ( july 18 , 2013 ) ; and weight change of -11.6 kg ( 17.9% loss ) over the most recent 3 months . examinations upon admission showed the following : total protein / albumin , 5.6/3.2 g / dl ; hemoglobin / hematocrit ( hgb / hct ) , 8.4 g / dl/25.4% ; total lymphocyte count ( tlc ) , 741.2 cells / mm ; cholesterol , 162 mg / dl ; blood urea nitrogen / creatinine ( bun / cr ) , 20/1.0 mg / dl ; ca / p , 8.3/2.3 mg / dl ; na / k / cl , 132/4.2/98 meq / l ; c - reactive protein , 6.28 mg / dl ; hemoglobin a1c , 9.7% ; and casual glucose , 470 mg / dl . the nutritional and physical examination data showed that the patient had stomatitis , nausea , and vomiting as side effects of chemotherapy and the blood pressure was 142/83 mmhg . in terms of food / nutrition - related diet history , the dietary prescription and diet - related experiences were such that the patient experienced a loss of appetite after chemotherapy , did not receive education intended for cancer patients ( due to being too tired ) . the patient 's food and beverage consumption over the most recent week ( after the fourth round of chemotherapy ) includes six glasses of cola ( 480 kcal / day ; 120 g of sugar ) due to stomatitis , nausea , and vomiting . at the hospital , the administration of other foods was not attempted since the patient experienced pain and vomiting after consuming soft foods , particularly gruel ( not eaten ) and intravenous alimentation ( nutriflex lipid peri eod ; energy , 465 kcal ; c : p : f ratio , 34.4:17.2:48.4 ; carbohydrate , 40 g ; protein , 20 g ; fat , 25 g ) . the patient stayed in the bed whole day . in the patient - generated subjective global assessment conducted for nutritional assessment , the patient scored 15 points , indicating the need for improved symptom and focused nutrition management . approximately 1600 - 1860 kcal of energy ( baseline weight , 53.2 kg ; based on calculation , 30 - 35 kcal / kg needed ) and 64 - 85 g of protein ( baseline weight , 53.2 kg ; based on calculation , 1.2 - 1.6 g / kg needed ) were set as the required nutritional intake . two nutritional diagnoses were made : frist , malnutrition arising from a decrease in food intake related to the side effects of chemotherapy ( stomatitis , nausea , vomiting ) as well as a lack of food / nutrition - related knowledge regarding food types and quantities . the evidence for this diagnosis being that the patient experienced a 17.9% weight loss over the most recent 3 months and consumed only 28% of the required nutrients during the most recent week ( after the fourth round of chemotherapy ) ; second , the patient 's lack of knowledge with regard to foods and nutrients which is possibly attributable to fear for food intake and this was observed through refusal to ingest food and tendency to ingest only cola after chemotherapy . as a nutritional intervention for the first diagnosis , the medical team was advised to change the patient 's diet to diabetic thin rice gruel ( figure 1 ) ( 1,700 kcal and 72 g of protein ) and supply any intravenous aliments according to the amount of oral intake , which would provide 30% of the hospital food and the intravenous aliment maintenance to the patients . the hospital - supplied food consisted of soft and non - irradiated foods considering the patient 's stomatitis , and in the case of this particular patient , it was decided that close monitoring after dietary changes would be required . as a nutritional intervention for the second diagnosis , the patient was consulted for the need to consume a diverse range of foods and the hospital diet ( diabetic thin rice gruel and consider the patient 's preferences and lactose intolerance ) . this intervention would stop for the patient consuming cola , which could worsen the nausea , and try the hospital - supplied foods . the second screening visit was conducted on october 24 , 2013 , and physical measurements , biochemical data , medical examinations and treatments , nutritional physical examination data , and food / nutrition - related diet history were examined . on follow - up , there was no change in physical measurements , and examinations revealed the following : total protein / albumin , 5.1/3.1 g / dl ; hgb / hct , 8.5 g / dl/25.7% ; tlc , 712 cells / mm ; cholesterol , 135 mg / dl ; bun / cr , 19/1.0 mg / dl ; ca / p , 7.8/1.6 mg / dl ; na / k / cl , 138/50/107 meq / l ; blood glucose ( fasting , postprandial 2 hour glucose # 3 ) , 177/208/363/309 mg / dl ( 10/22 ) , 270/206/206/120 mg / dl ( 10/23 ) , and 126/133/260/246 mg / dl ( 10/24 ) . nutritional and physical examination data , included gastroenterological symptoms such as nausea and vomiting after consuming certain foods . for example , the patient had a vomit after drinking orange juice while apple juice did not cause any gastrointestinal trouble . vital signs were 135/80 mmhg . in terms of food / nutrition - related diet history , the patient 's dietary prescription and diet - related experiences were such that he was informed about diabetic thin rice gruels , non - irritating foods , and nutrient supplement drinks . the patient 's food and beverage consumption consisted of the supplied hospital diet : 20% of rice gruel , 50% of the steamed eggs and nutritious beverages , and 100% of the soup and apple juice ( energy , 1,050 kcal ; c : p : f ratio , 56.0 : 14.4 : 29.6 ; carbohydrate , 147 g ; protein , 37.2 g ; fat , 34.5 g ) along with intravenous aliments ( hepasol inj 500 ml / day ; 200 kcal , 50 g protein ) . after the first screening , the patient tried various foods and started learning to move in a wheelchair . the patient 's nutrient requirements were the same as at the first screening . upon nutrition monitoring and assessment , the goal set after the first screening with regard to nutritional intervention was partially achieved considering that the hospital food consumption was at 62% of the overall consumption and the intravenous aliment had changed ( nutriflex lipid peri eod to hepasol / d ) . after the second nutritional intervention , the patient stopped consuming cola and began to consume a diverse range of hospital foods ; thus , the goal of the intervention was reached . after the second screening , the patient again had two nutritional diagnoses : first , excessive protein supplementation , because extra protein was administered via the intravenous aliments outside oral intake , and the patient was consuming 125% the required protein quantity ; second , the patient 's lack of knowledge regarding food and nutrition due to absence of education about chemotherapy and food preparation . with regard to nutritional interventions , that for the first nutritional diagnosis involved advising the medical team to stop supplying protein via the intravenous aliments . for the second nutritional diagnosis , advising the medical team educate the patient about food choices and preparation which are appropriate for managing side effects of chemotherapy . the goal of the intervention was consumption of energy and protein > 70% of the requirement and maintenance or increase of body weight . continuous monitoring was required , and even after discharged , the patient required proper outpatient care . in this case , due to side effects following chemotherapy , the patient experienced significant and unintentional weight loss as a result of stomatitis , nausea , and vomiting and malnourished with poor ingestion . afterward , through nutritional interventions via the nutrition care process , the patient 's quantity of food intake was increased and further increases were expected under continuous care ( figure 2 ) . similarly , in earlier studies , individualized nutritional education effectively increased caloric and protein intake in cancer patients . thus , it is speculated that , had this patient also received a more active education regarding chemotherapy side effects and food preparation before and after his colon cancer surgery , poor ingestion caused by stomatitis and prevent weight loss after the second round of chemotherapy could have been treated easily . hereafter , to address this issue , all patients undergoing chemotherapy should be educated about practices that could enhance response to treatment and undergo continuous observation during treatment . additionally , with regard to this patient , it is regrettable that , despite the fact that repeated malnutrition was diagnosed during several admissions to the hospital during his second round of chemotherapy , the patient was not administered active care for malnutrition .

in this case study , the process of nutritional diagnosis and intervention conducted at a hospital on a malnourished patient who underwent treatment for a chronic illness ( chemotherapy for cancer treatment ) was recorded . the patient received his first round of chemotherapy for colorectal cancer , and then a second round after the cancer metastasized to the liver . the patient was malnourished and had experienced weight loss ( 17% loss in the most recent 3 months ) due to side effects of chemotherapy including stomatitis , nausea , and vomiting . nutritional diagnosis and intervention via the nutrition care process were implemented through two screening rounds , and the quantity of oral intake increased from 28% to 62% of the recommended daily intake . the patient required continuous monitoring and outpatient care after hospital discharge . it is speculated that if a more active patient education and dietary regimen with respect to chemotherapy side effects had been offered after the patient 's first chemotherapy cycle , it might have been possible to treat ingestion problems due to stomatitis during the second cycle of chemotherapy and prevent the weight loss . henceforth , patients receiving chemotherapy should be educated about nutrition management methods and monitored continuously to prevent malnutrition .

however , ingestion of excessive fluoride during tooth development can cause dental fluorosis.1 dental fluorosis is a deficiency in enamel mineralisation due to excessive daily fluoride intake during tooth development and its severity is directly related to the absorbed dose of this ion . since the dose - effect relationship is not precisely known , the dose of 0.07 mg f / day / kg of body weight has been accepted as the upper limit in terms of the clinically acceptable risk of dental fluorosis.2 the main sources of fluoride intake are fluoridated water , powdered milk reconstituted with fluoridated water , inadvertent ingestion of fluoridated toothpaste , inappropriate use of dietary supplements , as well as foods and beverages processed with fluoridated water.35 during infancy and childhood ( 3648 months ) , the fluoride intake in diet deserves special attention . this period coincides with the calcification of different stages of the developing permanent teeth crowns . this is also a critical time for ensuring that the optimal levels of ingested fluoride are not exceeded.3 several previous studies have determined the fluoride content of children s foods , such as milk,6,7 dinners and desserts811 and beverages.5,12,13 however , the fluoride concentration in many child addressed products remains unknown . thus , the aim of this study was to evaluate the fluoride ion concentration in some fermented milks present in the market . three lots of six different brands of fermented milks , with 80 g each bottle , were analyzed : parmalat-uva , chamyto , paulista , batavito , yakult , vigor club. the products were opened on the day of the analysis and 2 ml of each fermented milk was used in this experiment . fluoride concentrations were determined after overnight hexamethyldisiloxane ( hmds)-facilitated diffusion14 as modified by whitford , using a fluoride ion - specific electrode ( model 9409 , thermo electron corporation , beverly , ma , usa ) and a miniature calomel reference electrode ( accumet , # 13 - 620 - 79 : fischer scientific , pittsburgh , pn , usa ) , coupled to a potentiometer ( 290a , orion research inc . , boston , ma , eua ) . during the diffusion process , which was conducted at room temperature , the solutions in the nonwetable petri dishes ( j.prolab ind . , so jos dos pinhais , pr , brazil ) were gently swirled on a rotary shaker . fluoride standards ( 0.4 , 0.8 , 1.6 , 3.2 and 6.4 gf / ml ) were prepared by serial dilution of a stock - standard containing 100 gf / ml of fluoride ( orion 940907 thermo orion , beverly , ma , usa ) in triplicate and diffused in the same manner as the samples . comparison with identical non - diffused fluoride standards showed that recovery after diffusion was > 99% . the mean repeatability of the fluoride readings , based on the duplicate samples was 94.9% . fluoride concentrations ( gf / g ) in the different brands of fermented milk analyzed are shown in the table 1 . the fluoride concentration in the fermented milk of parmalat ranged from 0.022 gf / g to 0.031 gf / g , nestl from 0.228 gf / g to 0.272 gf / g , paulista from 0.182 gf / g to 0.220 gf / g , batavo from 0.028 gf / g to 0.030 gf / g , yakult from 0.115 gf / g to 0.206 gf / g and vigor from 0.808 gf / g to 1.171 gf / g . it is important to know all sources of fluoride ingestion that contribute to the total intake once the dental fluorosis is systemic caused by the excessive fluoride ingestion . although the exact relationship between the consumption of industrialized beverages and dental fluorosis is not clear their ingestion contribute for the total fluoride intake specially the high fluoride content products , which never always highlight its content in the labels . many studies have demonstrated that it is necessary to know the fluoride concentration of infant foods , foodstuffs and beverages to estimate the total fluoride ingestion by children.8,12,15 although the total fluorine intake from the diet is difficult to be precisely determined , it is clear that there is substantial variation on the intake of different foods , foodstuffs and beverages , and in the fluoride content of these products . since products are not required to have their fluoride content displayed , only a fluoride assay is possible to determine the dietary fluoride intake.3 all of the fermented milks analyzed in this study presented varied concentrations of fluoride , although none of the packages indicated that information . the optimal level of systemic fluoride intake where it is believed to be active against caries and is not related to the development of dental fluorosis is not accurately known . using rough estimative of the types and quantities of foods and drinks ingested and a technique less sensitive than the fluoride electrode to measure levels of fluoride , mcclure16 estimated that the average daily diet provided no more than 0.05 to 0.07 mg fluoride / kg body weight / day and that it did not exceed 0.10 mg fluoride / kg body weight / day for children aged 1 to 12 years . , most of the fermented milks analyzed presented low concentrations of fluoride , less than 0.3 gf / g . however , the products of the brand vigor showed higher fluoride concentration ranged from 0.808 gf / g to 1.171 gf / g . considering that the optimal level of fluoride intake ranges from 0.05 to 0.07 mgf / kg body weight , those analyzed products can contribute significantly to the total ingestion of fluoride increasing the risk of the dental fluorosis development . in this sense , the consumption of 80 g of a fermented milk containing 1.171 gf / g a day can contribute with about 0.008 mgf / kg body weight for a child weighing 12 kg ( approximately 2 years old ) . it is worth emphasizing that this dose ( 0.008 mgf / kg ) would be reached with the ingestion of only this beverage , without considering the other foods and beverages consumed during the day . several reports have been showed high fluoride concentrations in foods typically consumed by children , such as powdered milks , ready - to - drink juices and chocolate milks , cereals and snacks.3,5,6,19,20,21 the variation in fluoride concentrations among the lots is another issue that should be observed . in the present study , the lots of different brands presented variations ranged 16% to 44% . in the brand yakult , it was observed the largest difference among the analyzed lots . for beverages with low fluoride concentrations however , for products with high fluoride levels , such as the lot 2 of the brand vigor , this variation can contribute significantly for the total intake of this ion . on the other hand , the bioavailability of milk is another point to be considered . due to the high calcium concentrations in milk , there is a possibility of diminution of fluoride absorption from gastro - intestinal tract.22,23 thus , the calculations made for these products could be overestimated . milk is also rich in fats , what are known to increase the lag time of the food or beverage in the stomach.23 although milk interferes with the rate of fluoride absorption , it was demonstrated that 67 to 82% of total fluoride in milk is absorbed.24 another crucial factor when the association between fluorosis and infant foods is the critical period of fluoride exposure to develop fluorosis . enamel fluorosis can occur following acute or chronic exposure to fluoride during tooth formation assuming a significant relevance and emphasizing the importance of monitoring fluoride intake by little children . in this study the presence of fluoride could be observed in all of the fermented milks analyzed which can contribute with the total fluoride daily intake .

objectives : to evaluate the fluoride ion concentration in some fermented milks present in the market.methods:three brands of 6 fermented milks ( parmalat-uva , chamyto , paulista , batavito , yakult , vigor club ) were analyzed . fluoride concentration was evaluated after facilitated microdiffusion by hdms.results : parmalat products ranged from 0.022 gf / g to 0.031 gf / g , chamyto from 0.228 gf / g to 0.272 gf / g , paulista from 0.182 gf / g to 0.220 gf / g , batavito from 0.028 gf / g to 0.030 gf / g , yakult from 0.115 gf / g to 0.206 gf / g and vigor club from 0.808 gf / g to 1.171 gf / g.conclusions : the presence of fluoride could be observed in all of the fermented milks analyzed which can contribute with the total fluoride daily intake .

severe generalized recessive dystrophic epidermolysis bullosa ( rdeb - sev gen ) is an autosomal recessive disease with a poor prognosis that is characterized by generalized blistering of the skin and mucous membranes in response to slight mechanical stimulation . the main causes of death in patients with rdeb - sev gen are cutaneous squamous cell carcinoma , renal failure , infectious disease , and dilated cardiomyopathy [ 1 , 2 , 3 ] . long - term prognosis is especially dependent upon complications such as renal failure and infectious disease . when patients with rdeb - sev gen progress to end stage kidney disease ( eskd ) , hemodialysis with a permanent vascular catheter or peritoneal dialysis is introduced . however , it can be difficult to introduce hemodialysis with an arteriovenous fistula ( avf ) because of easy blistering and vulnerability of the skin due to the need for repeated puncture of the avf and the pressure required to ensure hemostasis after completion of a dialysis session . we herein present a patient with rdeb - sev gen in whom we introduced hemodialysis with a native avf who survived 4 years and 3 months from the introduction of hemodialysis . a 32-year - old man with rdeb - sev gen was referred to our nephrology department because of proteinuria , hematuria , and renal dysfunction . his clinical condition and pathological findings of skin biopsy led to a definitive diagnosis of rdeb - sev gen at 0 years of age . he had mitten deformities of his hands and feet , oral cavity blisters and scars , and low body weight ( 28 kg ) at the initial visit to our nephrology department . his hemoglobin was 6.8 g / dl , and serum creatinine was 4.7 mg / dl . over the next 3 months , although renal biopsy was not performed , end stage renal failure due to secondary amyloidosis was suspected with the finding of a high serum amyloid a level ( 1,250 g / ml ) . at the age of 32 , native avf anastomosis between the left median antebrachial vein and left radial artery was made for hemodialysis as the skin of his left elbow was dry and without blisters . because uremia had developed and he and his family hoped for hemodialysis instead of peritoneal dialysis , single - needle hemodialysis was introduced . the needle was fixed with tape on a bandage , taking special care not to tape his skin directly ( fig . astriction after removing the needle also took special care at the hemodialysis . in order not to create shear stress these ways of fixing the needle and pressing the site to remove the needle had not formed any blisters in his puncture site . he received hemodialysis therapy for 3.5 h thrice weekly and his kt / v was more than 1.4 . maintenance hemodialysis was performed with native avf for 21 months with no vascular access troubles related to infection or reduction in blood flow . unfortunately , his native avf was completely obstructed by thrombi after 21 months of hemodialysis . when percutaneous transluminal angioplasty failed , a permanent vascular catheter was selected for vascular access . two cuffed , single - lumen catheters , bio - flex tesior cath ( medcomp , harleysville , pa . , usa ) , were inserted into a subcutaneous tunnel under the left subclavian chest wall and placed in the superior vena cava ( fig . neither of his internal jugular veins was appropriate for the puncture because his neck was very short and had severe erosion . when catheter thrombi prevented blood removal , urokinase was introduced into the catheter 30 min prior to hemodialysis . when one catheter was obstructed , single - mode hemodialysis was performed using the other catheter . in the case of obstruction of both catheters due to thrombi , in one instance , we tried to insert a two - lumen catheter , the split stream otw catheter ( medcomp ) . however , erosion at the catheter exit site worsened and long - term use of that catheter was difficult . catheter - related infections were a frequent recurrence , and antibiotics were administered each time . the patient died 51 months after hemodialysis introduction because of cachexia related to the recurrence of cutaneous squamous cell carcinoma . in the present case of a patient with rdeb - sev gen , we highlight two clinically important issues . first , because his hemodialysis was initiated with his native avf , the frequency of vascular access - related infection or reduction of blood flow was reduced . to the best of our knowledge , this is the first report of a patient with rdeb - sev gen introduced to hemodialysis using a native avf . in these types of patients , peritoneal dialysis or hemodialysis with a permanent vascular catheter for blood purification have previously been reported [ 5 , 6 , 7 , 8 ] , but most reports discussed difficulty establishing avf or arteriovenous graft . moreover , in these patients , significant problems with repeated punctures and maintenance of hemostasis of the shunt after the establishment of avf persisted . it is very important not to create shear stress in the dermis during the puncture , adjustment of the needle , and while attempting to achieve hemostasis of the shunt . in our case , the frequency of vascular access troubles , such as infection or reduction of blood flow , was obviously low as a result of hemodialysis with a native avf . although hemodialysis with native avf is not applicable to all patients with rdeb - sev gen , it can be of great benefit to appropriate candidates . second , long - term effective dialysis management of eskd is possible in patients with rdeb - sev gen . iida et al . reported a patient with rdeb - sev gen who underwent hemodialysis with a permanent vascular catheter and survived for more than 4 years . although the prognosis for patients with rdeb - sev gen who undergo dialysis therapy is still unclear , the mortality of rdeb - sev gen patients who do not undergo dialysis therapy is very high . fine et al . reported 5 of 141 patients with rdeb - sev gen who died due to eskd , and the cumulative risk for death from renal failure was 12.3% . the cause of death in our case was cachexia due to the recurrence of cutaneous squamous cell carcinoma . with respect to renal failure , as a result of maintenance hemodialysis with native avf , and later a permanent vascular catheter , this patient survived for 51 months . maintenance hemodialysis with a native avf can be introduced to patients with rdeb - sev gen complicated by eskd . although it may not be initiated in all cases , infection related to the vascular access with a native avf appeared to be less frequent than with a permanent vascular catheter . hemodialysis with a native avf could favorably affect the prognosis for patients with rdeb - sev gen and eskd .

renal failure and infectious disease are strongly associated with morbidity and mortality in patients with severe generalized recessive dystrophic epidermolysis bullosa ( rdeb - sev gen ) . however , it is reportedly difficult to introduce hemodialysis with an arteriovenous fistula ( avf ) . we encountered a 32-year - old man with rdeb - sev gen in whom hemodialysis with a native avf was introduced that favorably affected his long - term survival . this patient eventually died because of cachexia related to the recurrence of cutaneous squamous cell carcinoma 51 months after hemodialysis introduction . we believe that in this patient , the frequency of vascular access troubles related to infection or reduction of blood flow was probably low as a result of hemodialysis with his native avf . thus , it seems likely that patients with rdeb - sev gen with end stage kidney disease who are on hemodialysis can be successfully managed with a native avf .

many types of cells of myeloid lineage including macrophages , but also microglia in the brain , kupffer cells in the liver , and bone osteoclasts , are considered to be tissue resident . the source and maintenance of these cells are either ongoing population from circulating monocytes with varying rates of turnover or in some cases by population from yolk sac derived progenitors and maintenance by self - renewal . microglia represent a well - defined example of these self - renewing , stable , tissue resident cells . renewal of resident macrophages is also from macrophage precursors that develop in the bone marrow , then enter into the blood stream as mature monocytes , and , finally , migrate into a specific tissue where they develop into a resident macrophage population . in many tissues they develop from primitive macrophages existing in the yolk sac or fetal liver and are maintained independently from bone marrow derived monocytes in the steady - state condition.4 , 8 , 9 , 10 however , by tracking macrophage development from birth to adulthood in the mucosa and lamina propria , it was observed that although primitive macrophages are present in the neonatal intestinal mucosa and lamina propria they are not maintained in adulthood.11 , 12 this important finding suggests that in contrast to other tissues , macrophages that reside in the gi mucosa and lamina propria of adult mice are replaced continuously by blood monocytes and that yolk sac this is reasonable given that mucosal and lamina propria macrophages are thought to monitor and respond to multiple factors at the interface between the organism and the luminal environment , and that these factors and responses are variable and can change rapidly . the resident macrophages of the muscle layers have not been studied in as much detail . these cells are abundant throughout the gi tract and express a limited number of phenotypic markers of activation.13 , 14 , 15 , 16 , 17 it is clear that monocytes can be detected in the muscularis propria layers of control mice , an immune cell infiltrate derived from monocytes can be detected after inflammation in animal models,18 , 19 and that varying numbers of macrophages can be identified by mannose receptor , c type 1 ( cd206 , mrc1 ) immunoreactivity in human gastric muscularis propria . our recent data have indicated that population of the gastric muscularis propria in macrophage - depleted , osteopetrotic ( op / op ) mice can occur by monocyte invasion and can produce both cd206-positive and cd206-negative macrophages . these studies all identified macrophages that express markers that are not present on resident macrophages in healthy , gi smooth muscle , such as those in the gi tract of nonobese diabetic ( nod ) mice when they are not diabetic . thus , muscularis propria macrophages are derived at least partially from monocytes and it remains to be determined whether some resident macrophages are the equivalent of microglia in the brain , derived from yolk sac progenitors and sustained by self - renewal . determining the fate and source of all macrophages in the muscularis propria is an important research opportunity for the future . the phenotype of macrophages in healthy tissues appears to be determined in part by the original monocyte progenitor from which the macrophages were derived as shown in figure 2 and supplemental poster . this is shown most clearly in op / op mice , which show reduced numbers of macrophages in most tissues . monocyte development and survival depends on colony - stimulating factor 1 ( macrophage ) ( csf1 ) , and op / op mice have an inactivating mutation in the gene encoding csf1.15 , 22 in mice , monocytes can be identified by cd115 immunoreactivity and can be divided into 2 different groups according to differences in lymphocyte antigen 6c ( ly6c ) expression . ly6c monocytes are associated with an inflammatory phenotype and differentiation into conventionally activated , proinflammatory m1 macrophages or inflammatory dendritic cells when they enter tissues . in the gi mucosa and lamina propria , ly6c monocytes develop an intermediate phenotype once inside the tissue , defined as chemokine receptor 1 ( cx3cr1 ) before they differentiate into mature macrophages.12 , 23 it takes 45 days for monocytes to acquire the phenotype of resident mucosal and lamina propria macrophages characterized by the expression of f4/80 , cd64 , major histocompatibility complex ii , cd11c , and cx3cr1 , and are associated with a slower rate of cellular turnover . cx3cr1 macrophages are characterized by high levels of proinflammatory cytokines such as interleukin ( il)6 , inducible nitric oxide synthase ( inos , nos2 ) , il1 , and tumor necrosis factor ( tnf).12 , 24 in contrast , cd115 , ly6c monocytes do not express inflammatory markers , are long - lived , and contribute to the resident macrophage population in gi mucosa.25 , 26 ly6c monocytes appear to be a precursor of a subset of resident cx3cr1 macrophages that are characterized by lower levels of c - c chemokine receptor type 2 ( ccr2 ) and cd62l ( l - selectin ) , and have an anti - inflammatory signature with increased levels of il10 and heme oxygenase-1 ( ho-1 ) and cd206 . these ly6c monocytes are a possible alternative to yolk sac progenitors as a source of resident macrophages in gi muscularis propria , but this has not been tested and it should be noted that the resident macrophages in the healthy muscularis propria express very low levels of ho-1 and cd206 in , for example , nondiabetic nod mice . furthermore , flow cytometry and immunohistochemistry indicate that macrophages in healthy mouse intestinal muscularis propria are a homogeneous population of major histocompatibility complex ii , cd11ccd103 cd11b expressing high levels of cx3cr1 . further research is necessary in understanding the source and fate of muscularis propria macrophages , with close attention to the strain of the mice under study . the immune systems in mice do vary widely by strain and some of the differences in macrophage phenotype and markers identified by different studies likely can be ascribed to differences in the genetic background strains used . in human beings , 3 monocyte populations have been defined based on cd14 and cd16 expression with distinct differences from mice in the markers and likely fate of those cells . for example , the equivalent of the mouse marker ly6c is the gr1 epitope in human monocytes , but although mouse ly6c monocytes are proinflammatory , gr1 + human monocytes release il10 and do not express proinflammatory markers . these differences are of great importance because to translate the large body of work performed in mice to understanding human health and disease , it is necessary to fully characterize human macrophages . one effort to achieve this was a comprehensive survey of markers conserved on macrophages cultured and differentiated from human and mouse blood monocytes and then validated in human lung tissue . an 87-gene transcriptome was identified that can be used as a basis for characterizing human macrophages in other tissues including the gi mucosa , lamina propria , and muscularis propria . in particular , transglutaminase 2 ( tgm2 ) is identified as a novel marker of alternatively activated , anti - inflammatory m2 macrophages that is as reliable as cd206 for tissue macrophages , but that also labels circulating monocytes with an anti - inflammatory phenotype , unlike cd206/mannose receptor c1 . it will be very interesting to see how tgm2 expression correlates with disorders of gastrointestinal function , especially if it proves that the levels of circulating tgm2-positive monocytes correlate with the numbers of tgm2 or cd206-positive tissue macrophages . changes in macrophage phenotype or activation of macrophages frequently are evident in response to disease or injury as discussed later for gi disorders . a variety of factors initiate signaling cascades that alter the expression of phenotypic markers in tissue macrophages in human beings and in animal models of disease . recent position pieces and reviews have moved beyond a strict division of macrophages into defined inflammatory vs anti - inflammatory , m1 vs m2 , or conventionally vs alternatively activated phenotypes.29 , 30 it is clear that the tissue , strain , and species under study and the injury or disease that activated the macrophages can produce many different outcomes . in the gi tract , specific enzymes and cytokines have been identified when the tissues are challenged by injury or disease ; these include inos , tnf , various interleukins and cytokines , ho-1 , arginase-1 , or cd206 . the association of these molecules with inflammation , repair , or suppression of inflammation is summarized in table 1 . some of these markers are reliable when studied in some species but not others ; for example , inos is expressed robustly in mouse macrophages in inflammation but is repressed epigenetically in human macrophages . arginase-1 and ym1 are examples of other proteins that are expressed in mouse but not human m2 macrophages . many of the markers are autocrine factors that feed back to increase or decrease the level of inflammation in injured tissues , or paracrine factors that support or repress the invasion and differentiation of cells in the affected tissue . il10 is an example of an autocrine factor that is released from and promotes cd206-positive , anti - inflammatory macrophages , while suppressing proinflammatory markers in other macrophages and immune cells.33 , 34 for example , in toxoplasma gondii ileitis in the mouse , prostaglandin e2dependent il10 production after accumulation of ly6c monocytes inhibits production of tissue - damaging tnf by neutrophils . we discuss later the changing balance in proinflammatory and anti - inflammatory markers in macrophages of the mouse gastric muscularis propria that leads to discrete temporal patterns of cellular injury during diabetes and the development of gastroparesis . the eventual phenotype of activated macrophages in disease also is determined significantly by the cellular origin of the cells . the underlying mechanisms can involve activation of monocyte invasion , expansion of the resulting macrophages in situ after monocyte invasion , or expansion of resident macrophages . for muscularis propria macrophages , these mechanisms have not been studied extensively . on the other hand , there have been many studies on mucosal and lamina propria macrophages in animal models of inflammation and human tissues . in the mucosa and lamina propria of c57bl6 mouse colon , dextran sulfate sodium induced inflammation initiates a tissue invasion of ly6c monocytes that expand into cx3cr1 macrophages , characterized by high levels of il6 , tnf , ccr2 , il1 , il12 , and il23.12 , 36 at the same time , the cx3cr1 population , representing the resident macrophages , does not change because it is the infiltrating monocytes that develop into a cx3cr1 phenotype . in fact , during the same period of time , the cx3cr1 population maintains its anti - inflammatory phenotype , characterized by high production of cd206 , cd163 , and transforming growth factor- receptor ii . ccr2 activation by monocyte chemoattractive protein-1 ( mcp-1 ) has an important role in ly6c monocyte accumulation in inflamed tissues as indicated in studies on the ccr2 knockout mouse showing reduced systemic inflammation and low levels of monocyte invasion in atherosclerosis , type ii diabetes , and obesity . in the gi tract , ccr2 knockout mice have reduced mucosal inflammation and damage after dextran sulfate sodium colitis and in rats , 2,4,6-trinitrobenzene sulfonic acid induced injury to the muscularis propria is reduced by mcp-1neutralizing antibodies . in surveying the complexities of macrophage activation , it is evident that the mechanisms and regulation of activation are not worked out in sufficient detail and certainly not for the muscularis propria . however , there are real opportunities for understanding and treating disease by examining these important cellular processes . macrophages have been observed in the muscularis propria in proximity to neuronal processes and interstitial cells of cajal ( icc ) , cells that are fundamental for the maintenance of normal gi function.13 , 16 , 41 in this context , these muscularis macrophages have been proposed not only to be immune regulators , but also to play a housekeeping role and be involved in the maintenance of normal motility in the healthy gut . with respect to iccs , muscularis macrophages are observed to be enveloped by and in close association with icc in healthy mouse and human gi tract , suggesting a possible role for them in the regulation of icc network organization and integrity . in disease , the expression of proinflammatory markers such as inos is associated with icc network damage , whereas expression of anti - inflammatory markers is associated with preservation of icc networks in diabetic mice with delayed gastric emptying ( see later ) . in disease , although macrophages clearly are activated or polarized in some manner , it is not clear whether unpolarized , resident macrophages also have a role in icc network maintenance . the interaction of macrophages with neurons is indicated by the variety of neurotransmitter receptors that are present on macrophages that functionally regulate macrophage phenotype including neurokinin receptors , glycine receptors , nicotinic 7 acetylcholine receptors , and p2 purine receptors.43 , 44 , 45 , 46 the converse also is true , neurons express receptors for macrophage - derived signaling molecules that alter neuronal activity and survival including bone morphogenetic protein ( bmp)2 and tnf. nitric oxide produced by inos in mouse ( but not human ) macrophages suppresses neuronal and smooth muscle excitability . thus , neuronal excitability can be impaired significantly when adjacent macrophages in the muscularis propria are activated , especially in disease ; as discussed in the section below on known roles of macrophages in gastrointestinal function and disease . muscularis propria macrophages seem to interact preferentially with only specific subtypes of neurons , namely those expressing choline acetyltransferase and neuronal no . because those subtypes of neurons appear to be reduced with age,50 , 51 , 52 as well as in particular pathologies affecting gi function , it has been proposed that macrophages play a potential role in the regulation of the enteric nervous system plasticity and development . in support of this conclusion , op / op mice that lack muscularis macrophages have more total enteric neurons compared with wild - type littermates , although it has been not reported whether this phenomenon affects all neurons or only specific subtypes . it will be important to further elucidate the role of macrophages in pathologies affecting the gi tract and characterized by neuronal loss . gut microbiota and their products can affect gi motility by interacting with key cells that regulate gi motility including enteric neurons , glial cells , enterochromaffin cells , and muscularis propria macrophages.27 , 54 , 55 the ability of different microbes and their products to modulate immune responses in the lamina propria of the gut has been studied extensively . intestinal macrophages play an important role in sensing microbial signals and produce il1 , required for rar - related orphan receptor -positive innate lymphoid cell driven granulocyte monocyte colony stimulating factor ( aka csf2 ) production , which in turn regulate regulatory t - cell numbers . in addition to the effect on immune homeostasis , recently the mechanisms by which gut microbiota interact with muscularis propria macrophages and potentially influence gastrointestinal motility have been identified . muscularis macrophages secrete bmp2 , which signals constitutively to enteric neurons through the bmp receptor . in turn , enteric neurons contribute to the maintenance of muscularis macrophages through secretion of the macrophage growth factor , csf1 . antibiotic treatment diminishes the expression of bmp2 and the number of macrophages , as well as signaling via the bmp receptor and expression of csf1 in neurons , and results in changes in gi transit . these results strongly suggest a regulatory role for gut microbiota on the enteric neurons by affecting the muscularis macrophages , however , further work is needed to elucidate the specific signaling pathways by which microbes or their specific products mediate this effect . gut microbiota also can influence gastrointestinal motility by signaling the host to increase serotonin ( 5-hydroxytryptamine [ 5-ht ] ) biosynthesis in enterochromaffin cells via fermentative end products such as short - chain fatty acids and bile acids.55 , 58 5-ht is a neurotransmitter that activates a large variety of receptors on cells in the muscularis propria and plays an important role in gastrointestinal motility . macrophages as a target of serotonin have not been explored in the gi tract but human alveolar macrophages express both 5-ht2b and 5-ht7 serotonin receptors and activation of those receptors increases the expression of anti - inflammatory markers and reduces the expression of proinflammatory markers . we are not aware of any data indicating expression of serotonin receptors on gi tract macrophages and this serves to emphasize that a detailed transcriptional profiling of subsets of muscularis propria macrophages such as that performed by martinez et al is necessary and would be revealing . although the earlier - described studies highlight the effect of gut microbiota on intestinal macrophages , future work will shed light on the role of gut microbiota / microbial products on potential cross - talk between macrophages and other key cell types involved in gastrointestinal motility such as enteric glia and iccs . studies on a variety of gi diseases and disorders have indicated that the activation of specific macrophage - mediated pathways in different subsets of cells results in either sustained impairment of tissue function and morphology or activation of potential mechanisms for repair and recovery of function . the extreme heterogeneity of tissue - resident macrophages during homeostasis and inflammation shows that a macrophage often is not just m1 or m2 when residing in a specific tissue,29 , 60 , 61 therefore it is important to characterize these signatures , determine the important effector molecules , their mechanism of effect , and the principal cellular targets . progress has been made by the study of several conditions in which a contribution of the innate immune system is either a part of , or the major mediator of , alterations in function . in this section ileus is a prolonged decrease in the rate of movement of intestinal contents that can be induced after sepsis or after abdominal surgery . the bacterial product , lipopolysaccharide , is sufficient to reproduce the symptoms of sepsis - induced ileus including macrophage activation and reduced gi transit and motility.62 , 63 cannon and murphy identified that it is handling of the intestine that results in postoperative ileus , and although modern surgical techniques have reduced the likelihood of the development of severe ileus , it continues to be a complication of intestinal surgery that significantly extends hospital stays for the affected patients and can be fatal.65 , 66 the underlying mechanisms for the development of ileus have been studied extensively because of the existence of reliable animal models that recapitulate the pathophysiological changes observed for ileus in human beings . for postoperative ileus , these animal studies have shown that an initial , neuronally mediated inhibition of motility67 , 68 and also mast cell degranulation is followed by a late inflammatory phase caused by activation of resident macrophages in the muscularis externa , which release chemoattractant molecules that in turn stimulate the population of the tissue with large numbers of monocyte - derived macrophages . tnf , il6 , mcp-1 ccl2 , il1 , and other cytokines and chemokines produced by these macrophages suppress motility by effects on smooth muscle and both intrinsic and extrinsic nerves . neurons are both the targets and the mediators of the macrophage response in postoperative ileus . stimulation of the vagal nerve after induction of ileus has been shown to reduce the release of proinflammatory cytokines and , consequently , inflammation as a result of the effect of released acetylcholine on 7 nicotinic receptors expressed on macrophages.70 , 71 this effect can be reproduced by using selective 7 nicotinic receptors agonists to treat gastric ileus . because vagal innervation is most dense in the stomach and proximal small intestine , vagal nerve activity is most likely to regulate inflammation in those regions . in the distal gi tract , the interaction of other cholinergic nerves with macrophages , including possibly intrinsic enteric motor neurons , is plausible but has not been shown . it also is not clear whether release of anti - inflammatory cytokines from m2-like macrophages are affected by vagal nerve stimulation in the gi tract , and it is also not clear whether all macrophages express 7 nicotinic receptors . this requires further research because it was reported that il10 , a potent cytokine derived from m2-like macrophages , which suppresses expression of inflammatory cytokines by m1-like macrophages , is necessary for recovery from postoperative ileus . thus , parallel mechanisms may be exploited for suppression of proinflammatory macrophages and up - regulation of anti - inflammatory macrophages in treatment of ileus . up - regulation of ho-1 in macrophages of the intestinal muscularis externa is one effective treatment for ileus that exploits the potent cytoprotective effects of ho-1 and its active product , carbon monoxide . indeed , inhaled carbon monoxide is effective in the treatment of postoperative ileus in mice . activation of glycine receptors to reduce proinflammatory cytokine production by macrophages and the resulting impairment of smooth muscle contractility is another effective way to reduce the degree of ileus after surgery in rats . the animal studies have shown that macrophage activation is central in the cascade of cellular and molecular events that result in ileus , and that modifying macrophage phenotype can ameliorate or treat the symptoms of ileus . furthermore , the process of studying postoperative ileus in animal models has identified interactions between macrophages and the other cells in the gi tract that are relevant to other gastroenteropathies as well as normal physiology and the development of the gi tract . these observations in animal models should be translatable to treatment of ileus in affected patients , but at present this has not yet been exploited fully . in inflammatory bowel disease ( ibd ) , macrophages and dendritic cells have been identified as homeostatic regulators of mucosal integrity and function by interacting with t cells and responding to t - cell activation . because inflammation in the intestinal mucosa represents one of the defining pathologies of ibd , the mucosa is the focus of research into understanding the causes and opportunities for treatment of these diseases . genetic risk factors that impair the mucosal barrier and exposure to microbes appear to determine whether inflammation occurs at the mucosal level , but how this results in changes of the macrophage activation in the muscularis , as well as changes in motility , have received limited attention . inflammatory changes in the mucosa are a consequence of activation of t cells with lamina propria dendritic cells and cx3cr1-positive macrophages playing a central role in these responses . the effectiveness of tnf-neutralizing antibodies ( infliximab , adalimumab , dolimumab , and so forth ) in the treatment of ibd indicates that proinflammatory cytokines released from macrophages , such as tnf , contribute to the disease . in addition , tnf neutralization induces cd206-positive , regulatory macrophages , which can increase t - cell apoptosis and contribute to mucosal healing . several powerful anti - inflammatory molecules are generated by regulatory or alternatively activated macrophages including transforming growth factor ( tgf)- and il10.84 , 85 tgf- signaling is tightly linked to the development of colitis . for example , in patients with ibd , the absence of an anti - inflammatory response to tgf- was associated with up - regulation of smad7 , a suppressor of tgf- signaling . il10 promotes immune tolerance , suppresses th1 immune responses , and appears to be a central determinant or gatekeeper of the pattern of immune responses . the importance of il10 signaling in the mucosa is indicated clearly by the severe colitis that develops in il10-receptor knockout mice . however , administration of recombinant il10 to patients with inflammatory bowel disease has not proved effective in treating the disease,88 , 89 possibly because il10 signaling is most important in preventing the initiation of inflammation rather than resolving established responses . in il10-receptor knockout mice , knockout of toll - like receptor signaling in myeloid differentiation primary response gene 88deficient mice prevents colitis , which supports the suggestion that il10 mediates initiation of inflammation in response to toll - like receptors . interactions between dendritic cells and t cells also depend on which subtypes of dendritic cells are examined because , similar to macrophages , dendritic cells can show diverse phenotypes . cd103-positive dendritic cells suppress colitis , whereas e - cadherin positive dendritic cells appear to promote inflammation via increased th17 responses . the persistence of gi dysmotility during and after infection or inflammation is a well recognized but incompletely understood phenomenon . it may be mediated partly by molecules generated by immune cells . in this respect , persistent symptoms during remission from ibd are similar to postinfectious irritable bowel syndromes in which symptoms persist after an infection and the accompanying macroinflammation has resolved.94 , 95 changes to enteric neurons , glia , icc , and smooth muscle have been reported,96 , 97 as well as effects on extrinsic nerves . however , although macrophage activation in the muscularis is known to occur after immune cell infiltration in both type 1 and type 2 immune responses , the role of cytokines that are derived from macrophages and persistently up - regulated postinflammation is not well defined . for example , there may be a residual maintenance of changes to cellular function and activity mediated by macrophage - derived factors . symptoms usually include early satiety , nausea , vomiting , and abdominal pain.100 , 101 , 102 gastroparesis affects people with diabetes , but also can be idiopathic or iatrogenic . because of the development of animal models of diabetic gastroparesis53 , 103 in which delayed gastric emptying developed in a subset of diabetic mice , it has been possible to explore the cellular changes in gastroparesis and translate those observations into studies on human tissues . as a result , there is a growing body of evidence that macrophages are involved in the cellular changes that are evident in gastroparesis.20 , 21 , 105 changes in cd206 macrophage populations have been correlated directly with the development of delayed gastric emptying in diabetic nod mice with the expression of ho-1 in those cells appearing to provide a mechanism for protecting icc when diabetes develops and neuronal no activity from neurons has been down - regulated . in human subjects with diabetic gastroparesis , cd206 cell numbers in the muscularis propria of the gastric body correlated with icc numbers,20 , 106 and icc loss is the cellular defect that most strongly associates with delayed gastric emptying . loss of the anti - inflammatory cd206 cells causes the cellular injury in diabetic gastroparesis because the direct damaging effects of m1-like macrophage cytokines , oxidative stress , high glucose , and insulin resistance were not being negated by the cytoprotective effects of anti - inflammatory cytokines and the ho-1 metabolite carbon monoxide generated by the cd206 cells.104 , 107 however , studies in the op / op , macrophage - depleted mouse also have determined that in the absence of any macrophages , diabetic op / op mice do not develop delayed gastric emptying and icc networks are preserved . thus , the cd206 macrophages that appear to express inos and are abundant in diabetic nod mice with delayed gastric emptying must be up - regulated to result in cellular damage and delayed gastric emptying . the source of these cells and mechanisms for up - regulation require further investigation and the results will need translating into studies in human gastroparesis . there are few effective treatments for gastroparesis but the evidence that active regulation of macrophage phenotype contributes to the development of delayed gastric emptying in mice and icc loss in human beings suggests that targeting the polarization of macrophages might be an effective therapy . indeed , in mice , up - regulating ho-1 in cd206 gastric muscularis macrophages reverses delayed gastric emptying in diabetic nod mice and delivering the product of ho-1 activity , which is co , has the same effect . il10 also has shown promise in this animal model by increasing the expression of ho-1 in the gastric body of diabetic nod mice and returning delayed gastric emptying , electrical slow - wave abnormalities , and icc network changes to normal . this is consistent with the known effects of il10 in suppressing proinflammatory macrophages and increasing anti - inflammatory macrophages . il10 is known to be safe for use in human beings and could be more effective in treating a disorder such as diabetic gastroparesis , in which macrophage changes appear to be a cause of the damage and functional changes to the tissue as opposed to inflammatory bowel diseases such as colitis in which a large variety of immune responses are causing the disease and il10 did not produce a clear clinical response . the recent demonstration that proinflammatory macrophages are necessary for the development of delayed gastric emptying in diabetic mice also suggests that neutralizing the proinflammatory cytokines generated by conventionally activated , m1-like macrophages may be another opportunity for treatment . in conclusion , macrophages are involved in many processes that control gastrointestinal motility in health and disease . in addition to being the mediators of responses to injury and disease , they appear to have roles in the development and regulation of cells in the healthy gi muscularis propria . this diversity of roles is reflected in many mechanisms for macrophage activation and up - regulation of various phenotypic markers . the responses and phenotypes are tissue- and disease - dependent and further understanding of these intricacies represents opportunities for better understanding of gi motility disorders and potentially treating the disorders .

there is an increasing awareness of the role of macrophages in the regulation and maintenance of gastrointestinal function in health and disease . this work has proceeded in the context of an increased understanding of the complex phenotypic variation in macrophages throughout the body and has shown previously unidentified roles for macrophages in diseases such as gastroparesis , postoperative ileus , and inflammatory bowel disease . opportunities for exploiting the phenotypic modulation of tissue resident macrophages have been identified as possible therapies for some of these diseases . in addition , macrophages are an established component of the innate immune system and can respond to variations and changes in the intestinal microbiome and potentially mediate part of the impact of the microbiota on intestinal health . we reviewed the latest work on novel concepts in defining macrophage phenotype , discuss possible mechanisms of action for tissue - resident macrophages in the gut , address the significance of microbiome effects on macrophage phenotype , and review the known and possible roles of macrophages in motility disorders of the gastrointestinal tract .

prolonged and unnecessary delay in tracheal extubation result in increased complication rates for patients receiving mechanical ventilation including airway trauma , chronic lung disease , ventilator associated pneumonia , and increased hospital costs . on the other hand - premature discontinuation carries a set of problems involving difficulty in establishing airways and compromised blood gas exchange . different methods , including clinical trials and calculated indices , have been developed to evaluate patients on mechanical ventilation and predict the optimum time to make the weaning decision . these methods include , tolerances of spontaneous breathing trials ( sbts ) , counting the respiratory rate , observation of work of breathing , and many other calculated indices such as the oxygenation index , measurement of the tidal volume and dynamic compliance , and the commonly used rapid shallow breathing index . however , some of these indices may be misleading , cost - effective , and requiring highly sophisticated equipments . recently , a tolerance of a spontaneous breathing trial while the patient receives varying levels of ventilatory support including continuous positive airway pressure ( cpap ) , low - level pressure support ventilation ( psv ) , or very recently automatic tube compensation ( atc ) is a new clinical test that has been considered an evidence - based strategy to predict successful weaning from assisted ventilation . the level of support may be relevant to whether the breathing trial is tolerated , because it has been argued that , for some patients , weaning failure may be attributable to the respiratory load imposed by the endotracheal tube . automatic tube compensation ( atc ) is a recent weaning mode of mechanical ventilation that has been developed to overcome the imposed work of breathing due to artificial airways . it delivers the exact amount of resistive load of the endotracheal tube for the flow measured at time , without affecting the patient 's breathing pattern . it potentially simulates spontaneous breathing without endotracheal tube , so it has been designated as electronic extubation . this mode of ventilation thus seems ideally suited for use during the weaning period . psv has been also widely used in the performance of a spontaneous breathing trial and has been shown to compensate for the additional work of breathing imposed by the endotracheal tube . however , some studies have shown that compared with psv , atc was more effective in overcoming the work of breathing necessary to overcome endotracheal resistance and resulted in more significant predictive values for successful weaning and extubation . pediatric and adult studies evaluating the efficacy of sbts have not systematically extubated patients who failed the breathing trial . therefore , the ability of a failed sbt to predict the need for ventilator support was not formally assessed , except in a previous study by chavez et al . in pediatric population . in our study , we assessed the sensitivity of both atc and psv in predicting extubation outcome , and we also assessed the outcome of failed sbts . the study was conducted in cairo university pediatric hospital ; pediatric intensive care unit , 9-bed capacity . the study period extended from may 2011 to february 2012 . in this patients were eligible for enrolment in the study if they met the following criteria judged by the intensive care doctors : ( 1 ) required mechanical ventilation for more than 24 hours ; ( 2 ) fulfilling weaning criteria , which was defined in our picu as follows : low ventilator rate [ 68 ] or less ; fraction of inspired oxygen ( fio2 ) 40 ; level of positive end expiratory pressure ( peep ) [ 3 , 4 ] ; improvement of the cause of respiratory failure ; oxygenation index ( oi ) ( [ mean air way pressure fio2]/pao2 ) < 5 ; the need for bronchial suction is 2 for the last 8 hours ; with stable vital , neurological and metabolic status . included the following : ( 1 ) duration of mechanical ventilation before enrolment is 24 hours or less ; ( 2 ) patients receiving high doses of sedations or vasoactive drugs ; ( 3 ) patients with disturbed conscious level despite improvement of lung pathology ; ( 4 ) patients who developed laryngeal edema after extubation ; ( 5 ) patients with pulse oxygen saturation < 90% , ph < 7.3 and paco2 > 50 mm hg during the trial . full history and data analysis including sex , age , weight , cause of mechanical ventilation , period of mechanical ventilation , length of stay in the picu , bronchodilators , pediatric risk of mortality score ( prism iii ) on day 1 admission , blood gases , pretrial oxygenation index , and ventilator setting parameters including : level of positive end expiratory pressure ( peep ) ; pretrial ventilator rate patients screened were randomly assigned in a blinded fashion with the use of opaque , sealed envelopes , to undergo two - hours spontaneous breathing trial with atc ( patients breathed through the ventilator circuit using continuous positive airway pressure of 5 cmh2o , fio2 less than 0.5 with the addition of atc 100% ; the atc group ) or psv ( patients breathed through the ventilator circuit using flow triggering and continuous positive airway pressure of 5 cmh2o , fio2 less than 0.5 , ps adjusted for endotracheal tube size ( ett ) ( ett size 3.03.5 = ps of 10 cmh2o ; ett size 4.04.5 = ps of 8 cmh2o ; ett size 5.0 = ps of 6 cmh2o ) ; the psv group ) . the spontaneous breathing trial was performed using the puritan - bennett 840 ventilator which compensate automatically for air leaks ( we do not use cuffed etts ) and was newly introduced in our picu ; previously used ventilator was newport e150 . the sbt was conducted by a respiratory therapist and nurse in the absence of the attending or other intensive care staff . physical signs including heart rate ; respiratory rate ; mean arterial blood pressure ; spontaneous expiratory tidal volume ( ml / kg / sec ) ; evidence of work of breathing ; increased frequency of suction ; pulse oxygen saturation blood gases were recorded during the trial . features of poor tolerance and weaning failure included respiratory rate outside the acceptable range for their age ; increase in heart rate of more than 20% with respect to baseline on mechanical ventilation ; increase or decrease in mean blood pressure of more than 20% of baseline ; signs of increased respiratory work ( i.e. , retractions , use of accessory respiratory muscles , paradoxical breathing ) ; pulse oxygen saturation < 90% and/or ph < 7.3 and/ or pco2 > 50 mm hg . when one of these findings occurred during the trial , the respiratory therapist terminated the trial to the previous ventilator settings . for patients with metabolic or respiratory acidosis during the trial , weaning criteria was revised with icu doctors , and these patients were excluded from the study . patients who passed the 2 hr trial were extubated by the respiratory therapist , and patients who failed the trial and were included in our study were recorded to be extubated within the next 24 hr by the intensive care doctors who were blinded to the results of the trial and the study aims . weaning was considered successful if reintubation was not required within 48 hr of extubation ( successful extubation group ) . failure to wean was defined as reintubation within 48 hr of extubation ( extubation failure group ) . informed consent was obtained from at least one parent or legal guardian for each patient before enrollment . the study design conformed to the revised helsinki declaration of bioethics and was approved by the scientific ethics committee of department of pediatrics , faculty of medicine of cairo university . data was analyzed using statistical package for special science software computer program version 16.0 ( spss inc . , categorical variables were expressed as number ( n ) , percent ( % ) and were compared using the chi - square test or fisher 's exact test , as indicated . continuous variables were compared using mann - whitney test and kruskal - wallis anova , as indicated . multivariate regression analysis was used to test the association between multiple quantitative and qualitative independent variables with the dependent variable . out of 47 screened patients , only 36 were enrolled in the study , 6 patients were excluded as they were reintubated due to laryngeal edema , and in the other 5 trial was terminated due to metabolic or respiratory acidosis during the trial . of the 36 patients enrolled in the study , 17 were weaned from mechanical ventilation on atc ( atc group ) and 19 were weaned on psv ( psv group ) . admission diagnosis of patients enrolled was as follows : lower respiratory tract infections ( n = 9 ) ; interstitial lung disease ( n = 3 ) ; postoperative ( n = 5 ) ; status epileptics ( n = 2 ) ; encephalitis ( n = 2 ) ; after arrest ( n = 1 ) ; history of poison intake ( n = 2 ) ; autoimmune diseases ( n = 2 ) ; guillain - barr syndrome ( n = 2 ) ; endocrinal disorder ( n = 1 ) ; septic shock ( n = 3 ) ; gastroenteritis and shock ( n = 2 ) ; myocarditis ( n = 1 ) ; immunodeficiency ( n = 1 ) . there were no significant differences between the atc and psv groups in any of the compared items . the course and outcome of the study population are summarized in figure 1 . in the atc group 11 of 17 ( 65% ) passed the sbt compared with 10 of 19 ( 53% ) in the psv group , but this difference was not statistically significant , ( p = 0.69 ) . out of 36 causes of reintubation were hypoxemia ( n = 5 ) , disturbed conscious level ( n = 3 ) , and new sepsis and pneumonia ( n = 4 ) . there were no significant difference in the causes of reintubation between the two groups ( p = 0.46 ) . mean length of stay in atc group was 20.9 14.4 , while in psv group was 21.35 11.8 with no significant difference ( p = 0.78 ) . our study showed that successful completion of the sbt had a greater predictive value for successful extubation than the predictive value of failed trial for extubation failure ; successful completion of the sbt on atc showed a 91% , sensitivity with a positive predictive value of 91% and specificity was 83% with a negative predictive value of 83% and accuracy 88% . while successful completion of the sbt on ps showed a 69% sensitivity for predicting successful extubation with a positive predictive value of 90% and specificity was 83% with a negative predictive value of 56% and accuracy 74% with no significant difference between the two groups ( p = 0.52 ) . table 3 shows the univariate analyses comparing patients who were successfully extubated and patients who failed extubation and reintubated there was a statistically significant association between reintubation and the following risk factors during the breathing trial : tachypnea , tachycardia , increased work of breathing , and failing atc trial . by stepwise multivariate logistic regression analysis of significant risk factors among all the study group ( no = 36 ) in the current study , we compared extubation outcome using 100% atc versus psv during a spontaneous breathing trial for two hours . we found that 9 of 19 ( 47% ) patients in the psv group failed the breathing trial compared to only 6 of 17 ( 35% ) patients in the atc group . this observed difference of 12% between both groups however did not reach statistical significance ( p = 0.69 ) . the positive predictive values were nearly similar in both groups ; 9 of 10 patients in the psv ( 90% ) passed the sbt and successfully extubated ( maintained extubation for > 48 hours ) compared with 10 of 11 ( 91% ) patients in the atc group . these findings are similar to a study conducted by cohen and his colleagues comparing atc with psv during a spontaneous breathing trial in adults . they found that patients who failed sbt in psv group were higher than those in atc group ; however the difference was not significant . they found that psv had a higher ppv predicting patients with successful extubation than atc ( psv , 85% versus 80% ) ; however , the difference was not significant ( p = 0.87 ) . in another study comparing atc with psv , the author found no significant difference in extubation outcome between the two groups ; however , he did find that half of the patients who failed a breathing trial with psv tolerated a subsequent trial with atc and were successfully extubated . these findings can be explained by the fact that atc may provide more complete support . this is supported by a previous study in which the authors assessed the accuracy of the compensation provided by psv and atc relative to the endotracheal tube - related pressure dissipation . they found that the difference between the theoretical pressure required to overcome the endotracheal tube resistive properties and the actual pressure delivered by the ventilator was lower and negligible when atc was applied during a spontaneous breathing trial when compared with psv . in our study , we found that rate of patients who failed the sbt in atc group and reintubated was higher than that of psv group . the negative predictive values for successful extubation were 83% for atc versus 55% for psv ; however , this difference was not statistically significant ( p = 0.52 ) . this may be attributed to small number of cases in each group in addition to the nearly similar accuracy of the two groups ( 74% for psv and 88% for atc ) . the reason for this low negative predictive value in psv group may be secondary to mechanical factors such as endotracheal tube discomfort , increased work of breathing caused by the augmented resistive force imposed by a small endotracheal tube , and inability to overcome this load due to in part the use of a relative low - level pressure in the psv group used in this study . pressure support level for each ett size used in this study defined by randolph et al . may need to be reevaluated in another study with a large population size . these findings were similar to a study conducted in pediatric patients by chavez et al . , who found that failed sbts , using a flow - inflating bag to provide a low constant pressure of 5 mmhg , did not accurately predict extubation failure ; however , this study did not compare the outcome of different pressure supported breathing trials . the reintubation rate for the whole studied patient was 33.3% which was higher than recent suggestions , where extubation rate of 15 to 29% implies an acceptable balance between performing premature extubation and unsuccessfully prolonged mechanical ventilation [ 1618 ] . the finding that a significant number of patients who successfully passed the sbts and extubated subsequently required reintubation merits further considerations . patients with higher severity scores of illness at admission and those with higher incidence of nosocomial infections are at increased risk of extubation failure . our relatively high incidence of reintubation rate could be in part due to the high incidence of nosocomial pneumonia and the development of new sepsis among patients under study , a finding which is consistent with other similar studies [ 1922 ] . a potential limitation to our study was the small number of the study population which was related to the low turnover rate due to prolonged length of stay of our patients ; median length of stay in the picu in psv group was 19 days ( 452 days ) , and median length of stay in the picu in atc group was 20 days ( 450 days ) . finally , the use of either atc or psv for prediction of extubation outcome in general icu populations was , reliable and did not require special monitoring or complex data collections . both have accepted positive and negative predictive values for successful extubation .

background . automatic tube compensation ( atc ) has been developed to overcome the imposed work of breathing due to artificial airways during spontaneous breathing trials ( sbts ) . objectives . this study aimed to assess extubation outcome after an sbt ( spontaneous breathing trial ) with atc compared with pressure support ventilation ( psv ) and to determine the risk factors for extubation failure . methods . patients ready for extubation were randomly assigned to two - hour spontaneous breathing trial with either atc or pressure support ventilation . results . in the atc group ( n = 17 ) , 11 ( 65% ) patients passed the sbt with subsequent extubation failure ( 9% ) . while in psv group ( n = 19 ) , 10 ( 53% ) patients passed the sbt with subsequent extubation failure ( 10% ) . this represented a positive predictive value for atc of 91% and psv of 90% ( p = 0.52 ) . five ( 83% ) of the patients who failed the sbt in atc group were reintubated . this represented a higher negative predictive value for atc of 83% than for psv which was 56% . none of the assessed risk factors were independently associated with extubation failure including failed trial . conclusion . atc was equivalent to psv in predicting patients with successful extubation . a trial failure in atc group is associated with but does not definitely predict extubation failure .

legionella pneumophila is a facultative intracellular pathogen that causes a severe form of pneumonia termed legionnaires disease.l . pneumophila is primarily found in freshwater environments where it infects a wide array of amoeba host . through adaptation to the amoeba host , l. pneumophila has evolved elaborate mechanisms to replicate within amoeba and human macrophages . upon inhalation of l. pneumophila - containing aerosols , l. pneumophila enters alveolar macrophages , where it evades the endocytic pathway and resides in a rough endoplasmic reticulum - derived vacuole referred to as the legionella - containing vacuole ( lcv ) . biogenesis of the lcv is governed by the dot / icm type iv secretion system that delivers 300 effector proteins into the host cell . this repertoire of effector proteins manipulates a myriad of host cellular processes including evasion of lysosomal fusion , modulation of host cell pro - apoptotic and antiapoptotic pathways , and acquisition of nutrients through manipulation of host cell proteasome machinery . loss of single translocated dot / icm substrates rarely causes a detectable defect in intravacuolar proliferation of l. pneumophila . however , intracellular replication in amoeba and human macrophages is dependent on the eukaryotic - like f - box effector protein ankb in the aa100/130b and the paris strain but not in the philadelphia - derived lp02 strain . despite the presence of at least four additional f - box effector proteins in l. pneumophila , ankb is dot / icm - translocated and is anchored into the lcv membrane through host - mediated farnesylation , where the f - box domain of ankb directly interacts with the host scf1 ( skp1/cul1/f - box ) e3 ubiquitin ligase complex on the lcv membrane , to promote lysine - linked polyubiquitination of the lcv . in human cells and amoeba , the lysine - linked polyubiquitinated proteins on the lcv are degraded through ubiquitin - dependent proteolysis by the proteasome , generating an increased level of free cellular amino acids above the threshold required as a source of carbon and energy for intracellular replication of l. pneumophila . many intracellular pathogens form a replicative niche within specialized vacuoles in the host cell . the proteomes of yersinia pseudotubercuolosis and salmonella typhimurium - containing vacuoles have been partially characterized from j774a.1 mouse macrophages . the lcv proteome of the jr32 philadelphia - derived strain of l. pneumophila has been generated using the mouse macrophage cell line raw 264.7 . in addition , the proteome of the lcv of the corby strain of l. pneumophila in the amoeba host dictyostelium discoideum has been profiled . the raw 264.7 mouse macrophage cell line is permissive for l. pneumophila despite originating from the balb / c mouse , whose primary macrophages are nonpermissive to l. pneumophila infection . importantly , the molecular bases of permissiveness of human and mouse macrophages to infection by l. pneumophila are distinct . therefore , it is important to determine the lcv proteome within human macrophages , which are the only known mammalian host for l. pneumophila . because of the essential requirement of the polyubiquitinated proteins on the lcv for intracellular replication , their identification can be valuable to decipher biogenesis and dynamic remodeling of the lcv . we determined the complete and the ubiquitinated proteome of the lcv in u937 human macrophage cell line for the wt aa100/130b strain and the isogenic ankb mutant . the intravacuolar proliferation defect of the ankb mutant is due to its inability to decorate the lcv with lysine - linked polyubiquitinated proteins , but the mutant is localized within an er - derived lcv that evades lysosomal fusion similar to the wild - type strain . therefore , the ankb mutant is a unique and useful genetic tool to analyze the total and ubiquitinated proteome of nonreplicative lcv that is not decorated with polyubiquitinated proteins and contrasts that with the replicative polyubiquitinated lcv of the wt strain . we identified 1193 host proteins localized to the wt strain lcv and 1546 on the ankb mutant lcv with metabolism proteins , in particular , phophatidylinositol and carbohydrate , cellular signaling , and protein transport are significantly represented . the ankb mutant lcv proteome contained > 80% of the wt strain lcv proteome . the additional 354 proteins on the ankb mutant lcv are primarily involved in transcription / translation and the immune response . of the 24 ubiquitinated proteins on the wt strain lcv , a large portion ( 25% ) are involved in immune response signaling and regulation ( interferon regulatory factor 7 and interleukin-1 receptor - associated kinase 1 ) , while proteins involved in transport and intracellular trafficking were also identified . in contrast , the ankb mutant lcv contained 29 ubiquitinated proteins primarily involved in signaling ( integrin beta-1 , beta-2 , and alpha-5 ) and vesicle trafficking ( rab1a , rab14 ) . the wt and ankb mutant ubiquitinated proteomes each contained p97 , tubulin , and the neutral amino acid transporter slc3a2 . further analysis of the identified nature of native ubiquitinated host proteins on the lcv within human macrophages could prove valuable to determine how l. pneumophila exploits human macrophages . l. pneumophila strain aa100/130b ( atcc baa-74 ) and the isogenic ankb mutant were grown on bcye agar plates for 3 days at 37 c prior to use in infections as described previously . a total of 6 10 u937 macrophages were plated in t175 cm flask in 60 ml of rpmi media supplemented with 10% fbs . at a multiplicity of infection of 50 bacteria per cell , legionella - containing vacuoles were formed by internalization of bacteria diluted in 20 ml of rpmi media . internalization of the bacteria was performed for 30 min at 37 c under 5% co2 . cells were washed three times in pbs and then incubated in growth media for 4 h at 37 c under 5% co2 . after 4 h , cells were washed three times in pbs ( 4 c ) and scraped into a 50 ml screw cap centrifuge tube and pelleted at 4 c for 5 min at 450 g . cells were resuspended in homogenization buffer ( 250 mm sucrose , 20 mm hepes / koh ( ph 7.2 ) + 0.5 mm egta ( ph 8.0 ) and pelleted by centrifugation at 675 g for 6 min at 4 c . cells were then resuspended in homogenization buffer with protease inhibitors ( roche cocktail ) at 2 10/ml . cells were lysed with a dounce homogenizer on ice and visualized under light and confocal microscopy to ensure effective cell lysing and legionella vacuole integrity . whole cells and nuclei were then pelleted in an1.5 ml tube for 3.5 min at 344 g . the supernatant was placed in a new 1.5 ml tube and centrifuged for 3.5 min at 344 g , resulting in the postnuclear supernatant ( pns ) . the sucrose solutions for the gradient were made in w / v in 20 mm hepes / koh ( ph 7.2 ) . the sucrose gradient was made by layering the pns ( 39% sucrose ) onto 2 ml of 55% sucrose layered onto 1 ml of 65% sucrose in a 14 mm 89 mm beckman ultracentrifuge tube . we then layered 2 ml of 10% sucrose onto 2 ml of 25% sucrose solution onto the pns . the sucrose gradient was centrifuged for 1 h at 100 000 g at 4 c in a swinging bucket rotor ( beckman sw41 ) . the lcvs were isolated from the 5565% interface using a 16 g needle and not disturbing any other fraction . lcvs were placed into 10 ml of pbs ( 4c ) and centrifuged at 40 000 g ( sw41 ) for 30 min at 4 c . pelleted lcvs were solubilized in 1% triton x-100 in pbs for 30 min on ice . following centrifugation at 10 000 g for 5 min to pellet bacteria , the supernatant containing eukaryotic proteins associated with the lcv was stored at 80 c . isolated lcvs were plated onto 24-well coverslips pretreated with poly - l - lysine and allowed to adhere for 1 h. extravacuolar l. pneumophila were labeled with a rabbit anti - l . pneumophila antibody prior to permeabilization for 1 h. lcvs were then permeabilized with methanol ( 20 c ) for 5 min , blocked with 3% bsa for 1 h , and then labeled with a mouse anti - l . pneumophila antibody for 1 h. secondary antibodies used were alexa flour goat antimouse 488 and alexa flour goat antirabbit 555 . polyubiquitinated proteins present on the lcvs were labeled with a mouse antipolyubiquitin antibody ( enzo life sciences ) for 1 h prior to vacuole membrane permeabilization and visualized with alexa flour goat antimouse 488 . for mass spectrometry analysis , tca - precipitated proteins were resuspended in 8 m urea and 50 mm tris ph 8.5 . resuspended peptides were diluted 1:1 with 50 mm tris ph 8.5 to lower the urea concentration to 4 m prior to digestion with endoproteinase lys - c ( 10 ng / ul ) for 8 h. digestions were further diluted 4:1 with 50 mm tris ph 8.5 to lower the urea concentration to 1 m urea prior to digestion with trypsin ( 5 ng / ul ) for 8 h. peptides were desalted using a 50 mg tc18 sep - pak ( waters ) . ubiquitinated peptides were immunoprecipitated using ubiquitin motif immunoaffinity beads ( cell signaling technology ) , as recommended by the manufacturer with the following modifications . desalted peptides were dissolved in 1.01.4 ml of iap buffer ( 50 mm mops - naoh , ph 7.2 , 10 mm na2hpo4 , 50 mm nacl ) . the peptide solution was cleared by centrifugation for 510 min at 13 200 g . the supernatant was transferred to a new tube and cooled on ice for 10 min . the cooled peptide solution was transferred to the tube with the antibody beads and incubated on a rotator at 4 c for 60 min . the beads were washed four times with 1 ml of iap buffer , followed by one wash with 1 ml water . the peptides were eluted from the beads by adding 55 l of 5% formic acid ( fa ) , mixed , and incubated at room temperature for 10 min . the beads and elution buffer were transferred to a teflon spin column , and the eluate was collected by centrifugation . the beads were washed with 45 l of 0.1% tfa and combined with the first eluate . steve gygi , dr . ryan kunz , and ross tomaino at the harvard university taplin mass spectometry facility , mass spectrometry data were collected on an orbitrap fusion mass spectrometer equipped with an easy nano - lc 1000 for sample handling and liquid chromatography . peptides were separated on a 75 m 30 cm hand - pulled fused silica microcapillary column with a needle tip diameter less than 10 m and packed with 1.8 m 120 gp - c18 beads from sepax technologies . the column was equilibrated with buffer a ( 3% acn + 0.125% fa ) . an equal amount of proteins for all samples with 2 g / each was loaded onto the column at 100% buffer a. separation and elution from the column were achieved using a 90 min 325% gradient of buffer b ( 100% acn + 0.125% fa ) . survey scans of peptide precursors from 400 to 1400 m / z were performed at 120 k resolution ( at 200 m / z ) ; agc , 50k ; max injection time , 100 ms ; monoisotopic precursor selection turned on ; charge state , 26 ; dynamic exclusion , 45s with a 10 ppm tolerance . tandem ms was performed in top speed mode ( 2 s cycles ) starting with the most intense precursor having an intensity greater than 5k . parent ions were isolated in the quadrupole ( 0.7 m / z isolation window ) . collision - induced dissociation was performed in the ion trap with a rapid scan rate ; 35% collision energy ; agc , 10k ; max injection time , 35 ms ; parallelizable time was turned on . a suite of in - house software tools were used for .raw file processing , controlling peptide and protein false discovery rates , and assembling peptide level data into protein level data . the in house software refers to software used in the gygi laboratory . the ms / ms spectra were searched using the sequest algorithm against a composite protein database consisting of all protein sequences from the uniprot human database ( 88 501 proteins ) along with nonsense and common contaminating proteins ( 111 proteins ) in both the forward and reverse direction . sequest parameters used to search the ms data were : precursor tolerance , 50 ppm ; fragment ion tolerance , 1 da ; fully tryptic ; 2 missed cleavages ; variable modifications of oxidized methionine ( 15.9949 da ) , alkylation of cysteine ( 57.0214 ) , and diglycine motif on lysine ( 114.0429 ) . the peptide - level false discovery rate was restricted to < 1% by using linear discriminate analysis based on several different sequest parameters including xcorr 1.0 , deltaxcorr , charge state , and a minimum peptide length of seven amino acids . the localization score is based on the ascore algorithm , where a localization score 19 indicated > 99% certainty in site localization . protein identifications for the complete proteome of the wt strain and ankb mutant strain are based on identification of at least two unique peptides . the proteome list was obtained from analyses of two lcv samples of the wt strain and the ankb mutant , and only the proteins that were reproducible in both samples were included in our analyses . metacore by thomson reuters is an integrated software suite that is available online for functional analysis of various aspects of screening data . metacore software determines the most significant relationships among the proteins analyzed such as pathway maps , go processes , process networks , and metabolomic networks . for our analysis , the software determined the most significant relationships shared among the wt strain lcv proteome and a separate analysis for the ankb mutant strain . the excel spreadsheet containing the complete wt strain lcv proteome and the ankb mutant stain lcv proteome were separately uploaded into the start page , and an enrichment analysis workflow was generated . to determine the identity of host proteins on the lcv , we infected u937 human macrophages with l. pneumophila wt strain aa100/130b or its isogenic ankb mutant strain . the ankb mutant strain evades lysosomal fusion and is localized within an er - derived lcv , similar to the wt strain . however , the ankb mutant strain fails to replicate within human macrophages and amoeba due to the levels of cellular amino acids being below the threshold needed as the major source of carbon and energy to support intravacuolar proliferation of l. pneumophila . the u937 human macrophage cell line is widely used in studies on legionella - human macrophage interaction and was used in this study instead of primary human monocytes due to the need of a large number of cells to isolate lcvs , because only 20% of the cells become infected . following a 4 h infection of 0.6 10 u937 macrophages by each of the strains , the lcvs were purified according to previously reported protocols with minor modifications . cells were lysed through dounce homogenization , and the pns was used to isolate lcvs through density ultracentrifugation on a discontinuous sucrose gradient , which resulted in purified lcvs at the 5565% interface ( figure 1a ) . to ensure vacuole integrity following purification , isolated lcvs were evaluated using confocal microscopy after differential membrane permeabilization as well as vacuole marker staining to ensure the lcv membranes were intact . vacuoles were labeled with a polyclonal anti - legionella antibody prior to vacuolar permeabilization , which resulted in 20% of bacteria being labeled , while 100% of bacteria were labeled after vacuolar membrane permeabilization , indicating that the lcv membrane is intact on 80% of the isolated lcvs ( figure 1b ) . we next evaluated the presence of polyubiquitinated proteins , which showed that 70% of isolated lcvs were decorated with polyubiqutinated proteins ( figure 1c ) . isolated lcvs were solubilized in 1% triton x-100 , and the eukaryotic proteins associated with the lcv were identified by high - throughput liquid chromatography coupled to tandem mass spectrometry ( lc ms ) . a positive protein identification for the proteome was based on at least two unique peptides . although numerous proteins were identified , it is likely that some proteins present with scarce quantities on the lcv are not detectable by our analyses . u937 macrophages were infected with wt l. pneumophila or the isogenic ankb strain at an moi of 50 for 30 min washed and the infection proceeded for 4 h. cells were lysed through dounce homogenization , and the postnuclear supernatant was used to isolate lcvs through density ultracentrifugation on a discontinuous sucrose gradient . ( a ) diagram of the sucrose gradient showing the isolated lcvs at 5565% interface and ( b ) confocal microscopy of isolated lcvs labeled with mouse anti - l . ( c ) confocal microscopy of isolated lcvs labeled with rabbit anti - l . the proteome of wt strain aa100/130b lcv contained 1193 eukaryotic proteins , while the lcv of the ankb mutant strain contained 1546 eukaryotic proteins ( suppl . we profiled the proteins according to various cellular functions , including transcription / translation , vesicle trafficking , immune response , ribosomal proteins , ubiquitination , proteasome machinery components , signaling , cytoskeleton arrangement , and metabolism ( figure 2 ) . using these categories we identified the largest proportion of wt strain lcv proteins to be involved in metabolism ( 21% ) while cytoskeleton arrangement ( 12% ) , signaling ( 12% ) , and transcription / translation ( 11% ) were highly represented ( figure 2 ) . proteins involved in immune response ( 8% ) , ribosome machinery ( 6% ) , vesicle trafficking ( 6% ) , and ubiquitin - dependent proteolysis ( 6% ) were significantly represented among the wt strain lcv proteome ( figure 2 ) . despite the presence of additional proteins on the ankb mutant lcv , the overall distribution of the proteins based on cellular function was very similar to the wt strain lcv proteome ( figure 2 ) . the large numbers of shared proteins on isolated lcvs of wt strain and the ankb mutant were consistent with the findings that the ankb mutant strain is localized in an er - derived lcv that evades lysosomal fusion but lacks sufficient levels of amino acids for intravacuolar proliferation . the ankb mutant lcv contained 80% of the proteins on the wt strain lcv with an additional 354 proteins primarily involved in regulation and initiation of translation , transcription , apoptosis , and immune response signaling . it is likely that degradation of lysine - linked polyubiquitinated proteins on the wt strain lcv renders them undetectable by proteomic analysis . the degradation of lysine - linked polyubiquitinated proteins on the wt strain lcv also alters the relative abundance of identifiable proteins in the proteome of the two strains . the 1193 eukaryotic proteins localized to the wt strain lcv and the 1546 localized to the ankb mutant lcv identified by high - throughput lc ms were grouped according to their cellular function according to the uniprot and genecards database sets . proteome data of the wt strain lcv regarding metabolism identified proteins involved in neutral amino acid transport ( slc1a5 , slc38a2 , slc3a2 ) , cationic amino acid transport ( slc7a1 ) , and monocarboxylate transport ( slc16a1 , slc16a3 ) as well as a variety of atpases involved in calcium , sodium , and potassium transport . a large proportion of metabolism - related proteins are involved in carbohydrate metabolism , especially glycolysis or glucose transport ( 7% ) , while 5% are involved in lipid metabolism . both the ankb mutant and wt strain lcv proteomes harbor phosphatidylinositol phosphatases ( inpp5d , inppl1 ) , kinases ( pi4ka ) , phosphatidylinositol 3,4,5-trisphosphate - dependent gtpase - activating proteins ( arap1 , asap1 ) , and guanine exchange factors ( swap70 , prex1 , def6 ) ( suppl . there are also proteins that bind to membranes or vesicles enriched in phosphatidylinositol that function in early endosomal trafficking ( clint1 , rufy1 ) and receptor - mediated endocytosis ( epn1 ) . consistent with the findings that the lcv of the wt strain and the ankb mutant strain is rer - derived , the proteome data revealed 7080 ( 6% ) 40s and 60s ribosomal associated proteins present on wt and ankb mutant lcvs ( figure 2 ) . our data identified 6 to 7% of the wt , and ankb mutant lcv proteomes play a role in vesicular trafficking , which included eight small rab gtpases ( rab1 , rab2 , rab5 , rab6 , rab7 , rab10 , rab11 , rab14 ) as well as four sorting nexins and multiple adp ribosylation factors involved in endocytic recycling , vesicle transport , and vesicle budding ( suppl . the ankb mutant lcv had four additional rab gtpases ( rab8 , rab13 , rab27 , and rab35 ) ( suppl . the high similarity in ribosomal and vesicular trafficking proteins in wt and ankb mutant lcv proteomes is very consistent with the findings that biogenesis of the ankb mutant lcv is very similar to that of the wt strain . proteins involved in apoptosis and immune responses , which comprise 8% of the wt strain lcv proteome , include multiple major histocompatibility complex proteins , ligands , and receptors involved in t - cell adhesion ( intercellular adhesion molecule 1 , 3 ) and signaling ( leukocyte - associated immunoglobulin - like receptor 1 , hcls1 binding protein 3 ) , promoters of cell apoptosis ( bcl2-associated athanogene 6 , caspase recruitment domain family member 6 ) , inhibitors of apoptosis ( defender against cell death 1 , fam129b ) , and tnf- regulators and proteins , which promote lps - induced tnf- production ( thymocyte selection associated family member 2 ) ( suppl . the proteome of the ankb mutant lcv contained 84 of the 92 immune response proteins on the wt strain lcv with an additional 42 proteins . these include seven clusters of differentiation molecules ( cd109 , cd47 , cd58 , cd63 , cd82 , cd83 , cd84 ) , two bcl2-associated proteins ( bag3 , bax ) , and two negative regulators of apoptosis ( tnfaip8 and aven ) ( suppl . proteins involved in cellular signaling pathways on the wt strain and the ankb mutant lcv include tyrosine - protein kinases that play a role in the response to environmental stress and cytokines such as tnf- ( map4 ) , regulation of cell growth , differentiation , migration and the immune response ( csk ) , cytoskeleton remodeling in response to extracellular stimuli , cell motility and receptor endocytosis ( abl2 , arap1 ) . additional signaling proteins included rho gtpase activating proteins ( arhgap1 , arhgap17 , arhgap18 ) , rab gtpase activating proteins ( tbc1d10b , tbc1d15 , tbc1d5 ) , and guanine nucleotide - binding proteins ( g proteins ) . the wt strain and ankb mutant lcv proteomes contained proteins involved in cytoskeletal membrane integrity and organization ( actin , coronin ) and proteins that regulate actin and microtubule polymerization ( arp2/3 complex , kank1 , lasp1 ) . interestingly , molecular chaperones that play a role in the folding of actin and tubulin ( cct2-cct8 ) were present on the lcv , possibly indicating that following initial lcv formation various cytoskeleton proteins are constantly recruited to the lcv membrane . validation of many of the proteins detected in the proteome of the lcv in our analyses comes from many published studies that showed that at least 17 of the proteins identified in our proteome have been already shown to be localized to the lcv using different strategies , such as confocal microscopy ( suppl . the wt strain and ankb mutant lcv contain a significant portion ( 6% ) of proteins involved in ubiquitination and proteosomal degradation ( figure 2 ) . in agreement with findings of colocalization of skp1 and p97 with the lcv by confocal microscopy , our proteome data identified the skp1 component of the scf e3 ubiquitin ligase complex and the atpase p97 on the lcv ( suppl . in addition , both proteomes contained rad23a and rad23b , which serve as multiubiquitin chain receptors that bind to the 26s proteasome and deliver lysine - linked polyubiquitintated proteins for proteasomal degradation . four e2 ubiquitin conjugating enzymes and eight e3 ubiquitin ligases were identified on the wt strain lcv ( table 1 ) . the e3 ubiquitin ligases identified on the wt strain lcv regulate apoptosis , nf- activation , and ifn- production , while the e2 ubiquitin conjugation enzymes catalyze the synthesis of lysine and lysine - linked polyubiquitin chains ( table 1 ) . the eight ubiquitin - specific peptidases on the wt strain lcv can act as deubiquitinases ( vcpip1(p97 ) , usp14 , usp15 , usp9x ) , which are able to remove ubiquitin moieties from polyubiquitin chains , which prevent proteasomal degradation , or are involved in lysine - linked polyubiquitination disassembly ( usp5 ) ( table 1 ) . the ankb mutant lcv contained 9 e3 ubiquitin ligases of which three were found on the wt strain lcv ( table 1 ) . the e3 ubiquitin ligases on the ankb mutant lcv are involved in regulation of dna , p53 activation , and mtorc1 signaling pathway . the ankb mutant lcv contained four additional ubiquitin - conjugating enzymes including two atypical ubiquitin - conjugating enzymes ( ube2h , ube2o ) and ube2 m , which catalyze the attachment of the ubiquitin like protein nedd8 onto other proteins ( table 1 ) . bioinformatic analysis of the lcv proteome was performed using metacore by thomson reuters software . metacore software determined the most significant relationships among pathway maps , process networks , go processes , and metabolomic pathways from eukaryotic proteins localized to the lcv . the top pathway maps identified in wt and ankb mutant lcv proteomes include regulation of the actin cytoskeleton by rho gtpases , the role of pka in cytoskeleton remodeling , and integrin - mediated cell adhesion . signaling pathways including g - protein signaling - rhoa regulation pathway and g protein- 12 signaling pathways were also identified in the proteome pathway enrichment analysis . the generated pathway maps correlate with the proteome data that reveal a large proportion ( 12% ) of lcv proteins are involved in cytoskeletal formation of the lcv . bioinformatic analysis of metabolic networks in the wt strain lcv proteome revealed the phosphatidylinositol-4,5-diphosphate pathway and phosphatidylinositol-3,4,5-triphosphate pathway as two of the top five ( figure 3 ) . these data reflect the aforementioned results of a wide array of phosphatidylinositol - dependent proteins in the wt strain and the ankb mutant lcv proteomes . this is also consistent with findings that have shown the lcv is decorated with phospatidylinositol-4 phosphate ( pi(4)p ) to which multiple effector proteins ( sidc , sidm , sdca , ridl ) bind on the lcv membrane . carbohydrate metabolism ( glycolysis , gluconeogenesis , and glucose transport ) was the third most abundant metabolomic network identified , while valine , tryptophan , and methionine synthesis and transport were identified in the top eight metabolic pathways represented in the lcv proteome ( figure 3 ) . the complete wt strain lcv proteome was analyzed through metacore software , using the enrichment analysis function , to identify the most significant metabolic networks . when proteins found only on the wt strain lcv but not on the ankb mutant lcv were analyzed through metacore many of the pathways were consistent with the total wt strain lcv proteome . the finding that the ankb mutant proteome contained 80% of the proteins present in the wt strain lcv proteome is consistent with the metacore analysis of very similar pathways in both of the proteomes . interestingly , ubiquitin - proteosomal proteolysis was identified as the second most abundant process network involved in wt strain lcv - specific proteins despite the findings that the ankb mutant lcv proteome contains a majority of the proteins found in the wt strain lcv proteome . these results are consistent with the requirement of polyubiquitination of the wt strain lcv for intracellular replication . to identify ubiquitinated proteins on the wt and ankb mutant lcv , the solubilized lcv proteins were immunoprecipitated with antiubiquitin antibodies and identified by lc ms . we identified 24 ubiquitinated proteins on the wt strain lcv such as annexin a2 , plasminogen activator inhibitor , rab5 gdp / gtp exchange factor , and transitional endoplasmic reticulum atpase ( p97 ) ( table 2 ) . the polyubiquitinated proteins identified on the lcv were lysine- , lysine- , lysine- , and lysine - linked as evident from the findings that ubiquitin was ubiquitinated on the four lysine residues k , k , k , and k. interestingly , 6 ( 25% ) ubiquitinated proteins play a role in the immune response such as interleukin-1 receptor - associated kinase 1 and interferon regulatory factor 7 , which play a key role in the signaling and regulation of the immune system against pathogens . furthermore , we identified two amino acid transporters ( slc3a2 and slc1a4 ) and a sodium bicarbonate transporter ( slc4a7 ) to be ubiquitinated on the wt strain lcv . we analyzed these proteins through the mammalian ubiquitination site database , which contains a comprehensive list of all ubiquitination sites on mammalian proteins . the search revealed that 17 of the identified ubiquitinated proteins have been shown to be ubiquitinated on the same lysine residues , as identified in our lc ms analyses . however , three of the ubiquitinated proteins identified ( plasminogen activator inhibitor 2 , mast cell - expressed membrane protein 1 , isoform 13 of sodium bicarbonate cotransporter 3 ) were not in the database , while four ( vimentin , target of myb protein 1 , receptor - type tyrosine - protein phosphatase c , transient receptor potential cation channel subfamily v member 2 ) were in the database but have been previously shown to be ubiquitinated on other lysine residues than the ones we identified in our analysis . the ankb mutant lcv contained 29 ubiquitinated proteins primarily involved in signaling ( integrin beta-1 , beta-2 and alpha-5 , rho gdp - dissociation inhibitor 2 ) and ubiquitination ( cullin-5 , the e3 ubiquitin ligase laptm5 , and the e2 ubiquitin - conjugating enzyme e2n ) ( table 3 ) . the ankb mutant lcv had multiple ubiqutinated transporters , including the monocarboxylate transporter ( slc16a3 ) , the glucose transporter ( slc2a3 ) , and neutral amino acid transporters ( slc3a2 , slc1a5 ) as well as 2 atpases ( atp2b4 and atp13a3 ) ( table 3 ) . localization score19 is>99% certainty wt strain lcv - specific ubiquitinated proteins . while the wt strain and ankb mutant lcv had some common ubiquitinated proteins ( p97 , slc3a2 , tubulin ) , there was a significant difference in the ubiquitinated proteome among the two strains , as can be seen by the bolded proteins in the wt strain ubiquitinated proteome table . the ankb mutant lcv had multiple ubiquitinated proteins involved in intracellular trafficking ( rab1a , rab14 ) and two gtpases ( rac1 , rhog ) , while the wt strain contained only one ubiquitinated protein involved in intracellular trafficking ( rab5 gdp / gtp exchange factor ) . proteasomal degradation of lysine - linked polyubiquitinated proteins on the wt strain lcv may explain the major differences in the modified host proteins between the two strains . during the past several years characterization of the lcv , through cellular and biochemical studies , has provided some insights into how l. pneumophila form a replicative niche in the host cell . , we sought to identify the total lcv proteome as well as the ubiquitinated proteins present on the lcv within human macrophages . the cellular functions of the identified proteins were widely distributed with cellular metabolism proteins representing the largest proportion of the wt strain and ankb mutant lcv proteome at 20% . signaling and cytoskeletal proteins were also highly represented . the ankb mutant lcv contained 80% of the proteins on the wt strain lcv with 569 unique proteins primarily involved in immune response , transcription and translation . the additional proteins on the ankb mutant but not the wt strain lcv could be due to proteasomal degradation of lysine - linked polyubiquitinated proteins in the wt strain lcv . this may also explain the difference in the relative abundance of proteins in the two proteomes because the ankb mutant lcv contained 14 additional ribosomal proteins and 8 additional clusters of differentiation molecules compared with the wt strain lcv . however , this proteome analysis revealed proteins present on the wt strain and the ankb mutant strain lcv and not the relative abundance of the identified proteins . in addition , scarce proteins may not be detectable in our proteomic analyses of the lcv . the lcv proteome contains a unique set of antiapoptotic proteins that could enhance host - cell survival to enable intracellular bacterial growth and may explain the inhibition of apoptosis in l. pneumophila infected cells . we identified the inhibitor of apoptosis ( iap ) protein birc6 , which binds and inhibits effector caspases 3 and 7 . because caspase 3 has been shown to be triggered by l. pneumophila , the role of birc6 should be evaluated to determine the effect birc6 has during infection . this is of particular importance because activation of caspase 3 does not lead to apoptosis of l. pneumophila - infected macrophages , which exhibit a strong antiapoptotic phenotype . birc6 also possesses e3 ubiquitin ligase activity that has been shown to ubiquitinate apoptotic proteins resulting in their proteosomal degradation . in addition to birc6 , we identified the protein defender against cell death ( dad1 ) , which has been implicated in downstream effects of bcl2 to prevent cell death . because the lcv also harbors eukaryotic pro - apoptotic proteins ( bcl2-associated athanogene 6 , apoptosis - inducing factor ) further investigation into the dynamics , balance , and functions of these antagonistic proteins could prove valuable to understand how l. pneumophila modulates a fine balance between pro- and antiapoptotic events in the infected cells . further investigation into the function and recruitment of antiapoptotic proteins on the lcv could explain why permissive macrophages are particularly resistant to exogenous apoptotic stimuli during l. pneumophila infection . the wt strain and ankb mutant lcv contains a significant portion ( 6% ) of proteins involved in post - translational modifications such as ubiquitination and farnesylation . it has been shown that multiple l. pneumophila effector proteins undergo host - mediated farnesylation , which enable their anchoring into host membranes . there are no reports of l. pneumophila effector proteins undergoing ubiquitination . however , further characterization of the e3 ubiquitin ligases identified on the lcv could prove valuable to determine if they catalyze ubiquitination of l. pneumophila effector proteins through lysine or non - lysine - linked polyubiquitination . lysine - linked polyubiquitination of the lcv through the ankb effector is required for intracellular replication . previous studies have shown the valosin - containing protein ( p97 ) participates in the removal of polyubiquitinated proteins from the lcv . proteome data identified the skp1 component of the scf1 e3 ubiquitin ligase complex on the lcv as well as rad23a and rad23b , which serve as multiubiquitin chain receptors that bind to the 26s proteasome and deliver lysine - linked polyubiquitintated proteins for protesomal degradation . four e2 ubiquitin - conjugating enzymes ( ube2k , ube2n , ube2l3 , ubev1 ) were identified on the wt strain lcv ; however , only ube2k and possibly ube2l3 catalyze the synthesis of lysine - linked polyubiquitinated proteins . therefore , it could be speculated that the e2 ubiquitin - conjugating enzyme ube2k or ube2l3 delivers ubiquitin moieties to the scf1 e3 ubiquitin ligase complex for lysine - linked polyubiquitination of the ankb substrates and rad23a or rad23b along with p97 delivers the polyubiquitinated proteins to the proteasome to generate an increase in free cellular amino acids . l. pneumophila is auxotrophic for seven amino acids ; therefore , retrieval of host cell amino acids is essential for intracellular replication . it is not known how l. pneumophila transports amino acids through the lcv membrane to provide the carbon and energy source for intracellular replication . we have identified five amino acid transporters ( slc7a5 , slc3a2 , slc38a2 , slc1a5 , slc1a4 ) in the lcv proteome . it has been shown that the amino acid transporter slc1a5 is required for l. pneumophila replication in human macrophages . because of the essential need for the acquisition of host cell amino acids , further investigation into the localization and function of the host slc amino acid transporters and the specific amino acid transporter they import into the lcv lumen could provide insightful information on how intravacuolar l. pneumophila import amino acids from the host cell cytosol through the vacuolar membrane . although amino acids are the major sources of carbon and energy for l.pneumophila , the organism utilizes the entner pneumophila mutants deficient in the entner doudoroff pathway have a significant defect in intracellular growth in a549 human epithelial cells , a / j mouse macrophages , and acanthamoeba culbertstoni . the presence of the glucose transporters ( slc2a1 , slc2a14 , slc2a3 ) on the lcv is consistent with the import and utilization of glucose by intravacuolar l. pneumophila as a potential source of carbon and energy during intravacuolar growth . the lcv proteome contained two monocarboxylate transporters ( slc16a1 , slc16a3 ) that transport pyruvate across eukaryotic membranes . interestingly , pyruvate supplementation rescues the ankb mutant for intracellular replication in human macrophages and amoeba . therefore , host - derived pyruvate may constitute an important source of carbon and energy for intravacuolar l. pneumophila , and import of pyruvate by the lcv is likely mediated by slc16a1 and slc16a3 identified in the lcv proteome . we identified 24 ubiquitinated proteins on the wt strain lcv and 29 on the ankb mutant lcv . ubiquitination of these proteins could have a wide range of effects due to the complexity and altered location or function of proteins modified by various ubiquitin polymer linkages . of the 24 ubiquitinated proteins on the wt strain lcv , 6 are involved in the immune response ( t cell activation , il-1 , and toll - like receptor signaling ) . ubiquitin was found to be ubiquitinated on four lysine residues ( k , k , k , and k ) , which indicates polyubiquitin chains are formed on some of these ubiquitinated proteins through linkages other than lysine . interestingly , the transitional endoplasmic reticulum atpase ( p97 ) that participates in the removal of lysine - linked polyubiquitinated proteins from the lcv was found to be ubiqutinated on the lcv . some of the 21 ubiquitinated proteins specific to the wt strain are likely to be substrates for ankb . in addition , the ubiquitinated proteins on the lcv are likely ubiquitinated by various l. pneumophila ubiquitin ligases ( f - box , u - box ) and host e3 ligases identified in the proteome . a previous study analyzed ubiquitinated proteins from raw 264.7 macrophage whole cell lysates during l. pneumophila infection . importantly , our ubiquitinated proteome is of the lcv ; however , there were some common ubiquitinated proteins ( vimentin , slc4a7 , elongation factor 2 ) . further studies are needed to determine the lysine - linked polyubiquitinated proteins as well as the lysine- , lysine- , and lysine - linked polyubiquitinated proteins localized to the lcv to assess their role in the intracellular infection . rab5 is activated by the guanine nucleotide exchange factor ( rabex-5 ) , which was found to be ubiquitinated on the wt strain lcv . the l. pneumophila effector vipd interacts with activated endosomal rab5 and is required for endolysosomal evasion . because of the importance of rab5 during l. pneumophila infection , the ubiquitination of rabex-5 could pose another strategy for endolysosomal evasion by l. pneumophila . the lcv proteome has been generated for the jr32 philadelphia - derived strain of l. pneumophila in the mouse macrophage cell line raw 264.7 , which identified 1156 proteins . proteins involved in cellular metabolism are the most abundant in our proteome as well as the previously characterized jr32 strain proteome . however , in our lcv proteome within human macrophages , proteins involved in metabolism ( 20% ) were less abundant compared with previous reports ( 50% ) . solute carriers , 40s and 60s ribosomal proteins and proteins involved in vesicular trafficking ( sorting nexins , rab gtpases ) , were very similar in the lcv proteomes of the jr32 and aa100/130b strains within mice and human macrophages , respectively . the lcv proteome of the jr32 strain in mouse macrophages had a larger number of 28s and 39s mitochondrial ribosomal proteins as well as nadh dehydrogenase proteins compared with the aa100/130b strain in human macrophages . while phophatidylinositol - dependent proteins have been identified ( sac1 , ship1 ) in the jr32 strain lcv ; we identified phophatidylinositol - dependent gtpase activating proteins and guanine exchange factors that were not detectable in the lcv proteome of the jr32 strain within mouse macrophages . interestingly , the e3 ubiquitin ligases and e2 ubiquitin - conjugating enzymes we identified have not been detected in the lcv of the jr32 proteome within raw 264.7 mouse macrophages . the differences between the lcv proteome of the jr32 and aa100/130b strains could be attributed to the different l. pneumophila strains that exhibit plasticity in 30% of the genome or more importantly the difference in the lcv between mouse and human macrophages or both . this report outlines the total and novel ubiquitinated proteome of the lcv that include e2 ubiquitin - conjugating enzymes and e3 ubiquitin ligases . the phosphatidylinositol - dependent proteins in our lcv proteome and the metacore bioinformatic analysis of metabolomic networks in the wt strain lcv proteome clearly show the phosphatidylinositol-4,5-diphosphate pathway as the most abundant , while amino acid synthesis and transport are also highly represented in the lcv proteome . the amino acids , pyruvate , and glucose transporters in the lcv proteome show a unique mechanism of import of host sources of carbon and energy for intravacuolar nutrition and metabolism of l. pneumophila . further investigation into the ubiquitinated proteins and the various nutrient transporters localized to the lcv could prove valuable to understanding how l. pneumophila manipulates host cell machinery to acquire nutrients and circumvent a starvation response during infection . our findings should provide a rich resource for future investigations of numerous cellular processes and pathways involved in biogenesis and dynamic remodeling of the lcv within human macrophages .

within protozoa or human macrophages legionella pneumophila evades the endosomal pathway and replicates within an er - derived vacuole termed the legionella - containing vacuole ( lcv ) . the lcv membrane - localized ankb effector of l. pneumophila is an f - box protein that mediates decoration of the lcv with lysine48-linked polyubiquitinated proteins , which is essential for intravacuolar replication . using high - throughput lc ms analysis , we have identified the total and ubiquitinated host - derived proteome of lcvs purified from human u937 macrophages . the lcvs harboring the aa100/130b wt strain contain 1193 proteins including 24 ubiquitinated proteins , while the ankb mutant lcvs contain 1546 proteins with 29 ubiquitinated proteins . pathway analyses reveal the enrichment of proteins involved in signaling , protein transport , phosphatidylinositol , and carbohydrate metabolism on both wt and ankb mutant lcvs . the ankb mutant lcvs are preferentially enriched for proteins involved in transcription / translation and immune responses . ubiquitinated proteins on the wt strain lcvs are enriched for immune response , signaling , regulation , intracellular trafficking , and amino acid transport pathways , while ubiquitinated proteins on the ankb mutant lcvs are enriched for vesicle trafficking , signaling , and ubiquitination pathways . the complete and ubiquitinated lcv proteome within human macrophages illustrates complex and dynamic biogenesis of the lcv and provides a rich resource for future studies .

visual attention is the selection of visual information for purposes such as in - depth processing , perception , or action control . because we have to select information at all times , understanding attention is a key to an understanding of almost any form of cognition . to date , however , the mechanisms by which attention operates are not fully understood . one persistent debate in this area concerns the role of inhibition of irrelevant stimuli as one form of top - down control over attention . whereas some researchers believe that inhibition of attention is a response to initial capture of attention and , thus , follows preceding attentional capture by an irrelevant stimulus , other researchers believe that active inhibition of attentional capture by an irrelevant stimulus is possible right from the start of such a stimulus . to start with the first proposition , many researchers argued that salient objects capture attention in a bottom - up way ( cf . [ 3 , 4 ] ) . according to the salience model of attention , any visual stimulus that stands out among its surroundings by a strong feature contrast in color , orientation , or luminance may capture attention in an exogenous stimulus - driven way , regardless of the current goal of the observer ( cf . [ 5 , 6 ] ) . in line with this prediction , an irrelevant color singleton distractor that is a stimulus with a color different from its surrounding stimuli , such as one green circle among several red circles , interferes with finding a shape - defined target stimulus ( i.e. , the one rectangle among several circles ) ( cf . ) . this is the case although attending to the specific color of the singleton is neither necessary nor helpful for finding the target . such findings have been attributed to the bottom - up capture of attention by an irrelevant singleton . as a consequence , attention is thought to first be distracted away from the relevant target and only later be redirected towards the target . this is possible after a deliberate inhibition of the irrelevant stimulus , allowing attention to disengage from the distractor . findings by kim and cave are in general agreement with this late - inhibition or disengagement hypothesis . these authors used a probe stimulus as a second target in a combined search and probe reaction task . critically , the probe was shown after the search display with the probe either at the position of the search display 's shape - defined target or at the position of the search display 's color - singleton distractor . with an interval of 60 ms between search display and probe , kim and cave observed ( nonsignificantly ) faster responses to probes at color - distractor positions than to probes at shape - target positions . however , with a cue - target onset asynchrony ( ctoa ) of 150 ms between the color - singleton distractor ( cue ) and the probe ( target ) , responses to probes at the position of the color - singleton distractor were significantly delayed relative to responses to probes presented at the location of the shape target . like theeuwes et al . , kim and cave took their results as an indication of bottom - up capture by the color - singleton distractor with a short ctoa , giving way to disengagement and even active inhibition with a long ctoa . the core notion of the disengagement hypothesis , that is , the idea of active inhibition following initial allocation of attention towards a stimulus , is also at the heart of another well - known phenomenon called inhibition of return ( ior ) . ior denotes the finding that attracting visual attention toward one position in space by a cue delays a second attention shift to the same position at a later point in time [ 810 ] . ior is observed with long ctoas and corresponds to longer reaction times where cue and target are presented at the same position ( sp ) compared to cue and target at different positions ( dp ) . thus , the idea of stimuli initially triggering attentional capture and later inhibition is a very dominant notion found throughout the attention literature . however , recent findings by mcdonald et al . and by ansorge et al . are potentially in disagreement with this late - inhibition or disengagement hypothesis for irrelevant stimuli . ansorge et al . asked their participants to saccade to one out of four positions , varying randomly from trial to trial ( see figure 1 for an illustration of a similar stimulus and task sequence ) . prior to the saccades , participants were presented with a relevant or an irrelevant color singleton cue . participants only had to attend to the relevant cue because this cue indicated the position of a discrimination target later in the trial . in contrast , the participants were asked to ignore the irrelevant cue : when an irrelevant cue was presented , no target discrimination was required so that it was safe to ignore this cue . in addition , ignorance of the irrelevant cue was encouraged : because cue and saccade target positions were uncorrelated ( cf . ) and because saccades require a prior attention shift to the target position [ 13 , 14 ] , the participants could fully concentrate on the saccade task and ignore the irrelevant cues completely . in contrast , the participants were forced to shift their attention to the relevant cue for the encoding of its position for the discrimination task and at the potential cost of a suboptimal preparation of their saccades . all of these cues were nonpredictive of the saccade target position , and relevant and irrelevant cues had different fixed colors , so that the participants knew exactly which color they had to attend to ( e.g. , red ) and which color they could ignore ( e.g. , green ) . under these conditions , ansorge et al . studied the time course of selective attentional capture and/or inhibition by looking at the development of the saccadic latencies across the latency distribution , from quick to slow saccades . with a long ctoa , and with relevant cues , ior followed initial capture : initial capture among the fast responses was reflected in quicker saccades to a saccade target at the same position ( sp ) as the cue compared to slower saccades to a saccade target at a different position ( dp ) than the cue . with relevant cues , this pattern reversed into ior among the slower responses . in contrast , with a long ctoa and irrelevant cues , inhibition in the form of slower saccades to sp than dp targets was found right from the beginning and without preceding capture effects . these findings point to a form of proactive inhibition of irrelevant cues , completely preventing attentional capture by the irrelevant cues , rather than late disengagement . in fact , the only reliable capture effect for irrelevant cues showed up in a condition with a different procedure and no subsequent inhibition ( experiment 4 ) . thus , no conclusion could be drawn about transitions from capture to disengagement . a recent study by mcdonald et al . equally found evidence for proactive inhibition of irrelevant stimuli without a trace of preceding attention capture . to discern between capture and inhibition , mcdonald and colleagues used two lateralized components of the event - related potential ( erp ) : the n2pc ( cf . [ 15 , 16 ] ) or posterior contralateral negativity ( pcn ) and the pd [ 18 , 19 ] . the n2pc has been widely used to investigate both stimulus - driven capture and top - down contingent capture [ 2125 ] . importantly , when mcdonald et al . tested for initial capture of attention by irrelevant stimuli , all they found was a pd , that is , evidence for proactive inhibition of the irrelevant stimuli . this was found after splitting the erps into fast and slow responses : the quickest target responses of the participants indicated proactive inhibition of attentional capture by an irrelevant distractor . even so , it is not entirely clear whether the findings reflected only early inhibition or whether some capture of the irrelevant singletons occurred before it was suppressed . regarding the findings of ansorge et al . , these authors used saccadic latencies after relatively long ctoas ( > 200 ms ) . this method is relatively insensitive to the early attentional effects , so that preceding attention capture even by irrelevant cues might have gone unnoticed ( see their condition with a ctoa of 200 ms ) . regarding the findings of mcdonald et al . , it is possible that their observations reflected a mixture of weaker capture effects of the irrelevant distractors in some of the trials and of stronger inhibition of distraction in other trials . as a result , a net inhibitory pd effect could have masked evidence for early capture in the form of an n2pc in the study of mcdonald et al . at least in the slower responses , there was also clear evidence for this possibility : on slow response trials ( ) there was neither an early distractor ( ) nor a late target n2pc ( ) . the absence of either n2pc suggests that the target and distractor n2pc wave cancelled each other out ( ) thus , to test once more whether capture by irrelevant cues could precede subsequent inhibition , we combined the methods of ansorge et al . and of mcdonald et al . , using two different measures for early and late effects based on the very same trials . late inhibition was assessed through the presence of ior in saccadic reaction times after a sufficiently long ctoa of 1 s. this procedure allows for the registration of an early capture effect by all singletons , without masking by a concomitant n2pc by the targets . in this situation , in addition , we conducted a median - split of the erps on the basis of whether a fast or a slow saccade was given that allowed us to test whether the fastest responses were associated with a pd component , similar to mcdonald et al . . the aim of the current experiment was to investigate the connection between attention capture by relevant and irrelevant stimuli and ( subsequent ) inhibition . we examined within the same trials ( 1 ) the amount of initial capture of attention by an irrelevant cue and a relevant cue in the form of the n2pc and ( 2 ) the amount of inhibition in the form of an early pd and late saccadic inhibition of return . in detail , in the first display of each trial , we used one of two color - singleton cues : the first cue was relevant in half of the trials and it was irrelevant in the other half of the trials . a relevant first cue had a fixed color ( e.g. , it was green ) , known to the participant . the participant had to look for the relevant first cue and importantly , also covertly , attend to its location because it indicated the position of a subsequent discrimination target . we call this cue relevant to make clear that its color serves the same purpose as a searched - for feature of a target in a standard color - search task . in contrast , the irrelevant first cue had a different color ( e.g. , it was blue if the relevant cue was green ) , also known to the participants . therefore , the participants could have ignored this cue completely and were not required to shift spatial attention to its location . we call this cue irrelevant because its color served the same purpose as the color of an irrelevant distractor in a visual search display . that is , the color of the irrelevant first cue indicated with 100% certainty that this stimulus could be safely ignored . to test ior after the first cue , our participants had to encode the position of a second singleton cue in a second display for a saccade in a subsequent third display ( see figure 1 ) . importantly , positions of the first cue and of the second saccade cue were uncorrelated . because the participants have to allocate their attention to the position of the saccade target ( cf . [ 13 , 14 , 28 , 29 ] ) , they had to disengage their attention away from any first cue and to redirect it towards the position of the second cue in anticipation of the saccade target . also , the ctoa was 1 s long allowing for both inhibition ( or disengagement ) of attention and saccadic inhibition ( of return ) . we therefore expected saccadic inhibition with respect to the position of the first or covert cue ( [ 11 , 30 , 31 ] ; see also ) . the question is whether with irrelevant first cues a capture effect in the form of an n2pc precedes this inhibition effect or whether inhibition is observed from the start , in the form of a pd . also , in the relevant condition , an n2pc to the first cue was to be expected because an attention shift to this first cue was required to encode its position . twelve volunteers participated but one was excluded because her saccade latencies were more than three standard deviations slower than that of the other participants . the remaining participants ( with a mean age of 25 years and a male / female ratio of 6 : 5 ) reported normal or corrected - to - normal vision . written and informed consent was obtained from each participant before the experiment . visual stimuli were presented on a 19-inch crt color monitor ( sony multiscan g400 ) , with a screen resolution of 1,024 768 pixels and a refresh rate of 100 hz . the participants sat at a distance of 57 cm from the screen in a quiet , dimly lit room , with their head resting on a chin rest to ensure a constant viewing distance and a straight - ahead gaze direction . the first and second displays were presented for 50 ms and the last display for 1 s. all displays were separated by an interstimulus interval of 450 ms , such that the onset asynchrony between two displays was 500 ms . a gray central fixation cross was presented on a black background ( < 1 cd / m ) , visible throughout each trial . the first display consisted of six equidistant placeholders , each in the shape of the digital letter 8 ( with a size of 1.7 1 and with stroke strength of .3 ) . a placeholder was located per each of the positions at 0 , 60 , 120 , 180 , 240 , and 300 from the vertical meridian that is , the shape-8s were presented equally spaced on the circumference of a virtual circle centered on the screen , with an eccentricity of 7. five placeholders were presented in gray ( cielab color coordinates : 6.9 , 16.8 ) , and one was presented in a different color , either in green ( cielab : 30.2 , 24.9 ) or blue ( cielab : 46.9 , 89 ) . it was always shown at one of the four lateral positions but never presented above or below the fixation . the shape-8s were replaced by three letters e and three digits 3 , in digital notation . five of these shapes were presented in gray and one was presented in red ( cielab : 47.6 , 41.1 ) . this red stimulus was the second or saccade cue and it could also only appear at one of the four lateral positions . the red singleton was called a saccade cue because this second cue served as the cue for the saccade target in the subsequent display . also , in this display , one figure served as a discrimination target if it had been cued by a relevant first cue ( blue or green cue ) in the preceding display , with relevant first cue color fixed across trials and balanced across participants . positions of the discrimination target and second ( or red ) cue were uncorrelated across trials . consequently , in 25% of the trials the discrimination target and second or saccade cue were at the same position ( sp condition ) , and in 75% of the trials they were at different positions ( dp condition ) . this display consisted solely of six empty circles surrounding the stimulus positions as used in the preceding displays . the saccade display was presented for 1 s. the color of the first singleton cue in the first screen indicated whether the discrimination task in the second screen had to be performed on a given trial . for instance , a first green singleton was linked to the discrimination task while a first blue singleton could be ignored , or vice versa . in the discrimination task , participants had to encode and remember the shape of the digit presented in the second screen at the position of the relevant first singleton cue . this was necessary for the report of this figure at the end of the trials . as soon as the third display , the saccade display , appeared the saccade had to be executed . after the saccade was executed , in a relevant - cue trial , participants typed the identity of the discrimination target letter ( i.e. , whether the letter e or the digit 3 was presented ) by pressing the marked buttons # f and # j labeled left and right on a standard keyboard directly in front of the participants . if no discrimination was necessary ( i.e. , after irrelevant cues ) , this part of the trial was skipped . participants started the next trial in a self - pace manner , by pressing the space bar . participants were informed that the color singleton cues could only appear at the four lateral positions on the screen and that the position of the second or saccade singleton cue was independent of the position of the first singleton cue . blocks consisted of 64 trials and feedback was given about whether the target discrimination was correct and about whether the saccade was registered during the third screen . altogether ten blocks of trials were conducted , of which the first was training and not analyzed . each factor combination of the variables discrimination target ( e or 3 ) , first cue position ( above / left , above / right , below / left , and below / right ) , first cue color ( blue , green ) , and second cue 's position ( above / left , above / right , below / left , and below / right ) was equally likely and presented in a pseudorandom order within each block . saccades were recorded with an eyelink 1000 desktop mount system ( sr research , mississauga , on , canada ) with a 35 mm lens and eyelink software version 4.52 , sampling at 1,000 hz . a 9-point calibration was used to adjust the eye - tracker before the experiment and in advance of every single block . saccadic reaction time ( saccadic rt ) was calculated as the time between ( 1 ) the onset of the third display ( with the saccade - target stimulus circle ) and ( 2 ) the time of a local velocity minimum that immediately preceded the point in time at which eye velocity exceeded 80/s . a saccade counted as correct if it landed in an area of 1.5 around the center of the saccade target . saccade landing position was calculated as the x - y coordinates of the eye - tracker signal at the time at which eye velocity returned to a presaccadic baseline level . also , if the eyes started to move earlier than 100 ms after the saccade target , a trial was discarded . dc - eeg was recorded from 23 scalp electrodes mounted in an elastic cap at standard positions of the extended 10/20 system at sites fpz , f7 , f3 , fz , f4 , f8 , fc5 , fc6 , t7 , c3 , cz , c4 , t8 , cp5 , cp6 , p7 , p3 , pz , p4 , p8 , o1 , o2 , and oz . the continuous eeg was sampled at a rate of 1,000 hz with a digital low - pass filter of 50 hz . all scalp electrodes were online referenced to a noncephalic sternovertebral site , above the seventh vertebra and the right manilum sternum . the vertical eog ( electrodes below and above the left eye ) and the horizontal eog ( electrodes at the outer canthi ) were recorded bipolarly , so as to delete trials with eye movements during the critical eeg recording interval . trials with saccades earlier than 100 ms after the saccade target ( detected with the eye - tracker ) or muscular artifacts ( exceeding 80 v at any electrode ) , as well as trials in which the target was not correctly discriminated , were excluded from analysis . erps were calculated for 400 ms after the first cue 's onset relative to a 50 msec precue baseline . n2pc amplitudes in response to the first color cue were calculated separately for left and right and relevant and irrelevant cue , collapsed across all saccade target positions as mean erp amplitudes at locations p3/4 in the 160270 ms interval after cue onset . a switch box was implemented behind the parallel port of the master to send one unique synchronization trigger every 500 ms ( one for the onset of the first display , one for the second display , and one for the third display in each trial ) in parallel , separately to the two slaves , eye - tracker and eeg recorder . in total , 17.5% of all trials were excluded . trials with saccades faster than 100 ms and slower than 1 s after the saccade target accounted for 8.1% , trials with saccades towards the wrong target or with muscular artifacts for another 6.4% , and trials with a false identification of the discrimination target for 3% . to take the dynamics of the saccadic response into account , saccadic rts were sorted and grouped into five percentiles from fast to slow ( cf . ) . this was done to test our hypotheses about ior with differently fast responses because the amount of capture and of ior does vary over time and an effect that is absent in the average of all responses can well be present when looking at only the faster or only the slower responses ( e.g. , [ 11 , 34 ] ) . as can be seen in figure 2 , from fast responses on the left to slow responses on the right this was reflected in faster saccadic rts under dp conditions ( broken lines ) as compared to sp conditions ( solid lines ) , more so with the irrelevant cues ( red lines ) than with the relevant cues ( blue lines ) . a repeated - measures anova with the variables position ( same versus different position of first or covert cue and saccade cue / target ) , cue type ( relevant first cue or irrelevant first cue ) , and percentile ( 1st to 5th ) revealed inhibition at the location of the first or covert cue only among the slowest responses in the form of slower saccadic latencies in sp than dp conditions . this was reflected in a significant interaction of position and percentile , f(4,40 ) = 2.79 , p < .05 . from the 1st to the 5th quintile , saccadic inhibition ( saccadic rt in sp conditions minus saccadic rt in dp conditions ) was 0 ms , 1 ms , 4 ms , 3 ms , and 32 ms ( 1st to 4th quintile , all ts < 1 ; 5th quintile , t(9 ) = 2.29 , p < .05 ) . in addition , we found faster saccadic rts in trials with a relevant than an irrelevant cue in the first display ( 241 ms versus 273 ms ) , resulting in a marginally significant main effect for cue type , ( 1,10 ) = 4.72 , p = .055 . there was also a trivial main effect of percentile ( increasing saccadic rts with percentile ) , ( 4,10 ) = 94.56 , p < .01 . further , there was a numerically stronger inhibitory effect on saccades after the irrelevant cue ( 10 ms ) than after the relevant cue ( 2 ms ) , as would be expected based on an active inhibition explanation . however , the two - way interaction of relevance and position was not significant , ( 1,10 ) = .55 , p = .48 , as was the three - way interaction , f < 1 . in sum , saccadic inhibition was selectively present in the slowest saccades and it was largely independent of the type of cue that was used in the first display . figure 3 shows erps time - locked to the first cue 's onset at lateral posterior electrodes p3 and p4 contra- and ipsilateral to the first cue , separately for cues with a relevant color ( panel a ) , cues with an irrelevant color ( panel b ) , and difference waves ( i.e. , contra- minus ipsilateral activity for relevant and irrelevant cues , panel c ) . the differences are depicted together with topographical erp - difference maps for the time window of the n2pc ( 160 ms to 270 ms ) . all erps are relative to a baseline from 50 ms before the first cue to the onset of the first cue . as can be seen , there was an n2pc in the relevant and in the irrelevant cueing conditions . also , by looking at figure 3 , it seems as if the n2pc started later and was weaker in the irrelevant than in the relevant cueing condition . these observations were confirmed in a repeated - measures anova with the variables cue type ( relevant or irrelevant cue ) , laterality ( electrode ipsi- or contralateral to the first cue ) , and hemisphere ( right or left hemisphere ) . the analysis revealed a significant main effect for laterality , ( 1,10 ) = 7.3 , p < .05 , and a significant interaction of laterality and cue type , f(1,10 ) = 10.14 , p < .01 . however , if the cue was relevant , the n2pc was stronger ( contra- minus ipsilateral activity : 0.76 v ) and started earlier than if the cue was irrelevant ( .30 v ) , as was shown in section 2.2.3 . we were concerned that the choice of the electrode locations of the n2pc might have been unfortunate . therefore , we repeated our major analysis of the n2pc in a repeated - measures anova with the additional variable site ( p3/p4 , p7/p8 , and o1/o2 ) and the variables cue type ( relevant or irrelevant cue ) and laterality ( electrode ipsi- or contralateral to the first cue ) as before . besides replicating the main effect of laterality , f(1,10 ) = 6.86 , p < .05 , and an interaction of laterality and cue type , f(1,10 ) = 5.56 , p < .01 , there were no significant main effects , all fs < 1.40 and all ps > .28 , and no significant interactions including the three - way interaction of site , cue type , and laterality , all fs < 2.10 all ps > .15 . in addition , the anova was also repeated with the erps pooled across p3 , p7 , and o1 ( for the left side ) and across p4 , p8 , and o2 ( for the right side ) . this anova also confirmed a laterality effect , ( 1,10 ) = 6.86 , p < .05 , and an interaction of laterality and cue type , f(1,10 ) = 5.56 , p < .01 , and no main effect of cue type , f < 1 . to demonstrate the earlier onset of the n2pc with relevant cues than with irrelevant cues , we split the n2pc window into an early phase ( 160 ms to 215 ms after the cue onset ) and into a late phase ( 215 ms to 270 ms after the cue onset ; cf . ) . in the early window , an anova revealed a significant two - way interaction of laterality and cue type , ( 1,10 ) = 30.17 , p < .01 . post - hoc t - tests revealed that the contra - to - ipsilateral negativity difference ( .78 v ) was only significant in the relevant condition , ( 10 ) = 4.33 , p < .01 , but not in the irrelevant condition ( .02 v ) , t(10 ) = .08 , p = .93 . a similar anova of the late time window only led to a main effect of laterality , ( 1 , 10 ) = 8.10 , p < .05 . the contra - to - ipsilateral negativity difference was about similar in relevant ( .77 v ) and irrelevant ( .66 v ) cueing conditions . there was neither a main effect of cue type , nor of hemisphere , nor any interaction between the variables , all other fs < 2.10 all ps > .18 . recently , mcdonald and colleagues showed that irrelevant distractors elicited a pd among the fastest responses . we therefore also repeated our anova of the activity at p3 and p4 , with only the fastest 50% of the saccades and the two within - participant variables cue type ( relevant or irrelevant cue ) , and laterality ( electrode ipsi- or contralateral to the first cue ) . again , activity was more negative at contra- than ipsilateral electrodes , ( 1 , 10 ) = 11.16 , p < .05 . this time , however , the interaction was far from significant , f < 1 . in contrast to the findings of mcdonald et al . , a more prominent n2pc rather than a pd was observed with the irrelevant singleton cues during the fastest responses . in the present study , we tested whether irrelevant cues were proactively inhibited or whether they captured attention before being inhibited . in line with the latter possibility , relevant , and importantly also irrelevant , cues elicited an n2pc and both stimuli led to inhibition of saccades 1 s after the cues . this was reflected in slower saccadic rts to targets in sp than dp conditions . in other words , we found the typical ior effect , an observation in line with the late inhibition or disengagement hypothesis of theeuwes et al . . this finding is also in agreement with prior findings of ansorge et al . with relevant cues . in their study , these authors found a capture effect of the relevant cues when a ctoa of 200 ms was used . ansorge et al . also observed that ior started earlier with an irrelevant cue than with a relevant cue . this particular finding could not be observed in the present study . in the present study , among the slowest responses , ior with irrelevant cues this latter finding is thus also not so well in line with theeuwes et al . 's disengagement theory , according to which one would have expected stronger disengagement or ior after irrelevant than after relevant cues . according to disengagement theory , only the stronger disengagement of attention that follows irrelevant cues accounts for seemingly stronger capture effects by relevant than irrelevant cues . clearly , this prediction of the disengagement theory was not confirmed . in contrast , our results suggested a mixture of early capture differences with more capture by relevant than irrelevant cues and a later disengagement effect that was numerically stronger with irrelevant than relevant cues , as two sources contributing to stronger capture effects by relevant than irrelevant cues . concerning stronger capture by relevant than irrelevant cues , this was reflected in the n2pc . when we looked at the n2pc as an index of the initial capture of attention , we found a larger overall n2pc . this reflected on average an earlier start of the n2pc elicited by the relevant cue . these findings are in line with prior findings showing an earlier or temporally less variable capture effect and often even a selective capture effect for top - down matching than nonmatching cues [ 12 , 21 , 23 , 35 , 36 ] . this difference in capture for top - down matching as compared to nonmatching cues is typically assumed to reflect either of two processes : selective top - down tuning to sets of features so that initial capture is restricted to the cues matching the set [ 12 , 37 ] or less inhibition of attention captured by the top - down matching cue [ 1 , 38 ] . with the current procedure , we can not decide which of these interpretations holds true , that is , whether the temporally more variable or trailing onset of the n2pc by the irrelevant cues reflected less initial capture by these cues or a combination of initial capture by the irrelevant cues and proactive inhibition of the irrelevant cues . with respect to the latter , however , we did not find any evidence for strong early proactive inhibition of the irrelevant cues in the form of a pd . the trailing of the n2pc for irrelevant cues might be a tentative hint for some proactive inhibition . without any proactive influence , one would expect similar onset times of the n2pc for relevant and irrelevant stimuli ( although the initially smaller n2pc for irrelevant stimuli may camouflage its early onset ) . in particular , prior studies found proactive inhibition in the form of a pd when only looking at the fastest responses . in contrast to this finding , early or proactive inhibition was not associated with the fastest responses in the present study . this was evident when we sorted the erps as to whether they were recorded in a trial with a quick or slow saccade : among the fast saccades , the n2pcs of irrelevant and relevant cues became even more similar . this means that in the present study , more proactive inhibition would have counteracted the irrelevant cue 's n2pc onset in the trials with the slower saccades . which factors might account for the differences between the present study and the previous study by mcdonald et al . ? to reconcile the different findings , results from kiss and colleagues might be of interest . these authors presented target and distractor simultaneously ( similar to ) and found proactive inhibition of the irrelevant distractor in the form of a pd when the display was shown for 200 ms but an n2pc plus subsequent inhibition ( again in the form of a pd but occurring at a later point in time ) when the display was presented until a response was given . this might indicate that the irrelevant distractor elicits an n2pc and captures attention when the participants have time for their attention to shift to the target so that the distractor - elicited capture is not masked by a concomitant target - elicited n2pc . this might also explain why we found an n2pc of the irrelevant cues whereas most contingent - capture studies did not find any evidence for capture by irrelevant singleton distractors ( e.g. , [ 21 , 39 ] ) . with respect to the finding of an n2pc to the irrelevant cue in the present study and its absence in prior studies , a few other procedural differences might also play a role . first of all , the relevant cue was 100% valid ( 100% sp ) ; that is , it predicted the discrimination target position with certainty . although there was no discrimination target in the irrelevant target position it is possible that a bit of the general informative value of the relevant cues spilled over to the irrelevant cues . in other words , participants might have inadvertently attended to the irrelevant cue on at least some trials , for example , because they were not paying close enough attention to the color of the first cue . in support of this possibility , it would have been possible to find the relevant cues by the so - called singleton search strategy [ 40 , 41 ] . in fact , the use of two different relevant colors one ( e.g. , blue ) for the first display 's relevant cue and another one ( red ) for the saccade cue in the second display might have encouraged our participants to use a singleton search strategy rather than a feature search strategy . a few findings seem to indicate that the use of a top - down set containing two relevant colors leads to the erroneous capture of attention by an irrelevant color - singleton distractor in at least some trials ( cf . in addition , participants might have actively searched for even the irrelevant cues because these cues informed the participants that they would not have to discriminate between the different target orientations and keep the cue 's position in mind . the relatively long ctoa might have encouraged this strategy further because it would have allowed sufficient time to first willingly attend to each cue relevant and irrelevant and then to return attention to a neutral position after the irrelevant cue and before the onset of the target . even though this particularity of our procedure might explain why we did find an n2pc for both relevant and irrelevant cues , it is important to note that we were still able to ascertain two things : first , recording eeg we were able to demonstrate capture where behavioral measures only indicated inhibition . thus , although one might argue that the difference in the way participants processed relevant and irrelevant cues in our study was only small , our eeg measure was definitely sensitive to it . in sum , we might not have ended the debate over early proactive inhibition for complete prevention of capture once and for all with our study . however , we provide one more piece in the puzzle and another demonstration of the usefulness of combining eeg with behavioral measures to obtain a more complete picture of the processes engaged through a given paradigm . a further point that needs , it would be strange if different degrees of initial capture by relevant versus irrelevant cues ultimately lead to relatively similar degrees of ior by these stimuli . however , researchers had argued from very early on that capture and certain forms of inhibition could be partly independent processes . today , it is clear that nonattentional factors like motor inhibition and sensory habituation can also contribute to inhibition [ 8 , 49 , 50 ] . therefore , it is in principle possible to find similar degrees of late inhibition after different degrees of capture [ 5153 ] or even more inhibition following less capture by an irrelevant stimulus . along similar lines , prinzmetal et al . reported that attention capture and ior are differentially modulated by , on the one hand , the number of potential target locations and , on the other , the presence of distractor stimuli in the target display . dissociations of attention capture and ior are also in line with neurophysiological observations suggesting that the two effects arise at different stages of processing and may therefore be modulated differentially ( e.g. , ) . in more functional terms , prinzmetal et al . recently suggested that attention capture may best be described by a serial search mechanism , reminiscent of the attentional spotlight that ( at least for top - down matching cues ) is first allocated to the cued location and has to be redirected on invalid ( dp ) trials . ior , however , may better be accounted for by a decision process in a competitive accumulator model in which the decision to respond to a particular location previously visited by attention is systematically delayed ( see also ) . in conclusion , the two mechanisms proposed for attention capture and ior are very distinct , supporting the possibility for dissociations . previous studies such as gibson and amelio failed to find any evidence for ior with color singletons , a result that was ascribed to the special role of abrupt onsets for the occurrence of ior . here , we show that relevant and even irrelevant color singletons lead to ior when an eye movement instead of a manual response is used and when the saccadic rt distribution is taken into account . in line with this interpretation , godijn and theeuwes and more recently priess et al . and ansorge et al in conclusion , in line with the late inhibition or disengagement theory , we have shown that the irrelevant and the relevant distractor first both captured attention ( reflected in their n2pcs ) before they were actively inhibited ( reflected in saccadic ior ) . this lack of proactive inhibition was also found if only the fastest responses were analyzed . however , we found little indication that ior was stronger after irrelevant than relevant cues . therefore , it is not likely that disengagement was the only responsible process . early inhibition ( among the slower responses ) or contingent capture must have also contributed to the n2pc differences between relevant and irrelevant cues .

attentional capture is usually stronger for task - relevant than irrelevant stimuli , whereas irrelevant stimuli can trigger equal or even stronger amounts of inhibition than relevant stimuli . capture and inhibition , however , are typically assessed in separate trials , leaving it open whether or not inhibition of irrelevant stimuli is a consequence of preceding attentional capture by the same stimuli or whether inhibition is the only response to these stimuli . here , we tested the relationship between capture and inhibition in a setup allowing for estimates of the capture and inhibition based on the very same trials . we recorded saccadic inhibition after relevant and irrelevant stimuli . at the same time , we recorded the n2pc , an event - related potential , reflecting initial capture of attention . we found attentional capture not only for , relevant but importantly also for irrelevant stimuli , although the n2pc was stronger for relevant than irrelevant stimuli . in addition , inhibition of saccades was the same for relevant and irrelevant stimuli . we conclude with a discussion of the mechanisms that are responsible for these effects .

actinobacteria generate a large number of structurally diverse small molecules with potential therapeutic value . genomic analyses of this productive group of bacteria show that their genetic potential to manufacture small molecules exceeds their observed ability by roughly an order of magnitude , and this revelation has prompted a number of studies to identify members of the unknown majority . as a potential window into this cryptic secondary metabolome , pairwise assays for developmental interactions within a set of 20 sequenced actinomycetes were carried out . these assays revealed that amycolatopsis sp . aa4 , a so - called rare actinomycete , produces a novel siderophore , amychelin , which alters the developmental processes of several neighboring streptomycetes . using this phenotype as an assay , we isolated amychelin and solved its structure by nmr and ms methods coupled with an x - ray crystallographic analysis of its fe - complex . the iron binding affinity of amychelin was determined using edta competition assays , and a biosynthetic cluster was identified and annotated to provide a tentative biosynthetic scheme for amychelin .

the oral mucosa performs essential protective functions that significantly affect the general health of an individual . a decline in the protective functions of the oral mucosa could expose the individual to a variety of pathogens and chemicals that enter the oral cavity . of the myriad of white lesions found in the oral mucosa , a proficient clinician when examining a white lesion should establish the clinical diagnosis mostly by excluding all other lesions that appear white in oral cavity . linea alba , leukoedema , and traumatic keratosis have a chance of being misdiagnosed as leukoplakia . in visualizing a white lesion , one should also look for the etiologic factors that could cause constant trauma resulting in hypertrophy in the affected area . a 12-year - old female patient was referred to the dental outpatient department of hah centenary hospital with an asymptomatic swelling in left buccal mucosa . the swelling started insidiously 3 years ago without any antecedent trauma or dental infection and gradually increased in size . the patient sought multiple medical consultations , for which various diagnoses were made including leukoplakia and submucosal buccal cyst . however , the swelling recurred within 6 months of each surgery . on examination , patient was apparently healthy with noticeable facial asymmetry with prominence in lower third of face on left side . intra - oral examination revealed a diffuse swelling in left buccal mucosa extending from lower left second molar region to left commissure obliterating the buccal vestibule . dental examination revealed retained left lower deciduous second molar ( 75 ) with the upper left second premolar ( 25 ) in torsiversion . this resulted in an open bite ( lack of occlusion of teeth when the jaw is closed normally ) in the region [ figure 2 ] . radiographic findings revealed retained lower deciduous second molar ( 75 ) and congenitally missing second premolar in the region ( 35 ) [ figure 3 ] . based on clinical examination and past histopathology report , diagnosis of left buccal mucosal hypertrophy secondary to open bite in relation to 24 and 75 was made . open bite in relation to 75 and 24 intra - oral periapical radiograph showing congenitally missing 35 chronic irritation of the oral mucosa frequently results in abnormal fibroblast activation and keloid formation . this pathological scar formation is often associated not only with pain , functional disability but also disfigured esthetics of the tissue . the open bite was hypothesized as being the etiology of the chronic cheek bite and , therefore , its correction was planned before the excision of the lesion . fixed orthodontic therapy was suggested which was refused by the patient 's father owing to financial constraints . as an alternative , porcelain fused to metal crown on 75 was cemented to close the open bite which gave fair results . the lesion was excised under general anesthesia and wound coverage was done using collagen membrane ( kllagen , eucare pharmaceutical , india ) secured with 3 - 0 vicryl ( ethicon , johnson and johnson , india ) at wound margins [ figure 4 ] . surgical wound covered with collagen membrane the kollagen is sterile collagen sheets of bovine origin . two days postoperatively , a buccal shield made of acrylic wasinserted [ figure 5 ] . buccal shield inserted 48 hrs after surgery this acted as an aid to keep away the healing tissues from dental arches , and therefore , coming in bite . the patient was advised liquid and semisolid diet and to wear the shield throughout the day until complete healing . this normal diet was resumed , but shield was continued until 2 months and then tapered over the next 6 months to use of no shield . the patient was kept on quarterly follow - up . nearly 18 months postoperatively , there has been no recurrence of the lesion except for the scaring at left commissure which might require commissuroplasty for cosmetic reasons [ figure 6 ] . in a developing country like india where the ratio of health practitioners to the population is low , health professionals in tertiary centers are overburdened with their respective work . this violates the basic fundamentals of medicine , do not treat the symptom but the disease that is , identify and treat the root cause of a disease . buccal mucosal hypertrophy secondary to open bite was misdiagnosed as leukoplakia , submucosal cyst by some of the practitioners . recurrence within 6 months was seen after each surgery . based on the clinical examination and previous histopathological reports , we first treated the open bite responsible for the lesion by giving a crown on lower deciduous tooth . a buccal shield is similar to an oral screen that is a myofunctional orthodontic appliance that is indicated to restore nasal breathing , intercept premaxillary protrusion , and prevent habits such as finger - sucking and tongue - thrusting . we called it as a buccal shield as it was designed to shield the healing buccal mucosa after planned surgery from trauma . wounds , left uncovered , are prone to infection , contraction , and scarring with other clinical complications and require means to close the defects . the usefulness of collagen membrane dressing over the surgical defects of oral mucosa is well - documented in the literature . in our case forty - eight hours postoperatively buccal shield was delivered to ensure a protection of healing wound from any self - inflicted trauma . modifications of the design were performed on the basis of stability and retention to maintain the patient 's compliance . nevertheless , our patient adjusted to the appliance quickly and without incident . the shield was worn in continuity until 2 months and then tapered over the next 6 months to its abstinence . it has been 18 months postsurgery , and there has been no recurrence of the lesion . the un - identification of the etiology of this hypertrophic lesion led to multiple surgical interventions , increased treatment cost , time , and dissatisfaction . no one is immune from error but a quality time spent on patient examination is what every patient deserves and the moral duty of a clinician . establishing a definitive diagnosis prior to , or in conjunction with ,

timely and accurate diagnosis of a medical / dental condition is the first critical step to ensure appropriate treatment . lack of astuteness in diagnosis may assume many forms . each medical / dental practitioner is morally and legally bound to perform his or her duties to a specified standard of care . the cause of misdiagnosis may be hurriedness , lack of testing , or a simple mistake ; failure to diagnose can constitute a breach of that duty , making the medical / dental provider liable for any resulting damages . the following case report highlights one such misdiagnosed case .

signet cell carcinoma of the colon and rectum are rare aggressive tumors that are known for their spread to other organs and the peritoneum . while well documented in the adult population , the disease is seen more in males with a male to female ration of 1.1:1 ( 2 ) . the incidence is higher in the adult population and is usually diagnosed at a younger age when compared to other colorectal cancers . the infrequency of the disease among the adolescents makes the diagnosis more difficult and the prognosis less favorable . we report a case of signet cell colon cancer a in a 17 year old child . a 17 year old male child presented with one month of progressive right sided abdominal pain and swelling . he reported that eating and playing sports aggravated his pain . vital signs were all within normal limits on admission . physical examination revealed a pleasant child without any distress . laboratory examination revealed a white blood cell count of 11.5 per cubic millimeter , hematocrit of 35% , and platelet count of 395,000 cells / cubic millimeters . the ultrasound of the abdomen revealed a non peristalsing bowel loop which had a configuration strongly suggestive of intussusceptions of the right side of the abdomen . this ultrasound was followed by a ct scan of the abdomen , which showed significant abnormality involving the ascending colon characterized as marked mural thickening having a low density relative to muscle . the patient underwent right colon resection with ileocolic anastomosis followed by with 20 cycles of folfox-6.one year later , patient presented with symptoms and signs of bowel obstruction . follow up ct - scan was done which indicated cancer recurrence in distal pelvis warranting further investigations . fine needle aspiration was positive for malignant cells consistent with recurrence of signet ring cell carcinoma . signet cell cancer of the colon is a rare subtype of colon cancer , where abundant intracytoplasmic mucin pushes the nucleus to the periphery giving a signet ring appearance . in order to meet the definition , there are reports that signet ring cell carcinoma has been increasing in incidence ( 2 ) and currently signet cell carcinoma accounts for 0.7% of the colorectal cancer . patients are often noted to be younger compared to the non - signet cell tumors of the colon . median age is about 59 years when compared to the non signet cell cancer , where the median age is about 61 years ( 2 ) . our case is very significant because of very young age of presentation of this cancer . signet cell carcinoma is presumed to be caused by genetic mutations just like the other colorectal cancers . micro satellite instability ( msi ) is a well - recognized phenomenon in colorectal cancer . micro satellite unstable tumors can be divided into two distinct msi phenotypes : msi - high ( msi - h ) and msi - low ( msi - l ) . signet cell carcinoma has a high level of msi ( msi - h ) ( 3 ) . incidence of k - ras mutation is lower compared other colorectal cancer ( 4 ) . about half of the of signet cell ring cell carcinomas were found in the rectum and colon , most predominant site being the right hemi colon in about 29% . left colon comprises about 15% and approximately 9% of the tumors are noted in the transverse colon ( 5 ) . a majority of the signet cell carcinomas initially present with the distant metastasis in about half of the patients . surgery is part of the standard management of patients with colon and rectal cancer stages i , ii and iii . chemotherapy is the standard adjuvant therapy for stage iii colon cancer , and chemo radiotherapy is the standard adjuvant therapy for stage ii and iii rectal cancer . however , for stage iv colon and rectal cancer , chemotherapy alone may be appropriate and in bulky tumors surgery / radiation can be used for palliation . the median survival period is about 9 months . comparing the respective signet ring cell carcinoma to the high - grade non - signet cell rectal carcinoma the five year survival rates are nearly equal . one of the reasons for poor survival period is that most of the signet cell colon cancers are diagnosed at more advanced stages ( 7 ) . in conclusion early curative resection has significantly improved survival rates compared to the late curative resection groups and to the other groups .

signet cell carcinomas of the colon are well documented in the adult population , but this cancer incidence is very low in the adolescent population . a 17 year old male child presented with one month of progressive abdominal pain . ct scan of the abdomen showed significant abnormality involving the ascending colon characterized as marked mural thickening . biopsy results indicated signet ring cell carcinoma . signet cell carcinoma is presumed to be caused by genetic mutations just like the other colorectal cancers . treatment for signet cell carcinoma is the same as other colorectal cancer . surgery is part of the standard management of patients with colon and rectal cancer stages i , ii and iii . signet cell cancer has a poor survival with the median survival period of about 9 months . the incidence among adolescence is much lower than that of the adult population .

dermatitis herpetiformis ( dh ) is one of the subepidermal autoimmune bullous diseases characterized by skin and intestinal lesions . diagnosis of dh is established on the results of direct immunofluorescence test ( dif ) revealing granular deposits of iga in the papillae and the presence of circulating iga antibodies directed against endomysium and/or tissue and epidermal transglutaminase ( ttg , etg ) [ 2 , 3 ] . skin lesions in dh are histologically characterized by neutrophilic infiltrate leading to destruction of basement membrane zone ( bmz ) proteins , anchoring fibers , and blister formation [ 46 ] . bullous pemphigoid ( bp ) is a blistering disease , characterized by inflammatory infiltrate in the dermis , presence of igg and c3 deposits along the basement membrane zone , and circulating igg autoantibodies . autoantibodies binding to autoantigens ( glycoproteins : 230 kd ( bpag1 ) and 180 kd ( bpag2 ) ) localized in the basement membrane of the epidermis activate a series of immunological and enzymatic phenomena leading to destruction of basement membrane components and anchoring fibers and blister formation as in dh [ 7 , 8 ] . inflammatory infiltrates in the dermis , formed by eosinophils and neutrophils and bound in vivo deposits along the basement membrane in bp or in the top of papillae in dh , are observed . ultrastructural studies also confirmed the presence of intensive inflammatory infiltrate at dermo - epidermal junction , as well as destruction of hemidesmosomes and components of extracellular matrix . formation of the infiltrates is preceded by early accumulation of leukocytes , depending on activity of adhesion molecules . the binding of autoantibodies leads to activation of keratinocytes , release il-6 and il-8 , of activation of c5 component of the complement and metalloproteinases enzymes produced by eosinophils and neutrophils attracted to the basement membrane by selectins and integrins and chemokines [ 10 , 11 ] . some of them are highly specific whereas others may interact with more than one ligand [ 12 , 13 ] . few studies available suggested cytokines ' role in generation of inflammatory influx in autoimmune blistering diseases [ 1417 ] . there is increasing evidence that th17 cells and the cytokines they release such as interleukin-17 ( il-17 ) are important regulators of innate and adaptive immune responses in many th1- and/or th2-mediated autoimmune diseases such as rheumatoid arthritis , systemic lupus erythematosus , and allergic asthma . there is also evidence that th17 cells may have a role in pathogenesis of blistering skin diseases . interleukin-17 is important in initiation and maintenance of many autoimmune reactions and it is involved in production of proinflammatory cytokines , matrix metalloproteinases , neutrophils , and eosinophils , all of which are important pathogenic factors in bullous pemphigoid and dermatitis herpetiformis . the hypothesis is that interleukin-17 may have an important pathogenic role in dh and bp . it was previously reported that th17 cells are recruited to the lesional skin in pemphigus vulgaris ( pv ) and pemphigus foliaceus ( pf ) . immunohistochemical studies showed that both il-17 + and foxp3 + cells were present in higher numbers in cells in bp lesions , compared with control skin . the previous study suggests that , compared with pemphigus , bp shows more th17 cell - related inflammation and less treg - related regulation . there was no data about il-17 serum levels in blistering disease comparing this level with tissue expression . there was no literature data with reference to the role of il-17 in pathogenesis of dermatitis herpetiformis . il-17 can be assessed in the skin lesions and sera of patients and can be used as a marker of disease activity and response to therapy . the information obtained could also lead to the development of novel therapeutic strategies for this and other autoimmune blistering diseases . the goal of this study was to assess interleukin-17 expression in skin lesions and perilesional area and serum levels in patients with dh and bp . the study included 34 persons : 14 untreated patients with bp ( range : 58 to 84 years , average : 68,5 ) and 10 with dh ( range : 18 to 70 years , average : 49,8 ) in an active stage of the disease . a control group consisted of 10 healthy individuals ( range : 19 to 80 years , average : 52,6 years ) . all the patients signed informed consent before entering the study and the study protocol ( rnn/132/07/kb ) was approved by the local ethical committee of medical university of lodz . eight out of 10 dh patients had skin lesions characterized by vesicles and itching papules ; the others had erythematous papules . in all the cases histological pictures showed perivascular neutrophilic infiltrates , the presence of pierrard 's abscesses , and in all patients small subepidermal blisters . in 7/10 samples large unilocular blisters displaying multiple neutrophilic papillary microabscesses were found . direct immunofluorescence tests revealed the presence of granular deposits of iga in skin papillae and indirect immunofluorescence tests were positive for igaema ( oesophagus monkey igaema , medizinische labordiagnostica ) in all the patients ( titer 1 : 401 : 640 , median 1 : 80 ) . antitissue transglutaminase antibodies measured using an immunoassay ( celikey , pharmacia & upjohn ) were present in 7/10 cases ( median 5.1 iu / ml ( range : 0,0186,3 ; iu / ml ) . diagnosis of dh was established based on clinical presentation and results of histological and immunological examination . in 12 out of 14 patients skin blisters , vesicles , and itching papules were found , whereas others had only small vesicles and urticarial papules . in all the patients direct immunofluorescence test revealed igg / c3 linear deposits along bmz . in salt split test deposits were observed in epidermal part of the blister . using indirect immunofluorescence test circulating igg antibodies were found in 14/14 patients , whereas elisa test showed the anti - nc16 autoantibodies ( mbl , nagoya , japan ) present in serum of 11 out of 14 patients . typical histological features of bp including neutrophilic infiltrates , eosinophils , lymphocytes , and , in 12 cases subepidermal blisters supported the clinical diagnosis . the biopsies were taken from the buttock or trunk skin before administration of any ( topical or systemic ) treatment . paraffin - embedded sections ( 3 - 4 m thick ) were used for routine h&e staining and for immunohistochemistry with dako envision detection system using immunoperoxidase method . the following primary monoclonal antibodies were used : antiinterleukin 17 obtained from r&d , uk . for immunohistochemistry the paraffin - embedded sections were placed on adhesive plates , dried at 56c for 24 hours , and later deparaffinized in a series of xylenes and alcohols with decreasing concentrations . activity of endogenous peroxidase was inhibited with 3% hydrogen peroxide solution in methanol for 5 minutes . in order to retrieve the antigenicity of tissues and allow them to react with antibodies , specific procedures were used for each of the tested antibody , according to manufacturers ' instruction . after incubation with diluted antibodies for 60 minutes at room temperature , they were washed with tris buffer twice . dako envision double - step visualization system was then applied in order to visualize the antigen - antibody reaction . in cases of positive immunohistochemical reaction cellular nuclei were stained with the meyer haematoxylin for 2 minutes . after dehydration and processing through series of acetones and xylenes histological morphometry was performed by means of image analysis system consisting of a pc equipped with a pentagram graphical tablet , indeo fast card ( frame grabber , true color , real time ) , produced by indeo ( taiwan ) , and color tv camera panasonic ( japan ) coupled to a carl zeiss microscope ( germany ) . this system was programmed ( multiscan 8.08 software , produced by computer scanning systems , poland ) to calculate the number of objects ( semiautomatic function ) . the coloured microscopic images were saved serially in the memory of a computer , and then quantitative examinations had been carried out . the il-17-positive cells were counted ( semiautomatic function ) in a sequence of 710 consecutive computer images of 400 high power fields0.0047 mm each . the results were expressed as percentages of il-17-positive cells of all lymphocytes determined by their morphology . in each case differences between groups were tested using unpaired student 's t - test preceded by evaluation of normality and levene 's test . il-17 levels were measured in serum in all patients and healthy controls undergoing skin biopsy . five cc of venous blood were drawn from the ulnar vein and after centrifugation serum was stored at 20c for an immunoassay . il-17 levels are shown in pg / ml as mean + / sd whereas t - tn are presented in iu / ml . the mann - whitney test was applied in statistical analysis and results with p < 0.05 were considered statistically significant . in controls staining for il-17 was almost negative ( figure 1 ) . in both bullous pemphigoid and dermatitis herpetiformis groups il-17-positive cells ( lymphocytes ) were detected in the upper dermis of the skin along the basement membrane and around vessels ( figures 2 , 3 , 4 , and 5 ) . the quantitative data of il-17-positive cells expressed as percentages of all lymphocytes in bp and dh appear from table 1 . in biopsies of perilesional skin il-17 expression was present in all patients with bp . in majority of patients moderate intensity of il-17 expression was present in skin lesions , whereas in six patients strong il-17 expression was observed . expression in lesional skin and perilesional skin is statistically higher in skin lesions ( in bp p < 0.003 and in dh p < 0.05 ) . although il-17 expression was present in all patients , in one patient the expression was low . in skin biopsies of perilesional area and skin lesions taken from patients with dh , il-17 expression was weaker than in patients with bp ( 1.9 1.2 versus 2.3 2.1 and 3.9 2.8 versus 7.4 4.1 , p < 0.001 , resp . ) . il-17 levels were statistically higher in dh patients ( 28.10 + / 1.36 ) as compared to patients with bp ( 24.05 + / 0.78 , resp . il-17 concentrations in dh and bp patients compared to healthy subjects were statistically higher ( p < 0.001 ) ( table 2 ) . pathogenesis of bullous diseases is not known , although several aspects of their pathogenesis have been already elucidated [ 1 , 3 , 7 , 10 ] . bp and dh are diseases characterized by inflammatory changes in area surrounding skin lesions typical for the disease including ( but not limited to ) an increase in vessel and connective tissue adhesion molecules expression , an influx of various inflammatory cells , local as well as systemic increase in proinflammatory cytokine synthesis . due to differences in composition of an inflammatory cells in both dh and bp , it was of interest to analyze the il-17 expression in skin lesions , which might play an important role in selective recruitment of leukocytes to the skin . in this paper we were investigating the expression of il-17 in skin lesions and perilesional skin and blood concentration in well - described patients with bp and dh . the pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes , and one of these , il-1 , has been previously implicated in inflammatory skin disease . th17 cells , a subset of th cells involved in autoimmunity and inflammation , possess il-1 receptors and secrete cytokines such as il-17 and il-22 in response to il-1 stimulation . a mutation in the inflammasome protein nlrp3 ( nacht , lrr , and pyd domains containing protein 3 ) causes excess production of il-1 , resulting in an augmentation of th17-dominant pathology . these results indicate that cytokines from th17 cells may potentiate il-1-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism . tumor necrosis factor-(tnf- ) is known to play a pivotal role in the pathogenesis of psoriasis . tnf- has been shown to act directly on keratinocytes , thereby inducing the production of various kinds of chemokines , which contributes to the infiltration of leucocytes into the psoriatic lesions . recent studies have shown that both interleukin il-17 and il-27 are increased in psoriatic lesional tissue . however , the interactions between tnf- , il-17 and il-27 in chemokine production by keratinocytes have not been fully elucidated . this suggests that lesional balance of il-17 and il-27 is involved in the recruitment of t cells , natural killer cells , neutrophils , monocytes , or dendritic cells , thereby affecting inflammation in skin diseases . in our recent paper we have showed a high expression of tnf- in skin lesions in bp and dh . it has been also postulated that the process of blister development might be driven by many factors including local metalloproteinases overexpression , mediated by cytokines . various proinflammatory cytokines , that is , tnf , are involved in many biological processes , among others is overproduction of metalloproteinases , especially stromelysins , gelatinases , and matrilysins.17 . th17 cells , characterized by interleukin-17 production , play crucial roles in the pathogenesis of autoimmune diseases . a potential role of th17 cells in bp was recently suggested , because an increased recruitment of il-17 + cells in the lesional tissue was observed in mucous membrane pemphigoid ( mmp ) , another pemphigoid member . although the role of th17 cells in the pathogenesis of autoimmune diseases is unresolved , they may be the initiators of diseases . alternatively , th17 cells may possibly appear in a protective response to maintain epithelial homoeostasis . if the latter is the case , the more severe disruption in epithelial integrity in bp and pv , when compared with pf , may increase the number of regional th17 cells . the second intriguing issue is that the number of foxp3 + cells in bp was significantly smaller than that in pemphigus groups . in fact , a decreased number or impaired functions of circulating treg cells in several autoimmune diseases have been reported . there unfortunately is still vast amount of data supporting the role of il-17 in pathogenesis of autoimmune bullous diseases . according to our knowledge , these findings are in contrast with data concerning il-17 concentration in sera in patients . in dh patients il-17 serum level was higher in comparison to patients suffering from bp , although this difference in serum concentration between dh and bp patients was smaller than between dh and bp and control group . inflammatory changes in skin in both studied diseases are related not only to neutrophilic influx but also to eosinophils aggregation within area of involved skin . our data may suggest that different ccr1 expression may take part in eosinophils infiltration in bp . th2 cells are involved in all stages of immune response in the course of the allergic reaction [ 30 , 31 ] , and studies carried out so far indicate that these cells are also the source of many proinflammatory cytokines and chemokines involved in allergic inflammation : il-4 , -5 , -10 , and -13 [ 32 , 33 ] . it has been shown that expression of mrna for cytokines associated with lymphocytes th2 ( especially il-5 ) and the concentration of these cytokines in the tissues of an allergic reaction correlate with the number of excited eosinophil 's purposes [ 34 , 35 ] which indicates that the accumulation and activation of lymphocytes cd4 + t cells are directly related to the induction of eosinophilic infiltration . among the recently discovered cytokines ( 1993 ) proinflammatory synthesized by activated t lymphocytes , cd45ro + memory , both the auxiliary ( cd4 + ) and cytotoxic ( cd8 + ) , is a group belonging to the family of cytokines interleukin 17 ( il-17 ) [ 36 , 37 ] . there is numerous scientific evidence that these cytokines play an important role in acute and chronic inflammations [ 36 , 37 ] , including also the pathogenesis of allergic diseases . it is believed that il-17a ( which is a prototype cytokine that group ) can in the mechanism similar to that described above , the relationship between activity and the development of th2 cells infiltration , development of eosinophilic influx , an important link to in an indirect way it combines synthesizing cells t with an increased influx to the site of reaction inflammatory neutrophil [ 37 , 39 ] . numerous studies have shown that increased il-17a activity leads to local flow neutrophils for respiratory purposes , mainly through the influence of this cytokine to synthesize stromal cells chemotactic factors for neutrophils purposes ( such as cxc chemokines ) and growth factors for these cells ( g - csf , gm - csf ) [ 36 , 40 ] . during activation of eosinophils , these cells start secrete ltc4 , lipoxins , paf , txa2 and pge2 . in addition , proteins secreted by eosinophiles such as il-13 , il-5 , gm - csf , eotaxin and rantes activate an autocrine mechanism of the same cell . synthesis of eosinophilic cells , il-1 , il-4 , and il-16 suggests that they may affect the maturation and activation of other cells . similarly , in our study , it is confirmed that these cells are activated in the process of formation changes primarily in patients with bp , where the concentrations of eotaxin and other chemokines were significantly higher than in patients with dh and healthy subjects . extremely important is the impact of eosinophil activity markers on other factors such as the cytokine network of adhesion molecules and tissue factor . the efficacy and safety of neutralizing il-17 itself are under clinical investigation in autoimmune settings , but initial reports suggest different responses in psoriasis and rheumatoid arthritis . clearly more work is required to fully understand the relevance of the il-23/il-17 connection in the spectrum of autoimmune disease . action of autoantibodies in bp and dh can not fully explain several important features of the diseases such as the difficulty of transferring with the pathogenic autoantibodies or the presence of heavy lesional infiltration of eosinophils and neutrophils which is necessary for disease production , although it has a lot of immune mechanisms and relations between them in bullous diseases described . the hypothesis is that interleukin-17 has an important pathogenic role in bp and dh and can describe features of the disease not explained by the autoantibody theory . this cytokine can be assessed in the sera of patients and can be used as a marker of disease activity and response to therapy . the information obtained could also lead to the development of novel therapeutic strategies for this and other autoimmune blistering diseases . bp and dh are diseases with unknown pathogenesis , varying in clinical picture , and have many common issues regarding the inflammatory changes in skin lesions . inflammatory changes in skin in both studied diseases are related not only to neutrophilic influx but also to eosinophils aggregation within area of involved skin . there is vast amount of data supporting the role of il-17 in pathogenesis of autoimmune bullous diseases . in our hands these findings are in contrast with data concerning il-17 concentration in sera in patients . in dh patients il-17 serum level was higher in comparison to patients suffering from bp , although this difference in serum concentration between dh and bp patients was smaller than that between dh and bp and control groups . il-17 can be be used as a marker of disease activity and response to therapy .

dermatitis herpetiformis ( dh ) and bullous pemphigoid ( bp ) are skin diseases associated with eosinophilic and neutrophilic infiltrations . although cytokines are critical for the inflammatory process , there are single findings concerning concentration of il-17 in bullous diseases . the goal of this study was to assess il-17 expression in dh and bp patients . skin biopsies were taken from 10 dh , 14 bp patients and from 10 healthy subjects . the localization and expression of il-17 was studied by immunohistochemistry and the serum concentration was measured by immunoassays . expression of il-17 in the epidermis and in influxed cells in dermis was detected in skin biopsies . expression of il-17 was statistically higher in epidermis and infiltration cells in specimens from bp than from dh patients . examined interleukin expression was detected in perilesional skin of all patients but it was much lower than in lesional skin . the expression of il-17 was not observed in biopsies from healthy people . serum level of il-17 was statistically higher in bp and dh groups as compared to control group . our results provide the evidence that il-17 may play an essential role in activating and recruiting eosinophils and neutrophils , which ultimately contribute to the tissue damage in dh and bp .

conversion disorder has one or more symptoms that affect voluntary motor or sensory function suggesting a neurological or other medical condition , but they are inconsistent with known neurological or musculoskeletal pathologies . instead , the symptoms are due to an unconscious expression of a psychological conflict or need . the symptoms are often reinforced by social support from family and friends or by avoiding underlying emotional stress . the symptoms of patients with conversion disorder can be debilitating and include paralysis of one or more limbs , ataxia , tremors , tics , and dystonia . many other names are used to describe this disorder are functional gait disorder , hysterical paralysis , psychosomatic disorder , conversion reaction , and chronic neurosis . the disorder is more common in adolescence than in childhood . despite conversion disorders long - documented history , it is often confused with other psychological disorders conversion disorder , remain diagnostic challenges for the clinicians . a 17-year - old female coming from mses who was premorbidly maintaining well came with complaints of asymmetrical repetitive flickering like movement of the right hand which started on the day of her 12 grade board exams . she was observed to have reduced sleep since 1 week before her exams and had relatively less communication with family members . on the day of her exams , by the time she got the question paper her whole of her right arm started having repetitive flickering movement vigorously , and she had to support her right arm with the left to write the exam and had come out of exam hall without completing the exam . within a few days , the abnormal movements had progressed to her right leg . informant said that she use to have crying spells and appear sad most of the time as she had not given the exams . she was treated with promethazine and trihexyphenidyl neuroimaging was done which was found to be normal . she showed some improvement after 20 days , but she was not completely resolved , on the day before the day of admission , she developed shivering over her whole body and was admitted to the intensive care unit . it was not associated with loss of consciousness , no urine or fecal incontinence , no frothing from the mouth , no tongue biting , and no up rolling of eyeball . electroencephalogram computed tomography and magnetic resonance imaging brain were done and were found to be normal . , it was found that she was an above average student in her class and that her family had too much expectation from her . she also said that her younger sister was always given more attention by her mother . her episodes were provoked when asked to write or hold a pen with her right hand , also when she was asked to walk without assistance . she was also observed to flex her right toe while walking and during stay in the hospital she was observed to be having a sudden onset of asymmetrical repetitive jerky movements of bilateral legs . routine hemogram , renal function test , liver function test blood sugar , lipid profile , and thyroid function were found to be normal . the patient was prescribed diazepam 4 mg per days and after 2 days it was increased to 6 mg per day , she showed gradual improvement . on the initial days of sessions , her symptoms got aggravated during the sessions and session had to be stopped in between . after few attempts , the patient had ventilated to us how her mother gives less importance to her when compared to her younger sister who is 6 years younger to her . the patient had also said that from her toddler stage till 10 standard she was living with her paternal grandmother and father , and now she moved to a different house along with her parents . her parents were also included in the sessions and her issues with her mother were discussed . the patient had gradually started walking without difficulty and frequency of abnormal movements had reduced . diazepam was tapered and stopped within a week and had been stable at the time of discharge . patient came for a follow - up after 2 weeks , and she had been maintaining well . the clinical picture is indicative of dissociative motor disorder f44.4 according to icd 10 . after taking a detailed history , it was clear that her parents were giving her more pressure to attain high marks in board exam . during interview whenever a patient was asked to hold a pen or to write her symptoms increased . patient was started on therapy sessions as well as low - dose diazepam . in the sessions , possible causes of these symptoms were discussed , and she was encouraged to hold the pen and write . her parents were also psychoeducated about the psychosomatic nature of the symptoms and advised to encourage her for a symptom - free lifestyle . they were also given an instruction not to pay attention to her complaints of physical nature . , patient was explained to her that if there is any problem in her nerves , it will be recorded in that study , along with that dose of diazepam was also increased to 6 from 4 mg . after the study , patient showed a dramatic improvement in her symptoms . failure of treatment in dissociative disorders occurs mostly when we can not identify the primary stressor or gain . by taking proper history and early identification of the stressor there are case reports where dissociative disorders are managed alone with therapy sessions , and the patient is taught to how to deal with stressful situations . nerve conduction study is a diagnostic test to evaluate the function , i.e. the electrical conduction of motor or sensory nerve of the human body . it can also be done along with needle electromyography to measure both nerve and muscle function . in this , the study is performed by electrical stimulation of a peripheral nerve and recording of a muscle supplied by this nerve . the time taken for electrical impulses to travel from the stimulation to the recording site is measured . in this patient , this procedure was well explained to the patient , and she showed marked improvement . conversion disorder , somatoform disorder , and malingering always remain a diagnostic challenges for the clinicians . the prompt history taking , identification of stressors , use of appropriate and validated physical examination manoeuvres , and coordination of care and information exchange between all family members and medical team may facilitate the expeditious care of these patients in a cost - effective manner . the existing literature supports a multidisciplinary treatment approach , with specific interventions , such as cognitive behavior therapy for cognitive restructuring and psychodynamic therapy for addressing symptom connections to trauma and dissociation .

conversion disorders are more prevalent in childhood and adolescence , especially in females . they are usually associated with stressors and symptoms usually reflect a means to avoid the stressor , or also with a primary and secondary gain . this case report involves a similar situation where a young girl was treated successfully with diazepam , therapeutic nerve conduction study , and behavioral psychotherapy .

in the past , the endoplasmic reticulum was viewed as a single , continuous , and homogeneous compartment with a uniform ca store . however , such a simplistic view does not explain the functional heterogeneity of this complex organelle . in particular , the endoplasmic reticulum must provide a pool of rapidly exchanging ca for signal generation , while concurrently maintaining areas within its lumen with stably high ca levels for proper protein folding and processing ( rooney and meldolesi , 1996 ) . today it is widely accepted that although the endoplasmic reticulum is physically continuous ( i.e. , lumenally connected ) ( subramanian and meyer , 1997 ) , it is spatially and functionally heterogeneous ( villa et al . , 1993 ; meldolesi and pozzan , 1998 ; baumann and walz , 2001 ; blaustein and golovina , 2001 ) . this heterogeneity may be established by the nonuniform distribution of endoplasmic reticulum ca - handling proteins : ( 1 ) the ca - binding proteins , such as calreticulin , calsequestrin , glucose - regulated protein 78 and 94 ( grp78/bip and grp94 ) , protein disulfide isomerase ( pdi ) , * and proteins belonging to the pdi - like family , erp72 , erp57 , and the newly identified erp29 ( sargsyan et al . , 2002 ) ; ( 2 ) the ca uptake channels , sarcoplasmic / endoplasmic reticulum ca - transporting atpases ( sercas ) , and ( 3 ) the ca release channels , inositol 1,4,5-trisphosphate ( insp3 ) receptors and ryanodine receptors . a nonuniform distribution of these ca - handling proteins necessarily divides the endoplasmic reticulum into subdomains , which extend beyond the classical divisions of rough endoplasmic reticulum , smooth endoplasmic reticulum , and nuclear envelope . unique accumulations of ca - handling proteins in the endoplasmic reticulum may determine special areas of this membrane system , involved in either ca signaling , ca homeostasis , or protein folding and processing . spatially and temporally complex ca signals generated by the endoplasmic reticulum underlie a diversity of cellular processes ( berridge et al . , 2000 ; johnson and chang , 2000 ) . such ca - dependent pathways include muscle contraction , secretion , proliferation , apoptosis , cell adhesion , differentiation , motility , cellular metabolism , fertilization , and control of gene expression ( johnson and chang , 2000 ) . it is an enormous task for the cell to be able to encode information in the form of ca waves and oscillations , such that signal fidelity is maintained and the correct cellular outcome is achieved . the endoplasmic reticulum is able to solve this problem by establishing a nonuniform distribution of its ca - handling proteins , thus creating spatially heterogeneous ca stores within its lumen ( pezzati et al . heterogeneity in endoplasmic reticulum lumenal ca has previously been demonstrated using electron energy loss imaging ( pezzati et al . , 1997 ) , and such store heterogeneity is sufficient to generate ca signals with enough complexity to control various ca - dependent cellular processes ( johnson and chang , 2000 ) . furthermore , these ca signals may play a critical role not only in the cytoplasm but also in the endoplasmic reticulum lumen ( corbett and michalak , 2000 ) . the pancreatic acinar cell is a model cell type used to study the effects of the spatial heterogeneity of ca - handling proteins on ca - dependent events and has been extensively characterized by the group of petersen ( belan et al . , 1996 ; petersen et al . , 1999 ) and others ( kasai et al . , 1993 ; nathanson et al . , 1994 ; lee et al . the pancreatic acinar cell has special ca release sites in its apical region , termed the trigger zone , which are enriched in insp3 receptors ( nathanson et al . , 1994 ; lee et al . , 1997b ; petersen et al . , 1999 ) , whereas its uptake sites , enriched in sercas , are located in the basal region ( lee et al . , 1997a ; petersen et al . , 1999 , the focus has been on the distribution of ca pumps and channels and their critical role in the generation of spatially complex ca waves and oscillations . we suggest here , however , that ca signal generation and regulation is an intricate process , which likely also requires input from the endoplasmic reticulum lumenal environment and its ca - binding proteins ( simpson et al . , 1997 ; johnson and chang , 2000 ) . the major ca - binding protein of the endoplasmic reticulum of smooth muscle and nonmuscle cells is calreticulin ( milner et al . , 1991 ) , calreticulin may influence the action of both sercas and insp3 receptors ( corbett and michalak , 2000 ) , thereby modulating ca uptake and release , respectively , and thus regulating ca signals . in particular , camacho 's group has shown that calreticulin , in the lumen of the endoplasmic reticulum , inhibits ca uptake by the serca2b pump , thus inhibiting the continued generation of ca waves ( camacho and lechleiter , 1995 ) . this may be due to a direct interaction between calreticulin and the lumenal cooh - terminal tail of the serca2b isoform . in support of this interaction , calreticulin has been shown to colocalize with serca2b ( john et al . , 1998 ) . in addition , a recent report indicates that a mathematical model , developed from single cell ca dynamics , has predicted an interaction between calreticulin and the serca pump ( baker et al . , 2002 ) . this model suggests that calreticulin alters the pump 's affinity for ca , thus regulating ca oscillations . cell subfractionation experiments have revealed that calreticulin copurifies with insp3 binding sites ( enyedi et al . , 1993 ) , whereas double immunolabeling experiments have shown that calreticulin colocalizes with the insp3 receptor in the acrosome , in the equatorial segment , and in cytosolic vesicles of human spermatozoa ( naaby - hansen et al . , 2001 ) . in summary , although the strategic placement of ca pumps and channels is imperative in generating spatially complex ca signals ( johnson and chang , 2000 ) , it is the responsibility of ca - binding proteins to provide a releasable pool of ca near the release channels , and to modulate the activity of the pumps and channels to regulate ca waves and oscillations . the arrangement of ca - handling proteins in the endoplasmic reticulum creates specialized ca - handling subdomains . for example , in astrocytes and oligodendrocytes , ca wave amplification sites exist along the endoplasmic reticulum , which are enriched in calreticulin , sercas , and insp3 receptors and thus exhibit elevated ca release kinetics ( simpson et al . , 1997 , 1998 ) . this organization between the insp3 receptor and calreticulin is similar to the specialization seen in the junctional sarcoplasmic reticulum in striated muscle between calsequestrin and the ryanodine receptor ( allen and katz , 2000 ; gatti et al . , 2001 ) . calreticulin is excluded from these junctional areas , and is found in the longitudinal sarcoplasmic reticulum , from where calsequestrin is excluded ( allen and katz , 2000 ) . the mechanisms underlying the heterogeneous distribution of endoplasmic reticulum ca - binding proteins have only recently been elucidated for calsequestrin . the term condensation is used to refer to the head - to - tail oligomerization of calsequestrin , which is responsible for creating dense cores of the protein that are heterogeneously located throughout the sarcoplasmic reticulum ( gatti et al . , 2001 ) . other endoplasmic / sarcoplasmic reticulum resident proteins may utilize a similar mechanism to establish a nonuniform distribution . the heterogeneous distribution of ca - handling proteins organizes the endoplasmic reticulum into various functional domains , some responsible for ca signaling , some for ca homeostasis , and others for protein folding and quality control . for example , some parts of the endoplasmic reticulum that are enriched in insp3 receptors , sercas , and certain ca - binding proteins may be responsible for rapid ca uptake and release , whereas other regions enriched only in ca - binding proteins may be left to carry out the housekeeping functions of the endoplasmic reticulum ( i.e. , protein processing ) , which are also ca - dependent but are shielded from fluctuations in ca concentration ( rooney and meldolesi , 1996 ) . therefore , the distribution of resident ca - binding proteins may also be potentially important in protein folding and quality control , as most of these proteins are involved in aspects of protein folding and maturation ( i.e. , chaperoning ) , and are enriched in the rough endoplasmic reticulum , the site of protein synthesis ( opas et al . , 1991 ; baumann and walz , 2001 ) , but are nonuniformly concentrated within this compartment ( baumann and walz , 2001 ) . chaperones are weakly associated with one another and form a matrix in which they become embedded , resulting in their increased local concentration ( baumann and walz , 2001 ) . 1 compares the distribution of calreticulin to that of pdi and grp94 within the endoplasmic reticulum of mouse embryonic fibroblasts . pdi exhibits an overlapping yet distinct distribution with calreticulin , whereas grp94 exhibits virtually complete overlap with calreticulin ( fig . 1 , a and b , respectively ) . the differential distribution of various lumenal ca - binding proteins may be of great physiological importance for ca homeostasis . calreticulin and grp94 are two major ca storage proteins of the endoplasmic reticulum , by virtue of their extremely high ca capacity ( milner et al . , 1991 ; argon and simen , 1999 ) , which is not matched by any other endoplasmic reticulum protein . fluctuations of ca concentration in the lumen of the endoplasmic reticulum , which are ultimately regulated by ca - handling proteins , may have profound effects on the structure and function of integral and lumenal ( peripheral ) membrane proteins and likely contribute to the functional heterogeneity of the endoplasmic reticulum ( corbett and michalak , 2000 ) . furthermore , endoplasmic reticulum functions involved with protein processing have been shown to underlie various diseases such as alzheimer 's disease , parkinson 's disease , and 1-antitrypsin deficiency ( kopito and ron , 2000 ; paschen and frandsen , 2001 ; rutishauser and spiess , 2002 ) . the distribution of ca - binding proteins within the endoplasmic reticulum of mouse embryonic fibroblasts , in relation to calreticulin ( crt ) , as shown by double immunofluorescence labeling and visualized by confocal microscopy . for methods , see mesaeli et al . ( 1999 ) . left to right : crt distribution ( red ) , pdi , or grp94 distribution ( green ) . the last column of each row is an overlay of the two previous images ; yellow represents areas of overlap . ( b ) the distribution of grp94 shows virtually complete overlap compared with that of crt . additionally , it was recently postulated that the endoplasmic reticulum and sarcoplasmic reticulum may play different roles in cells in which they coexist ( jaconi et al . , 2000 ; for example , in cardiomyocytes , the sarcoplasmic reticulum is involved in the classical role of excitation contraction coupling , whereas the endoplasmic reticulum has been suggested to perform housekeeping functions , such as protein turnover ( mesaeli et al . , 2001 ) . the heterogeneous distribution of calsequestrin and calreticulin in the heart , along with their respective release channels , ryanodine receptors in the sarcoplasmic reticulum and insp3 receptors in the endoplasmic reticulum , may be responsible for this duality of function . studies on calreticulin deficient cardiomyocytes show that these cells exhibit spontaneous contraction , thus suggesting a functional sarcoplasmic reticulum ( mesaeli et al . , 1999 ) . however , calreticulin null fibroblasts show impaired ca homeostasis by the endoplasmic reticulum ( nakamura et al . , 2001 ) , and this may also be the case for calreticulin - null cardiomyocytes . at the early stages of development , the endoplasmic reticulum may play a critical role in protein and lipid synthesis as well as in the regulation of ca - dependent transcriptional processes . mature muscle , the sarcoplasmic reticulum becomes responsible for regulating cauptake and release and excitation contraction coupling . interestingly , heterogeneity in compartmentalization may already be evident during development . in human oocytes , for example , the endoplasmic / sarcoplasmic reticulum associated ca - binding proteins calreticulin is predominant in the cell cortex , whereas calsequestrin is found throughout the entire cytoplasm ( fig . such differential distribution of calreticulin and calsequestrin indicates that oocytes have two distinct ca storage compartments : one enriched in calreticulin ( and the insp3 receptor ) , and the other in calsequestrin ( and the ryanodine receptor ) . calsequestrin may localize to certain regions by the condensation mechanism , whereas calreticulin may utilize specific protein interestingly , calnexin , like calreticulin , is also predominant in the cell cortex where it is found in a peculiar trilaminar arrangement ( fig . calnexin is a chaperone , calsequestrin is a ca storage protein , whereas calreticulin carries out both functions . thus , certain regions of the endoplasmic reticulum may be involved in intensive protein processing required for oocyte maturation and embryo development , whereas other regions may be involved in ca homeostasis . in conclusion , the spatial heterogeneity of the endoplasmic reticulum may be established early on in development , and this warrants further investigation . deciphering the organization of ca - handling proteins in the endoplasmic reticulum may hold a clue to our understanding of the generation of the multifunctionality of this membrane system . human oocytes showing distribution of calreticulin ( crt ) , calnexin ( cnx ) , and calsequestrin ( csq ) . crt predominates in the cell cortex ; cnx is also in the cell cortex , but in a trilaminar arrangement ; and finally , csq is found spread throughout the cell .

the endoplasmic reticulum is a heterogeneous compartment with respect to the distribution of its ca2 + -handling proteins , namely the ca2 + -binding proteins , the ca2 + pumps and the ca2 + release channels . the nonuniform distribution of these proteins may explain the functional heterogeneity of the endoplasmic reticulum , such as the generation of spatially complex ca2 + signals , ca2 + homeostasis , and protein folding and quality control .

the setting was the kaiser permanente medical care program of northern california ( kpnc ) , a large group practice prepaid health plan where gdm is diagnosed in accordance with the american diabetes association ( ada ) guidelines ( 2,13 ) . the study was conducted in collaboration with the kpnc regional perinatal service center , which provides supplemental prenatal care over the telephone to women with gdm . women with gdm according to the ada criteria ( 13 ) were eligible to participate . exclusion criteria included : age < 18 years ; multiple gestation ; diagnosis of diabetic retinopathy ; high - risk pregnancy ( i.e. , drug or alcohol abuse , chronic health problems , or pregnancy complications ) ; thyroid diseases diagnosed in the last 30 days ; and non - english speaker . eligibility was assessed through the review of electronic medical records and telephone interviews ( fig . women were randomly assigned to the lifestyle intervention condition or usual care control condition upon completion of their baseline clinic visit . a computer randomization program was used to ensure that the conditions remained balanced with regards to the following characteristics : age ( < 30 and 30 years ) , parity ( 1 and > 1 ) , and pregravid bmi ( < 27.0 and 27.0 kg / m ) . the institutional review board at kpnc approved the study protocol . the intervention was called diet , exercise and breastfeeding intervention ( debi ) for women with gdm . two trained dietitians delivered the intervention , which was adapted from the dpp curriculum and incorporated behavioral constructs from social cognitive theory ( 14 ) and the transtheoretical model ( 15 ) of behavior change . the prenatal phase started soon after the diagnosis of gdm and consisted of one in - person session and two individual telephone counseling contacts . the dietitians explained to the women their elevated risk of developing type 2 diabetes and advised the women to comply with the iom guidelines ( 16 ) for gwg that were in place at the time . for obese women , the iom guidelines did not provide an upper gwg limit , thus it was recommended that obese women not exceed a gwg of 11.4 kg , as advised for overweight women . they were encouraged to follow the ada diet ( 17 ) and engage in moderate intensity physical activity for 150 min per week ( 18 ) . written intervention materials about portion size , foods with low glycemic index or low fat , and how to read food labels were discussed during telephone counseling contacts . toward the end of pregnancy , intervention women were referred to a lactation consultant who discussed the benefits of breastfeeding , offered a breast pump , and encouraged the women to exclusively breastfeed for 6 months ( 19 ) . the lactation consultant then scheduled calls ( 14 calls , as needed ) in the first 6 weeks after delivery to evaluate latch and feeding techniques and to review the maintenance of milk supply . the early postpartum phase began 6 weeks after delivery and ended at 7 months postpartum . for women whose pregravid bmi was < 25.0 kg / m , the postpartum weight goal was to reach prepregnancy weight . for women whose pregravid bmi was 25.0 kg / m , the postpartum weight goal was to lose an additional 5% of their prepregnancy weight . women were asked to reach their weight goal during the first 12-months postpartum and were given the dpp handbook that contained written materials organized in 16 sessions ( 20 ) . there was a core curriculum of 8 sessions with up to 8 additional sessions offered to those who desired more contact . the sessions were conducted over the telephone except for the first and the last , which were conducted in - person . women were encouraged to perform 150 min of moderate or harder physical activity per week and to consume 25% or less of total calories from fat per day . fat and calorie intake and physical activity goals were tailored based on dietary and physical activity assessments . women were given self - monitoring diaries for recording the fat grams consumed and the minutes of physical activity and were asked to return these diaries weekly by mail . these diaries were used by the dietitians to help the women meet their goals . during counseling contacts , dietitians would review the previous week s diary and written material designated for that session . the maintenance phase began soon after the early postpartum phase and ended at 12 months postpartum . it consisted of three telephone contacts that reinforced the positive behavioral changes achieved during the core curriculum and addressed relapse . women randomized to the usual care condition received printed educational materials that included publicly available information on gdm . in the postpartum period , they received two newsletters focusing on issues related to infant safety and general health . participants were asked to attend four clinic visits for data collection : one during pregnancy soon after the diagnosis of gdm ( baseline ) and three follow - up visits at 6 weeks , 7 months , and 12 months postpartum . baseline and follow - up data were collected by research assistants who were unaware of the condition assignment . height was measured at baseline with a standard stadiometer , and weight was measured at each visit with a tanita wb-110 digital electronic scale . information on diet during the previous month ( assessed via a 120-item food frequency questionnaire ) and physical activity during the previous week ( 22 ) was collected at baseline , 6 weeks , and 7 months postpartum . total gwg was calculated as the difference between the latest weight measured during pregnancy ( obtained from medical records ) and self - reported pregravid weight . the use of diabetes medications , number of perinatal clinic visits during pregnancy , and infant birthweight were obtained from the medical records . all data analyses were by intention - to - treat . for the primary dichotomous outcome of meeting the postpartum weight goal and the secondary outcome of breastfeeding , we used repeated - measures logistic regression with estimation via generalized estimating equations to account for the within - person correlation in repeated binary responses . for continuous secondary outcomes ( such as diet and physical activity ) , linear mixed - effects models were used to examine the overall average difference between the intervention and usual care conditions in the means at each visit . condition difference in outcomes at each clinic visit were presented , with associated significance probabilities obtained from the longitudinal regression models that included condition - visit interaction terms . heterogeneity in intervention effect over time was assessed via a test of the condition - clinic visit interaction terms . women with gdm according to the ada criteria ( 13 ) were eligible to participate . exclusion criteria included : age < 18 years ; multiple gestation ; diagnosis of diabetic retinopathy ; high - risk pregnancy ( i.e. , drug or alcohol abuse , chronic health problems , or pregnancy complications ) ; thyroid diseases diagnosed in the last 30 days ; and non - english speaker . eligibility was assessed through the review of electronic medical records and telephone interviews ( fig . women were randomly assigned to the lifestyle intervention condition or usual care control condition upon completion of their baseline clinic visit . a computer randomization program was used to ensure that the conditions remained balanced with regards to the following characteristics : age ( < 30 and 30 years ) , parity ( 1 and > 1 ) , and pregravid bmi ( < 27.0 and 27.0 kg / m ) . the institutional review board at kpnc approved the study protocol . the intervention was called diet , exercise and breastfeeding intervention ( debi ) for women with gdm . two trained dietitians delivered the intervention , which was adapted from the dpp curriculum and incorporated behavioral constructs from social cognitive theory ( 14 ) and the transtheoretical model ( 15 ) of behavior change . the prenatal phase started soon after the diagnosis of gdm and consisted of one in - person session and two individual telephone counseling contacts . the dietitians explained to the women their elevated risk of developing type 2 diabetes and advised the women to comply with the iom guidelines ( 16 ) for gwg that were in place at the time . for obese women , the iom guidelines did not provide an upper gwg limit , thus it was recommended that obese women not exceed a gwg of 11.4 kg , as advised for overweight women . they were encouraged to follow the ada diet ( 17 ) and engage in moderate intensity physical activity for 150 min per week ( 18 ) . written intervention materials about portion size , foods with low glycemic index or low fat , and how to read food labels were discussed during telephone counseling contacts . toward the end of pregnancy , intervention women were referred to a lactation consultant who discussed the benefits of breastfeeding , offered a breast pump , and encouraged the women to exclusively breastfeed for 6 months ( 19 ) . the lactation consultant then scheduled calls ( 14 calls , as needed ) in the first 6 weeks after delivery to evaluate latch and feeding techniques and to review the maintenance of milk supply . the early postpartum phase began 6 weeks after delivery and ended at 7 months postpartum . for women whose pregravid bmi was < 25.0 kg / m , the postpartum weight goal was to reach prepregnancy weight . for women whose pregravid bmi was 25.0 kg / m , the postpartum weight goal was to lose an additional 5% of their prepregnancy weight . women were asked to reach their weight goal during the first 12-months postpartum and were given the dpp handbook that contained written materials organized in 16 sessions ( 20 ) . there was a core curriculum of 8 sessions with up to 8 additional sessions offered to those who desired more contact . the sessions were conducted over the telephone except for the first and the last , which were conducted in - person . women were encouraged to perform 150 min of moderate or harder physical activity per week and to consume 25% or less of total calories from fat per day . fat and calorie intake and physical activity goals were tailored based on dietary and physical activity assessments . women were given self - monitoring diaries for recording the fat grams consumed and the minutes of physical activity and were asked to return these diaries weekly by mail . these diaries were used by the dietitians to help the women meet their goals . during counseling contacts , dietitians would review the previous week s diary and written material designated for that session . the maintenance phase began soon after the early postpartum phase and ended at 12 months postpartum . it consisted of three telephone contacts that reinforced the positive behavioral changes achieved during the core curriculum and addressed relapse . women randomized to the usual care condition received printed educational materials that included publicly available information on gdm . in the postpartum period , they received two newsletters focusing on issues related to infant safety and general health . participants were asked to attend four clinic visits for data collection : one during pregnancy soon after the diagnosis of gdm ( baseline ) and three follow - up visits at 6 weeks , 7 months , and 12 months postpartum . baseline and follow - up data were collected by research assistants who were unaware of the condition assignment . height was measured at baseline with a standard stadiometer , and weight was measured at each visit with a tanita wb-110 digital electronic scale . information on diet during the previous month ( assessed via a 120-item food frequency questionnaire ) and physical activity during the previous week ( 22 ) was collected at baseline , 6 weeks , and 7 months postpartum . total gwg was calculated as the difference between the latest weight measured during pregnancy ( obtained from medical records ) and self - reported pregravid weight . the use of diabetes medications , number of perinatal clinic visits during pregnancy , and infant birthweight were obtained from the medical records . all data analyses were by intention - to - treat . for the primary dichotomous outcome of meeting the postpartum weight goal and the secondary outcome of breastfeeding , we used repeated - measures logistic regression with estimation via generalized estimating equations to account for the within - person correlation in repeated binary responses . for continuous secondary outcomes ( such as diet and physical activity ) , linear mixed - effects models were used to examine the overall average difference between the intervention and usual care conditions in the means at each visit . condition difference in outcomes at each clinic visit were presented , with associated significance probabilities obtained from the longitudinal regression models that included condition - visit interaction terms . heterogeneity in intervention effect over time was assessed via a test of the condition - clinic visit interaction terms . of the 710 women with gdm , 695 ( 97.9% ) were contacted ( fig . there were no differences between these two groups with regard to age , race / ethnicity , and gestational age ( data not shown ) . among those who completed the eligibility screening , 83 ( 23.1% ) were not eligible . among the remaining 235 women , 222 ( 94.5% ) agreed to participate and made a baseline visit appointment ; 197 ( 83.8% ) women attended the baseline visit , gave informed consent , and were randomized . participant retention at each follow - up visit in the intervention and usual care conditions , respectively , were as follows : 95 and 97% at 6 weeks , 80 and 91% at 7 months , and 80 and 90% at 12 months postpartum ( fig . no differences in baseline characteristics were observed between women in the intervention and usual care conditions except that the 1-h glucose value from the diagnostic 100-g oral glucose tolerance test was lower among women in the intervention condition compared with women in the usual care condition and small differences in smoking and employment status ( table 1 ) . baseline characteristics of women with gdm by randomization condition , kpnc , 20052008 data are % unless otherwise indicated . ogtt , oral glucose tolerance test . * p values from tests that exclude the missing category . the proportion of women who reached their postpartum weight goal was higher in the intervention condition ( table 2 ) . although this difference was not statistically significant , the condition difference in absolute proportion was 16.1% ( p = 0.07 ) at 12 months postpartum . similar results were obtained after adjusting for 1-h glucose levels and in subgroup analyses where women were stratified by smoking or employment status ( data not shown ) . proportion of women with gdm meeting the postpartum weight goals by randomization group and time since delivery all p values for treatment condition and follow - up visit interaction are > 0.05 . because the postpartum weight goals differed between women with pregravid bmi < 25.0 kg / m and women with pregravid bmi 25.0 kg / m , the efficacy of the intervention within these two groups was examined ( table 2 ) . the magnitude of the absolute condition difference in the proportion of women meeting the postpartum weight goal was similar in normal weight and overweight / obese women . because the rationale for starting the intervention during pregnancy was that preventing excessive gwg would result in reduced postpartum weight retention , the efficacy of the intervention was assessed separately in those who did and did not exceed the recommended gwg ( table 2 ) . the intervention appeared to be more effective among women who did not exceed the guidelines for gwg ; the absolute difference between the intervention and the usual care conditions in the proportion of women reaching the weight goal at 12 months postpartum was 22.5% ( p = 0.04 ) . in a sensitivity analysis where women who did not attend the postpartum visits were categorized as not having reached the weight goal at 12 months postpartum , the intervention condition remained more likely to reach the postpartum weight goal than usual care ( absolute difference = 11% ) despite a slightly greater loss to follow - up among women in the intervention condition . as compared with the usual care arm , the women in the intervention at 7 months postpartum showed a statistically significant decrease in dietary fat intake ( condition difference in the mean change in percent of calories from fat : 3.55% , p = 0.002 ) ; a greater but nonsignificant increase in physical activity ( condition difference in the mean change in minutes of moderate - to - vigorous physical activity per week : 25.3 , p = 0.91 ) ; and a higher but not significant likelihood to partially or exclusively breastfeed ( condition difference in proportion : 15.0% , p = 0.09 ) ( table 3 ) . breastfeeding and changes in dietary fat intake and physical activity by randomization condition and time since delivery all p values for treatment condition and follow - up visit interaction are > 0.05 except for change in percent of calories from fat ( p = 0.001 ) . ninety - three percent of the women randomized to the intervention completed the first prenatal session , and 79% completed two or more . on average , the pregnancy telephone sessions lasted 31.2 17.7 min , while the in - person sessions lasted approximately 1 h. for the early postpartum phase , 74% of the women completed the 8 core intervention sessions . the telephone sessions lasted on average 32.6 15.7 min each , while the in - person sessions lasted approximately 1 h. women returned a median of 3 self - monitoring diaries , with 30.9% returning 6 or more . overall , the women were very satisfied with the intervention ( 97% ) and most ( 92% ) would recommend the program to others with gdm . a majority ( 82% ) said they were likely to continue to set weight goals . two focus groups , each with 8 intervention participants , were conducted upon completion of the study . for physical activity , the following themes emerged : 1 ) the need for support for physical activity from family and others in their social network ; 2 ) the addition of a website to connect with other gdm women ; and 3 ) tips on how to exercise with a new infant . reported barriers to physical activity were personal and child illnesses , returning to work , and bad weather . for diet , the following themes emerged : 1 ) the need for information on the optimal type of carbohydrate for the transition from the pregnancy diet to the low - fat postpartum diet and 2 ) the need for low - fat recipes . the proportion of women who reached their postpartum weight goal was higher in the intervention condition ( table 2 ) . although this difference was not statistically significant , the condition difference in absolute proportion was 16.1% ( p = 0.07 ) at 12 months postpartum . similar results were obtained after adjusting for 1-h glucose levels and in subgroup analyses where women were stratified by smoking or employment status ( data not shown ) . proportion of women with gdm meeting the postpartum weight goals by randomization group and time since delivery all p values for treatment condition and follow - up visit interaction are > 0.05 . because the postpartum weight goals differed between women with pregravid bmi < 25.0 kg / m and women with pregravid bmi 25.0 kg / m , the efficacy of the intervention within these two groups was examined ( table 2 ) . the magnitude of the absolute condition difference in the proportion of women meeting the postpartum weight goal was similar in normal weight and overweight / obese women . because the rationale for starting the intervention during pregnancy was that preventing excessive gwg would result in reduced postpartum weight retention , the efficacy of the intervention was assessed separately in those who did and did not exceed the recommended gwg ( table 2 ) . the intervention appeared to be more effective among women who did not exceed the guidelines for gwg ; the absolute difference between the intervention and the usual care conditions in the proportion of women reaching the weight goal at 12 months postpartum was 22.5% ( p = 0.04 ) . in a sensitivity analysis where women who did not attend the postpartum visits were categorized as not having reached the weight goal at 12 months postpartum , the intervention condition remained more likely to reach the postpartum weight goal than usual care ( absolute difference = 11% ) despite a slightly greater loss to follow - up among women in the intervention condition . as compared with the usual care arm , the women in the intervention at 7 months postpartum showed a statistically significant decrease in dietary fat intake ( condition difference in the mean change in percent of calories from fat : 3.55% , p = 0.002 ) ; a greater but nonsignificant increase in physical activity ( condition difference in the mean change in minutes of moderate - to - vigorous physical activity per week : 25.3 , p = 0.91 ) ; and a higher but not significant likelihood to partially or exclusively breastfeed ( condition difference in proportion : 15.0% , p = 0.09 ) ( table 3 ) . breastfeeding and changes in dietary fat intake and physical activity by randomization condition and time since delivery all p values for treatment condition and follow - up visit interaction are > 0.05 except for change in percent of calories from fat ( p = 0.001 ) . ninety - three percent of the women randomized to the intervention completed the first prenatal session , and 79% completed two or more . on average , the pregnancy telephone sessions lasted 31.2 17.7 min , while the in - person sessions lasted approximately 1 h. for the early postpartum phase , 74% of the women completed the 8 core intervention sessions . the telephone sessions lasted on average 32.6 15.7 min each , while the in - person sessions lasted approximately 1 h. women returned a median of 3 self - monitoring diaries , with 30.9% returning 6 or more . overall , the women were very satisfied with the intervention ( 97% ) and most ( 92% ) would recommend the program to others with gdm . a majority ( 82% ) said they were likely to continue to set weight goals . two focus groups , each with 8 intervention participants , were conducted upon completion of the study . for physical activity , the following themes emerged : 1 ) the need for support for physical activity from family and others in their social network ; 2 ) the addition of a website to connect with other gdm women ; and 3 ) tips on how to exercise with a new infant . reported barriers to physical activity were personal and child illnesses , returning to work , and bad weather . for diet , the following themes emerged : 1 ) the need for information on the optimal type of carbohydrate for the transition from the pregnancy diet to the low - fat postpartum diet and 2 ) the need for low - fat recipes . this pilot study , which evaluated a dpp - based lifestyle intervention that aimed to reduce diabetes risk factors and started soon after the diagnosis of gdm and continued into the postpartum period , was shown to be feasible . thus , the dpp lifestyle intervention delivered by telephone was successfully adapted for women with gdm . the intervention appeared to reduce type 2 diabetes and gdm risk factors such as body weight and dietary fat intake ( 7,23 ) and increased breastfeeding . the intervention helped women meet their postpartum weight goals by reducing postpartum weight retention in normal - weight women and helping overweight or obese women to lose weight . only two randomized controlled trials have included women with a pregnancy complicated by gdm . in both trials , the affected pregnancies occurred several years prior to study initiation . in the troglitazone in the prevention of diabetes ( tripod ) trial ( 8) , treatment with troglitazone reduced the incidence of diabetes by over 50% . subgroup analyses ( 24 ) among dpp women with a history of gdm demonstrated that intensive lifestyle intervention and metformin both decreased the risk of type 2 diabetes by approximately 50% . our study provides feasibility data suggesting that a dpp - based intervention delivered by telephone in pregnancy through the postpartum period is feasible and effective in women with gdm . the higher proportion of women reaching their postpartum weight goal is mostly because of the postpartum intervention , however the short pregnancy intervention may have promoted some initial behavioral changes , making the postpartum intervention more effective as shown by our subgroup analyses according to gwg . given the strong association between gwg and postpartum weight retention ( 9,11,25 ) , it may be difficult to intervene and reduce postpartum weight without first preventing excessive gwg . in our stratified analyses , the intervention appeared to be more effective among women who met the guidelines for gwg , either because the postpartum intervention was easier for such women to adopt as they may be more adherent to health recommendations or because of the biological connection between gwg and postpartum weight retention . the limitations are familiar to feasibility studies : a relatively small sample size and an intervention that needed additional refinement , specifically with input from the target population . information learned from this feasibility trial will inform a future , adequately powered , randomized lifestyle intervention trial . there was also a small differential loss to follow - up between the intervention and the usual care conditions . this difference may be because of the translational nature of the study , which did not require participants to attend at least two baseline study visits before randomization , as is common practice in efficacy trials . in conclusion , our study suggests that an adaptation of the dpp lifestyle intervention , delivered primarily by telephone , is feasible for women with gdm . it may also reduce pregnancy weight retention and may help overweight or obese women lose weight in the postpartum period . although the intervention was effective in reducing dietary fat intake , strategies for helping postpartum women to overcome barriers and increase their physical activity are needed . future randomized control trials should focus on developing such strategies , particularly for soliciting social support for increasing physical activity .

objectiveto pilot , among women with gestational diabetes mellitus ( gdm ) , the feasibility of a prenatal / postpartum intervention to modify diet and physical activity similar to the diabetes prevention program . the intervention was delivered by telephone , and support for breastfeeding was addressed.research design and methodsthe goal was to help women return to their prepregnancy weight , if it was normal , or achieve a 5% reduction from prepregnancy weight if overweight . eligible participants were identified shortly after a gdm diagnosis ; 83.8% consented to be randomly assigned to intervention or usual medical care ( 96 and 101 women , respectively ) . the retention was 85.2% at 12 months postpartum.resultsthe proportion of women who reached the postpartum weight goal was higher , although not statistically significant , in the intervention condition than among usual care ( 37.5 vs. 21.4% , absolute difference 16.1% , p = 0.07 ) . the intervention was more effective among women who did not exceed the recommended gestational weight gain ( difference in the proportion of women meeting the weight goals : 22.5% , p = 0.04 ) . the intervention condition decreased dietary fat intake more than the usual care ( condition difference in the mean change in percent of calories from fat : 3.6% , p = 0.002 ) and increased breastfeeding , although not significantly ( condition difference in proportion : 15.0% , p = 0.09 ) . no differences in postpartum physical activity were observed between conditions.conclusionsthis study suggests that a lifestyle intervention that starts during pregnancy and continues postpartum is feasible and may prevent pregnancy weight retention and help overweight women lose weight . strategies to help postpartum women overcome barriers to increasing physical activity are needed .

cavernous carotid artery ( cca ) aneurysms are considered a distinct group from all other intracranial aneurysms as the completeness of the cavernous sinus ( cs ) walls is responsible for their low subarachnoid hemorrhage ( sah ) risk.1 their etiology can be idiopathic , iatrogenic , traumatic , and rarely infectious.2 in 1924 , barth described the classic triad of signs for traumatic cavernous carotid pseudoaneurysms ( ccpa ) ; it includes severe epistaxis often years following the injury delaying diagnosis by blurring the link between them , monocular vision loss , and skull base fracture.3 direct trauma to the wall of the cca can lead to the development of a carotid cavernous fistula ( ccf ) , however ; additionally injuring the wall of the cs will lead to pseudoaneurysm formation by the fibrous organization of the locally contained hematoma within the remains of the cs.4 5 knowledge of skull base surgical anatomy hand in hand with superb surgical dexterity grants us the safety in drilling , a cornerstone during the exposure part of a skull base surgery . nevertheless , vascular laceration can occur ; more commonly during the dissection phase if the tumor encases a vessel . we describe the incidental discovery of a ccpa during a routine contrast - enhanced computed tomography ( ct ) imaging done as part of the fever workup on an 11-year - old patient , 21 days following craniotomy for a skull base rhabdomyosarcoma tumor . we emphasize the importance of active search for these aneurysms following surgery as aneurysmal formation can occur even in the absence of obvious intraoperative vessel injury hemorrhage or clinical symptoms . an 11-year - old child diagnosed with a rhabdomyosarcoma in his left infratemporal fossa and parapharyngeal space underwent tumor resection that included neoadjuvant chemotherapy , combined transcervical a follow - up magnetic resonance imaging scan a year later disclosed a new lesion involving the lateral part of the left cs and foramen ovale ( fo ) necessitating reoperation ( fig . ( a f ) preoperative and ( g and h ) postoperative ct and mri showing the tumor . b : contrast - enhanced axial ct and mri [ c and d ] : scans showing left cs mass . [ g and h ] : axial ct scans showing no bleeding or brain infarcts ) . ct , computed tomography ; cs , cavernous sinus ; mri , magnetic resonance imaging . a left frontotemporal craniotomy combined with zygomatic osteotomy was followed with ligation of the middle meningeal artery at the foramen spinosum via a subtemporal extradural approach . the anterolateral wall of the fo was drilled out to allow mobilization of the tumor that invaded the mandibular division of the trigeminal nerve ( v3 ) . proximal vascular control was achieved via unroofing the petrous internal carotid artery ( ica ) near the junction of the glasscock and kawase triangles . the greater and lesser petrosal nerves were sectioned to prevent facial nerve injury and to improve petrous carotid exposure . elevating the external leaf of the cs posterolaterally exposed the tumor and v3 within meckel cave . the lateral bony wall of the superior orbital fissure was drilled out starting from the foramen rotundum ( fr ) to its upper free edge . to achieve mobilization of trigeminal 's nerve maxillary branch at fr its anterolateral aspect the three branches of the trigeminal nerve were incised at their foramina 's entry point and the trigeminal nerve at its entry point to meckel cave . an intradural approach to the cs was used to allow medial mobilization and dissection of the tumor . the dura was split just below the dural entry point of the oculomotor nerve , allowing identification of the cca ; the tumor was separated from the medial cs and cca from proximal to distal using blunt and sharp dissection allowing a complete mobilization and resection of the tumor from the cca with preservation of the middle layers of the cs , oculomotor , and trochlear nerves . however , due to tumor invasion , the abducens nerve has been incised adjacent and lateral to the cca . during the dissection of the tumor from the medial part of the cs a minor arterial and venous bleeding were encountered and controlled with application of surgicel ( ethicon , somerville , new jersey , united states ) and gentle pressure . after complete tumor resection , meckel cave entry point was sealed with muscle piece and tachosil patch ( takeda austria gmbh , linz , austria ) , dural closure was achieved using 4/0 surgilon suture ( medtronic , dublin , ireland ) and a vascularized temporalis muscle and pericranial flap were mobilized to the middle cranial fossa . the patient recovered well and a postoperative ct scan disclosed minor fluid collection in the temporal fossa , but no bleeding or brain infarcts ( fig . three weeks following the surgery the patient presented with fever and as a part of the fever workup a contrast - enhanced ct scan was done , which disclosed a mass at the left cs ( fig . 2e ) revealed a left ccpa measuring 18 mm 3.5 mm . the pseudoaneurysm was treated endovascularly using a single - covered stent allowing preservation of the lumen with no further complication ( fig . c ) . ( a ) contrast - enhanced axial ct show left cs mass . ( e and f ) left carotid digital subtraction angiography before and after stenting of the pseudoaneurysm . ct , computed tomography ; cta , computed tomography angiography ; cs , cavernous sinus . saccular traumatic pseudoaneurysms occur due to arterial wall laceration , contained by an organized extraluminal hematoma thus lacking a true wall , a fact that predispose them to rupture more easily than idiopathic or traumatic fusiform aneurysms in which a true wall exists , but adventitial thinning caused by peeling the tumor off the adventitia causes vascular dilatation ; their rupture risk is lower than saccular aneurysms.6 hemorrhagic or embolic complications7 8 9 10 are common for these rare pseudoaneurysms and they face the patient at great risk if being unrecognized , thus , the immediate recognition of the above symptoms and treatment is prerequisite to prevent fatal complications . iatrogenic ccpa is mostly described in relation to trans - sphenoidal procedures due to the proximal relation between the cca , the wall of the sphenoid sinus and the sella turcica.9 11 12 it is rarer following skull base surgery due to the relative paucity of the procedure , nevertheless , its life - threatening consequences such as rupture , severe hemorrhage , pituitary apoplexy , stroke , and death necessitate urgent diagnosis and treatment even for an incidentaloma , such as our case . one report exists , where a patient refusal to treat his pseudoaneurysm resulted in his death from severe epistaxis 2 years following surgery , thus making the always optional conservative management treatment irrelevant.9 when suspicion of vascular injury arises during surgery , it is logical to perform a postoperative vascular imaging , such as cta or dsa.5 13 14 in our case , in the absence of major intraoperative bleeding or direct injury to the arterial wall , such suspicion did not arise . despite the uneventful operative and postoperative course , a pseudoaneurysm measuring 18 mm 3.5 mm was discovered and later stented with a single stent . knowing that postsurgical changes can blur minute vascular abnormalities and that that the natural history of pseudoaneurysms is to grow over time we recommend to perform a delayed vascular imaging , specifically in search for this feared complication even in the absence of suggestive symptomatology and a negative immediate postoperative imaging . in the absence of intact cs walls after surgical resection , the aneurysm will not be contained within the cs wall and might rupture directly into the epidural space leading to the formation of acute epidural hematoma and subsequently brain herniation and death within a very short period of time . rapid formation of ccpa can occur following cs surgery , even with uneventful surgery and without obvious injury to the ca wall . the formation is asymptomatic and can end in a catastrophic bleeding as the aneurysm is not contained in the cs walls . therefore , postoperative imaging directed to identify aneurysmal formation should be conducted for early diagnosis and treatment .

iatrogenic cavernous carotid pseudoaneurysms are a special group among other intracranial aneurysms . they can occur during the dissection phase of the surgery if the tumor encases a vessel . complications of their rupture as hemorrhage or stroke are life threatening . early recognition and treatment is mandatory to avoid catastrophic sequelae . we present the successful diagnosis and endovascular treatment of a postoperative cavernous carotid pseudoaneurysm following radical cavernous sinus resection .

endoscopic treatments , including endoscopic mucosal resection ( emr ) and endoscopic submucosal dissection ( esd ) , for early gastric cancer ( egc ) have markedly progressed and are widely accepted [ 13 ] . emr can be safely performed for egc conditioned by differentiated mucosal egc smaller than 20 mm in diameter , and the general indications were proposed by the japanese gastric cancer association . recently , esd is frequently used for the treatment of egc because esd has the advantage of curability on achieving successful en - block resection [ 13 ] . furthermore , extended indications of emr / esd for egc have been clinicopathologically investigated [ 58 ] . the expanded criteria of emr / esd for egc are still controversial , and the expanded criteria need to be confirmed yet . most mucosal egc showed low - level lymph node metastasis while approximately 1520% of submucosal egc showed node metastasis . there are cases that underwent additional gastrectomy after emr / esd , because cancer invades the submucosal layer of the stomach with / without lymphovascular invasion histopathologically detected in specimens obtained by emr / esd . regarding criteria for egc treatment , patients sometimes hope for endoscopic treatment , as it is less invasive compared to surgery . reported that endoscopic therapy in patients who are inoperable or have a high surgical risk appears effective . there have recently been reports regarding additional treatments , including gastrectomy , after incomplete emr / esd [ 12 , 13 ] . patients with gastrectomy after prior emr / esd may have several problems regarding the surgical management and long - term outcome . especially , the clinicopathologic issues , excluding the histopathologic features of gastric cancer , in gastrectomized patients after prior esd have not yet been investigated . we retrospectively investigated the clinicopathologic issues concerning gastrectomized patients after esd for egc , and the reasons for esd and further surgery for egc , and pre- and postoperative problems were determined from a surgical viewpoint . a total of 252 patients with gc were newly diagnosed and surgically treated between 2004 and 2008 at shinshu university hospital . eighteen patients ( 15 men and 3 women , 7.1% : group a ) were additionally gastrecomized after esd for egc . sixty - four patients ( 49 men and 15 women , 25.4% : group b ) were routinely and electively gastrectomized for egc with submucosal invasion . the following parameters were investigated : ( 1 ) problems before treatments , ( a ) comorbidities , ( b ) multiple cancers of other organs , and ( c ) history of major abdominal surgery and invasive treatment for other diseases ; ( 2 ) reason for treatment of egc , ( a ) reason for receiving esd , and ( b ) reason for additional gastrectomy ( pathologic findings of esd ) ; ( 3 ) pathologic findings after additional gastrectomy ; ( 4 ) postoperative complications ; ( 5 ) clinical outcome after additional gastrectomy . finally the clinicopathologic features in group a were compared with those of group b. esd for egc was performed by several gastrointestinal endoscopists in shinshu university hospital and other hospitals in nagano prefecture . one hundred and fifty - three patients received endoscopic treatments in the same period , and 15 ( 9.8% ) of the 153 patients with additional gastrectomy received prior endoscopic treatments at our hospital . the remaining 3 visited from other hospitals for endoscopic treatment of egc in our hospital . esd for egc was carried out employing a few methods using hook and flex knives or an insulation - tipped diathermic knife . the obtained sections were histopathologically examined and were routinely evaluated according to the japanese classification of gastric carcinoma established by the japanese gastric cancer association . when the histopathologic findings after esd showed submucosal invasion with / without lymphatic or venous invasions , additional gastrectomy was considered . gastrectomy with regional node dissection for egc with / without prior esd was performed employing an open or laparoscopy - assisted approach . when egc was located in the lower and middle thirds of the stomach , distal gastrectomy was performed . when egc was located in the upper third of the stomach , total or proximal gastrectomy was performed . 7 ) or ( with no . 7 , 8a , and 11p ) in patients with egc . in advanced gc , d2-node dissection was usually performed . these resected specimens and lymph nodes were examined according to the routine histopathologic procedures for diagnosis and staging . the clinicopathologic features of gc were described according to the japanese classification of gastric carcinoma . all 18 patients with additional gastrectomy were performed d1-lymphadenectomy : 14 with node dissection surrounding left gastric arteries and 4 with additional dissection of the nodes surrounding the common hepatic and splenic and celiac arteries . for 5 years after surgery , these patients were followed in the outpatient clinic of shinshu university hospital in order to check for recurrence / metastasis of the tumors by esophagogastroduodenoscopy every year and computed tomography ( ct ) of the abdomen and chest every 6 months and/or annually . death caused by recurrence / metastasis of gc or by other diseases after surgery was investigated . data are shown as the prevalence , or mean and ordinal data were compared by the mann - whitney u and chi - square or fisher 's exact probability test . five - year survival rates after surgery were calculated employing the kaplan - meier method . comorbiditiesall 18 patients had one or more comorbidities before treatments for egc ( table 1 ) . additionally , two of 4 patients with diabetes mellitus had not yet been treated , and insulin treatment was started for surgery . one with chronic obstructive pulmonary disease and one with idiopathic interstitial pneumonia routinely underwent home - oxygenic treatment . all 18 patients had one or more comorbidities before treatments for egc ( table 1 ) . additionally , two of 4 patients with diabetes mellitus had not yet been treated , and insulin treatment was started for surgery . one with chronic obstructive pulmonary disease and one with idiopathic interstitial pneumonia routinely underwent home - oxygenic treatment . multiple cancers in other organsother primary cancers were observed in 8 patients ( 44.4% : table 2 ) . nine antecedent cancers in other organs were observed in 7 patients , while one synchronous cancer was observed in the liver . in these patients , nine antecedent cancers in other organs were observed in 7 patients , while one synchronous cancer was observed in the liver . in these patients , history of major surgery and invasive treatmenthistories of major abdominal operations were observed in 6 patients ( 33.3% ) . hepatectomy for hepatocellular carcinoma and the extirpation of retroperitoneal liposarcoma had been performed in one . a history of invasive treatment / operation for other disorders was observed in 3 patients ( 16.7% ) : surgical clipping of cerebral aneurysm , coronary artery bypass grafting , or percutaneous coronary intervention for ischemic heart disease . these invasive treatments with / without surgery were performed in 9 patients ( 50.0% ) . hepatectomy for hepatocellular carcinoma and the extirpation of retroperitoneal liposarcoma had been performed in one . a history of invasive treatment / operation for other disorders was observed in 3 patients ( 16.7% ) : surgical clipping of cerebral aneurysm , coronary artery bypass grafting , or percutaneous coronary intervention for ischemic heart disease . these invasive treatments with / without surgery were performed in 9 patients ( 50.0% ) . reason for receiving esdregarding the patients ' opinions , 14 ( 77.8% ) of the 18 patients hoped for esd treatment for egc . they gave the following reasons : their advanced age , comorbidities , history of abdominal surgery , and social convenience . regarding the doctors ' opinions , egc was suspected as submucosal cancer , but diagnostic treatment was conducted in 4 patients ( 22.2% ) by esd . regarding the patients ' opinions , 14 ( 77.8% ) of the 18 patients hoped for esd treatment for egc . they gave the following reasons : their advanced age , comorbidities , history of abdominal surgery , and social convenience . regarding the doctors ' opinions , egc was suspected as submucosal cancer , but diagnostic treatment was conducted in 4 patients ( 22.2% ) by esd . reason for additional gastrectomy ( pathologic findings of esd)twenty - three egc were removed by esd in the 18 patients ; double lesions patients were removed in 5 patients ( 27.8% ) . cut - end margins of 11 lesions in 10 patients ( 55.6% ) were positive : the positive lateral margin in 3 patients ( 16.7% ) and the positive vertical margin in 8 patients ( 44.4% ) . submucosal invasion less than 500 m from the muscularis mucosa ( sm1 ) was shown in 5 lesions of 4 patients ( 22.2% ) , and submucosal invasion deeper than 500 m from the muscularis mucosa ( sm2 ) in 14 lesions of 14 patients ( 77.8% ) . histological vessel invasions , including lymphatic and/or venous invasions , were shown in 11 lesions : 2 lesions with sm1 and 9 lesions with sm2 . based on the histopathologic findings of egc specimens after esd , residual cancer was suspected in 10 patients , and node metastasis was suspected in 11 patients because of presence of histologic lymphatic and/or venous invasion . the remaining 2 showed a regional node swelling ( over 10 mm in diameter ) on abdominal ct in the followup after esd . twenty - three egc were removed by esd in the 18 patients ; double lesions patients were removed in 5 patients ( 27.8% ) . cut - end margins of 11 lesions in 10 patients ( 55.6% ) were positive : the positive lateral margin in 3 patients ( 16.7% ) and the positive vertical margin in 8 patients ( 44.4% ) . submucosal invasion less than 500 m from the muscularis mucosa ( sm1 ) was shown in 5 lesions of 4 patients ( 22.2% ) , and submucosal invasion deeper than 500 m from the muscularis mucosa ( sm2 ) in 14 lesions of 14 patients ( 77.8% ) . histological vessel invasions , including lymphatic and/or venous invasions , were shown in 11 lesions : 2 lesions with sm1 and 9 lesions with sm2 . based on the histopathologic findings of egc specimens after esd , residual cancer was suspected in 10 patients , and node metastasis was suspected in 11 patients because of presence of histologic lymphatic and/or venous invasion . the remaining 2 showed a regional node swelling ( over 10 mm in diameter ) on abdominal ct in the followup after esd . cancer remnant was observed in only one patient , while no remnant was observed in 17 patients ( 94.4% ) . new cancer was detected in the resected stomach after gastrectomy in 4 patients ( 22.2% ) ; 2 of the 4 patients had a third cancer . these lesions were mucosal and less than 10 mm in diameter ; majority of them shows 24 mm in size . in addition , helicobacter pylori was observed in 17 ( 94.4% ) , but the presence was same between the patients with single and multiple gastric cancer in group a. postoperative complications were observed in 9 patients ( 50.0% ) in group a. liver dysfunction was observed in 3 patients ; one of them showed massive ascites , approximately 1,000-ml drainage , every day after surgery . other postoperative complications were acute pancreatitis , pericarditis with heart failure , angina pectoris , and atrial fibrillation with tachycardia . these complications were conservatively treated , and consequently improved . in group b , no case with anastomotic leakage was also observed . there was no difference of postoperative morbidity between group a and group b. no postoperative mortality was observed . in addition , there was no difference in operative blood loss and operating time between the two groups ( table 3 ) . in the followup mental / neurological diseases , including depression and dementia , were observed in 4 patients over 75 years old . other primary cancers were subsequently observed in 2 patients ( 11.1% ) : hepatocellular carcinoma 26 months after and lung cancer 50 months later . death due to other diseases was observed in 4 patients ( 22.2% ) without the recurrence of egc : respiratory failure due to amyloidosis , hepatic failure due to cirrhosis , metastatic lung cancer detected after gastrectomy , and the recurrence of esophageal cancer treated by chemoradiotherapy before esd . a significant difference between group a and group b was observed in the age , tumor size , histologic differentiation , preoperative complications , surgical risk , and death by other diseases ( table 3 ) . new disorders after surgery were frequently observed in group a than group b although this was not statistically significant . no case with gastric cancer recurrence was observed in group a as well as group b. group a showed a less favorable outcome after surgery in terms of overall survival than group b ( p = 0.0087 ) . however , the cancer survival rate after additional gastrectomy was the same in the two groups . there are several clinicopathologic issues in patients with submucosal cancer treated by additional gastrectomy after esd for egc . two major points regarding diagnostic and therapeutic issues in these patients with additional gastrectomy were considered . one is a problem regarding egc and metastasis , including incomplete resection by esd , the presence of node metastasis before esd , and the oversight of small egc and metachronous cancer after esd . the other is a problem regarding patients involving the surgical risk , other primary cancers , and new disorders after gastrectomy . on consideration of these issues , it is important that additional gastrectomy is employed based on the histopathologic findings after esd . there was only one case with a cancer remnant on the removed stomach although 9 showed positive lateral and/or vertical margins after esd . piecemeal resections of egc by emr have been reported to be associated with a high risk of local recurrence , and esd for egc has the advantage of being associated with a lower frequency of recurrence than emr . furthermore , yokoi et al . reported that esd facilitates the curative resection of locally recurrent egc . in the present study , this finding may be explained by burn degeneration at the cut - end of egc treated by esd . tanabe et al . reported that the mean width of burning degeneration at the cut ends of egc treated by esd was 1,203 m . goto et al . reported that preceding esd for egc had no negative influence on the prognosis when additional gastrectomy was performed , and it may be permissible to remove some egc by esd as a first step to prevent unnecessary gastrectomy . therefore , cases without local recurrence of egc may undergo the omission of additional gastrectomy when no node metastasis can be definitely shown . mutual features in the cases with node metastasis detected after additional gastrectomy were considered as follows : ( 1 ) a protruding type tumor , ( 2 ) over 25 mm in tumor size , ( 3 ) moderately differentiated type , ( 4 ) sm2 , ( 5 ) positive lymphatic invasion , but ( 6 ) no cancer remnant after esd . furthermore , the 3 had preoperative comorbidities and a surgical history of laparotomy . reported that 18.6% of submucosal egc showed node metastasis histopathologically , while 91% of surgically treated egc did not have node metastasis . oda et al . reported that 6.3% of noncurative patients with a possible risk of node metastasis after emr / esd for egc showed regional node metastasis after gastrectomy . the present cases had to receive gastrectomy initially , because their histopathologic findings after esd were considered to be associated with a high risk of node metastasis in egc . however , they desired not to undergo surgery because of their advanced age , presence of physical and social complications , and history of laparotomy . it is hard to detect regional nodes with metastasis employing abdominal ultrasonography , endoscopic ultrasonography , ct , and conventional magnetic resonance imaging . a new modality is necessary for an accurate diagnosis of node metastasis in patients with egc before emr / esd and surgery . additional gastrectomy based on the histopathologic findings after esd is unnecessary in two thirds of the present cases without cancer remnants or node metastases , while the fact that additional gastrectomy is necessary in one third of the patients may be important . from the findings of the three cases with node metastasis detected after additional gastrectomy , we suggested that additional gastrectomy should be performed in egc patients with co - morbidity showing over 25 mm in tumor size , sm2-invasion , and lymphatic invasion . however , it is possible that additional gastrectomy after esd is avoided in the other patients , when no small cancer is detected endoscopically . regarding multiple gastric cancer after emr / esd , in the present study , 33.3% of the cases synchronously showed multiple egc . nasu et al . reported the characteristics of metachronous and synchronous egc on initial emr . takenaka et al . reported metachronous cancers of the gastric remnant after distal gastrectomy and the utility of esd for egc of the gastric remnant . although egc newly detected after additional gastrectomy was mucosal and small cancer , these lesions may have the potential to become metachronous cancer after esd in the future . most of the patients undergoing additional gastrectomy hoped for treatment of egc by esd as a less - invasive procedure because they had several underlying diseases / preoperative comorbidities and surgical histories for other diseases . they were elderly and had a higher surgical risk in gastrectomy after esd than routine gastrectomy for submucosal cancer . furthermore , the nonelderly patients under 70 years old with additional gastrectomy also had several preoperative comorbidities . hirasaki et al . reported that elderly patients over 75 years old treated by esd for egc frequently had underlying diseases , but there were no differences in complications after esd and the complete resection rate . kakushima et al . reported that the complete resection rate in esd for egc and the complication rate after esd in elderly patients were not significantly different from those of younger patients . because the number of elderly egc patients with / without underlying diseases and surgical risk has been steadily increasing worldwide , we should pay attention to these egc patients , treated by esd and/or additional gastrectomy , regarding the clinicopathologic issues of age and comorbidities . postoperative complications were frequently observed in the gastrectomized patients after esd compared to those treated with routine gastrectomy ; however , no mortality was observed in this series . no data from large - scale studies was found in postoperative morbidity and mortality comparing between egc patients receiving additional and routine gastrectomy . the disease free - survival rate after additional gastrectomy in patients with incomplete esd for egc was not worse , similar to the present study . no data were found for other primary cancers and new disorders , including mental / neurological disorders , in a long - term followup of egc patients treated by additional gastrectomy after esd . approximately 1.55.4% of patients with subsequent cancer in other organs developing metachronously were detected after egc treatment [ 2325 ] . the number of patients with other subsequent cancers after surgery for egc may not be so high , but a high frequency ( 16.7% ) of other subsequent cancers was observed in the present study . furthermore , no data were found regarding new disorders after additional gastrectomy for egc , although the clinicopathologic studies of esd in elderly patients with egc were identified [ 21 , 22 ] . the fact that the present cases with additional gastrectomy frequently had comorbidities / underlying diseases may affect the capacity to discover new disorders after additional gastrectomy . from these findings , it was considered that overall survival of cases with additional gastrectomy was poorer than in those receiving routine gastrectomy for submucosal egc . egc patients with a number of comorbidities , including a surgical history and multiple cancers in other organs , may hope for less - invasive treatment by esd . consequently , additional gastrectomy may be recommended in one third , and we should consider several issues , including surgical problems as well as the complete resection of cancer and node metastasis before / after additional gastrectomy . it is possible that additional gastrectomy may be avoided in the other patients with comorbidities , when another small egc may not be detected endoscopically . regarding the followup , an issue that some of these patients died of other diseases remains . furthermore , new modalities for diagnosis of lymph node metastasis may need for omission of additional gastrectomy .

purpose . we investigated the clinicopathologic features of early gastric cancer ( egc ) patients who have undergone additional gastrectomy after endoscopic submucosal dissection ( esd ) because of their comorbidities . methods . eighteen ( 7.1% ) of 252 gc patients were gastrectomized after prior esd . reasons for further surgery , preoperative and postoperative problems , and the clinical outcome were determined . results . the 18 patients had submucosal egc and several co - morbidities . other primary cancers were observed in 8 ( 44.4% ) . histories of major abdominal operations were observed in 6 ( 33.3% ) . fourteen patients ( 77.8% ) hoped for endoscopic treatment . due to additional gastrectomy , residual cancer was suspected in 10 , and node metastasis was suspected in 11 . a cancer remnant was histologically observed in one . node metastasis was detected in 3 ( 16.7% ) . small egc was newly detected in 4 . consequently , additional gastrectomy was necessary for the one third . no patient showed gc recurrence . however , 9 ( 50% ) had new diseases , and 4 ( 22.2% ) died of other diseases . the overall survival after surgery in these patients with additional gastrectomy was poorer than those with routine gastrectomy for submucosal egc ( p = 0.0087 ) . conclusions . additional gastrectomy was safely performed in egc patients with co - morbidities . however , some issues , including presence of node metastasis and other death after surgery , remain .

according to mantini et al . , the left hepatic vein ( lhv ) draining into the coronary sinus ( cs ) is an extremely rare finding classified as type ia2 of congenital abnormalities involving the coronary sinus . the anomalous vein is usually a single vessel that arises from the left hepatic lobe , passes through the diaphragm and joins the body of the coronary sinus close to its orifice . in most published cases , the anomaly is of no clinical or hemodynamic significance , unless the patient is qualified for heart surgery . we report a case of an anomalous connection of lvh to cs in the child operated on for partial anomalous ( right ) pulmonary vein drainage ( papvd ) . a two - year - old male patient was admitted for open heart surgery with preliminary diagnosis of a partial anomalous connection of the middle and inferior right pulmonary veins to the right atrium and 9 mm atrial septal defect ( asd ) , based on ultrasound and computed tomography ( ct ) scan evaluation . the transthoracic echocardiography performed in our department found posterior rotation of the right margin of the intraatrial septum resulting in papvd , but did not confirm the presence of asd . the left pulmonary veins as well as right superior pulmonary vein were found to drain in a normal way to the left atrium . during surgery , while dissecting the inferior vena cava for direct bi - caval cannulation , we found a 5 mm diameter vein passing through the diaphragm and draining into the coronary sinus . it was located leftward and parallel to the inferior vena cava ( ivc ) within the distance of 1.5 cm . any major tension applied to the vein resulted in cardiac rhythm disturbances and drop in blood pressure . after commencing moderate hypothermic cardiopulmonary bypass ( cpb ) ( 31c ) , the aorta was cross - clamped for a single period of 21 minutes , and the right atrium was opened and inspected . the intracardiac findings consisted of two right pulmonary vein orifices within the right atrium and no atrial septal defect . ostium of the coronary sinus was markedly enlarged , whereas the orifice of the anomalous ( left hepatic ) vein was clearly visible in the most distal part of the coronary sinus , close to the right atrial chamber . a large part of the atrial septum was resected and a patch of autogenous pericardium was then used to re - direct the right pulmonary veins to return to the left atrium . the postoperative period was uneventful and the patient was discharged home on the 9 day after surgery . considering the intraoperative findings and possible surgical consequences of unrecognized anomalous connection of the hepatic vein into the coronary sinus , we decided to reevaluate the patient and look for the morphological evidence of the left hepatic vein to the coronary sinus connection . the purpose of this procedure was to determine whether it was possible to make the proper diagnosis prior to surgery . the analysis of preoperative echocardiographic recordings carried out anew did not show the vein directly . it was most likely due to the markedly increased , turbulent flow caused by an abnormal connection of the right pulmonary veins joining the lowermost part of the right atrium close to the ivc - ra junction and cs ostium . we expected that redirecting the flow from both right pulmonary veins to the left atrium with the patch would diminish blood turbulence within the area of interest and make the task easier . indeed , the four - chamber apical view revealed the dilatation of the distal ( closest to the ostium ) part of the coronary sinus , whereas more proximal parts of the cs were normally narrow ( fig . the thorough , gradual inspection of the cs , seen in transverse cut with modified parasternal short axis views , revealed the ostium of the abnormal vein joining the dilated part of the coronary sinus . the 3 mm diameter vein that originated from the liver , was passing the diaphragm , and joined the coronary sinus perpendicularly ( fig . 2 and 3 ) proximal part of coronary sinus is narrow while distal segment is enlarged ( asterisk ) . right ventricle , lv left ventricle modified parasternal short axis view showing transversal cut of distal ( ostial ) coronary sinus . 2 . cs normal sized proximal part of coronary sinus ( dotted circle ) , mpl posterior leaflet of mitral valve , thao thoracic aorta , sinus venosus ( sv ) , which comprises two horns , is located on the caudal end of the primary heart tube and receives blood from three different sources : vitelline or omphalomesenteric veins , umbilical veins , and common cardinal veins . in the complex process of normal heart development , during the 5 week of gestation , the left horn of the sinus venosus is reduced to the coronary sinus and oblique vein of the left atrium . persistent patency of connections between the left vitelline vein system and left horn of sv could be the main reason for the left hepatic vein to the coronary sinus communication . inferior vena cava , lhv left hepatic vein , cs ostium of coronary sinus , tv tricuspid valve , fo right ventricle direct venous drainage of a single hepatic vein into the coronary sinus should not have any clinical or hemodynamic consequences . nevertheless , it has been suggested that it may be associated with atrial arrhythmias and could lead to technical difficulties during coronary sinus approach procedures . while undiagnosed prior to surgery , the anomaly might be found during inspection of the inferior vena cava region either by placing a loop around the vessel , or preparing the site for direct caval cannulation . it is worth mentioning that any excessive tension of lhv should be avoided because of possible rhythm disturbances as well as reduced cardiac output , due to cs blood flow changes . the blood inflow through lvh during the intracardiac procedure is easily controlled by temporary occlusion with tourniquet ( like in the reported case ) , or direct cannulation . the lvh to cs connection is an important issue in patients qualified for fontan type surgery because of the significant right to left shunt 's volume into low - pressure pulmonary venous atrium .

left hepatic vein ( lhv ) that drains blood into a coronary sinus ( cs ) is an extremely rare congenital anomaly of systemic vein drainage with only single reports published . in most of these cases the unusual venous connection was found incidentally during diagnostics or surgery.the case of a two - year - old boy in whom the anomaly was discovered during open heart surgery for partial anomalous pulmonary venous drainage ( papvd ) is presented . difficulties in obtaining proper diagnosis preoperatively are confronted with postoperative echo findings.embryology and evolution of sinus venosus are discussed to explain the persistent connection between hepatic venous circulation and a coronary sinus . the authors attempt to recapitulate the possible surgical consequences of lhv - cs continuity .

t regulatory ( treg ) cells are a t lymphocyte subpopulation that control the balance between immune activation and tolerance . treg cells can originate from two main sources : thymus - generated natural tregs ( ttreg ) and peripheral inducible tregs ( ptreg ) , generated during immune priming . several factors are required for ttreg generation ; these cells are strongly dependent on tcr and cd28 signals and on several cytokines . cytokines contribute to treg maintaining via chain signaling of il-2 and il-15 , and tgf increases foxp3 expression . however , certain cytokines like as tnf- have a controversial role in ttreg generation [ 2 , 3 ] . on the other hand , ptreg generation requires stimulation in an anti - inflammatory milieu , a process where dendritic cells are critically involved [ 3 , 4 ] . according to cytokine production , tregs have been further classified ; for instance , th3 cells are characterized by tgf production ; tr1 cells produce il-10 , and tr35 cells produce il-35 . while the expression of the transcription factor foxp3 can be transient in humans , this factor is associated with a suppressive function in mice . several action mechanisms by which treg cells control the immune response have been reported : ( 1 ) inhibition by immunoregulatory cytokines such as tgf , il-10 , and il-35 ; ( 2 ) inhibition by cytolysis of effector cell by producing granzyme and perforin ; ( 3 ) metabolic interruption , including an inhibition of the proliferative response via il-2 receptor , camp - mediated metabolic inhibition , and immunomodulation mediated by the a2 adenosine receptor ; ( 4 ) interaction with dendritic cells that modulates their function and maturation . il-10 is an 18-kd protein produced by tr1 cells . while its production is not restricted to this cell line since monocytes , dendritic cells , neutrophils , other t lymphocytes , and b lymphocytes are able to release it , tr1 cells are the only regulatory cells to produce il-10 [ 5 , 6 ] . il-10 has been described as the main immunomodulatory cytokine ; additionally , it can act as a paracrine or autocrine signal and can be induced by catecholamines [ 7 , 8 ] . il-10 inhibits the production of inflammatory cytokines such as il-12 , causing a decrease in the th1 response and in inf- production ; it also promotes the phagocytic activity , increasing the removal of cellular debris at the inflammation site . one of the best known molecular mechanisms of il-10 is the action on effector cells . the costimulatory molecule cd28 is involved in the interaction between effector cells and antigen - presenting cells . by binding its receptor , il-10 inhibits tyrosine phosphorylation in cd28 , inhibiting pi3k / akt activation , which in turn inhibits the signaling cascade leading to nf-b translocation ( figure 1 ) [ 10 , 11 ] . as shown in figure 1 , il-10 exerts its biologic function by activating jak1 and tyk2 , both proteins associated to stat1 , stat3 , and in certain cases stat5 ( figure 1 ) [ 10 , 12 ] . in rats , the activation of the il-10 receptor in dendritic cells has been observed to promote the jak1-tyk2-stat3 pathway . when activated , this pathway inhibits dendritic cell maturation , decreasing the expression of mhc ii , cd11b / c , cd80 , and cd86 . in monocytes , il-10 has been demonstrated to induce the expression of the socs3 suppressor gene , which has influence on ifn - induced tyrosine phosphorylation in stat1 . on the other side , in a lps - induced inflammation model , il-4 has been observed to promote c - maf expression in activated macrophages ; c - maf binds the promoter of the il-10 gene , favoring its production ( figure 1 ) [ 14 , 15 ] . by binding its receptor in tr1 , il-10 induces jak1 and tyk2 phosphorylation , activating stat3 , which is translocated to the nucleus , promoting socs3 ; in turn , socs3 inhibits the nf-b - induced factor myd88 , resulting in the inhibition of the il-1 , il-6 , and tnf- cytokines ( figure 1 ) . moreover , it has been observed that il-10 promotes stat3 phosphorylation and its translocation to the nucleus in treg cells , mediating a further il-10 production ( figure 1 ) . il-10 exhibits a wide range of biological activities , including immunosuppression , anti - inflammation , and immunomodulation . il-10 is able to inhibit mhc i expression in b and t cells and also in dendritic cells , all of them involved in the inflammatory response . the transforming growth factor - beta ( tgf ) family includes several structurally and functionally related proteins acting as multifunctional factors in a wide range of biological processes . tgf is a 25 kda protein and a multifunctional cytokine , given its different effects on the cell [ 18 , 19 ] . during secretion , tgf undergoes proteolysis by an endopeptidase , which cleaves the peptide bond linking the mature factor and the latency - associated peptide ( lap ) . for tgf to be activated this process can be triggered by a number of factors , like temperature and ph changes . three tgf isoforms are known in humans , 1 , 2 , and 3 , each coded in a different chromosome . functional tgf receptors are types i and ii ( tgf ri and tgf rii ) , and nonfunctional receptors include types iii , iv , and v. tgf ri and rii bind tgf-1 and tgf-3 with greater affinity than tgf-2 . tgf ri and tgf rii are responsible for the biological effects of tgf-1 in mammal cells . however , tgf riii is also capable of binding tgf-1 . while tgf riii has no role in signal transduction , it has been suggested that it controls the availability of tgf-1 in the local extracellular microenvironment and regulates its active presentation to the functional receptors [ 21 , 22 ] . in the extracellular space , tgf binds tgf riii , which then recruits tgf rii and phosphorylates itself ; alternatively , tgf can bind directly to membrane - anchored tgf rii and induce the attraction of tgf ri and its ensuing phosphorylation ( figure 2(a ) ) . both tgf ri and tgf rii have a cysteine - rich extracellular region , a transmembrane region , and a cytoplasmic region ; the latter includes a serine - threonine kinase domain . there is a functional dependence between tgf rii and tgf ri , since tgf ri requires tgf rii to bind the ligand , while tgf rii requires tgf ri for a functional signaling . when tgf binds tgf rii , the latter phosphorylates tgf ri in the serine- and glycine - rich domain near the transmembrane region . the activation of the ligand - receptor complex allows the direct interaction of smad proteins with the kinase domain of the type - i receptor , recruiting smad2 and smad3 and phosphorylating them , thus activating the canonical pathway and forming a complex with smad4 . this protein acts as a convergence node for the signaling pathways induced by members of the tgf superfamily ( figure 2(a ) ) . the active complex formed by smad2 , smad3 , and smad4 is translocated to the nucleus , where it acts as transcriptional coactivator and regulates the transcription of several tgf-responder genes [ 21 , 23 ] . tgf activates tak-1 , a kinase of serine and threonine residues of the map kinase family , by the noncanonical pathway . ras , a member of the small g proteins , has also been involved in signaling . certain mapks , including those kinases regulated by extracellular signals such as erk-1 and erk-2 and stress - activated kinases such as jnk and pi3k , are also activated in some cell types ( figure 2(a ) ) . a number of mechanisms have been proposed : ( i ) it suppresses effector t cell differentiation ; ( ii ) it promotes the differentiation of nave t cells into regulatory t or th17 cells ; ( iii ) it inhibits t and b cell proliferation ; ( iv ) it inhibits the activity of macrophages , dendritic cells , and nk . the inhibitory activity of tgf on treg cells is due to the high lap / tgf expression in treg membrane . unlike t cells , monocytes and dendritic cells express tgf receptors , thus allowing for cell - to - cell interaction . tgf has been demonstrated as necessary to generate tolerogenic dendritic cells ( dcs ) by inducing ido , an enzyme that inhibits t cell proliferation . as another relevant suppressive mechanism , tgf inhibits the production of il-2 ( figure 2(b ) ) . the cis - actin protein , which suppresses il-2 production , is called tob ; this protein binds smad2 , blocking the activation of runx 1/3 . tgf modulates cell proliferation by controlling the expression of cell cycle - regulating factors , including cyclin - dependent kinases ( cdks ) like p15 , p21 , and p27 and cell cycle promoters like cmyc , cyclin d2 , cdk2 , and cyclin e. tgf also promotes t nave cell differentiation into treg cells by inducing foxp3 expression , or into th17 cells in the presence of il-6 ( figure 2(c ) ) . foxp3 then induces the transcription of inhibitory cytokines such as tgf ( figure 2(c ) ) . on the other hand , th17 differentiation from nave t cells signaling via il-6 activates stat3 , which induces the expression of ror. ror then induces the transcription of il-17 ( figure 2(c ) ) . interleukin 35 ( il-35 ) is a heterodimeric cytokine in the interleukin 12 ( il-12 ) family . this cytokine family can be composed of one to five subunits ( p19 , p28 , p35 , p40 , and another from epstein - barr virus gene 3 , also called ebi3 ) . il-35 , an immunosuppressor cytokine formed by the p35 and ebi3 subunits , is produced by treg cells . il-35 has two known functions : to suppress the proliferation of t helper cells and to promote the conversion of nave t cells into highly suppressor treg cells ( itr35 ) . recently , this cytokine was demonstrated to be capable of inducing the conversion of b lymphocytes into b regulatory cells . the il-35 receptor is formed by the il-12r2 and gp130 subunits ; it can be heterodimeric or homodimeric . while gp130 is expressed in practically all cells , il-12r2 is expressed predominantly in activated t lymphocytes , nk cells , and to a lesser extent dcs and b cells . the il-35 signaling path has not been completely described yet ; however , it is known that the heterodimeric receptor activates stat1 and stat4 , which induces ebi3 and p30 expression , causing nave t cells to convert into il-35-producing ( itreg ) cells , suppressing cell proliferation , blocking the shift to a th1 profile , and mediating il-10 production . treg cells are able to release the immunomodulatory cytokines il-10 , tgf , and il-35 . this action may target several immune cells expressing receptors for such cytokines . this way , several immune cells in different stages ( activated , effector , or resting ) could be affected by the receptor - ligand interaction . the effect of cytokines is probably not only local , since they could spread by the bloodstream throughout the body . thus , these cytokines play a role in the polarization of immune response during various pathologies . regulatory t ( tregs ) cells produce a serine protease called granzyme b , which allows them to induce apoptosis in effector t cells [ 2832 ] . during treg - effector cell interaction , directed exocytosis from treg granules to the extracellular space of both cells takes place ; these granules contain granzymes and perforins . once released from the treg , perforin molecules insert themselves into the lipid membrane of the target cell and polymerize in the presence of calcium ions to form a transmembranal cylinder ; each cylinder forms a pore through which granzymes enter the cell ( figure 3(a ) ) . granzyme can also enter the cell by an endocytosis process mediated by the manose-6-phosphate receptor . in this case , granzyme is sequestered in endosomes into the cytosol , and perforin acts to release it ( figure 3(b ) ) [ 3337 ] . additionally , granzymes can bind to the cell surface in a way that granzyme recruitment is stimulated by perforin - mediated membrane damage ( figure 3(c ) ) . once within the target cell , granzyme b could induce apoptosis by caspase - dependent or independent mechanisms , as discussed elsewhere [ 3840 ] . cytolysis allows treg cells to act on several immune cell populations by cell - to - cell interaction . this mechanism is highly effective , since treg cells induce death by apoptosis on effector cells , thus decreasing the number of effector cells and controlling the immune response . interleukin 2 ( il-2 ) , chiefly secreted by t cells in response to antigenic stimuli , is the main cytokine for t cell proliferation . il-2 receptor is expressed by t lymphocytes , nk cells , b cells , macrophages , and monocytes ; however , only t lymphocytes are capable of producing this cytokine . il-2 receptor is a complex formed by three subunits : alpha chain ( cd25 ) , beta chain ( cd122 ) , and gamma chain ( cd132 ) ; each subunit plays an important role in facilitating the transduction of il-2-dependent signals . alpha chain ( il-2r ) has a very short cytoplasmic domain and does not participate in signal transduction ; however , it is required to increase the affinity of il-2 to its receptor . on the other hand , beta ( il-2r ) and gamma ( il-2r ) chains play a crucial role in signal transduction . treg cells constitutively express high levels of il-2 alpha chain , having thus a higher affinity to il-2 , and compete for this growth factor with proliferating cells . by depriving proliferating effector cells from il-2 , treg cells do not only prevent them from continuing the proliferative process but also leave them without a vital cytokine , causing metabolic interruption and cell death ( figure 4(a ) ) [ 3 , 43 ] . cyclic adenosine monophosphate ( camp ) is a second messenger , capable of regulating the functional activity of effector cells and antigen - presenting cells . the high camp content in treg cells is due to the 50-fold higher expression of adenylyl cyclase 9 ( ac9 ) . additionally , the high cd25 expression in treg cells has been observed to favor adenylyl cyclase 7 activation and camp accumulation . the differential expression of cd25 and adenylyl cyclase in treg cells is controlled by foxp3 . tregs show a low camp degradation rate due to a diminished expression of the phosphodiesterase 3b enzyme in this cell subpopulation . recent findings indicate that camp concentration is controlled by mir-142 - 3p , a microrna that regulates foxp3 transcription activity , and thus the expression of adenylyl cyclase . while mir-142 - 3p inhibits the production of adenylyl cyclase 9 in conventional t cells , this effect is not seen in treg cells since the foxp3 transcription factor negatively regulates mir-142 - 3p expression and keeps the ac9/camp pathway active . treg cells transfer camp to target cells by intercellular communications called gap junctions ( figure 4(b ) ) . in mammal cells , communicating junctions are used for the bidirectional traffic of ions , metabolites , and other molecules weighing less than 1 kd . the increase in intracellular camp activates protein kinase a ( pka ) , since camp binds to the pka regulating subunit , activating it by releasing its catalytic subunit . on the cell membrane inner face , phosphorylation of tyrosine kinase c - src ( csk ) by pka increases its activity ; then , csk phosphorylates and inactivates the lymphocyte - specific protein tyrosine kinase ( lck ) , an important protein in the proximal activation of t cell receptors . a number of signaling pathways can be regulated by pak . for instance , the camp response element - binding ( creb ) protein is phosphorylated by pak at serine 1343 , which prevents it from forming a complex with the csk binding protein ( cbp ) and from binding to camp response elements ( cre ) ; these can be found in genes coding for t cell receptors or in other genes involved in t cell activation . additionally , pak regulates the activity of the nuclear factor in activated t cells ( nf - at ) . when nf - at is phosphorylated by pak , binding sites for another protein called 14 - 3 - 3 are created . this new complex decreases nf - at transcription activity ( figure 4(b ) ) [ 4749 ] . when cells are in resting state , nf-b is found in the cytoplasm , complexed with its inhibitor , ib , which prevents it from translocating to the nucleus . during cell activation , ib is phosphorylated by an ib kinase , which induces the nf-b / ib complex to split apart . conversely , when pka is phosphorylated , its catalytic subunit ( pka - c ) binds to the nf-b / ib complex , stabilizing it and keeping it inactive ( figure 4(b ) ) . nf-b regulates the transcription of a large number of genes , including those coding for the proinflammatory cytokines tnf- il-1b , il-6 , il-8 , vegf , and metalloproteases ( mmp1 , mmp2 , mmp3 , and mmp13 ) . while the main target for camp is pak , camp has also been demonstrated to directly activate the exchange protein directly activated by camp ( epac1 and epac2 ) . this protein regulates the activation of a gtpase called rap-1 , responsible for activating erk , thus inhibiting cell proliferation and differentiation ( figure 4(b ) ) . cd39 and cd73 are ectoenzymes , highly expressed on the surface of treg cells ( figure 4(c ) ) . cd39 is a nucleoside triphosphate diphosphohydrolase-1 ( ntpdase 1 ) that degrades atp into amp . the expression of cd39 in tregs is regulated by the foxp3 transcription factor , and its catalytic activity is enhanced by tcr compromise . in turn , extracellular amp is rapidly degraded into adenosine by cd73 , an ecto-5-nucleotidase bonded to the membrane of treg cells ( figure 4(c ) ) . the adenosine resulting from amp hydrolysis binds four different surface receptor subtypes coupled to gs proteins , called a1 , a2a , a2b , and a3 . the a2ar receptor is the main adenosine receptor associated to t and b lymphocytes , nk cells , macrophages , dendritic cells , and granulocytes ( figure 4(c ) ) . the outcome of stimulating these receptors is an intracellular amp accumulation ; through the camp - dependent protein kinase , these signals phosphorylate and activate creb . the latter binds the nuclear cofactor p300 , producing a complex that regulates the expression of several genes in their promoter regions . creb is able to regulate indirectly the transcription of some inflammatory genes , competing with nf-b / p65 and then suppressing the expression of proinflammatory cytokines like tnf- ( figure 4(c ) ) [ 51 , 5355 ] . amp can also activate other substrates like epac1 and other kinases such as erk and jnk , altering the expression of proinflammatory genes through transcription factors responsible for synthesizing interleukins like il-12 and tnf- ; it promotes as well the production of anti - inflammatory cytokines like il-10 ( figure 4(c ) ) . the fact that tregs produce adenosine and respond to it at the same time means that this molecule acts as an autocrine factor to optimize the anti - inflammatory response . it also increases treg suppressor capacity , inhibits the expression of costimulatory molecules in dendritic cells , and inhibits the activation of effector cells [ 52 , 55 ] . treg - mediated metabolic disruption occurs by competition for il-2 , a growth factor for effector cells . under these conditions , treg cells control the immune response in a nonspecific but effective manner , since by consuming il-2 treg cells inhibit effector cell proliferation , actually impairing the immune response . on the other hand , immunosuppression mediated by camp and the 2a adenosine receptor induce several signaling pathways in effector cells that in turn impact transcription factors , controlling the effector response . this molecule has a high affinity by dendritic cell - expressed cd80 and cd86 ; thus , tregs compete with effector cells to bind these ligands . this interaction involves several events , including the production of inf- , a potent inducer of indoleamine 2,3-dioxygenase ( ido ) . ido degrades tryptophan in a metabolic pathway starting with tryptophan oxidation to n - formylkynurenine , immediately followed by hydrolysis to kynurenine . this initial product follows various degradation pathways whose final products are kynurenic acid , 3-hydroxykynurenine , anthranilic acid , 3-hydroxyanthranilic acid , and quinolinate ( figure 5(a ) ) [ 5961 ] . once tryptophan is degraded by ido , a signaling cascade starts . since there is a local tryptophan deficit , the level of transfer rna without amino acid load increases , and as a result the general control nonderepressible kinase 2 ( gcn2 ) is activated , and it hyperphosphorylates the translation initiation factor ( eif2 ) , leading to a suppression in protein synthesis . the union of the ternary complex formed by eif2 , gtp , and the initiation transfer rna for methionine ( met - rnai ) to the 40s ribosome subunit is inhibited , preventing the release of the binary complex eif2/gtp and inhibiting the eif2-promoted gtp / gdp exchange . the result is a cell cycle arrest in the g1 phase and thus anergy of effector t cells ( figure 5(a ) ) [ 60 , 62 , 63 ] . although the mechanism is not well understood , 3-hydroxyanthranilic acid and quinolinic acid have been demonstrated to induce apoptosis in th1 cells by directly activating caspase-8 [ 64 , 65 ] ; this truncates the bid protein , producing a small proapoptotic fragment called truncated bid ( tbid ) . this active fragment can translocate to the mitochondria , causing the release of cyt c by interaction with bax , another member of bcl-2 family . in the cytosol , cyt c interacts with the proteins apaf1 and caspase-9 in the presence of atp , building the apoptosome , a catalytic complex that starts the caspase cascade activation , leading to the digestion of structural proteins , degradation of chromosomal dna , and phagocytosis of apoptotic bodies ( figure 5(a ) ) [ 66 , 67 ] . the metabolite 3-hydroxyanthranilic acid has also been reported to induce death in activated t cells by depleting glutathione , one of the main antioxidant molecules in animal cells . a decrease in glutathione promotes a misbalance between ros production and the antioxidant capacity . on the other side , t cell apoptosis due to oxygen free radicals produced by kynurenine and 3-hydroxykynurenine has been observed , leading as well to changes in the oxidant - antioxidant balance and promoting oxidative stress . oxidative stress causes a global collapse of mitochondrial function , reducing energy production and therefore contributing to cell death [ 64 , 69 ] . another immunosuppression mechanism involves kynurenine binding to the aryl - hydrocarbon receptor ( ahr ) , a transcription factor expressed in cell populations like t cells , resulting in a shift to a t regulatory cell phenotype ( figure 5(a ) ) [ 70 , 71 ] . lymphocyte - activation gene 3 ( lag3 , cd223 ) is a cell surface molecule expressed in tregs . structurally , lag3 is homologue to the cd4 receptor and binds to mhc ii with a significantly higher affinity than cd4 [ 7375 ] . the lag3-mhc ii union induces a signaling cascade starting with plc2 and p72syk phosphorylation and the activation of pi3k / akt , p42/44erk , and p38mapk ( figure 5(b ) ) . the latter kinase has been involved in the maturation process of dendritic cells ; these exhibit an increase in the expression of costimulatory molecules and a decrease in antigen capture [ 76 , 77 ] . another study demonstrated that , after crosslinking mhc in the presence of lag3 , itam - mediated inhibitory signals are induced , involving erk sequential activation and ship-1 recruitment similar to those proposed for fcr. the inhibitory signaling started by coengagement of mhc with lag3 along with cd4 and tcr could be mediated by several components , including ship-1 , which may not be recruited by themselves by the cd4 or tcr signaling pathways . ship-1 has been labeled as a nf-b negative regulator and as a negative factor in cell activation . additionally , the conjoint union of ctla-4 and lag3 promotes a tolerogenic phenotype in dcs ( figure 5(b ) ) . dendritic cells being a key component of the immune response , the action implying cell - to - cell contact with dcs is one of the most important mechanisms for treg cells . depending on its phenotype , when interacting with a treg cell , a dc acquires a tolerogenic phenotype , which in turn promotes further treg cell generation , providing a suppressor microenvironment . the competition for dc ligands between effector and treg cells allows for an additional control mechanism of the immune response . the action mechanisms of tregs described above can act together or independently , according to the requirements of the immune system and homeostasis maintenance , or during the progression of various pathological processes . treg cells have been used as part of an escape mechanism by several pathogens , including viruses and helminthes , and they also modulate the immune response in noninfectious pathologies such as tumors . knowing the modulation molecular mechanisms and their effect according the pathological situation could help in identifying the therapeutic targets allowing an effective immune response .

t regulatory cells play a key role in the control of the immune response , both in health and during illness . while the mechanisms through which t regulatory cells exert their function have been extensively described , their molecular effects on effector cells have received little attention . thus , this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective .

elearning has been formed in a knowledge - based community and is the product of fast and expanding developments of modern technologies ( 1 - 5 ) . elearning possesses special components including ease and speed of updating , storage , retrieval , distribution , and sharing with network - based data , conducting learning process and live communication with the learners via computer and by means of internet standard technology , focus on comprehensive paradigm in learning , educational flexibility and learning , learning redundancy capability and learner- centered learning ( 6 ) . actually , reduction in educational costs is deemed as one of the reasons , which have caused e - learning to be highly noticed today ( 7 ) . while developing e - learning products and preparation of opportunities for e - learning has been posited as one of the fields of higher education that is increasingly expanded , our knowledge and awareness about the effectiveness of these new attitudes about learning are restricted because of the shortage of valid scientific evaluations ( 2 ) . anyway , some studies have been so far conducted about evaluation of e - learning , in each of which certain goals and perspectives have been addressed . in a study conducted to examine and explain a framework for qualitative guarantee of e - learning all over taiwan , the attitude of participants in virtual educational program was investigated . in this survey , two types of educational programs were explored including e - learning service certification ( e - lse ) and e - learning course- ware certification ( e - lcc ) . the analysis signified that the quality of the framework had adequate validity and authenticity and it showed that the participants in both e - lse and e - lcc curricula felt satisfied with their educational program . the behavioral analysis indicated that in e - lcc method , the positive and growing attitudes were only directed toward improving the acceptance of e - lsc among the participants ; also , in e - lcc , positive orientations referred to further preferences and adaptability to improve and facilitate the acceptance and agreeableness of e - lcc ( 8 - 11 ) . the multiplicity of effective factors , variables and diverse classifications , which have been implemented from several perspectives , is one of the paramount challenges in evaluation of e - learning . to assess an e - learning setting , it necessitates identifying the main and effective key factors in implementation of electronic programs successfully including the elements . in this regard , the main success factors may be due to activities and elements which should be confirmed in order to ensure the successful implementation of curricula ( 2 ) . khan s p3 ( people , process and product ) model assumes several factors as effective in creation of an appropriate e - learning environment . the presented continuum in this model includes eight dimensions as follows : institutional , pedagogical , technological , interface design , management , evaluation , resource support and ethical . the present study was designed to evaluate the virtual course of medical education in shiraz university of medical sciences based on khan s p3 e - learning model to answer the quality of different elements of the course in students and directors perspective . this evaluation research study with post single group was designed to determine the effectiveness of a virtual program in master degree at shiraz university of medical sciences ( sums ) . all students ( 60 ) who had participated in this virtual program in more than one year and 21 experts including teachers and directors participated in this evaluation project . according to p3 e - learning model , an evaluation tool ( questionnaire ) with 5-point likert rating scale ( from 0 never to 4 perfectly exists ) was designed and applied to collect the data . content validity of the questionnaire was checked by 3 professors in e - learning and its total reliability ( r=0.92 ) was confirmed . internal consistency of the eight dimensions was measured through cronbach s alpha coefficient ( table 1 ) alpha coefficient in eight dimensions of the questionnaire the final questionnaire was emailed to all 110 students who had participated in the virtual education for more than one year ; all students 60 filled out the form and returned the email . the researchers asked all 21 directors and teachers who were involved in the program to fill out the questionnaire and they accepted . the students perspectives on different dimensions and criteria of the virtual course are shown in table 2 . students perspective on e - learning dimensions teachers perspectives on different dimensions and criteria of the virtual course are shown in table 3 . based on the findings from the students , resource support of the virtual program was in inappropriate condition in both online and offline sources ( with means of 0.73 and 0.77 ) . similarly , it can be inferred from the results that e - learning in shiraz university of medical sciences is not at a favorable level in all dimensions . according to the teachers and directors views , technological domain with mean 3.18 , especially in hardware and software criterion was in a better condition than other domains . however , this superiority is also relatively shown in subordinated parts of other domains as well ( including accessibility and guidance in interface design ) . the best condition was related to technological dimension while the worst status was related to the ethical dimension ( 12 ) . the best advantages of applying khan s p3 model was related to its comprehensiveness that includes all effective cornerstones in e - learning system and also the other one is that khan s continuum was based on features and requirements of learning environment . thus , presence of such framework in this regard and using it within the format of appropriate tools for evaluation of e - learning in universities and higher education institutes , which present e - learning curricula in the country , may contribute to implementation of the present and future e - learning curricula efficiently and guarantee its implementation in an appropriate way .

introduction : in e - learning , people get involved in a process and create the content ( product ) and make it available for virtual learners . the present study was carried out in order to evaluate the first virtual master program in medical education at shiraz university of medical sciences according to p3 model . methods : this is an evaluation research study with post single group design used to determine how effective this program was . all students 60 who participated more than one year in this virtual program and 21 experts including teachers and directors participated in this evaluation project . based on the p3 e - learning model , an evaluation tool with 5-point likert rating scale was designed and applied to collect the descriptive data . results : students reported storyboard and course design as the most desirable element of learning environment ( 2.300.76 ) , but they declared technical support as the less desirable part ( 1.171.23 ) . conclusion : presence of such framework in this regard and using it within the format of appropriate tools for evaluation of e - learning in universities and higher education institutes , which present e - learning curricula in the country , may contribute to implementation of the present and future e - learning curricula efficiently and guarantee its implementation in an appropriate way .

human pancreata were harvested from adult heart - beating , brain - dead donors and designed to be processed for islet isolation and transplantation . for different reasons ( prolonged ischemia time , suspicion of tumors , etc ) , some pancreata were not processed for islet isolation and small specimens were taken for histology . sixteen specimens were from nondiabetic donors with a mean age of 40.1 16.2 years ( range : 1569 ) and a mean bmi of 25.2 2.9 kg / m ( range : 2029 ) . five specimens were from five type 2 diabetic donors with a mean age of 57.8 9.1 years ( range : 4265 ) and a bmi of 32.3 10.0 kg / m ( range : 2650 ) . glycemia at the time of hospital admission was 8.9 3.8 mmol / l ( mean sd , n = 12 ) for nondiabetic donors and 13.9 5.3 ( mean sd , specimens from nondiabetic donors were obtained from head ( n = 4 ) , body ( n = 2 ) , tail ( n = 5 ) and unspecified regions ( n = 9 ) of the pancreas ( for four pancreata , specimens from both the head and the tail were available ) . specimens from type 2 diabetic donors were obtained from head ( n = 1 ) , body ( n = 2 ) , and tail ( n = 2 ) of the pancreas . however , by double immunofluorescence for insulin and amylin ( using a rabbit anti - amylin antibody purchased from progen biotechnik , heidelberg , germany ) , several -cells were found to be positive for amylin , in all five specimens . in one specimen , mouse pancreata were harvested from c57/bl6 mice ( janvier laboratories , le genest - saint - isle , france ) . to this end , human islet isolations were performed as previously described according to the ricordi method with local adaptations ( 13,14 ) . the use of human islets for research was approved by our local institutional ethics committee . after purification , islets were cultured overnight at 37c , and at 25c thereafter , in cmrl medium containing 5.6 mmol / l glucose and supplemented with penicillin , streptomycin , glutamine , hepes , and 10% fcs ( hereafter referred to as complete cmrl ) . islets used for morphologic analysis were cultured for a total of 3648 h. for islet cell dispersion , cultured islets were rinsed in pbs and incubated in accutase ( sigma , st louis , mo ) for 9 min with occasional pipetting . single - cell suspensions ( 90% single cells ) were rinsed with complete cmrl , and aliquots of 10 cells were incubated for 24 h at 37c in nonadherent 60-mm diameter petri dishes containing 6 ml complete cmrl . pancreas samples were incubated for 24 h in ( methanol - free ) 10% formalin , dehydrated , and embedded in paraffin . isolated islets were incubated for 24 h in methanol - free 10% formalin , then deposited at the bottom of flat - bottomed tubes , embedded in agar to immobilize them , dehydrated , and finally embedded in paraffin . all pancreata and islet samples were sectioned at 5 m . for one pancreas sample , 80 consecutive sections were performed . islet cells cultured 24 h in complete cmrl were harvested and injected into poly - l - lysine coated cunningham chambers ( 15 ) . after 1 h at 37c , chambers were rinsed to remove unattached cells and filled with methanol - free 10% formalin . after 20-min incubation at room temperature , they were rinsed with pbs and stored at 4c before being used for immunofluorescence . before immunofluorescence , sections were deparaffinized and rehydrated with a series of alcohol solutions of decreasing concentration . incubations were done at room temperature , except as indicated below . for double staining ( insulin + glucagon , insulin + somatostatin , or insulin + pp ) , slides were treated 20 min with 0.5% triton x-100 and 20 min with 0.1% bsa , at room temperature . slides were then incubated for 1 h with a mouse anti - glucagon antibody ( 1:3,000 ; sigma ) , a rabbit anti - somatostatin antibody ( 1:300 ; dako , carpinteria , ca ) , or a rabbit anti - pp antibody ( 1:300 ; dako ) . after rinsing , slides were incubated for 1 h with either an alexa 488conjugated anti - mouse antibody ( 1:500 ; invitrogen , basel , switzerland ) or a fluorescein isothiocyanate conjugated anti - rabbit antibody ( 1:400 ; jackson immunoresearch laboratories , west grove , pa ) . then , slides were rinsed and incubated for 1 h with a guinea pig anti - insulin antibody ( 1:1,000 ; dako ) and successively for 1 h with a rhodamine - conjugated anti guinea pig antibody ( 1:100 ; jackson immunoresearch laboratories ) . for triple staining ( insulin , glucagon , and cd34 ) , deparaffinized sections were treated for 10 min at 37c with a 0.1% trypsin solution , for 20 min with 0.5% triton x-100 , and for 20 min with 0.1% bsa , at room temperature , and incubated successively for 1 h with a mouse anti - cd34 antibody ( 1:50 ; serotec , oxford , u.k . ) , for 1 h with a alexa 488conjugated anti - mouse antibody ( 1:500 ; invitrogen ) , for 1 h with rabbit anti - glucagon ( 1:50 ; dako ) and guinea pig anti - insulin ( 1:1,000 ; dako ) antibodies , and for 1 h with a rhodamine - conjugated ( 1:750 ) anti - rabbit and amca conjugated anti microscopic sections and islet cells were analyzed with an axioskop microscope ( zeiss , feldbach , germany ) equipped with ultraviolet illumination and filters for blue , red , and green fluorescence . images were captured with an axiocam color ccd camera ( zeiss ) and recorded on computer through the axiovision software ( zeiss ) . isolated cells were also analyzed by confocal microscopy ( zeiss lsm510 meta ) configured for simultaneous analysis of red and green fluorescence . analysis of red and green fluorescence on digital images was performed using the offline metamorph imaging software for microscopy ( universal imaging , west chester , pa ) . this software was programmed to automatically quantify red- and green - stained areas within defined regions of interest . differences between means were assessed either by the student t test and when required by one - way anova . when anova was applied , scheff least - significant difference post hoc analysis was used to identify significant differences . human pancreata were harvested from adult heart - beating , brain - dead donors and designed to be processed for islet isolation and transplantation . for different reasons ( prolonged ischemia time , suspicion of tumors , etc ) , some pancreata were not processed for islet isolation and small specimens were taken for histology . sixteen specimens were from nondiabetic donors with a mean age of 40.1 16.2 years ( range : 1569 ) and a mean bmi of 25.2 2.9 kg / m ( range : 2029 ) . five specimens were from five type 2 diabetic donors with a mean age of 57.8 9.1 years ( range : 4265 ) and a bmi of 32.3 10.0 kg / m ( range : 2650 ) . glycemia at the time of hospital admission was 8.9 3.8 mmol / l ( mean sd , n = 12 ) for nondiabetic donors and 13.9 5.3 ( mean sd , specimens from nondiabetic donors were obtained from head ( n = 4 ) , body ( n = 2 ) , tail ( n = 5 ) and unspecified regions ( n = 9 ) of the pancreas ( for four pancreata , specimens from both the head and the tail were available ) . specimens from type 2 diabetic donors were obtained from head ( n = 1 ) , body ( n = 2 ) , and tail ( n = 2 ) of the pancreas . however , by double immunofluorescence for insulin and amylin ( using a rabbit anti - amylin antibody purchased from progen biotechnik , heidelberg , germany ) , several -cells were found to be positive for amylin , in all five specimens . in one specimen , mouse pancreata were harvested from c57/bl6 mice ( janvier laboratories , le genest - saint - isle , france ) . to this end , human islet isolations were performed as previously described according to the ricordi method with local adaptations ( 13,14 ) . the use of human islets for research was approved by our local institutional ethics committee . after purification , islets were cultured overnight at 37c , and at 25c thereafter , in cmrl medium containing 5.6 mmol / l glucose and supplemented with penicillin , streptomycin , glutamine , hepes , and 10% fcs ( hereafter referred to as complete cmrl ) . islets used for morphologic analysis were cultured for a total of 3648 h. for islet cell dispersion , cultured islets were rinsed in pbs and incubated in accutase ( sigma , st louis , mo ) for 9 min with occasional pipetting . single - cell suspensions ( 90% single cells ) were rinsed with complete cmrl , and aliquots of 10 cells were incubated for 24 h at 37c in nonadherent 60-mm diameter petri dishes containing 6 ml complete cmrl . pancreas samples were incubated for 24 h in ( methanol - free ) 10% formalin , dehydrated , and embedded in paraffin . isolated islets were incubated for 24 h in methanol - free 10% formalin , then deposited at the bottom of flat - bottomed tubes , embedded in agar to immobilize them , dehydrated , and finally embedded in paraffin . all pancreata and islet samples were sectioned at 5 m . for one pancreas sample , 80 consecutive sections were performed . islet cells cultured 24 h in complete cmrl were harvested and injected into poly - l - lysine coated cunningham chambers ( 15 ) . after 1 h at 37c , chambers were rinsed to remove unattached cells and filled with methanol - free 10% formalin . after 20-min incubation at room temperature , they were rinsed with pbs and stored at 4c before being used for immunofluorescence . before immunofluorescence , sections were deparaffinized and rehydrated with a series of alcohol solutions of decreasing concentration . incubations were done at room temperature , except as indicated below . for double staining ( insulin + glucagon , insulin + somatostatin , or insulin + pp ) , slides were treated 20 min with 0.5% triton x-100 and 20 min with 0.1% bsa , at room temperature . slides were then incubated for 1 h with a mouse anti - glucagon antibody ( 1:3,000 ; sigma ) , a rabbit anti - somatostatin antibody ( 1:300 ; dako , carpinteria , ca ) , or a rabbit anti - pp antibody ( 1:300 ; dako ) . after rinsing , slides were incubated for 1 h with either an alexa 488conjugated anti - mouse antibody ( 1:500 ; invitrogen , basel , switzerland ) or a fluorescein isothiocyanate conjugated anti - rabbit antibody ( 1:400 ; jackson immunoresearch laboratories , west grove , pa ) . then , slides were rinsed and incubated for 1 h with a guinea pig anti - insulin antibody ( 1:1,000 ; dako ) and successively for 1 h with a rhodamine - conjugated anti guinea pig antibody ( 1:100 ; jackson immunoresearch laboratories ) . for triple staining ( insulin , glucagon , and cd34 ) , deparaffinized sections were treated for 10 min at 37c with a 0.1% trypsin solution , for 20 min with 0.5% triton x-100 , and for 20 min with 0.1% bsa , at room temperature , and incubated successively for 1 h with a mouse anti - cd34 antibody ( 1:50 ; serotec , oxford , u.k . ) , for 1 h with a alexa 488conjugated anti - mouse antibody ( 1:500 ; invitrogen ) , for 1 h with rabbit anti - glucagon ( 1:50 ; dako ) and guinea pig anti - insulin ( 1:1,000 ; dako ) antibodies , and for 1 h with a rhodamine - conjugated ( 1:750 ) anti - rabbit and amca conjugated anti guinea pig antibodies ( 1:150 ; jackson immunoresearch laboratories ) . microscopic sections and islet cells were analyzed with an axioskop microscope ( zeiss , feldbach , germany ) equipped with ultraviolet illumination and filters for blue , red , and green fluorescence . images were captured with an axiocam color ccd camera ( zeiss ) and recorded on computer through the axiovision software ( zeiss ) . isolated cells were also analyzed by confocal microscopy ( zeiss lsm510 meta ) configured for simultaneous analysis of red and green fluorescence . analysis of red and green fluorescence on digital images was performed using the offline metamorph imaging software for microscopy ( universal imaging , west chester , pa ) . this software was programmed to automatically quantify red- and green - stained areas within defined regions of interest . differences between means were assessed either by the student t test and when required by one - way anova . when anova was applied , scheff least - significant difference post hoc analysis was used to identify significant differences . when pancreas sections were double stained for insulin and glucagon , apparently small islets ( 4060 m in diameter ) showed a segregated cell type distribution with -cells localized in the mantle and -cells in the core of the islets ( fig . c ) , -cells were found in the mantle as in 40- to 60-m diameter islets , and also along simple or ramified empty areas ( fig . islets with apparent diameters of 50100 m generally displayed one simple empty area ( fig . 1a ) , whereas islets with apparent diameters larger than 100 m displayed multiple and/or ramified empty areas ( fig . 1a c ) . labeling for cd34 revealed that vessels surrounded the periphery of islets and localized inside these empty areas , hereafter referred to vascular channels ( fig . careful examination showed that -cells were always lining the vascular channels and the mantle region of the islets , and juxtaposed to endothelial cells ( fig . interestingly , no cd34 staining was observed inside the -cell bulk , suggesting that vessels did not penetrate the -cell bulk . because in this analysis apparently small islets could be tangential sections of bigger islets , we also examined serial sections , which permitted us to explore the thickness of several islets ( n = 8) in their entirety . thus , after a triple immunofluorescence for insulin , glucagon , and cd34 , we confirmed that no vascular channel penetrated the core of 40- to 60-m diameter islets or the clusters of -cells in bigger islets . sections of human pancreata with islets of different sizes were either double - labeled for insulin ( red ) and glucagon ( green ) ( a c ) or triple - labeled for insulin ( blue ) , glucagon ( red ) , and cd34 ( green ) ( g i ) . l : outlines of endocrine tissue labeled for insulin and glucagon were drawn . except for the 40- to 60-m diameter islets , all islets displayed one or several unstained empty areas ( vascular channels ) at their core . most glucagon - expressing cells were located around vascular channels and at the mantle of islets , independent of their size . insulin - expressing cells seemed clustered into discrete ovoid areas surrounded by -cells . in triple - labeled sections ( g i ) , vascular channels displayed staining for cd34 , indicating that they contained vessels . islets shown are representative of at least 200 islets observed on sections from 16 different pancreata . ( a high - quality digital representation of this figure is available in the online issue . ) when double staining was performed for insulin and somatostatin or insulin and pp , we observed that - and pp - cells had roughly the same topographic position as -cells ( not shown ) . to confirm segregation of - and -cells , areas labeled for glucagon ( green = a ) and insulin ( red = b ) were quantified in three different subregions of each islet profile . these subregions were 1 ) the mantle , defined as a region of 20-m deep that follows the external perimeter of islets , 2 ) the core , defined as the total islet area minus the 20-m mantle area , and 3 ) the vessel area , defined as a region of 20-m deep that follows the contour of vessels ( fig . the results expressed as b/(a+b ) ratios were 0.71 for the core and 0.50 for the mantle and the vessel area of islets ( fig . 2b ) , showing that -cells preferentially localized at the core compared with mantle and vessel area . considering that single - cell section areas are roughly similar between - and -cells , one can extrapolate that the core contains three times more -cells than -cells , whereas the mantle and the region around vascular channels contain roughly the same number of - and -cells . when subregions were selected with a 10-m instead of a 20-m rim , the differences in ratios among subregions were amplified ( not shown ) , correlating with the observation that the -cells were lining the islet mantle and vessels , as a single layer ( 10 m ) . the mean apparent diameter of all islets analyzed here was 154 4 m ( means sem of 255 islets from 21 different pancreata ) . a : pancreatic islet labeled by immunofluorescence for insulin ( red ) and glucagon ( green ) ; rims ( white lines ) delimiting mantle and core subregions and one vascular channel area are shown ; scale bar , 10 m . after immunofluorescence , areas labeled for insulin ( b ) and glucagon ( a ) were measured and results expressed as b/(a+b ) ratio . this ratio was calculated for areas measured in the different subregions ( mantle , core , and vessels ) and in the whole islets ( whole ) . analyses were performed on histologic sections from nondiabetic human ( b ) , type 2 diabetic human ( c ) , and control mouse ( e ) pancreata and cultured isolated human islets ( d ) . ( a high - quality digital representation of this figure is available in the online issue . ) when pancreatic islets from type 2 diabetic donors were similarly analyzed , an equivalent pattern of islet cell distribution was observed ( fig . 2c ) . however , the range of values was lower in diabetic compared with control islets . this last observation is in agreement with published works showing a decrease of -cell mass in type 2 diabetes ( 1619 ) . after enzymatic digestion , the islet vasculature is damaged and cell arrangement is expected to be perturbed after culture . total profile area of these structures was eight times lower in isolated islets compared with profile area of vascular channels in islets in situ . morphometric analysis indicated similar b/(a+b ) ratios between the core and mantle of cultured isolated islets ( fig . when analysis of control pancreatic islets was performed according to their apparent diameter , b/(a+b ) ratios in the mantle remained constant ( fig . 3a ) , as ratios in the core gradually decreased with the increasing islet apparent diameter ( fig . this observation confirmed that apparently small islets ( 50100 m in diameter ) had a core - mantle organization close to that of rodent islets ( fig . 2d ) , and that apparently bigger islets had elevated numbers of non-cells in their core . after immunofluorescence , insulin- and glucagon - labeled areas were measured and results expressed as b/(a+b ) ratio , where a and b were representing glucagon and insulin labeled areas , respectively . a : b/(a+b ) ratio calculated for areas measured in the 20-m mantle part of islets . b : b/(a+b ) ratio calculated for areas measured in the core of the islets . n = 39 for islets with 50- to 100-m diameter , 60 for islets with 100- to 150-m diameter , 58 for islets with 150- to 200-m diameter , 24 for islets with 200- to 250-m diameter , and 12 for islets with 250- to 300-m diameter . to gain an insight into the islet cell organization , we performed consecutive serial sections through pancreas specimens and a three - dimensional analysis of islets ( fig . it appeared evident that islet cells were not organized into discrete core - mantle subunits as previously described ( 9,11 ) . rather , islet cells were organized into a continuous structure , like a folded epithelial plate ( board ) , that spans the three - dimensional ( 3d ) network of the whole islet . the epithelial plate , whose thickness did not vary much , had a triple - laminated section with a central part that contained -cells and was lined at both sides with - and other islet cells . moreover , vessels were juxtaposed at both sides of the epithelial plate structure , invaginating in and following its folds . in 54 islets from five different pancreata , we measured the distance between vascular channels , equivalent to the thickness of the trilaminar plate . the value observed of 42 13 m ( mean sd ) roughly fits the diameter of the smallest islets . -cells , because of their position within the epithelial plate , are exclusively aligned along vessels . -cells were also found aligned directly along vessels in alternation with -cells . where -cells were the most numerous , they formed a fence between endothelial cells and -cells . in this case , -cells appeared to form a second layer of cells over the first layer of -cells . however a careful examination of these cells revealed that they develop extensions that infiltrated between -cells and progressed until the surface of endothelial cells ( fig . confocal microscopic analysis showed that narrow strips of insulin staining between -cells corresponded to true cytoplasmic extensions and not to insulin released into extracellular compartment . indeed , confocal analysis revealed that insulin staining between -cells had a granular pattern similar to that observed elsewhere inside -cells , and colocalized with actin staining ( supplementary fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1177/dc1 ) . as a consequence of this cell organization , the heterologous contacts between - and -cells around vessels are most numerous than homologous contacts between - and -cells ( fig . when association between - and -cells was assessed in cultured isolated islet cells , we observed many -cells wrapped by -cells and rarely the contrary ( fig . 6 ) . of all -cells contacting a -cell , 38 8% ( means sem of three experiments ) were round and had a perimeter almost completely wrapped by a -cell . this result revealed a unique plasticity of -cells , which were able to spread around -cells , suggesting that this characteristic was intrinsic to -cells and not dictated by some islet coercions , such as extracellular matrix or islet vasculature . consecutive sections through an entire pancreatic islet labeled for insulin ( red ) and glucagon ( green ) . images show that insulin ( red) and glucagon ( green)stained cells are organized into continuous 3-d networks that span the entire islet . sections at both ends show islet profiles with an apparent similar core - mantle structure to that of 40- to 60-m diameter islets . these consecutive images also reveal that vascular channels were in fact continuous ramified structures that were connected in places with the surrounding islet tissue ( arrowheads ) . ( a high - quality digital representation of this figure is available in the online issue . ) unique association between - and -cells in pancreatic islets . pancreatic islets labeled for glucagon ( green ) and insulin ( red ) are shown at low magnification ( a and d ) . boxed areas are shown at higher magnification in b and e. - and -cells lining vascular channels in b are depicted in c. - and -cells lining the islet mantle in e are depicted in f. arrowheads point to cytoplasmic extensions of -cells that span -cells . scale bars , 10 m in a and d , and 5 m in b and e. heterologous contacts between - and -cells ( beta - alpha ) and homologous contacts between -cells ( alpha - alpha ) and -cells ( beta - beta ) around empty areas were scored . their relative frequencies are shown in g. columns are means sem ; n = 52 islets from 10 pancreata . ( a high - quality digital representation of this figure is available in the online issue . ) unique association between - and -cells in cultured islet cells . human islet cells were isolated and cultured for 24 h. after a double immunofluorescence for insulin ( red ) and glucagon ( green ) , islet cells were analyzed by confocal microscopy . a c : images showing a cell pair composed of one -cell ( a ) surrounded by one -cell ( b ) ; the merged image is shown in c. the cell pair shown here is representative of cell pairs observed in different human cell preparations from at least 10 different pancreata . d : one series of consecutive merged images of a cell pair composed of one -cell ( green ) surrounded by a -cell ( red ) . e : all heterologous contacts between - and -cells were scored according to their type of association : a -cell wrapping an -cell ( beta wrapping alpha ) , neutral apposition between - and -cells ( alpha - beta ) , and an -cell wrapping a -cell ( alpha wrapping beta ) . results are shown as relative frequencies and columns are means sem of five islet cell preparations from five different pancreata . from all heterogeneous contacts between - and -cells , the percentage of -cells whose profile was round and perimeter almost completely wrapped by a -cell as in d was 38 8 ( means sem of three experiments ) . ( a high - quality digital representation of this figure is available in the online issue . ) the specimens used to analyze the pancreatic islet architecture were from different regions of the pancreas , including head , body , and tail . we did not observed obvious variations in islet structures according to the regional origin of the pancreas specimens . to further investigate this point , we compared head and tail specimens from the same pancreas ( n = 4 ) . islets with the trilaminar plate structure were observed in both head and tail regions of all pancreata . in this morphologic analysis , we show that islet cells are organized into a trilaminar plate comprising one layer of -cells sandwiched between two -cell enriched layers . a : -cells ( green ) and -cells ( red ) are organized into a thick folded plate lined at both sides with vessels ( blue ) . -cells are mostly at the periphery of the plate and in close contact with vessels . -cells occupy a more central part of the plate and most of them develop cytoplasmic extension that runs between -cells and reaches the surface of vessels . b : the plate with adjacent vessels is folded so that it forms an islet . no evident vascular channel penetrates the central layer of -cells inside the trilaminar plate , but many vascular channel profiles are found adjacent to the -cell enriched layers . absence of cd34 staining within the -cell layer strongly suggest absence of endothelial cells . if some endothelial cells unlabeled for cd34 were nevertheless present , they should form tiny and collapsed vessels because no structures resembling typical vessels were observed . there is no doubt that most , if not all , -cells are in direct contact with vasculature . with regard to -cells other -cells sit on a layer of -cells and display cytoplasmic extensions that run between -cells to reach the vessel surface as well . other -cells are more distant from vessels and doubts persist as to their ability to reach the vasculature . no endothelial cells were observed in the core of 40- to 60-m diameter islets presenting a core - mantle organization , suggesting that vessels do not penetrate these islets . but here again , -cell extensions intercalated between peripheral -cells were observed , and it is possible that most -cells reach the surrounding vasculature in this way . our description of human islet cell organization is innovative but does not contradict the different views reported so far about the human islet architecture ( 6,8,9,11,12 ) , including the diametrically opposed opinions describing islets with either segregated or intermingled cell types . indeed , a clear segregation between - and -cells was observed , and this was particularly true in small human islets where -cells surrounded a core of -cells . in larger human islets , a kind of cell segregation was also observed because -cells were confined mostly at the periphery of the trilaminar plate . from another point of view , human islet cells can be considered intermingled , because we clearly showed that intercellular contacts between - and -cells predominate . this is certainly a consequence of the trilaminar organization and cytoplasmic extensions of -cells that intercalate between -cells . this is not the case in rodent islets where the higher degree of cell segregation clearly favors - and -cell homologous contacts . the potential physiological significance of heterologous contacts between - and -cells is suggested by studies showing that glucagon positively affected insulin release ( 20,21 ) . in this respect , we recently demonstrated that insulin secretion from individual -cells was increased when they were in contact with -cells ( 22 ) . these results , combined with those showing that heterologous contacts predominate in human islets , suggest that glucagon is a more potent regulator of insulin secretion in human than in rodent islets . heterologous interactions between - and -cells in human islets are not only frequent but also unusual , because -cells most often wrap the neighboring -cells . this behavior of -cells toward -cells was also observed in vitro in cultured isolated islet cells , demonstrating that it is innate to cells and not commanded by some islet constraints . most likely , cytoskeleton and adhesion molecules expressed by islet cells may play a role . a physiological relevance of this unique plasticity of -cells is possible , especially considering that glucose - induced insulin secretion in vitro is improved in spreading -cells compared with round - shaped -cells ( 23 ) and that cytoskeleton plays a role in insulin secretion ( 24 ) . it remains to be demonstrated whether this modification has a direct impact on insulin secretion . islets within pancreata of type 2 diabetic donors have roughly the same structure as islets from nondiabetic donors . indeed , we have found that the associations between - and -cells were roughly similar in type 2 diabetic and nondiabetic islets , suggesting that the -cell dysfunction observed in type 2 diabetes is not related to a clear abnormality of islet cell organization . certainly , more studies are required to understand whether a more subtle defect in cell organization is involved . our quantitative analysis demonstrated that -cell mass relative to -cell mass was substantially decreased in type 2 diabetic islets . these results are in agreement with some previous reports showing a decrease of the absolute -cell mass or of the volume of islets relative to the volume of pancreas in type 2 diabetic subjects ( 1619 ) . we have shown that the unique organization of islet cells observed within human pancreata is not maintained in cultured isolated islets . this is not surprising considering the insults sustained by islets during the isolation procedure , including disruption of vascularization , enzymatic digestion of extracellular matrix , and mechanical shaking of the tissues . finally , the handling , fixation , and paraffin embedding of islets may also affect the cellular structure of the islets . it would be interesting to investigate whether islet architecture is maintained in freshly isolated islets and the real effect is of the culture on islet morphology . many components of the medium could affect islet morphology in vitro . among these components , there are the insulin secretagogues , such as glucose , that have been shown to affect expression of adhesion molecules , including e - cadherin and integrins , in islet cells ( 23,25 ) . in rodents , dissociated islet cells readily reaggregate in culture and after a few days are able to form pseudoislets with a core - mantle organization similar to that of native islets . this indicates that in rodents , information that decides the islet architecture is foremost provided by islet cells themselves ( 26,27 ) . our observations suggest that islet cells by themselves are not responsible for the unique cellular organization of human islets . in humans , the more complex organization of islets could involve more complex mechanisms . the role of vessels and extracellular matrix components in the maintenance of human islet architecture remains to be investigated . furthermore , it will be particularly interesting to determine whether islet morphology is restored after transplantation and whether the unique arrangement between - and -cells is critical for the function of transplanted islets .

objectiveit is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets . however , a clear description of human islet architecture has not yet been reported . the aim of this work was to describe our observations on the arrangement of human islet cells.research design and methodshuman pancreas specimens and isolated islets were processed for histology . sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells.resultsin small human islets ( 4060 m in diameter ) , -cells had a core position , -cells had a mantle position , and vessels laid at their periphery . in bigger islets , -cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets . as a consequence of this organization , the ratio of -cells to -cells was constantly higher in the core than in the mantle part of the islets , and decreased with increasing islet diameter . this core - mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets . three - dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates , folded with different degrees of complexity and bordered by vessels on both sides . in epithelial plates , most -cells were located in a central position but frequently showed cytoplasmic extensions between outlying non-cells.conclusionshuman islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between - and -cells , while permitting homologous contacts between -cells .

mirizzi syndrome ( ms ) , an unusual complication of gallstone disease , was first described by the argentinean surgeon pablo mirizzi based on cholangiography in 1948.1 it is rare and occurs in only 0.7%1.4% of gallstone patients ; the typical signs are intermittent or constant jaundice , fever , and right upper quadrant abdominal pain . although these symptoms are found in approximately 80% of cases , there is no specific clinical picture of ms . as a result , it is difficult to identify it preoperatively.2 ms can be confirmed only when the typical cholangiography revealed a smooth structure of common bile duct ( cbd ) . although some advanced technologies , such as magnetic resonance cholangiopancreatography ( mrcp ) , multidetector computed tomography ( mdct ) , and ultrasound ( us ) can assist in preoperative diagnosis , ms with fistula is mainly diagnosed during operation . the cbd or hepatic duct may be involuntarily injured during duct operation , and the former is easily injured by technical failures , causing an aberrant duct . therefore , more appropriate surgical procedures need to be adopted for ms with an aberrant duct fistula . we herein reported a case of ms with an unusual type fistula that was successfully treated with the combined approach of partial hepatectomy , cholecystectomy , and t - tube drainage . the patient , a 76-year - old asian female , was detected with liver gallstones ( lithiasis ) by ultrasonic diagnosis during a health examination and was admitted to our department . at that time , she did not show fatigue , jaundice , intermittent high fever , and right upper quadrant abdominal pain . moreover , according to the abdominal findings via physical examination , no icteric scleras and right upper quadrant abdominal tenderness in deep palpation were found , nor were the rebound tenderness or abdominal muscle rigidity and palpable masses found . the following were the testing indexes : blood tests showed alt : 313 u / l ( normal : 040 u / l ) , alp : 67 u / l ( normal : < 106 u / l ) , and -ggt : 93 u / l ( normal : < 50 u / l ) with normal bilirubin levels : 20 mol / l ( normal values for total bilirubin : 025 mol / l).serologic tests for hepatitis b and c were all negative . tumor markers including -fetoprotein , carcinoembryonic antigen , and carbohydrate antigen 19 - 9 were within normal limits.us examination showed cholelithiasis with a distended gallbladder , multiple calculi within its lumen , and moderate intrahepatic biliary dilatation.abdominal enhanced multidetector computed tomography scan revealed an atrophic right posterior liver and a dilated hepatic duct and right posterior sectional duct with multiple stones inside.mrcp indicated the presence of a sinus tract between the gallbladder fossa and right posterior hepatic duct . cholecystobiliary and aberrant right posterior hepatic duct fistula were also observed ( figure 1 ) . computed tomographic ( ct ) scans showed multiple dilated bile ducts in the right posterior segment with stones ( figure 2 ) . u / l ) , alp : 67 u / l ( normal : < 106 u / l ) , and -ggt : 93 u / l ( normal : < 50 u / l ) with normal bilirubin levels : 20 mol / l ( normal values for total bilirubin : 025 mol / l ) . tumor markers including -fetoprotein , carcinoembryonic antigen , and carbohydrate antigen 19 - 9 were within normal limits . us examination showed cholelithiasis with a distended gallbladder , multiple calculi within its lumen , and moderate intrahepatic biliary dilatation . abdominal enhanced multidetector computed tomography scan revealed an atrophic right posterior liver and a dilated hepatic duct and right posterior sectional duct with multiple stones inside . mrcp indicated the presence of a sinus tract between the gallbladder fossa and right posterior hepatic duct . cholecystobiliary and aberrant right posterior hepatic duct fistula were also observed ( figure 1 ) . computed tomographic ( ct ) scans showed multiple dilated bile ducts in the right posterior segment with stones ( figure 2 ) . although the patient was suffering from severe lithiasis , she did not show any symptoms . on the basis of the aforementioned data , the initial diagnosis before operation was ms complicated with intrahepatic lithiasis associated with aberrant right posterior hepatic duct fistula . surgery remained the exclusive choice of curative therapy , and the definitive diagnosis was confirmed intraoperatively . an exploratory laparotomy through a right subcostal incision was performed . during cholecystectomy , two severe anatomic alterations were observed . the first was an impact stone found in the cystic duct , which firmly adhered to the aberrant right posterior hepatic duct and the cholecystobiliary fistula , and involved less than one - third of the circumference of the duct . the other was a wide range of tissue adhesion , which was associated with elongated cystic duct , parallel to the cbd . multiple cystic dilations of the intrahepatic biliary tree and black pigment stones adherent to the bile duct wall were observed in the resected specimen ( figure 3 ) . the cbd was lavaged , and the defect of the cbd was sutured in a one - layer manner . the postoperative course was uneventful and the patient was discharged a week after the operation . ms , a rare complication ( ~1% ) of chronic cholecystitis and cholelithiasis , was first described in 1923 by hans kehr and was characterized by impaction of stones in the cystic duct or neck of the gallbladder , resulting in mechanical compression or erosion of the cbd.1 as there is no specific clinical or laboratory finding for the syndrome , ms was always characterized with jaundice , abdominal pain , and alterations during the serum tests of hepatic function . however , these symptoms of ms are seen in ~80% of cases.2 the patient we reported here had no significant clinical picture . even with the availability of modern imaging techniques , majority of cases are not identified preoperatively . mcsherry et al3 classified ms into two types based on endoscopic retrograde cholangiopancreatography ( ercp ) findings : type i ms when there is extrinsic compression of the common hepatic duct and type ii ms when the gallstones erode the common hepatic duct wall , resulting in a cholecystocholedochal fistula . the most recent and important classification was made by nagakawa et al,4 while probably the best one was done by csendes et al.5 according to the classification of csendes et al5 , ms is divided in four types depending on the size of the destruction of the cbd.6 we consider this case as a special kind of ms where intrahepatic stones complicated patient s condition . in general , a preoperative diagnosis is crucial for us to choose the appropriate surgical approach . us was always utilized as a primary investigation in the treatment of patients with abdominal pain and jaundice . ms was confirmed via the evidence of dilated intrahepatic ducts and common hepatic duct by observing the narrowness of the compressed external bile duct that caused by the calculus.7 it was regarded as a criterion used to diagnose ms that cholangiographic evidence of stone affected compression of the bile duct in the gallbladder neck or cystic duct , or the presence of a cholecystcholedochal fistulam , which were detected by ct , mrcp , and ercp . in addition , direct cholangiography is the gold standard for locating subtle abnormalities within the bile ducts and detecting small calculi.8,9 nevertheless , the patient refused ercp , even though it remains the gold standard for preoperative diagnoses of ms . a recent study demonstrated that mrcp , a noninvasive alternation to ercp , is a useful preoperative diagnostic modality for ms with a higher accuracy and better interobserver agreement when combined with ct.10 in this case , mrcp demonstrated a possibility of an already existing abnormal fistula between the gallbladder and the common hepatic duct . noninvasive imaging modalities such as ultrasonography , ct , and magnetic resonance imaging can also accurately depict the presence of intrahepatic stones . the patient was diagnosed with intrahepatic lithiasis that caused liver fibrosis , atrophy , and fibrotic stricture of right posterior hepatic duct and her second / third branches . the incidence of hepatolithiasis has been reported to be 20%31% in the patients with gallstone disease , and it is a prevalent disease in east asia . compared to nonoperative treatment , hepatic resection is a preferable choice for unilateral stones treatment.11 it was reported that surgical therapy is a safe and effective management for hepatolithiasis . however , ms hinders the complex operative procedure further , by making it much more difficult on the condition of dangerous anatomical alteration in the area of calot s triangle . as the principle of which is to remove the stones as well as the biliary stricture and to reduce the risk of recurrent stones , hepatectomy seems to be the optimal treatment for hepatolithiasis among selected patients.1214 tabrizian et als15 investigation showed that hepatic resection is a safe and definitive treatment option in the control of primary hepatolithiasis , and it could achieve excellent short- and long - term effect . furthermore , according to jiang et als11 study , anatomical hepatectomy can produce better clinical outcome than limited hepatectomy about regional hepatolithiasis . in our study , hepatolithiasis was limited to a right posterior lobe ( segment ) irrespective of lobular atrophy or fibrosis . by receiving anatomical hepatectomy , she may get a radical cure so as to minimize the action on hepatic function . during operation , ms was confirmed only after the extraction of large gallstone that compacted on gallbladder neck and right posterior hepatic duct , and under this circumstance , the cystic duct usually ran parallel to the cbd ( 10% of cholangiograms).16 ms exhibited a dangerous and unexpected variation in calots triangle . thus , preoperative recognition of this variation is significantly important to avoid inadvertent ligation or severance of the bile duct . the fistula s excision and repair of the hepatic flexure with primary closure was finally performed without any further postoperative complications . a t - tube was routinely inserted after hepatectomy for postoperative cholangiography and choledochoscopy via the t - tube route . this protocol was approved by the institutional review board of the first people s hospital of wujiang . written informed consent was obtained from the patient for the publication of this case report and any accompanying images . preoperative evaluation of patient s condition , adequate recognition of variation , right approach , and minimal surgical maneuvers at the time of the operation were important key points for successful treatment of these clinical entities .

mirizzi syndrome ( ms ) is a rare complication of chronic cholelithiasis , which is always caused by a calculus in the cystic duct or neck of the gallbladder , resulting in mechanical compression of common bile duct and the gallbladder . it is clinically characterized by abdominal pain , fever , as well as obstructive jaundice . during cholecystectomy , ms is seen as a dangerous adherent and inflammatory tissue in the area of calot s triangle . in the general population , aberrant right posterior hepatic duct , one of the causes of bile duct injury during duct surgery , is present in 4.8%8.4% of people . herein we report a rare case of a 76-year - old female patient , with hepatolithiasis of right posterior lobe and cholecysto - aberrant right posterior hepatic duct fistula . this is a special type of ms ; however , interestingly , she did not have any symptoms , and the disease was found by physical examination incidentally . this case highlights another situation , namely , there may be difficulty in diagnosing ms and dissecting for operation . therefore , to avoid the complication associated with this special situation , the surgeons need to diagnose carefully and adopt an optimal treatment strategy .

the zoonotic intestinal disease giardiasis is caused by the enteric protozoan parasite giardia lamblia , one of the most common causes of intestinal infections worldwide . infection with giardia is acquired by the ingestion of viable cysts due to inadequate sanitation or poor treatment of drinking water . giardiasis is a disease of main concern as it affects children , adults , hypogammaglobulineamic , malnourished , and immunocompromised individuals leading to either acute or chronic diarrhea , nausea , malabsorption , weight loss , steatorrhoea , and growth retardation particularly in young children [ 13 ] . however , antibiotic therapy is associated with many unpleasant side effects ( e.g. , metallic taste ) , poor patient compliance , and enhanced occurrence of resistance leading to subsequent treatment failure . thus , this has encouraged research on alternative biotherapeutic strategies such as plant extracts ( phytomedicine ) , products derived from bees , and probiotics that are safe , inexpensive , and effective in improving the cause of intestinal parasitosis [ 46 ] . normally , the generation of immunophysiologic regulation in the gut depends on the establishment of indigenous microflora and has led to the introduction of novel therapeutic interventions based on the consumption of cultures of beneficial live microorganisms , the probiotics . probiotics are live microorganisms that beneficially affect the gastrointestinal balance if ingested in sufficient numbers , and provide health benefits that go beyond normal nutritional effects . probiotics , also known as microbial interference therapy ( mit ) , offer an attractive supportive therapy for gastrointestinal infections by acting as surrogative normal flora via various mechanisms such as production of antimicrobial substances , modification of toxins , interference with attachment , stimulation of immune system or a combination of mechanisms [ 9 , 10 ] . in our earlier studies , we have observed that the probiotics l. casei and l. acidophilus both modulate the murine giardiasis by reducing the severity and duration of the disease [ 1113 ] . however , it has also been observed that different strains of lactic acid bacteria ( lab ) have different properties and may beneficially influence the composition and metabolic activity of the endogenous microbiota [ 7 , 14 ] or inhibit the growth of a wide range of enteropathogens in intestinal diseases , as not all the strains are able to adequately survive the acidic ph or to adhere and colonize the gut . moreover , till date no effective probiotic has been reported for giardiasis , thus it is pertinent to delineate an effective probiotic for giardiasis . giardia lamblia trophozoites ( portland strain i ) were grown axenically in tyi - s-33 medium supplemented with antibiotic solution , and ph was adjusted to 6.9 before sterilization with 0.22 m seitz filter . for experimental inoculation , actively growing trophozoites ( 4872 h old culture ) were sedimented after chilling the tubes in ice for 15 min and finally suspended in phosphate buffer saline ( pbs-7.2 ) to contain 1 10 trophozoites/0.1 ml . various lactobacilli strains ( lactobacillus gg , l. acidophilus , l. plantarum , and l. casei ) were procured from microbial type culture collection ( mtcc ) , institute of microbial technology ( imtech ) , chandigarh , india . thereafter , the cultures were centrifuged , washed , and suspended in pbs-7.2 to contain 1 10 lactobacilli/0.1 ml and were fed via orogastric gavage . balb / c mice aged 5 - 6 weeks old ( 1820 gm ) were obtained from central animal house , panjab university , chandigarh , india . these were housed under standard conditions of light and dark cycle and were fed with laboratory diet and water ad libitum . water and feed before supplementation to animals were monitored for any bacterial or parasitic contamination by gram 's staining and lugol 's iodine staining techniques . animals were also screened for giardia infection via simple microscopic stool examination for three consecutive days . care and use of animals were in accordance with the guidelines of the institutional ethical committee . group i ( giardia infected ) : these mice were challenged orally with a single dose of 1 10 giardia trophozoites via orogastric gavage . group ii ( l. casei - giardia ) ; group iii ( l. acidophilus- giardia ) ; group iv ( l. plantarum - giardia ) ; group v ( l. rhamnosus gg- giardia ) . animals belonging to groups ii , iii , iv , and v were fed orally with a single dose of respective lactobacilli strains ( 1 10 lactobacilli/0.1 ml ) for 7 days . on the 8th day , a single challenge dose of giardia trophozoites ( 1 10 trophozoites ) was given orally along with a single dose of probiotic treatment . briefly , one gram of freshly passed faecal samples was dissolved in 10 ml of normal saline and homogenized using pestle and mortar mixer . slide was prepared , and cysts stained with iodine were counted on every third day using a hemocytometer and were expressed as cysts ml . to confirm if the lactobacilli species were able to survive the stress and colonize within the gastrointestinal tract , freshly voided faecal samples of mice belonging to groups i and v were homogenized in normal saline and serially diluted . the diluted homogenates ( 0.1 ml ) were spread plated on mrs agar and incubated at 37c for 2448 hrs , and cfu were counted . mice were sacrificed , and the proximal 10 cm section , mainly the jejunum , was removed and placed in 5 ml of the ice - chilled isotonic saline solution . the small intestine sections were minced and kept for 1520 minutes in ice - chilled saline , and trophozoites were counted using a haemocytometer . results were expressed as mean sd . the inter - group variation was assessed by student 's t - test and one way analysis of variance ( anova ) with equal number of observations followed by tukey 's multiple comparison test . giardia - infected mice ( group i ) voided cysts gradually from day 1 onwards and was significantly ( p < .05 ) highest ( 306.4 10 10.63 ) on day 7 postinoculation ( pi ) . thereafter , the cyst count started decreasing , and mice became giardia - free by day 25 pi ( figure 1 ) . however , oral feeding either with l. acidophilus , l. plantarum , l. casei , l. acidophilus , or l. gg significantly ( p < .05 ) reduced the cyst excretion in mice belonging to all the groups ( ii , iii , iv , and v ) from the beginning and became giardia - free by days 17 pi ( groups ii and iii ) and 13 pi ( groups iv and v ) , respectively . interestingly , mice fed orally with l. gg ( group v ) showed significantly ( p < .05 ) least cyst count from the beginning of the infection ( i.e. , 5 days pi onwards ) and became giardia - free by day 13 pi ( figure 1 ) . however , none of the mice from any of these groups showed any clinical symptoms like diarrhea , weight loss , and death . the faecal lactobacilli counts increased significantly ( p < .05 ) in l. gg - giardia mice ( group v ) from the beginning and were significantly higher ( p < .05 ) at each point of observation compared with giardia - infected mice ( group i ) that had least lactobacilli count ( figure 2 ) . it was found that oral administration of giardia trophozoites resulted in the establishment of infection as assessed by the number of trophozoites in the jejunum and cyst counts in faeces . interestingly , trophozoite counts were significantly ( p < .05 ) reduced in the gut of l. gg - treated mice ( group v ) compared with giardia - infected mice ( group i , figure 3 ) . it is generally accepted that the enteric bacterial environment represents a physiological factor that can interfere with the process of a g. lamblia infection . therefore , the present study was designed to assess the effect of various probiotic supplementations in modulating the giardia cycle in balb / c mice . it was found that orally administered giardia trophozoites in mice could transiently colonize the gut , and infection was self - limiting . it is very well evident that the supplementation of various probiotics has the potential to modulate murine giardiasis to variable extent . amongst all the lactobacilli , l. gg was found to be the most effective probiotic in modulating the giardiasis , both in terms of duration of giardia cycle and rate of cyst excretion . the present observation of reduced cyst count may be either due to better survival of l. gg in stomach or effective adherence and colonization in the gut compared with other lactobacilli strains . moreover , l. gg has also been found to be an effective treatment therapy for various bacterial and viral diarrheal diseases and is in accordance with the earlier studies [ 8 , 1113 ] . the ability of the l. gg to adhere to gastrointestinal tract and persistence was further evaluated by monitoring the lactobacilli count in faeces . it was observed that mice fed with l. gg had more lactobacilli in faeces compared with giardia - infected mice alone . interestingly , it was also found that l. gg feeding reduced the active number of giardia trophozoites in the gut leading to early resolution of giardia infection by day 13 ( pi ) . this may again be suggestive of effective colonizing ability of l. gg and interaction with enterocytes , thus enriching the endogenous microbiota and is in accordance with earlier studies [ 11 , 1820 ] . the reduced duration of infection may be partly either due to effective colonization of lactobacilli , production of secretary substance or competition for nutrients , and so forth . taken together , it can be concluded that various lactobacilli species have variable effects in intestinal diseases . among the four lactobacilli species , thus it can be said that l. gg is the ideal , safe , and stronger barrier against intestinal pathogens than other lactobacilli strains , leading to improved core health , that is , healthier digestion and improved immune system , and it may also serve as an alternative mode for the prevention of giardiasis .

the gut microflora is an important constituent in the intestinal mucosal barrier and has been introduced as the concept of probiotic therapy that beneficially affects the host by improving its intestinal microbial balance . therefore , the main objective of the study was to explore the protective potential of various lactobacilli strains for murine giardiasis . by experimentation , it was found that the probiotic supplementation of either lactobacillus casei , l. acidophilus , l. plantarum , or l. rhamnosus gg , 7 days prior to inoculation with g. lamblia trophozoites , reduced the rate of cyst excretion compared with giardia - infected mice . interestingly , l. gg was found to be the most effective probiotic in reducing the duration of giardia cycle and acts as an effective prophylactic probiotic for murine giardiasis but needs to be clinically correlated due to entirely different human microflora .

it is over 50 years since the first license for a fibrinogen concentrate ( fibrinogenio humano lio . ) historically , fibrinogen came into focus because of reports suggesting it was a cardiovascular risk factor , and there is still no final answer as to whether it is a mediator , or just a marker or predictor , of cardiovascular disease . it is only recently that research started focusing on the positive role of fibrinogen as a hemostatic agent , particularly in the management of perioperative bleeding . before we discuss the development of fibrinogen concentrate , we take a historical look at where the term fibrinogen first came from . as far back as the fifth- to fourth - century bc , however , it was not until the end of the 17th century that malpighi , using a single lens microscope , observed that whole blood clots have a fibrous and corpuscular component . the term he demonstrated that plasma contained soluble substances and that the precursor of fibrin was present in plasma but not serum . a few decades later , in 1847 , the term fibrinogen was invented by virchow where he stated should one want to give it a name , it could be called fibrinogen ( translated from german : vielmehr , wollte man sie benennen , so knnte man sie hchstens fibrinogen taufen . ) . some consider that he created the term out of the words fibrin and oxygen , since he believed that fibrin was a homogeneous mass formed from soluble fibrin after exposure of blood or exudates to oxygen , and others suggest that he composed the term by adding the syllable ogen for a precursor substance instead of the prefix pro . at the time , virchow was experimenting with exudates , rather than with whole blood , and therefore mistakenly applied the term fibrinogen to decaying exudates rather than to the precursor of fibrin in whole blood as it was understood later . in fact , virchow had not discovered fibrinogen , instead denis ( in his mmoire sur le sang in 1859 ) was the first to recognize that plasma contained a clottable substance different from fibrin and went on to attempt to purify and characterize this protein . i believe that i should repeat once more that i do not mean to define a liquid fibrin , plasma fibrin , but rather a substance which is not at all fibrous and which is the origin of fibrin , clottable lymph , a substance which i would like to call nevertheless , it took a further 20 years before fibrinogen was first isolated by hammarsten ( 1879 ) who precipitated and purified it from horse plasma by salting - out with sodium chloride in what has become the classical procedure for fibrinogen preparation . nowadays , bleeding management encompasses , among other things , the assessment of bleeding risk prior to surgery , and monitoring and managing blood loss during surgery , including appropriate management of bleeding . historically , the development of therapeutic blood management can be traced back to the 17th century where the first animal - to - human blood transfusion was conducted in 1667 . however , it was not until 1818 that james blundell , a british obstetrician , performed the first human - to - human transfusion to save a patient from exsanguination . the introduction of blood transfusion into the medical armamentarium was further advanced with the groundbreaking discovery of the abo blood group by the austrian american immunologist and pathologist karl landsteiner in 1901 , a feat for which he was later awarded the nobel prize . over the last 100 years one of the most relevant developments is the shift from using whole blood to using specific blood components ( e.g. , red blood cells , platelets , and fresh frozen plasma [ ffp ] ) and further to the purified , virus - inactivated , plasma - derived products ( such as factor concentrates ) . this latter development was made possible by the work of edwin joseph cohn who discovered that the complex mixture of components in whole blood could be separated through a process of fractionation , commonly referred to as in 1947 , he described 6 major plasma fractions , with fraction i containing most of the fibrinogen . cohn emphasized the importance of separating different plasma components , as it was more efficient and less wasteful to use a particular component for a specific need rather than whole plasma . this approach proved of great value during world war ii with the development of products such as fibrin foam helping to solve major problems connected with hemostasis . this change from the use of specific blood components to purified plasma - derived products for bleeding management represents a continuing paradigm shift . during major bleeding and replacement with red blood cell concentrates , fibrinogen is the first clotting factor to reach critically low levels . furthermore , low perioperative fibrinogen levels have been shown to be associated with increased bleeding . therefore the standard practice of replacing fibrinogen has focused on infusions of therapeutic plasma ( eg , ffp ) or cryoprecipitate ; however , there are differences concerning the volume of each product . therefore , with the progression from using whole blood to allogeneic blood products ( eg , plasma , red blood cells , platelet concentrates , and cryoprecipitate ) , the next consideration should be moving toward more purified , specific products such as factor concentrates ( eg , fibrinogen concentrate ) . this represents the move from the use of a liquid organ to a pharmaceutical product . notably , fibrinogen concentrate and freeze - dried fraction i ( prepared by cohn fractionation of plasma ) were already available before licensure of this product became obligatory . the european union birth date of the product is january 4 , 1966 , when fibrinogen concentrate ( human - fibrinogen behringwerke konzentrat ) was approved in europe for the first time by the german federal ministry of health . however , behringwerke ( a predecessor company of csl behring gmbh ) had already commenced production of human fibrinogen concentrate in 1956 , before registration of a product became mandatory with the medicinal products act coming in to force in 1961 in germany . in 1985 , the production process was modified to include pasteurization as a virus inactivation step , and the product was renamed haemocomplettan p ( csl behring gmbh ) . currently , all formulations of fibrinogen concentrate manufactured by csl behring are pasteurized , and over 3 million grams have been used since 1985 . at the time of writing , csl behring s fibrinogen concentrate is approved for treatment and prophylaxis of acquired and congenital fibrinogen deficiency under the trade name haemocomplettan p in several countries ( table 1 ) . in israel , haemocomplettan p is indicated for the treatment of acute bleeding episodes in patients with congenital fibrinogen deficiency , including afibrinogenemia and hypofibrinogenemia . the same product , licensed under the trade name riastap , is also available for the treatment of acute bleeding episodes in patients with congenital fibrinogen deficiency , including afibrinogenemia and hypofibrinogenemia , in australia , canada , mexico , new zealand , puerto rico , united states , and several european countries ( table 1 ) . additional countries received licenses to use the product that are no longer active , for example , pre-1985 ( colombia , jamaica , pakistan ) and post-1985 ( croatia , india ) . other trade names have been used for this product during the licensing history and in other countries , for example , fibrinogenio humano liof , fibrinogenio humano , fibrinogenio , haemocomplettan , haemocomplettan hs , human - fibrinogen behringwerke konzentrat . current evidence suggests that fibrinogen concentrate has a good safety profile and delivers a standardized dose of fibrinogen . haemocomplettan p / riastap has been shown to be effective and well tolerated in a number of clinical studies of patients with congenital or acquired fibrinogen deficiency . a postmarketing surveillance study of haemocomplettan p / riastap covering a 27-year period ( 1986 - 2013 ) found 28 spontaneous reports of possibly related thrombotic events for 2 611 294 g of distributed product . the use of haemocomplettan p / riastap to control bleeding and to decrease the use of allogeneic blood products during cases of acquired bleeding has been reported in a variety of clinical settings , including surgery , trauma , liver transplantation , and obstetrics . furthermore , recent systematic reviews confirm that the use of fibrinogen concentrate may reduce allogeneic blood product transfusion ; however , very often the trials are of low quality with a high risk of bias . moreover , the measured outcomes , such as transfusion requirements , may be prone to performance bias . therefore , more randomized controlled trials are warranted before it can be confirmed whether fibrinogen concentrate has a definitive role in the management of bleeding . additionally , some controversy still exists around the use of fibrinogen concentrate with regard to the appropriate level of fibrinogen to trigger treatment . ranucci and solomon are right to note in their editorial that there is currently a discrepancy among licences , guidelines , and clinical use and that there are open issues and gaps in knowledge that need to be addressed . four fibrinogen concentrate products are currently available in various regions and according to public sources more are in development . other developments include the production of recombinant fibrinogen , first engineered by lord et al in 1993 . the functional properties of a recombinant fibrinogen have been tested ex vivo in whole blood samples from a group of patients undergoing cardiac surgery . according to the clinical trials registries ( clinicaltrials.gov and clinicaltrialsregister.eu ) , there are currently 11 ongoing clinical studies investigating the use of fibrinogen concentrate in acquired bleeding . the results of these studies will potentially provide further evidence on the role of fibrinogen concentrate in the management of bleeding . these ongoing investigations will provide additional evidence of fibrinogen concentrate in acquired bleeding , but fibrinogen may have a number of different applications beyond bleeding management . thrombocytopenia is a common complication in hospitalized patients which affects blood clotting , and studies have shown that fibrinogen concentrate can increase the clot strength , independent of platelet count . further clinical investigations are necessary to confirm whether bleeding complications associated with thrombocytopenia could be managed through supplementation with fibrinogen concentrate . the role of fibrinogen and interference with the immune system is also worthy of further scrutiny , as a significant decrease in sepsis - induced mortality was observed in animals treated with fibrinogen concentrate in a rat model of sepsis - induced disseminated intravascular coagulation . advances in science and medicine have led to improvements in blood and blood products and in the way patients are treated . fibrinogen concentrate seems to offer a specific option in the management of bleeding ; however , further investigations are required to clearly define its role . fibrinogen concentrate was described in 2009 as a potential universal hemostatic agent ; the next 50 years will demonstrate whether this is the case .

march 2013 represented the 50th anniversary of the first license granted for a fibrinogen concentrate . in this review , we look at the history of bleeding management that led to the development of fibrinogen concentrate , discuss its current use , and consider future developments for this product .

an ideal spine injury classification system is able to both guide treatment and facilitate clear communication between the surgeons , researchers , and trainees . early classifications such as the denis classification and magerl classification described the thoracolumbar spine and were later extended to describe cervical spine injuries.1 2 more recently , dedicated classification systems for the thoracolumbar and subaxial cervical spine have been published or presented,3 4 including the aospine thoracolumbar spine injury classification system and the aospine subaxial cervical spine injury classification system.5 6 the aospine thoracolumbar spine injury classification system initially separates fractures into one of three morphologic types ( fig . 1)5 : type a , the compression injuries ; type b , the injuries associated with a failure of the anterior or posterior tension band without evidence of translation ; and type c , the translational injuries resulting from the failure of all the stabilizing elements leading to a complete disruption of the bony and soft tissue hinge . next , the patient is assigned one of five neurologic grades5 : n0 , the patient is neurologically intact ; n1 , the patient had a transient neurologic deficit ; n2 , the patient has persistent symptoms or signs of a nerve root injury ; n3 , the patient has an incomplete spinal cord injury or a cauda equina injury ; n4 , the patient has a complete spinal cord injury ; and nx , the neurologic examine of the patient can not be obtained . finally , the classification allows for patient - specific modifications : m1 denotes a possible ligamentous injury to the tension band that may necessitate surgery , and m2 denotes injury - specific morbidities , such as ankylosing spondylitis , polytrauma , significant medical comorbidities , among others , that may alter the recommended treatment.5 the three types of thoracolumbar fractures : type a , compression injuries ; type b , injuries associated with a failure of the anterior or posterior tension band without evidence of translation ; and type c , translational injuries resulting from the failure of all elements leading to complete disruption of the bony and soft tissue hinge.5 the subtypes of compression injures : a0 , spinous / transverse process fracture ; a1 , compression / wedge fracture ; a2 , pincer fracture ; a3 , incomplete burst / burst with a single end plate involved ; a4 , complete burst / burst with both end plates involved.5 the subtypes of tension band injuries : b1 , transosseous disruption ; b2 , posterior tension band injury ; b3 , anterior tension band injury.5 initially substantial inter- and intraobserver reliability of the aospine thoracolumbar spine injury classification system was reported in a group of highly trained academic spine surgeons ( 0.72 and 0.77 , respectively),5 and later moderate interobserver reliability ( kappa = 0.56 ) of the system was reported in 100 surgeons from all regions of the world who had no prior knowledge of the system.7 utilizing an injury severity score for each variable in the classification,8 kepler et al proposed a spine injury score ( table 1 ) to accompany the new classification.9 although the surgical threshold has yet to be established , the spine injury score will be used to help guide treatment . given the regional variation in treatment of thoracolumbar trauma , significant research has been performed attempting to determine the need for a regional interpretation of the spine injury score ; however , little has been written about the possible need for altering the surgical threshold based on the injury location within the thoracolumbar spine . there are significant differences in both the bony morphology and the neural elements between the upper thoracic spine and the lower lumbar spine , and these differences may significantly affect the ideal treatment algorithm . this aim of this narrative review , which collates the expertise of a multidisciplinary group of experienced spine surgeons in the aospine international trauma knowledge forum , is to highlight two possible difficulties in the development of a single surgical algorithm for the entire thoracolumbar spine . although a universally accepted definition of the cervicothoracic junction has not been defined , with different authors reporting c7t1 , c6t2 , and c6t3 as the cervicothoracic junction , this review will consider fractures between c6t3 as the cervicothoracic junction . fractures at this region account for 2.4 to 4.5% of spine fractures,10 and although these injuries may technically occur in the thoracic spine , they often behave similar to subaxial cervical fractures with 60 to 83% of these injuries resulting in neurologic impairment.11 12 the higher incidence of neurologic injuries at this level is likely multifactorial . first , the upper thoracic spinal canal is particularly narrow , leading to an increased risk of neurologic compression with any canal compromise . additionally , the cervicothoracic junction down to t4 is a watershed area for the blood supply to the spinal cord . the terminal branches of the vertebral , thyrocervical , and costoclavicular branches of the subclavian artery supply the caudal cervical spinal cord , and the radicular vessels from the proximal intercostal arteries provide the blood supply to the cephalad thoracic spinal cord.11 comparatively , significant canal compromise in the lumbar spine may or may not be associated with a major neurologic injury , as the spinal cord terminates around l2 and the cauda equina has a much higher tolerance for compressive lesions.13 the upper thoracic vertebrae also are morphologically more similar to cervical vertebrae than lumbar vertebrae . stanescu et al reported on the bony characteristics of 128 vertebrae in 16 spines between c5 and t5 , and they found a similar pedicle length and laminar height and width between the c7 vertebrae and the t1 vertebrae.14 additionally , despite a statically significant increase in the width of the t1 pedicle compared with the c7 pedicle ( 7.8 versus 6.5 mm , p < 0.05 ) , the pedicle width decreases between t2 and t5 , such that it more closely resembles that of the cervical spine than the lumbar spine.14 15 16 furthermore , the lamina pedicle angle is fairly consistent from c7 ( 88.3 degrees ) to t2 ( 90.5 degrees ) , but then it slowly increases to 96.7 degrees at t5.14 the clinical relevance behind these morphologic similarities can be seen in the comparable prevalence of injuries such as fracture - dislocations , burst fractures , and facet subluxation / dislocations in the subaxial cervical and upper thoracic spine.10 although the upper thoracic spine more closely resembles the subaxial cervical spine than the lumbar spine , it also has some properties more consistent with the remainder of the thoracic spine than the cervical spine . the facet orientation undergoes a significant change at the cervicothoracic junction , with the facet angle changing from 86 degrees at c7 to 69 degrees at t2 ( fig . 4b ) changing from 123 degrees at c6 to 107 degrees at t2.17 additional morphologic similarities between the upper thoracic spine and the remainder of the thoracic spine include the prominent transverse processes and the stabilizing costovertebral joint . ( a ) the facet angle and ( b ) the disk facet angle.17 ( i = the right facet angle ; ii = the left facet angle ; theta = the disk facet angle . ) although the upper thoracic spine has characteristics of both the subaxial cervical spine and the caudal thoracic spine , there are anatomic features unique to the upper thoracic spine that present specific challenges to the treating surgeon . the change from cervical lordosis to thoracic kyphosis , along with the significant increase in the stiffness of the thoracic spine , makes fractures in this region particularly prone to developing progressive kyphosis18 ; the successful stabilization of these injuries is difficult as the placement of upper thoracic pedicle screws is challenging given the small pedicle size . additionally , the presence of the heart and great vessels prohibit an anterior approach to t2t4 ; however , performing a complete posterior decompression around the spinal cord is challenging . manipulation of an already damaged spinal cord can result in permanent neurologic injuries , but the failure to expeditiously decompress the spinal cord will lead to a decreased rate of meaningful neurologic recovery.19 low lumbar burst fractures ( l3l5 ) are relatively rare fractures , accounting for 1.2% of all spine fractures,20 and this is due to the unique anatomy of the lower lumbar spine . the stout iliolumbar ligaments afford significant stability to the caudal lumbar vertebrae , and the segments caudal to the pelvic brim and the apex of the lumbar lordosis are somewhat protected from trauma.21 22 23 24 25 however , recently there has been an increased interest in the treatment of these injuries after lehman et al reported that lower lumbar burst fractures account for 60% of combat - related thoracolumbar fractures in the united states military.24 the authors postulate that this is due to the advent of a new body armor , which provides increased rigidity in the upper lumbar spine , effectively moving the transition zone between the rigid and stable segments into the low lumbar spine . furthermore , the improvised explosive devices used by combatants in afghanistan and iraq result in a violent axial force.24 historically , many authors have advocated the nonoperative treatment of low lumbar burst fractures , as the biomechanical environment of the low lumbar spine is significantly different from that of the thoracolumbar junction.20 21 25 at the thoracolumbar junction , the immediate transition from the rigid thoracic spine to the more mobile lumbar spine creates a fulcrum that may lead to an increased risk of instability affecting the upper lumbar spine more than the better - protected lower lumbar spine.21 23 24 moreover , the center of gravity at the thoracolumbar junction is located in the anterior column of the spine , therefore predisposing fractures at this level to develop progressive kyphosis.24 in contrast , caudal to l2 the center of gravity is located posterior to the vertebral body , leading to a more evenly distributed axial force across the fractured vertebra.24 butler et al reported on 14 neurologically intact patients with an l5 burst fracture.21 ten were treated nonoperatively and four underwent an l4s1 instrumented posterolateral fusion . no substantial radiographic difference was identified , with nonoperative patients having 10 degrees of kyphosis compared with 11 degrees in the operative group . however , the nonoperative group reported less pain and improved function at a mean follow - up of 71 months.21 similarly , seybold et al reported on 42 patients with low lumbar burst fractures ; 22 patients underwent either an anterior , posterior , or combined decompression and fusion.25 surgical patients had a nonsignificant ( p < 0.40 ) improvement in radiographic alignment ( 9.8 degrees of lordosis compared with 1.9 degrees ) , but there was no difference in the ability of the patient to return to work or the functional outcomes between the groups . although historic studies have not demonstrated a short or midterm benefit to operative treatment of low lumbar burst fractures , the importance of sagittal balance is a relatively recent concept that has not been reported in any of the aforementioned studies . a fracture at the thoracolumbar junction resulting in neutral or slightly kyphotic focal alignment represents little change from the natural alignment , as the normal lordosis between t12 and l2 is 1.7 degrees.26 however , the normal alignment between l4 and s1 is 49.2 degrees of lordosis,26 so an injury leading to the neutral alignment in the l5 vertebra significantly alters the overall alignment of the spine and dramatically affects the patient 's sagittal balance . although the long - term effects of this altered alignment have not been reported in the spine trauma literature , the deformity literature is clear in demonstrating that a pelvic incidence - lumbar lordosis mismatch leads to significantly inferior health - related quality - of - life outcome measures.27 28 29 undoubtedly , further studies evaluating the sagittal balance of patients with low lumbar burst fractures are needed ; however , it is unclear if this is improved with acute surgical intervention . both butler et al and seybold et al reported no significant difference in the radiographic parameters of patients treated with or without surgery.21 25 however , in a study that included 365 neurologically intact patients with a thoracolumbar compression or burst fracture , reinhold et al reported an improved ability to restore and maintain lordosis in patients who underwent a combined anterior and posterior procedure.30 similarly , in a study of 45 patients with thoracolumbar fractures , schnake et al reported the ability to correct and maintain the alignment over a 5-year period if a combined anterior and posterior approach was used.31 in addition to low lumbar burst fractures , fractures of the l5s1 facet joint present a unique set of challenges to the treating physician . although these fractures may occur in isolation , it is also possible that they are the cephalad extension of a sacral fracture resulting in spinopelvic instability.32 the treatment algorithm for these complex fractures is significantly different than for the fractures throughout the rest of the thoracolumbar spine , and a separate aospine sacral classification system is being developed . specifically , although the need for early decompression in patients with an injury leading to a neurologic deficit in the thoracic and lumbar spine is clear,19 the role for an urgent formal decompression in fractures at the lumbosacral junction is debated in the literature . in one study , schildhauer et al reported on 22 patients with a neurologic deficit from vertically unstable sacral fractures , and the authors did not perform a formal decompression.33 however , in a separate study published the same year , schildhauer et al reported on 18 patients with complete bowel and bladder dysfunction from a u - type sacral fracture resulting in lumbopelvic dissociation , and all patients underwent a formal decompression.34 although there is significant heterogeneity in the two patient populations with regard to the severity of injury and the timing of surgery , only 6/22 patients ( 27% ) who did not undergo a formal decompression had any signs of neurologic recovery , whereas 15/18 patients ( 84% ) who underwent a formal decompression had at least partial neurologic recovery.33 34 in addition to the neurologic differences , the surgical technique for fractures of the lumbosacral junction varies significantly from fractures of the cephalad thoracolumbar spine . spinopelvic fixation is required for these fractures , and cadaveric studies have demonstrated an enhanced stability with a triangular osteosynthesis ( a technique utilizing both spinopelvic fixation and either sacroiliac or trans - sacral screw fixation).35 36 the new aospine thoracolumbar spine injury classification system was developed to facilitate the communication and guide the treatment for thoracolumbar trauma . although the classification and spine injury score have been published , the surgical threshold is still being investigated.5 9 however , the current review details two areas that will present a challenge to the forthcoming global algorithm for the management of thoracolumbar trauma . fractures at the cervicothoracic junction and the lumbosacral junction have some properties similar to the remaining thoracolumbar spine , but they also have properties unique to these junctional locations . the criteria to decide on the outcome of injuries in these separate areas may be different . a survey is now being conducted by the aospine among spinal surgeons to define which factors are deemed relevant for the short- and long - term outcome in these different areas . because of the unique properties of fractures at the cervicothoracic junction and lumbosacral junction , injuries at these areas will require surgeons to use their clinical judgment to determine if the fracture is best classified with the new aospine thoracolumbar spine injury classification system , the aospine subaxial cervical spine injury classification system , or the upcoming aospine sacral injury classification system.5 6

study design literature review . objective the aim of this review is to highlight challenges in the development of a comprehensive surgical algorithm to accompany the aospine thoracolumbar spine injury classification system . methods a narrative review of the relevant spine trauma literature was undertaken with input from the multidisciplinary aospine international trauma knowledge forum . results the transitional areas of the spine , in particular the cervicothoracic junction , pose unique challenges . the upper thoracic vertebrae have a transitional anatomy with elements similar to the subaxial cervical spine . when treating these fractures , the surgeon must be aware of the instability due to the junctional location of these fractures . additionally , although the narrow spinal canal makes neurologic injuries common , the small pedicles and the inability to perform an anterior exposure make decompression surgery challenging . similarly , low lumbar fractures and fractures at the lumbosacral junction can not always be treated in the same manner as fractures in the more cephalad thoracolumbar spine . although the unique biomechanical environment of the low lumbar spine makes a progressive kyphotic deformity less likely because of the substantial lordosis normally present in the low lumbar spine , even a fracture leading to a neutral alignment may dramatically alter the patient 's sagittal balance . conclusion although the new aospine thoracolumbar spine injury classification system was designed to be a comprehensive thoracolumbar classification , fractures at the cervicothoracic junction and the lumbosacral junction have properties unique to these junctional locations . the specific characteristics of injuries in these regions may alter the most appropriate treatment , and so surgeons must use clinical judgment to determine the optimal treatment of these complex fractures .

rheumatoid arthritis ( ra ) is an autoimmune systemic disease in which the body 's immune system attacks against normal tissues and produces antibodies to destroy the joints . it affects 1 - 2% of the population ; women are more affected than men and between 30 to 50 years of age are more likely to develop the disease ( 1).in the systemic disease , such as ra , deposition of immune complexes in other organs can cause extra articular manifestations . involvement of the inner ear and sensorineural hearing loss ( snhl ) has also been seen in about 24 to 60% of patients . vasculitis , neuropathy , and medications are the suggested causes of snhl in these patients ( 1 - 5 ) . demonstrated cochlear impairment in patients with ra and found an inverse correlation between the duration of ra and inner ear injury ( 6 ) . takatsu ( 2005 ) et al . reported that snhl is more common in ra patients than controls , they also observed a relation between snhl and erythrocyte sedimentation rate ( esr ) in patients with ra ( 4 ) . ferrara ( 1998 ) et al . reported that electronystagmography ( eng ) revealed central vestibular disorders in patients with ra ( 7).yilmaz ( 2007 ) et al . contrasted the result of eng test results and accounted the association of ra with vestibular system dysfunction as well as auditory impairment ( 5 ) . kakani ( 1990 ) et al . evaluated the results of the saccade and the bithermal caloric test on ra patients but did not find abnormalities in the test results ( 2 ) . ( 2002 ) investigated vestibulo - ocular reflex ( vor ) , optokinetic reflex ( okr ) and postural function in patients with ra and compared with controls , and concluded that ra patients do not present substantial deficits in visual - vestibular function ( 1 ) . the studies that assessing the vestibular system in ra patients are limited and the results are controvential . previous studies used caloric , saccade and optokinetic tests to assess the vestibular system but these tests evaluated just superior vestibular nerve and the function of inferior vestibular nerve remains unknown . cervical vestibular evoked myogenic potentials ( cvemp ) reflect saccular and inferior vestibular nerve ( ivn ) function ( 8) . vemp is a brief reduction in strenocleidomastoid ( scm ) tone following appropriate acoustic stimuli . high intensity abrupt sounds activate the saccule and a biphasic ( positive and negative ) response appears , the first wave of the contract , p1 or p13 and the second wave is shown as n1 or n23 ( 9 ) . although medications can not stop vestibular dysfunction in ra patients , but early diagnosis and early rehabilitation can reverse damage . the aim of this study was to compare vemp between ra patients and normal people . twenty five patients with ra ( 19 female and 6 male : mean age , 40.007.92 years ) and 20 healthy volunteers ( 15 female and 5 male : mean age , 35.3510.48 years ) enrolled in this study . the ra patients were recruited from the rheumatology outpatient s clinic , imam khomeini hospital , tehran , and control subjects were studied through the rehabilitation faculty of tehran university of medical sciences . patients with an additional systemic disease such as diabetes mellitus , prior ear surgery , all kinds of conductive hearing loss , and limitation of neck movements were not included in this study . after skin preparation , the active surface electrode was placed over the middle of sternocleidomastoid muscle ( scm ) , and the reference electrode was placed over the upper sternum . vemp recordings were performed with an evoked potentials machine ( ics charter ep , gn otometrics , us ) . subjects were instructed to sitting and turning their head to opposite side of the stimulated ear ( 11 ) . we used manometer ( feedback method ) to monitor electromyographic ( emg ) level in the two sides ( 12 ) . the tone burst ( 500 hz ) , with intensity of 95 db nhl , rarefaction polarity and repetition rate of 5.1/ sec was delivered monaurally via an er3a - inserted earphone . acquisition parameters include : the amplifier gain5000 , analysis time 100 ms and filter with a bandwidth of 10 - 1500 hz ( fig . 1 ) . we analyzed the amplitude of the first positive - negative peak , p13-n23 ipsilateral to the stimulated ear and the latencies of p13 and n23 ( 13 ) . for the assessment of the amplitude , the percentage of vemp asymmetry ( va ) was calculated as 100[|ar al|/ ( ar + al ) ] , where ar is the p13-n23 amplitude on the right and al is the p13-n23 amplitude on the left and |ar al| is the absolute value of ( ar al ) ( 14 ) . as the parameters of the latency , we measured the peak latencies of p13 and n23 . p13 is the first positive peak of vemp , and n23 is the first negative peak following p13 . latency is the time from the onset of the stimulus to the peak ( 13 ) . cvemp responses obtained from a patient diagnosed with ra and a normal subject the data were analyzed using spss version 16.kolmogrov-smirnov test was used to check the normality assumption of data and independent sample t - test was used to compare the means . this study was approved by the ethics committee of tehran university of medical sciences . significant difference was observed between the mean peak latency of p13 between groups ( p<0.001 in the right ear and p<0.001 in the left ear ) . also , significant difference was observed between the mean peak latency of n23 in the left ear ( p=0.03 ) , but in the right ear it was not significant ( p=0.08 ) . there was no significant difference between the mean absolute amplitudes of the two groups ( p=0.52 in the right ear and p=0.09 in the left ear ) . in addition , there was no significant difference between the mean of the interaural amplitude difference ratio between groups ( p=0.24 ) . the latency of p13 was significantly higher in ra patients than control group . also , n23 latency was higher in patients than the control group , but it was statistically significant just in left ear . the sd of n23 was greater than that of p13 , resulting in a wider normal range of n23 than p13 . therefore , p13 is a better parameter to evaluate the latency of vemp ( 15 ) . to our knowledge , this is the first report of evaluating the vestibular system in ra patients with vemp test , but in systemic lupus erythematosuswhich is an autoimmune systemic disorder , latency of vemp is significantly higher than controls ( 16 ) . in 2001 murfushi et al . investigated vemp in patients with multiple sclerosis ( ms ) and concluded demyelinating lesions can cause slow conduction along the vestibulospinal pathway and lead to eliminate vemp response or prolonged latency . vestibular nerve and brainstem can be involved in acoustic neuroma , and ms is a representative disease of the central nervous system but meniere disease is representative of labyrinthine lesions . it is unlikely that prolonged vemp latencies are a sign of inner ear lesions , because in meniere disease latency is not affected . vemp latencyprolongation is an abnormality characteristic of centralvestibulopathy . in midbrain and upper pontine lesions , however , in medullary and mid to lower lesions vemp abnormalities were present ( 15).there are an evidence of the involvement of peripheral and central nervous system in inflammatory connective tissue diseases such as rheumatoid arthritis , lupus erythematosus and behcet syndrome ( 17 ) . probable mechanisms for central nervous involvement are : vascular lesions ( vasculitis and thrombosis ) ; production of autoantibodies to neuronal antigens , ribosomes , and phospholipids ; and the inflammation induced by local cytokine production , and maybe an inflammatory demyelinating process of the spinal cord which occurs in this disease . demyelinating lesions , ( lipoid sclerosis ) can cause clinical characteristics and imaging findings that are very similar to ms ( 17 , 18).central vestibular involvement in ra has been reported in previous studies ( 5 , 7 ) . ( 1998 ) reported that eng revealed central vestibular disorders in ra patients ( 7 ) . ( 2007 ) compared the results of smooth pursuit , saccade , positional , and caloric tests between ra patients and control group and reported that both central and peripheral system can be involved in ra ( 5 ) . in the present study , ( 2001 ) reported that absent vemp could occur in ms , meniere and vestibular schwannoma ( 15).although vemp is present in ra patients , but there is abnormality in latency , thus , maybe the severity of the lesion was not enough to be able of eliminating responses . there was no statistically difference between groups in absolute amplitude and interaural amplitude difference ratio . ( 2001 ) reported that the patients with meniere disease showed decreased amplitudes or absent responses ( 15 ) . the range of amplitude is from 25 v to 250 v . due to the large variation range , therefore , to investigate the unilateral vestibular dysfunctions , comparing the interaural amplitude difference ratio is more reasonable . abnormal asymmetry in the amplitude of the vemp is the indicator of unilateral vestibular dysfunction ( 19 , 20 ) . in the present study for this parameter these findings support the possibility of the symmetrical function of the vestibular system in both sides in the patient group . autoimmune inner ear disease ( aied ) is a progressive bilateral snhl that is accompanied by vestibular symptom ( 21 ) . various connective tissue diseases are such as wegener 's granulomatosis , polyarthritis nodosa , ra , systemic lupus erythematosus ( sle ) and cogan 's syndrome with involvement of the inner ear , auditory and vestibular dysfunction ( 22).immune mechanisms proposed for these symptoms include attacks of humoral antibodies to inner ear antigens , ototoxicity , and cell antigen - induced immune complex disease of the inner ear in small vessels ( 23 ) . one of the main important limitations of our study , was using just one otolitic test . moreover , the large distance between sampling and performing test led to lose a lot of patients . the significant finding in the present study is prolongation of latency . prolonged latency is abnormal characteristic finding in central vestibulopathy and representive of vestibulospinal tact lesions . therefore , the authors suggested monitoring vestibular function in these patients . future studies with more samples are recommended . tehran university of medical sciences and health services grant 92 - 03 - 32 - 24252 have supported this research . farahani and all members of the department of audiology ( tehran university of medical sciences ) and imam khomeini hospital ( rheumatology center ) .

background : rheumatoid arthritis ( ra ) is an autoimmune systemic disease . most common autoimmune diseases are multisystem disorders that may also present with otological manifestations , and autoimmune inner ear disease accompanied by vestibular dysfunction . this study aimed to compare the vestibular function between ra patients and normal subjects using cervical vestibular evoked myogenic potentials ( cvemps ) . methods : in this cross- sectional study , 25patients with ra ( 19 female and 6 male : mean ( sd ) age , 40.00 ( 7.92 ) years ) and 20 healthy subjects ( 15 female and 5 male : mean ( sd ) age , 35.35 ( 10.48 ) years ) underwent cvemps , using 500 hz - tone bursts at 95 db nhl intensity level . data were analyzed using independent sample t - test through spss software v. 16 . results : the mean peak latency of p13 was significantly higher in ra patients ( p<0.001 ) . the mean peak latency of n23 was significantly higher in patients in the left ear ( p=0.03 ) . vestibular evoked myogenic potential ( vemp ) responses were present in all ( 100% ) of the participants . there were no significant differences in mean peak to peak amplitude and amplitude ratio between the two groups . conclusion : according to the prolonged latency of vemp responses in ra patients , lesions in the retrolabyrinthine , especially in the vestibulospinal tract are suspected .

liver metastases from colorectal carcinoma ( crc ) commonly form nodular lesions in the liver parenchyma . however , there have been reports of unusual cases of metastatic liver tumors from crc that predominantly extend along the intrahepatic bile duct . such unusual tumors generally mimic intrahepatic cholangiocarcinoma ( ihcc ) clinically , radiographically , and pathologically . importantly , it remains unclear whether anatomical or partial hepatectomy is more suitable to treat such tumors . herein , we report a case of liver metastasis from crc presenting as an intrabiliary growth , and discuss the clinicopathological considerations that inform selection of the surgical procedure for such tumors . the patient was a 62-year - old japanese man who had undergone low anterior resection for rectal carcinoma 9 years earlier . histologically , the tumor was a well - differentiated adenocarcinoma with lymphovascular invasion ( figure 1a , bfigure 1the primary rectal adenocarcinoma . macroscopically , an irregular , reddish , ulcerated mass measuring 6 5 cm in size was found on the mucosal surface of the rectum ( a , arrow ) . the tumor was diagnosed as a well - differentiated adenocarcinoma ( b , hematoxylin and eosin , 200 ) . ) . three years and 2 months after the surgery , a metastatic tumor was found in liver segment vi . the patient chose radiofrequency ablation therapy ( rfa ) over surgery . during a post - rfa follow - up period of 6 years and 1 month , his serum carcinoembryonic antigen ( cea ) and carbohydrate antigen 19 - 9 ( ca19 - 9 ) levels remained within the normal ranges . however , 1 week before admission , serum cea and ca19 - 9 levels were elevated to 6.8 ng / dl ( normal range , < 5.0 ng / dl ) and 40.3 u / ml ( normal range , < 37.0 u / ml ) , respectively . the primary rectal adenocarcinoma . macroscopically , an irregular , reddish , ulcerated mass measuring 6 5 cm in size was found on the mucosal surface of the rectum ( a , arrow ) . the tumor was diagnosed as a well - differentiated adenocarcinoma ( b , hematoxylin and eosin , 200 ) . on admission to our hospital , the patient exhibited no abnormalities on physical examination . abdominal contrast - enhanced computed tomography revealed distention of the superior branch of the bile duct in segment iii of the liver ( b3 ) ( figure 2afigure 2preoperative computed tomography findings . abdominal contrast - enhanced computed tomography reveals distention of the superior branch of the bile duct of segment iii of the liver ( b3 ; arrow in [ a ] ) and a nodule in the same segment ( arrow in [ b ] ) . a slightly enhancing lesion extends along the inferior branch of b3 ( arrowhead in [ b ] ) . ) as well as a nodule in the same segment ( figure 2b ) . a slightly enhancing lesion extended along the inferior branch of b3 ( figure 2b ) . abdominal magnetic resonance imaging revealed a tumor along the inferior branch of b3 , with a low - intensity signal on t1-weighted images ( figure 3afigure 3preoperative magnetic resonance imaging findings . abdominal magnetic resonance imaging shows a tumor ( indicated by the arrow ) along the bile duct in the left lateral segment , with a low - intensity signal on t1-weighted images ( a ) , an isointense signal with background liver parenchyma on t2-weighted images ( b ) , and a high - intensity signal on diffusion weighted images ( c ) . ) and an isointense signal with background liver parenchyma on t2-weighted images ( figure 3b ) . on diffusion - weighted images , the lesion in the inferior branch of b3 exhibited a high - intensity signal ( figure 3c ) . based on these findings , the differential diagnoses were metastatic liver tumor from rectal carcinoma and ihcc . hence , we performed a left hepatectomy with dissection of the lymph nodes in the hepatoduodenal ligament . abdominal contrast - enhanced computed tomography reveals distention of the superior branch of the bile duct of segment iii of the liver ( b3 ; arrow in [ a ] ) and a nodule in the same segment ( arrow in [ b ] ) . a slightly enhancing lesion extends along the inferior branch of b3 ( arrowhead in [ b ] ) . abdominal magnetic resonance imaging shows a tumor ( indicated by the arrow ) along the bile duct in the left lateral segment , with a low - intensity signal on t1-weighted images ( a ) , an isointense signal with background liver parenchyma on t2-weighted images ( b ) , and a high - intensity signal on diffusion weighted images ( c ) . macroscopically , a whitish nodule , measuring 1.5 1.0 cm , was found in the parenchyma of segment iii adjacent to the inferior surface of the liver ( figure 4figure 4macroscopic findings of the liver tumor . macroscopically , a whitish nodule measuring 1.5 1.0 cm is observed in segment iii adjacent to the inferior surface of the liver ( arrow ) . the figure shows that the tumor involves the inferior branch of the bile duct in segment iii and predominantly extends along it ( arrowheads ) . the superior branch of b3 and the bile duct in segment ii were preserved . macroscopically , a whitish nodule measuring 1.5 1.0 cm is observed in segment iii adjacent to the inferior surface of the liver ( arrow ) . the figure shows that the tumor involves the inferior branch of the bile duct in segment iii and predominantly extends along it ( arrowheads ) . histological examination revealed that an adenocarcinoma showed predominantly intraductal papillary growth replacing the bile duct epithelium ( figure 5a , b , cfigure 5histological findings of the liver tumor . the histological appearance of the area highlighted by the solid box in figure 4 is shown . on gross appearance ( a ) , the tumor presented with intrabiliary growth . histological examination shows an adenocarcinoma with intrabiliary growth replacing the bile duct epithelium ( b , c ) . the boxes with solid and dashed borders in ( a ) are highlighted in panels ( b ) and ( c ) , respectively . high - power magnification shows that the cells of the liver tumor contained pencil - like and hyperchromatic nuclei ( d ) . insets in ( b ) and ( c ) are digital enlargements of the highlighted areas in each respective figure . hematoxylin and eosin : ( a ) loupe view ; ( b ) 20 ; ( c ) 20 ; ( d ) 100 . ) . the tumor cells showed abrupt transition to the adjacent bile duct , which in turn showed no cellular atypia ( figure 5b , c ) . the tumor cells of the liver contained pencil - like hyperchromatic nuclei ( figure 5d ) . the tumor cells are negative for cytokeratin ( ck ) 7 expression ( arrow in [ b ] ) , while the neighboring biliary epithelial cells show positive ck7 staining ( arrowhead in [ b ] ) ( b ) . both the tumor cells ( arrow in [ c ] ) and biliary epithelial cells ( arrowhead in [ c ] ) are negative for ck20 ( c ) . the tumor cells ( arrow in [ d ] ) are negative for carbohydrate antigen 19 - 9 ( ca19 - 9 ) while the biliary epithelial cells ( arrowhead in [ d ] ) are positive for ca19 - 9 ( d ) . ( a ) hematoxylin and eosin ; ( b ) ck7 ; ( c ) ck20 ; ( d ) ca19 - 9 100 . ) were negative for cytokeratin ( ck ) 7 and ck20 on immunohistochemical analysis ( figure 6b , c ) , whereas the normal biliary epithelium was positive for ck7 and negative for ck20 ( figure 6b , c ) . ca19 - 9 was absent in the tumor cells , but present in the biliary epithelial cells ( figure 6d ) . because both the original and metastatic tumors demonstrated strikingly similar histological appearances , the tumor was diagnosed as a liver metastasis from rectal carcinoma . the histological appearance of the area highlighted by the solid box in figure 4 is shown . on gross appearance ( a ) , the tumor presented with intrabiliary growth . histological examination shows an adenocarcinoma with intrabiliary growth replacing the bile duct epithelium ( b , c ) . the boxes with solid and dashed borders in ( a ) are highlighted in panels ( b ) and ( c ) , respectively . high - power magnification shows that the cells of the liver tumor contained pencil - like and hyperchromatic nuclei ( d ) . insets in ( b ) and ( c ) are digital enlargements of the highlighted areas in each respective figure . hematoxylin and eosin : ( a ) loupe view ; ( b ) 20 ; ( c ) 20 ; ( d ) 100 . the tumor cells are negative for cytokeratin ( ck ) 7 expression ( arrow in [ b ] ) , while the neighboring biliary epithelial cells show positive ck7 staining ( arrowhead in [ b ] ) ( b ) . both the tumor cells ( arrow in [ c ] ) and biliary epithelial cells ( arrowhead in [ c ] ) are negative for ck20 ( c ) . the tumor cells ( arrow in [ d ] ) are negative for carbohydrate antigen 19 - 9 ( ca19 - 9 ) while the biliary epithelial cells ( arrowhead in [ d ] ) are positive for ca19 - 9 ( d ) . ( a ) hematoxylin and eosin ; ( b ) ck7 ; ( c ) ck20 ; ( d ) ca19 - 9 100 . he has not exhibited any signs of recurrence in the liver or any other organ , and is functioning well 3 years after the most recent surgery . in the present case , a metastatic tumor from crc involved the inferior branch of b3 and extended predominantly along this branch , with an intraductal papillary growth pattern . comparison between computed tomography images obtained 6 months before admission and those obtained on admission showed the growth of the tumor ( figure 7a , bfigure 7comparison between the computed tomography images obtained 6 months before ( a ) and on admission ( b ) . six months before admission ( a ) , a slightly low attenuated nodule measuring 1.5 1.0 cm in diameter was found close to the inferior surface of the liver ( arrow in [ a ] , far right panel ) . on admission ( b ) , the nodule ( asterisk in [ b ] , far right panel ) extended along the inferior branch of the bile duct in segment iii ( b3 ) ( arrowheads in [ b ] , center and right panels ) . owing to a tumor embolism in the bifurcation of b3 , the superior branch was enlarged on admission ( box in [ b ] , left panel ) . neither enlargement of the superior branch of b3 nor tumor extension along the inferior branch of b3 was observed 6 months before admission ( [ a ] , left , center , and right ) . ) . on images obtained 6 months before admission , a nodule with a slightly low attenuation , measuring 1.5 1.0 cm in diameter , was retrospectively discovered close to the inferior surface of the liver ( figure 7a , right ) . neither enlargement of the superior branch of b3 nor tumor extension along the inferior branch of b3 was observed 6 months prior to admission ( figure 7a ; left , center , and right ) . at the time of admission , this nodule had already involved the inferior branch of b3 ( figure 7b ) . owing to a tumor embolism in the bifurcation of b3 , the superior branch of b3 was enlarged on admission ( figure 7b ) . the increased tumor volume and bile duct obstruction may explain the slight elevation of the serum cea and ca19 - 9 levels , respectively . comparison between the computed tomography images obtained 6 months before ( a ) and on admission ( b ) . six months before admission ( a ) , a slightly low attenuated nodule measuring 1.5 1.0 cm in diameter was found close to the inferior surface of the liver ( arrow in [ a ] , far right panel ) . on admission ( b ) , the nodule ( asterisk in [ b ] , far right panel ) extended along the inferior branch of the bile duct in segment iii ( b3 ) ( arrowheads in [ b ] , center and right panels ) . owing to a tumor embolism in the bifurcation of b3 , the superior branch was enlarged on admission ( box in [ b ] , left panel ) . neither enlargement of the superior branch of b3 nor tumor extension along the inferior branch of b3 was observed 6 months before admission ( [ a ] , left , center , and right ) . liver metastases from crc occasionally involve the bile duct and show intraductal papillary growth . according to a study by okano et al . similarly , kubo et al . reported that 10.6% ( 23/217 ) of liver metastases from crc presented with macroscopic intrabiliary growth . a report from jannelyn et al . on liver metastasis from crc concluded that 3.6% ( 41/1144 ) and 10.6% ( 18/170 ) of retrospectively and prospectively analyzed cases , respectively , presented with intrabiliary growth . liver metastasis from crc with intrabiliary growth is associated with a better prognosis than other forms of liver metastases . reported that patients with macroscopic bile duct invasion had a better 5-year survival rate ( 80% ) than those with microscopic bile duct invasion ( 48% ) or no bile duct invasion ( 57% ) . moreover , kubo et al . reported a significant difference between patients with and without macroscopic intrabiliary extension in terms of the interval between initial colectomy and hepatectomy ( 37.4 25.4 vs. 6.1 7.2 months , respectively ) . these different outcomes may be explained by the fact that most tumors with intrabiliary growth are well - differentiated adenocarcinomas . this feature is characteristic of less aggressive tumors and results in tumor colonization of the bile duct ; the normal biliary epithelium is replaced with tumor cells , which grow along an intact basement membrane without penetrating it . the clinical course of the patient presented here was consistent with that of a less aggressive tumor ; he had a well - differentiated adenocarcinoma with intrabiliary extension and has survived for 3 years after the hepatic surgery , for 9 years after rfa , and for 12 years after the primary surgery of the rectum . preoperative differentiation between liver metastasis from crc with intrabiliary growth and ihcc is difficult . there are no clinical symptoms that are highly characteristic of liver metastasis from crc with intrabiliary growth . while elevated cea and serum alkaline phosphatase levels are common findings on laboratory tests , , these markers can also be elevated in ihcc patients , . the computed tomography findings of liver metastasis from crc with intrabiliary growth are usually nonspecific , although a thickened portal tract , intrahepatic bile duct dilatation , and a wedge - shaped area with contrast enhancement are characteristic features of this type of tumor . on the other hand importantly , this case provides evidence for the superiority of anatomical hepatectomy over partial hepatectomy for metastatic liver tumors with intrabiliary growth arising from rectal adenocarcinomas . when liver metastases from crc form nodular lesions in the liver parenchyma , partial hepatectomy may be a sufficient treatment ; however , when the tumor shows intrabiliary spreading , as seen in the present case , partial hepatectomy alone may not completely excise the tumor because of the extension along the bile ducts . although there is no consensus regarding the most appropriate procedure for such patients , anatomical hepatectomy tends to be preferred over regional liver resection , . this is primarily to guard against the possibility of residual tumor cells on the cut margin of the bile duct , . moreover , anatomical hepatectomy is the logical choice because of the aforementioned difficulty in preoperatively differentiating between a liver metastasis with intrabiliary growth arising from crc and ihcc . in the present case , we performed a left hepatectomy with sufficient margins in the bile duct that resulted in good postoperative outcome ; the patient has survived for a relatively long period . pathological discrimination between liver metastasis from crc and ihcc should be performed carefully , based on both conventional histological examination using hematoxylin and eosin staining and immunohistochemical staining with antibodies against ck7 and ck20 . this procedure is particularly important to determine the need for , and the selection of , chemotherapy . metastatic liver tumors from crc with macroscopic intrabiliary growth show a histologically abrupt transition from the adjacent bile duct epithelium to tumor tissue : cellular atypia is absent in the normal bile duct epithelial cells . on immunohistochemical analysis , a typical ck7/ck20 + expression pattern 9% ( 18/206 ) of metastatic liver tumors from crc were negative for both ck7 and ck20 expression . similarly , sasaki et al . reported that 16% ( 4/25 ) of liver metastases from crc were ck7/ck20 , whereas rullier et al . reported that no ck7/ck20 cholangiocarcinomas were found among their cases ( 0/29 ) . in the present case , the liver tumor showed a histological appearance similar to that of the primary tumor , as well as an abrupt change from the normal bile duct epithelium to the adenocarcinoma . however , intrahepatic recurrence of the segment vi metastatic lesion treated with rfa 6 years before admission can not be completely ruled out . intrahepatic recurrences after treatment of metastatic liver tumors from crc show diverse patterns , including local recurrence , multiple hepatic nodules , and intrahepatic distant metastases . therefore , it is possible that ours is a rare case of intrahepatic recurrence . in conclusion , we report an unusual case of liver metastasis from rectal adenocarcinoma that presented with intrabiliary growth ( figure 8figure 8schematic representation of the tumor extension in the present case . the metastatic nodule formed in segment iii of the peripheral liver in , and involved the inferior branch of the bile duct in segment iii , resulting in distention of the superior branch of the bile duct in segment iii . abbreviations : chd , common hepatic duct ; rhd , right hepatic duct ; b2 , bile duct in segment ii ; b3 , bile duct in segment iii ; b3a , superior branch of b3 ; b3b , inferior branch of b3 . ) . during follow - up , physicians should consider the possibility of liver metastasis with intrabiliary growth for patients with a history of crc . moreover , our patient s long survival time suggests that anatomical hepatectomy is a more effective treatment than partial hepatectomy for metastatic liver tumors from crc with intrabiliary growth . the metastatic nodule formed in segment iii of the peripheral liver in , and involved the inferior branch of the bile duct in segment iii , resulting in distention of the superior branch of the bile duct in segment iii . abbreviations : chd , common hepatic duct ; rhd , right hepatic duct ; b2 , bile duct in segment ii ; b3 , bile duct in segment iii ; b3a , superior branch of b3 ; b3b , inferior branch of b3 .

liver metastases from colorectal carcinoma commonly form nodular lesions in the liver parenchyma . we report a case of liver metastasis from rectal adenocarcinoma that extended predominantly into the bile duct . a 62-year - old japanese man underwent low anterior resection for rectal adenocarcinoma 9 years ago . approximately 3 years later , he underwent radiofrequency ablation therapy for a metastatic liver tumor . nine years after surgery , a tumor in liver segment iii exhibiting intrabiliary extension was discovered ; it was unclear if this was a metastatic liver tumor or intrahepatic cholangiocarcinoma . accordingly , we performed a left hepatectomy with lymph node dissection . the tumor was negative for cytokeratins 7 and 20 , and was histologically similar to the primary rectal adenocarcinoma ; it was diagnosed as rectal carcinoma metastasis . the patient has survived for 3 years after the hepatic surgery , for 9 years after radiofrequency ablation therapy , and for 12 years after the primary surgery . this case shows that liver metastasis from colorectal carcinoma can present as a predominantly intrabiliary growth that mimics intrahepatic cholangiocarcinoma on imaging . moreover , our case provides evidence for the superiority of anatomical hepatectomy over partial hepatectomy for metastatic liver tumors with intrabiliary growth arising from rectal adenocarcinomas .

service user involvement ( sui ) is emphasized in many strategies , plans , and declarations . it is a recognized value in social work and in health care , yet in practice , sui is not always achieved [ 13 ] . there is a dissonance between the philosophies of sui and the existence of these philosophies in the reality of mental health nursing practice . sui is a difficult and complex concept to define , and it is often used as a synonym for participation . on the other hand , a distinction can be made between these two concepts . service user involvement entails preconditions of the service user 's impact on services in some way while user participation can mean users merely taking part in an activity or acting as an informant . consumerism or the ethos of markets sees service users as customers , consumers or stakeholders whose views need to be taken into account . approach where service user involvement serves the interests of the organizations , service systems , and markets . in contrast , the democratic , or empowerment model is concerned with service users having a voice in services , civil rights , and equal opportunities . the empowerment model is more about the bottom - up interests of service users themselves , and involvement is seen and valued as an end in itself . state that user involvement has the ability to improve the quality of care , but equally important is the potential effect it has on the service users ' personal and collective identity , sense of self - worth , and civil rights . in finland , sui is exercised mainly through local democratic mechanisms , elections , and patient organizations . there are also acts covering the rights and status of patients , social service clients , and complaints systems . the national plan for mental health and substance abuse work was introduced in 2009 , and it outlines common national objectives for mental health and substance abuse work . the plan emphasizes that the client 's status must be reinforced ; user experts and peers should be included in the planning , implementation , and evaluation of mental health and substance abuse work . as goodwin and happell point out , published research about service user involvement and participation is limited , and there is a lack of research reflecting the views and opinions of service users themselves . the purpose of this study was to describe service user involvement in mental health and substance abuse work from the viewpoint of clients . the following study question was addressed : what are service users ' conceptions of sui in mental health and substance abuse work ? this approach was first developed in the field of education to describe qualitatively different ways in which people conceive learning . phenomenography is a research approach which aims to study and describe different conceptions people have of various phenomena in the world around them [ 17 , 18 ] . in the phenomenographic approach , conception has two intertwined aspects : the referential aspect , which denotes the meaning of the object conceptualized , and the structural aspect , which shows the specific combination of features that have been discerned and focused on . the phenomenographic approach makes a distinction between how something is , and how it is perceived to be . the first - order perspective is directed towards the phenomenon as such and it is interested in facts , whereas the second - order perspective is about a person 's conceptions of that phenomenon . the second - order perspective is studied in phenomenography [ 18 , 22 , 23 ] . when applying a phenomenographic approach , when study questions are formulated , data is gathered and the analysis is done . at every stage , the researcher has to reflect and be conscience of her or his own conceptions of the phenomenon of interest . it means taking the place of the respondent , trying to see the phenomenon and the situation through her eyes , and living her experience vicariously ( page 121 ) . however , it is impossible to approach and analyse empirical data totally without preconceived ideas . an empirical study is guided by a specific research interest , and a researcher must be acquainted with previous theory in order to be able to pose relevant questions and to interpret and analyse the data . the phenomenographic analysis is not very structured , and it is always based on empirical data . the first stage involves reading the data repeatedly in order to form an overall picture of participants ' conceptions . meaningful and adequate expressions are searched for from the data , and meaning units are formed . in the second stage of the analysis , the third stage concerns grouping the similar meaning units into categories . in the fourth stage , categories or subcategories are combined into categories of description . the participants received information both in written and verbal forms and every participant signed a letter of informed consent . the cds , memory - cards and written interviews were stored in a locked cabinet and could be accessed only by the first author ( m.l ) . the electronic versions of the interviews were saved on a computer safeguarded by a password . the participants were persons who had used mental health and/or substance abuse services or were using these services during the time of the study . a total of 27 service users participated in the study . when selecting the participants , the purpose was to maximize the variation in description of the phenomenon ( sui ) . according to patton , the strength of this kind of purposeful sampling lies in selecting information - rich cases . maximum variation , snowball , and criterion sampling were applied . in the study , maximal variation was applied when including service users with experience of mental health and/or substance abuse services , inpatient and/or outpatient services , service users of both sexes and of different ages , from urban and rural areas . the participants were not obliged to talk about their background or medical history but the majority of them did talk to the interviewer about their background ( incl . five participants were under the age of 30 years , nine were between 31 and 50 years and four participants were older than 50 years . as described by the participants themselves , twelve of them had only used mental health services , four only substance abuse services , and eleven had used both mental health and substance abuse services . snowball sampling was used to get in contact with persons , who were no longer actively using mental health or substance abuse services . after one member of a group or organization had participated in the study , he or she could recommend another participant . the selection criteria were age ( 1865 years ) and the patient 's ability to give informed consent evaluated by nursing staff . when applying a phenomenographic approach , the study material is usually collected by means of interviews . in this study , a brochure of the study was given to the clients , and the persons interested in participating then contacted the interviewer . to get in contact with the users of services in in - patient care , the personnel of these units was consulted first . the personnel then evaluated the patients ' ability to give informed consent and to participate in the study . those service users willing and able to participate received a brochure about the study and contacted the interviewer , with the help of the staff , to arrange the interview . before these interviews , participants were told that participation was voluntary and participating had no effect on their care and treatment , that anonymity was guaranteed , and that they were able to withdraw from the study at any point . the interviews with the service users in in - patient care took place at the ward premises . otherwise the participants ' interviews took place at their homes or in other place reserved for that purpose . the interview themes were selected beforehand and special attention was paid to the entry question , while the subsequent dialogue proceeded according to the participant 's answers . the themes and the questions of each interview dealt with the participant 's conceptions of sui in mental health and substance abuse work . the first question in the interviews was in your opinion , what does service user involvement in mental health and/or substance abuse services mean ? during the interviews , some participants found it difficult to answer the entry question , so additional questions such as have you had an opportunity to participate in your own care ? or in what ways have you participated in your care ? were asked to clarify the concept of user involvement . the interviews were audio - taped excluding four cases ; in one case the recorder did not work , and in three cases the participant did not give consent for recording . in these cases , the purpose of data analysis is to find and define meanings expressed in the interviews , the meanings are then grouped into categories describing the data and the conceptions participants hold of the phenomenon of interest [ 18 , 20 ] . in this study , the analysis of the participants ' conceptions of sui in mental health and substance abuse work was carried out in three phases by the first author ( table 1 ) . the interviews were transcribed verbatim and the material was compared with the tapes . every interview was then listened to and read several times in order to get an overall impression and familiarity with the material . meaningful and adequate expressions related to the study questions were searched for and identified from the material . the meaning units were compared with each other with a focus on similarities and differences . the meaning units were also compared repeatedly with the original material in order to ensure the accuracy of the interpretations made . it is important that the researcher recognizes his or her own preconceptions and experiences and is able to bracket them . in the third phase , if a category of description would consist of subcategories , they would be vertically related to it and specify it [ 19 , 20 ] . as a result of the data analysis , four qualitatively different categories describing the participants ' conceptions of sui in mental health and substance abuse work emerged : service users have the best expertise , opinions are not heard , systems make the rules , and courage and readiness to participate ( figure 2 ) . the participants talked about service user involvement in personal care and treatment and in the development and delivery of mental health and substance abuse services . excerpts from the original interviews are included in the description of the categories to convince the reader of the validity of the categories . the letters before the quotations have the following meanings : s : service user , i : interviewer . according to the participants , involving service users in the planning and development of mental health and substance abuse work was necessary in order to achieve change . if plans were made without experts through experience , all you would achieve would be paper . to know what to develop and in which direction is difficult without personal knowledge of mental health or substance - related problems . in the interviewees ' opinion , persons with personal knowledge and experience had the best expertise concerning the content of mental health and substance abuse services . s : well , you can collect fees from the meetings and spend ten million euros , and all you get is zero . all you get is a pile of paper collecting dust in the archives , that 's all . sure , it 's important that professionals think about these things , but those who have to deal with them should be involved . this is a kind of disease you can only comprehend if you have experience of your own . s : well , it ( involving users ) could not do any harm , could not it ? they know better than the staff . how would i put it ? if you have been a patient yourself , you know better what 's been done to you and what has not . even though service users have useful and valuable expertise , the participants had the conception that sui did not always happen in practice . the opinions and experiences of service users were ignored . listening and valuing service users ' expertise required time and giving up paternalistic thoughts about service users . according to the participants , service users in mental health and substance abuse services still encountered negative attitudes and bias . sui entailed a division of power ; those in power were not willing to share it . it 's easier to make decisions when you do not know the problem , or the heart or the core of the problem . maybe it would be more difficult to make decisions if those concerned had their voice heard . i : you said that service users ' opinions are not often asked for . in your opinion , why is it so ? s : these kinds of ( involving ) activities are just evolving , but they are becoming more and more common , and more and more often people are asking users ' opinions . even persons recovering from mental health problems have many kinds of experiences and things to say . and in the end , they ( services ) should work on the patients ' conditions as much as possible . the kind of top down dictating is over . the mental health and substance abuse service system and organizational culture limited the achievement of sui . many laws and acts regulated mental health and substance abuse work ; the organizations were often hierarchical and inflexible . certain regulations existed in outpatient and inpatient care , and a service user had to adapt to those rules . according to the participants ' concepts , especially in inpatient care , there were many rules to obey . the meaning or the purpose of those regulations was not always clear to service users . i : have you been able to participate in your own care , for example ? but the time you stay in the hospital and things like that , they are decided mostly by the doctor . i have noticed that a doctor might have a certain style ; for example , in one ward the time spend in the hospital was three weeks no matter what your diagnosis or condition was . i : have you been able to participate in your own care , for example ? but the time you stay in the hospital and things like that , they are decided mostly by the doctor . i have noticed that a doctor might have a certain style ; for example , in one ward the time spend in the hospital was three weeks no matter what your diagnosis or condition was . s : to be able to influence something , that 's something that does not fit here in hospital . is not influencing something the politicians do ? yes , here you have certain rules and they wo n't be changed because of one patient , and these things you just have to accept . and nothing to it . the courage of users of services and their readiness for involvement and participation varied . mental health and substance abuse problems still leaded to stigma , bias , and prejudices . because of that , some service users did not want to take part , for example , in peer groups . a service user 's own mental and physical condition , medication and recovery affected their ability and readiness to participate . in finland , people are reserved , and active involvement and participation or public expressions of opinions are not common . in their opinion , it was better to turn to professionals and trust them . but if you are on medication , if you have many drugs , you do not have the strength to participate because you have enough problems of your own . these persons should be noticed . in my opinion , they are willing to participate and work as peers . yes , i 'd say 50 percent of all service users would be willing to participate . they see it as part of their recovery . but if you are on medication , if you have many drugs , you do not have the strength to participate because you have enough problems of your own . these persons should be noticed . in my opinion , they are willing to participate and work as peers . yes , i 'd say 50 percent of all service users would be willing to participate . methodologically , it can be argued that the phenomenographic approach is well suited to research concerned with different conceptions people hold of diverse phenomena . when applying an inductive approach , it was possible to form the categories of description from the rich and extensive data . this study confirmed that service users ' opinions are worth listening to , and that they have experiential knowledge to be used in the development of mental health and substance abuse work . even severely ill persons and patients in inpatient care can participate in studies if data collection methods are appropriate . the phenomenographic approach does not attempt to achieve an absolute truth , such a truth does not exist . the credibility of the study is discussed in the light of concepts introduced by fridlund and fridlund and hildingh . there are four concepts that are of general importance for scrutinizing a study : applicability , concordance , security , and accuracy . when applying a qualitative method other concepts such as identification , reasonableness , trustworthiness , and conscientiousness should be used . to ensure identification ; the data was acquired by means of interviews and the participants were selected by purposeful sampling . the aim was maximal variation , that is , to get as many qualitatively different conceptions of sui as possible . trustworthiness is connected with the reliability of the study . the accurate and detailed description of the data analysis and the direct quotations illuminating the categories of description add to the trustworthiness of this study . trustworthiness is also strengthened by the fact that the first author conducted all the interviews . conscientiousness means that the researcher is aware of his or her own preconceptions and experiences throughout the research process . in this study , the data was read and reflected on repeatedly , and the meaning units and categories were continuously compared to the original data in order to ensure the accuracy of the interpretations made . this study provided new information about sui in mental health and substance abuse work from the service user 's viewpoint . methodologically , the phenomenographic approach enabled the service users ' involvement and participation in this study . with an inductive approach mental - health and substance - abuse - related problems are a significant challenge to both public health and finances . in the eu , for example , about 11 percent of the population ( almost 50 million citizens ) are estimated to encounter mental health problems , depression being the leading health problem in many eu countries . in europe , promoting mental health , reducing stigma , discrimination , and social exclusion , and preventing mental health problems are priorities for the next ten years . on a practical level , involving service users in planning , development and service delivery can support the addressing of these challenges within mental health and substance abuse work . the results of this study can be utilized to understand the essence of sui , to analyze the barriers to its achievement , and to work towards its implementation . thus , these results can contribute to improving the education , practice , and management of mental health and substance abuse work . according to the results , service users have deep , experiential knowledge that should be used in individual care planning , as well as in the development , evaluation , and organizing of mental health and substance abuse services . the need to utilize this expertise has also been highlighted in previous studies [ 3335 ] . sui is particularly important in the planning , implementation , and management of services [ 34 , 36 ] . the results of this study confirmed that the achievement of sui in mental health and substance abuse work is still insufficient . this gap between the rhetoric of involvement and the reality in mental health and substance abuse practice has also been discovered in other studies [ 3 , 4 , 34 , 37 ] . the state of mental and physical well - being , medication , and personal recovery all affect the service user 's capability to be involved , as also argued in previous studies [ 5 , 38 , 39 ] . some service users are not willing or motivated to participate ; they would rather trust in professionals and their decisions . found in their study that not all users of drug treatment services felt the need to be involved , but rather wanted to concentrate on their own care . also , a lack of interest and general apathy and passivity may affect the readiness for involvement and participation . hence a challenge for sui is how to involve users who are passive and not interested in involvement and participation . in order to encourage diverse service users to participate , different , flexible , and innovative forms of involvement are needed [ 13 , 42 ] . according to the results an organization 's commitment to sui , and continuing support [ 43 , 44 ] can promote the staff 's commitment to involvement . the users of mental health and substance abuse services still encounter negative attitudes and prejudices . in previous studies , the importance of attitudes has also been highlighted [ 9 , 36 , 40 , 45 ] . special attention should be paid to the paternalist or negative attitudes of the staff [ 4 , 38 ] . the participants described conceptions of how the stigma related to mental health and substance abuse problems hindered involvement and participation . the results of this study support the views presented in earlier studies [ 4750 ] indicating that stigma and self - stigma are related to mental health and substance abuse problems . this study revealed that service users did not always know the meaning or purpose of various rules and decisions . services users need adequate and comprehensible information in order to be able to participate and to involve themselves . sufficient information promotes service users ' possibilities to participate in their individual care [ 5 , 38 , 51 , 52 ] . information sharing alone is not enough ; information should be available repeatedly and in a form that service users can understand [ 40 , 54 ] . tritter and mccallum point out that service users may emphasize different questions and issues than professionals . sui requires power - sharing and a new kind of expertise on the part of professionals . in this study , the participants reported that service users ' opinions are not always heard or taken into account . poulton stresses professionals ' need to get away from professional protectionism and medical paternalism to be able to share information and power with service users . as long as sui remains in control of services providers service users conceptualize sui in mental health and substance abuse services from four different perspectives . service users have deep , experiential knowledge that should be used in individual care planning , as well as in the development , evaluation and organizing of mental health and substance abuse services . there are several obstacles to sui , and different forms of involvement are needed for different kinds of service users to be involved . special attention should be paid to the provision of adequate information and genuine opportunities to participate . sui challenges professionals to share power and to develop a new kind of expertise . within mental health and substance abuse services , hence they have excellent opportunities to promote involvement and to encourage service users to get involved and participate .

service user involvement ( sui ) is a principal and a guideline in social and health care and also in mental health and substance abuse work . in practice , however , there are indicators of sui remaining rhetoric rather than reality . the purpose of this study was to analyse and describe service users ' conceptions of sui in mental health and substance abuse work . the following study question was addressed : what are service users ' conceptions of service user involvement in mental health and substance abuse work ? in total , 27 users of services participated in the study , and the data was gathered by means of interviews . a phenomenographic approach was applied in order to explore the qualitative variations in participants ' conceptions of sui . as a result of the data analysis , four main categories of description representing service users ' conceptions of service user involvement were formed : service users have the best expertise , opinions are not heard , systems make the rules , and courage and readiness to participate . in mental health and substance abuse work , sui is still insufficiently achieved and there are obstacles to be taken into consideration . nurses are in a key position to promote and encourage service user involvement .

the last two decades , the optical properties of single wall carbon nanotubes ( swnts ) have gained a great deal of interest [ 1 - 3 ] . the one - dimensional ( 1d ) nature of nanotubes offers unique properties to their excitonic spectrum with many revolutionary applications . many experimental techniques such as raman scattering and electrical conductivity have been employed for the investigation of optical and electronic properties of nanotubes with variable diameters and angle chilarities . relaxation dynamics and nonlinear properties in these nanostructures are key issues in understanding and developing their optoelectronic properties . ultrafast studies of carrier dynamics have been performed in swnts reporting that this system has a dynamic response ( < 1 ps ) one order of magnitude slower than in graphite ( ~130 fs ) . when mixing the swnts with conjugated polymers , the donor / acceptor interfaces of polymer / nanotube act as dissociation heterojunctions for photoexcited excitons . these bulk heterojunction structures are presently believed to be the best approach for organic photovoltaics and the advantage of this photoinduced charge generation is evident with the enhancement of photocurrent in organic solar cells . particularly , -conjugated poly(3-hexylthiophene ) ( p3ht ) has been of interest because of high carrier mobility , mechanical strength , thermal stability , and compatibility with fabrication process . however , the literature is lacking a comprehensive study of exciton and dissociated carrier ( polarons and electrons ) dynamics in these very promising composites for photovoltaic and optoelectronic applications . in this letter , transient absorption measurements with femtosecond resolution ( ~150 fs ) provides a means to investigate the ultrafast electron transfer from conjugated polymer to nanotubes and the involved radiative or nonradiative relaxations . we resolve the relaxation of excitons and dissociated carriers in swnt / p3ht composites as a function of nanotube concentration . we have found that carrier relaxation within the valence and conduction bands of p3ht is beyond our resolution time ( ~150 fs ) whereas the exciton dynamics have a double exponential relaxation . furthermore , the electron phonon interactions at the vibronic sidebands quench the radiative emission by introducing nonradiative relaxation channels . in addition , based on the observed ultrafast relaxation we present a comparison of swnts and -phenylc61-butyric acid methyl ester ( pcbm ) as mixture materials in the p3ht polymer matrix for their photovoltaic performance . the utilized experimental technique in this work is a noncollinear super - continuum pump probe configuration in conjunction with a regenerative ti : sapphire amplifier system with 100 fs pulses at 800 nm . this system amplifies the pulses to approximately 1 mj at a repetition rate of 1 khz . the temporal resolution of our experimental setup over the entire probing wavelength range has been measured to be better than 150 fs . the temporal variation in the optical absorption was monitored as a change in the reflectivity and transmission , which was a direct measure of the photo - excited carrier dynamics within the probing region . in this work , optical pumping at a fluence of 2 mj / cm was used to excite the composites and determine their temporal behavior . here , we should point out that around this fluence nonlinear effects such as exciton exciton annihilation were not observed in our experimental studies . for the preparation of the samples in this work , p3ht ( 5 mg ) was dissolved in 10 ml of dichlorobenzene inside a quartz pot which was kept over a hot plate at medium temperature . the initial volume of dichlorobenzene was noted and solvent was added if needed to replenish the evaporated amount . one milligram of hipco swnts ( obtained from cni ) was separately dispersed in 40 ml of dichlorobenzene . hrem ( using a jeol 4000exii ) observations showed that the nanotube material was free from catalytic remnants and formed bundles containing of up to seven nanotubes each . according to current literature , the hipco swnts are 1/3 metallic and 2/3 semiconducting . we have not performed any additional purification . appropriate amounts of p3ht and swnts were mixed from solution and the composites were ultrasonically agitated so long as to reach a uniform solution . the total mass of the deposited materials and the surface of the quartz substrates were kept the same to insure that the resulting films had similar thicknesses . the dispersion of swnts in the composites was examined by hrem using the jeol 2000ex ii microscope and we did not notice any difference compared to the pure swnts . in addition , iv measurements revealed a percolation threshold of 0.75 wt.% which denotes good dispersion of the swnts in line with current bibliography . 1 ) that upon increasing the nanotube concentration in our composites , the nanotubes form ropes and bundles with measured nanotube diameters about 1.4 0.1 nm . the effect of the swnts bundles formation on the optical excitonic transitions for pure swnts material has been experimentally studied . the interactions between swnts in close proximity with one another , and the corresponding changes in their electronic structure , have received much attention [ 20 - 23 ] . in addition to the inherent interest in understanding interacting 1d systems , intertube interactions are of substantial technological importance because swnts naturally form bundles in typical syntheses and bundling has the effect of both shifting and broadening the electronic transition energies . high resolution electron microscopy images of ( a ) pure swnts and ( b ) swnts dispersed in p3ht polymer ( swnt concentration 50% ) 2 , we present the optical absorption spectrum as a function of wavelength for the pure p3ht polymer and swnt / p3ht composites . it is obvious that the p3ht polymer absorbs in the visible spectra region depicting a singlet exciton transition ( 600 nm ) and two vibronic sidebands ( 520 and 560 nm , respectively ) . upon increasing the concentration of nanotubes , the absorbance of the composites decreases ( see the inset of fig . optical absorption measurements at room temperature of swnt / p3ht composites as a function of wavelength . the inset shows the absorption peaks as a function of nanotube concentration at the singlet exciton transition and the vibronic sidebands the decrease in the p3ht absorption is most likely due to the fact that there is less p3ht in the sample ( 65% swnt means that only 35% of the sample is p3ht , and the total sample mass is kept constant ) . due to various interactions that exist between the two materials ( donor acceptor system ) 3 , we present the experimental data of transient absorption for the pure p3ht polymer when it is excited by ultrashort laser pulses ( 150 fs ) at 400 nm . from these data , it is obvious that when we probe at resonant with the singlet exciton transition ( 600 nm ) we observe a pulse - width limited drop of absorption which is attributed to state filling by the coulomb - correlated electron this pulse width limited fast drop suggests that the exciton relaxation within the valence and conduction bands of polymer is beyond our time resolution . the first one is fast with a time constant < 1 ps and represents the fast relaxation of excitons with energies close to the separation between the gaussian - like higher occupied molecular orbital ( homo ) and lower unoccupied molecular orbital ( lumo ) states . the second decay can be described with a stretched exponential and most likely corresponds to the radiative emission of the p3ht polymer . the latter is more pronounced when we probe very close to resonant with the first or second vibronic sidebands ( see the curves of 550 and 500 nm in fig . the fast relaxation remains approximately the same , but the second stretched decay becomes faster due to the enhanced coupling of electronic with vibrational states ( electron phonon interactions ) . this coupling quenches the radiative recombination opening nonradiative relaxation paths by transferring the energy to the lattice via phonons emission . normalized transient absorption measurements for the pure p3ht polymer at probing wavelengths 500 , 550 , 600 , and 700 nm . the arrow indicates the time where the signal becomes positive for probing wavelength of 500 nm . the solid black line represents the mirror image of transient absorption signal with probing wavelength of 700 nm for comparison purposes in addition to monitoring state filling and the subsequent exciton relaxation , the probing beam may cause secondary re - excitations to energetically higher energy states . secondary absorption probably is present at all probing wavelengths , but it is more pronounced at 550 nm , a wavelength close to the strong second vibronic sideband absorption ( see fig 2 ) . the photoinduced absorption ( pa ) signal for delay times longer than 50 ps ( see 500 nm probing wavelength in fig . increasing the probing wavelength to 700 nm the energy of the probing photons is less than the homo - lumo energy gap . since the density of states follows a gaussian - like distribution there are states in the energy gap which result in weak absorption ( see fig . 2 , arrow ( 4 ) ) . following excitation with the 400 nm laser pulse , we observe an increase in absorption at 700 nm ( 1.77 ev ) . this means that we are re - exciting carriers from an energy level that is occupied after the absorption by the excitation photons . however , if we create a mirror image of the transient absorption at 700 nm with respect to the time delay axis , the resulting curve ( shown in fig . 3 as a continuum black line throughout the data ) depict similar dynamics as the 600 nm transient absorption data . this suggests that the 700 nm probe re - excites carriers between the lumo and a state 1.77 ( 700 nm ) above it ( for electrons ) . nanoengineered composites of semiconducting polymers offer opportunities to realize desirable different optical and electronic properties based on exciton energy transfer or dissociation phenomenon across the nano - interface between swnts and p3ht . figure 4 shows the relaxation dynamics of composites when we probe at resonant with the singlet exciton transition of p3ht matrix ( 600 nm ) . it is apparent from these data that the donor / acceptor interfaces in composites enhance the dissociation of excitons across the heterojunctions . normalized transient absorption measurements for the p3ht polymer , pure swnts , and swnt / p3ht composites at probing wavelength of 600 nm . the inset shows the fast decay time as a function of nanotube concentration following the initial excitation by the 400 nm photons , the probing 600 nm beam monitors the population of excitons at the energy state located 2 ev above the homo . as the nanotube concentration increases , we propose that exciton dissociation is amplified at the nanotube - polymer bulk heterojunctions due to the presence of the inherent field at these junctions . from the experimental data in fig . 4 , it is obvious that the nanotubes act as dissociation centers for the excitons minimizing the radiative recombination ( smaller stretched decay ) . the variation in exciton dissociation efficiency can be represented numerically by plotting the fast decay time as a function of nanotube concentration in the inset of fig . , we note that this behavior remains the same for all the probing wavelengths used in this work . the transient absorption signal of a pure carbon nanotube sample is also shown for comparison in fig . the signal consists of two contributions : a fast negative transient lasting for a very short period of time and a positive contribution that lasts for the remaining of the measured delay time period . absorption of the pump pulse ( 3.1 ev ) creates a population of excitonic states which gradually relax to lower energies before electrons and holes recombine . the fast recovery to positive absorption suggests subsequent secondary excitations by the 600 nm probing wavelength . one can not be certain the starting ( base ) and final energy levels involved here , we can only be certain that the energy difference is about 2 ev . in the remaining of delay times , our transient provides an account of the decrease in the population of the base energy level of the re - excitation . a detail analysis of these two antagonistic contributions for a broad spectrum of probing wavelengths between 480 and 980 nm for pure swnts 5 . ultrafast transient absorption measurements for pure swnts at probing wavelengths ranging between 480 and 980 nm . the inset shows a simple band diagram of carrier relaxation at the probing wavelength of 980 nm ( 1.26 ev ) , we only observe the photobleaching of excitonic states . pa , however , does not become significant until the probing wavelength becomes 600 nm ( 2 ev ) where we observed delayed pa as described above . interestingly , for probing wavelengths of 550 nm ( 2.25 ev ) or less , our signal is dominated by pa . as depicted by the simple mechanism depicted in the inset of fig . 5 , pa between states with energy difference between 2 and 2.58 ev is very strong . the dominant negative contribution at lower probing photon energies ( between 1.26 and 2 ev ) suggests the existence of an almost continuous density of states at these energies . when probing the same behavior in composites with high nanotube concentration ( 65 wt.% ) , fig . 6 shows that the re - excitation at high probing energies ( see 550 and 600 nm ) is not reproduced . the detection of excitonic state populations in swnts is therefore completely masked , except for the 700 nm ( 1.76 ev ) probing wavelength where we most likely see a contribution from the swnts and the polymer . the initial drop in absorption is due to probing state filling in swnts , a trend obvious in fig . however , the polymer shows a strong pa contribution at 700 nm , a feature we have observed for all composites . therefore , it is likely that pa contribution sets and overwhelms the negative contribution from the swnts absorption . normalized ultrafast transient absorption for the highest concentration composite ( 65% swnts ) for probing wavelengths of 550 , 600 , and 700 nm , respectively . in conclusion , we have studied ultrafast transient absorption on p3ht / carbon nanotube composites up to 65% swnt concentration . linear absorption measurements in these composites give an important insight of excitonic and vibronic sidebands . the experimental transient absorption along with the optical absorption measurements reveal that state filling effect and pa take place in these composites . we have found that carrier relaxation within the valence and conduction bands of p3ht is beyond our resolution time ( ~150 fs ) whereas the exciton dynamics have a double exponential relaxation . phonon interactions at the vibronic sidebands quench the radiative emission by introducing nonradiative relaxation channels . the addition of nanotubes in these composites alters the relaxation dynamics of formed excitons dissociating these at short time scale and introducing new free - carrier relaxation paths for electrons and polarons through nanotubes and p3ht chains , respectively . exciton dissociation is accelerated with the concentration of carbon nanotubes strongly suggesting that dissociation takes place at the nanotube - polymer heterojunctions . furthermore , even at high nanotube concentrations , the pump pulse is predominantly absorbed by the polymer albeit a strong influence by polymer - nanotube heterojunctions on transient absorption of the probe beam . this behavior could be justified by comparing the absorption strength of both materials at 400 nm . finally , it is well known in the filed of photovoltaic applications that solar cells based on swnt - p3ht composites do not work well , although swnt coatings might be useful as transparent electrodes . the evidence in this work suggests that this failing of the swnts is not due to lack of exciton dissociation , since we observe shorter exciton lifetimes as the amount of swnt is increased . this means that maybe other factors , like recombination of charge carriers in nanotubes or polymer chains , are responsible for their poorer performance in these photovoltaics . on the other hand , in preview work we have reported that pcbm - p3ht composites have also a fast exciton dissociation time which quenches the radiative recombination of the polarons / excitons , and increases the yield of photogenerated charged excitations from the pcbm - related states . with increasing the pcbm concentration in the blended materials in that work , we have observed that the relaxation times increase as opposed to the relaxation dynamics upon increasing the swnt concentration in the same p3ht matrix . we believe that this important difference is responsible for the higher photovoltaic performance of pcbm - p3ht compared with the swnt - p3ht composite . the work in this article was partially supported by the research programs eryan/0506/04 and eryne/0506/02 funded by the cyprus research promotion foundation in cyprus .

femtosecond transient absorption measurements on single wall carbon nanotube / poly(3-hexylthiophene ) composites are used to investigate the relaxation dynamics of this blended material . the influence of the addition of nanotubes in polymer matrix on the ultrashort relaxation dynamics is examined in detail . the introduction of nanotube / polymer heterojunctions enhances the exciton dissociation and quenches the radiative recombination of composites . the relaxation dynamics of these composites are compared with the fullerene derivative - polymer composites with the same matrix . these results provide explanation to the observed photovoltaic performance of two types of composites .

adolescence is a transitional period of life in which lifestyle including food habits are formed . adolescents tend to gain more money and to have more freedom to choose and buy their own foods and snacks . they also spend more time with their friends and imitate their behaviors , either healthy or unhealthy and newly formed ones establish gradually . recognizing food choices , behaviors and their related factors in adolescents are crucial as they affect adulthood health . as they make considerable portion of iranian population , investment on their health issues however , studies on dietary patterns of iranian adolescent indicated that they have inappropriate dietary patterns and food habits , include skipping meals , mainly breakfast , choosing unsuitable and non - nutritious snacks like sweet beverages , high fat and salty food item and low consumption of milk , fruits and vegetables that may cause growth retardation , metabolic changes resulting in reduced iq ( intelligence quotient ) , decreased concentration power , learning abilities and physical activities that totally affect reaching educational goals . adolescents spend lots of their day time at school and regarding their problem of skipping breakfast that effect their snack selection , school 's cafeteria are one of the major sources of providing their daily nutritional needs . many of these food centers supply unhealthy and low quality foods that can not meet nutritional needs of the students . so providing favorable , nutritious and healthy snacks , available via cafeterias , is an approach that may affect their food habits . further more , most of these food centers do not meet the minimum required standard of food services . in recent decade , there are trends toward qualitative methods in addition to quantitative researches . qualitative research is an easy , rapid and cost effective way of investigating ideas and understanding attitudes of studied population that in which , complexes statistical analysis is not used to state the results ; so it is an accepted technique for gathering information to plan appropriate intervention and policy making . so this qualitative study was done as a part of intervention program for training nutrition and health in order to improve schools cafeteria and food behavior of students to evaluate students snack habits regarding to their schools cafeteria status in tehran . this qualitative study was conducted on 240 students ( 12 - 15 years old ) using focus group discussion ( fgd ) technique . focus group discussion is the most important and efficient method of data collection in qualitative studies . subjects were selected randomly among the students , from 12 middle - schools in tehran ( n = 6 girls school and n = 6 boys schools ) . the students were informed about the importance of study and invited for participation as volunteers making them sure that all their opinions will be kept secret . field study consisted of 24 fgds was carried out . in each school , 2 fgds were holding separately , team including a moderator , a co - moderator , an observer and two note takers . focus group questions , including 10 questions , was designed according to two specific objects of the study ( breakfast and snacks consumption evaluation , cafeterias necessity and their evaluation ) by research team , then reviewed by an expert committee and modified based on their comments . after each session , records of two note takers were compared ; possible defects were completed and controlled by a recorder . before starting the field study , a pilot fgd was done with some students from a middle school which was not included into the main subjects of study , to match all team members performance and to check the questions intelligibility , study 's procedure and efficacy . finally , collected data were coded , categorized and analyzed using constant comparative method . data collection and analysis methods were performed and reviewed to make them valid . at the end of each meeting this study was approved by the research ethics committee ( no : 4230 ) of national nutrition and food technology research institute and all participants completed informed consent forms . why?what do you buy from school 's cafeterias?what is your opinion about school 's cafeteria ? based on the objectives , findings are presented in two parts : majority of students thought that eating breakfast is required and most of them consumed it . reason for skipping breakfast was mainly being late for school and poor appetite . in their opinion , honey , butter , jam , milk , bread and cheese , were nutritious for breakfast and about half of them , had bread with cheese , butter and walnut , as breakfast . their main reasons in this regard were : being energetic , useful to better understanding the lessons , prevent osteoporosis , increasing intelligence , having protein and vitamin contents , increasing height and making good mood . over half of their most important reasons were : preventing fatigue and hunger , providing energy , helping for better understanding the lessons , increasing intelligence , compensating for breakfast escape , having it as an entertainment and promoting health . the most consumed snacks among the students were cookies , fruits , bread and cheese , sandwiches , chocolate milk , fruit juices and potato chips . they thought that these snacks are nutritious ( having vitamins , protein , calcium and energy ) , useful for bone strength , prevent osteoporosis , and supply water for their body . majority of students stated that their schools cafeterias are not good . in their point of view , most of cafeteria are small , messy , and majority of the students mentioned about long queues for buying snacks and the cafeterias staff being unhygienic , with no uniforms and gloves . all of the students expect their schools cafeterias to be clean , larger and more organized , with suitable environment and equipments . majority of them believed that the staff must be hygienic , non smokers , wearing uniforms and gloves . majority of the students liked to have cafeterias as a necessary part of school , in the case of forgetting to bring snacks they can be beneficial to release their hunger . the most purchased items were : cookies , sandwiches with mayonnaise or ketchup , chocolate milks , soft drinks , potato chips , pop corn , chocolate , ice cream , puffed rice , fruit bars , nuts , dried fruits , jelly , cooked lentils and beans , roasted chickpeas , raisins , salted crackers , gum , tea , candy , fruits . their main reasons were making students satisfied by implementing their ideas and helping staffs for an efficient cafeteria . sources of nutritional knowledge for majority of the students were television , magazines , newspapers , parents , physicians , and teachers . their own experience , radio , media , relatives , classmates , friends , internet were other useful sources majority of students thought that eating breakfast is required and most of them consumed it . reason for skipping breakfast was mainly being late for school and poor appetite . in their opinion , honey , butter , jam , milk , bread and cheese , were nutritious for breakfast and about half of them , had bread with cheese , butter and walnut , as breakfast . their main reasons in this regard were : being energetic , useful to better understanding the lessons , prevent osteoporosis , increasing intelligence , having protein and vitamin contents , increasing height and making good mood . over half of their most important reasons were : preventing fatigue and hunger , providing energy , helping for better understanding the lessons , increasing intelligence , compensating for breakfast escape , having it as an entertainment and promoting health . the most consumed snacks among the students were cookies , fruits , bread and cheese , sandwiches , chocolate milk , fruit juices and potato chips . they thought that these snacks are nutritious ( having vitamins , protein , calcium and energy ) , useful for bone strength , prevent osteoporosis , and supply water for their body . majority of students thought that eating breakfast is required and most of them consumed it . reason for skipping breakfast was mainly being late for school and poor appetite . in their opinion , honey , butter , jam , milk , bread and cheese , were nutritious for breakfast and about half of them , had bread with cheese , butter and walnut , as breakfast . their main reasons in this regard were : being energetic , useful to better understanding the lessons , prevent osteoporosis , increasing intelligence , having protein and vitamin contents , increasing height and making good mood . their most important reasons were : preventing fatigue and hunger , providing energy , helping for better understanding the lessons , increasing intelligence , compensating for breakfast escape , having it as an entertainment and promoting health . the most consumed snacks among the students were cookies , fruits , bread and cheese , sandwiches , chocolate milk , fruit juices and potato chips . they thought that these snacks are nutritious ( having vitamins , protein , calcium and energy ) , useful for bone strength , prevent osteoporosis , and supply water for their body . majority of students stated that their schools cafeterias are not good . in their point of view , most of cafeteria are small , messy , and majority of the students mentioned about long queues for buying snacks and the cafeterias staff being unhygienic , with no uniforms and gloves . all of the students expect their schools cafeterias to be clean , larger and more organized , with suitable environment and equipments . majority of them believed that the staff must be hygienic , non smokers , wearing uniforms and gloves . majority of the students liked to have cafeterias as a necessary part of school , in the case of forgetting to bring snacks they can be beneficial to release their hunger . the most purchased items were : cookies , sandwiches with mayonnaise or ketchup , chocolate milks , soft drinks , potato chips , pop corn , chocolate , ice cream , puffed rice , fruit bars , nuts , dried fruits , jelly , cooked lentils and beans , roasted chickpeas , raisins , salted crackers , gum , tea , candy , fruits . their main reasons were making students satisfied by implementing their ideas and helping staffs for an efficient cafeteria . sources of nutritional knowledge for majority of the students were television , magazines , newspapers , parents , physicians , and teachers . their own experience , radio , media , relatives , classmates , friends , internet were other useful sources . majority of students stated that their schools cafeterias are not good . in their point of view , most of cafeteria are small , messy , and majority of the students mentioned about long queues for buying snacks and the cafeterias staff being unhygienic , with no uniforms and gloves . all of the students expect their schools cafeterias to be clean , larger and more organized , with suitable environment and equipments . majority of them believed that the staff must be hygienic , non smokers , wearing uniforms and gloves . majority of the students liked to have cafeterias as a necessary part of school , in the case of forgetting to bring snacks they can be beneficial to release their hunger . the most purchased items were : cookies , sandwiches with mayonnaise or ketchup , chocolate milks , soft drinks , potato chips , pop corn , chocolate , ice cream , puffed rice , fruit bars , nuts , dried fruits , jelly , cooked lentils and beans , roasted chickpeas , raisins , salted crackers , gum , tea , candy , fruits . their main reasons were making students satisfied by implementing their ideas and helping staffs for an efficient cafeteria . sources of nutritional knowledge for majority of the students were television , magazines , newspapers , parents , physicians , and teachers . their own experience , radio , media , relatives , classmates , friends , internet were other useful sources . in this study , fgd results showed that most of the students are aware of the necessity of breakfast and snack consumption and most of them eat breakfast which is consistent with other studies ; it seems that the students follow their own traditional patterns for their breakfast which is different from other countries patterns . reasons of skipping breakfast is nearly similar to findings of other studies in iran and other countries . morning snack consumption was a common fact among nearly all the students . despite their awareness of eating suitable snacks , consumption of non nutritious snacks was high at school . the most common consumed foods were biscuits , cookies , fruits and sandwiches which are consistent with other studies in iran . in italy , northern ireland and united state , variable cake , milk and soft drinks and sweet snacks were the most popular choices of students . it seems that being hungry is the most common reason of choosing cake and biscuits with milk or soft drinks as snacks by students . food availability , time , flavor and price could be also mentioned as determinant factors ; as it is shown that all these items are sweet and inexpensive to buy . croll and et al . , ( 2001 ) and sztainer and et al . , ( 1999 ) also mentioned hunger , food craving and cost in addition to parental influence on eating behaviors ( including the culture or religion of the family ) , benefits of foods ( including health ) , situation - specific factors , mood , body image , habit , media , time considerations of adolescents and parents and food availability were effective factors on food choices and eating behaviors . although , standardized cafeterias are important and necessary in schools , it seems that more than half of all cafeterias over the country are not acceptable neither environmental and equipments status nor food items . in study of cullen and et al . , ( 2007 ) , students declared that they prefer fresh and healthy foods , while they did nt have permanent access to these items . instead of plenty of unhealthy food , authors suggest that cafeterias in schools may cause eating less fruits , vegetables and milk and eating more soft drinks and chips . it seems that availability of healthy foods in cafeterias affect students snack habits . in conclusion , our findings showed that schools cafeterias are significant sources of supplying adolescents snacks which have undeniable impact on their snack habits . in the other words , although our students were enough aware of suitable foods to provide their nutritional needs , which resulted in their unsatisfaction with their cafeteria status , they continued to purchase their daily snacks , mainly because of hunger or peers impact and having no alternative option , which is consistent to other studies . schools cafeteria determine students food availability , as a result developing hygienic cafeterias containing healthy and nutritious food items is a key element to affect adolescents snack habits . reaching this goal requires administration of a multi disciplinary approach : it needs students cooperation in cafeterias , related activities as a part of nutrition education programs in which cafeterias would make informational and supportive environment , in addition to school principals and staff and parents help ; community and media support . these findings could be considered as an agenda for policy and decision makers of school nutrition programs , especially in respect of preventing non - communicable disease and promoting health of next generation .

background : the aim of this study was to evaluate patterns of students snacks habits regarding to their schools cafeteria status in tehran by focus group discussion ( fgd ) technique.methods:participants were 240 students ( 12 - 15 years old ) , selected from12 middle - schools in tehran . the field study consisted of 24 fgds sessions ; involving 8 - 10 participants . collected data were coded , categorized and analyzed using constant comparative method.results:over half of the students believed that snack consumption is necessary . although , majority of students believed that their schools cafeterias are not acceptable , they noted them as one of the necessary parts of school . nearly half of the children were complaining of unvaried and expensive food items . the most purchased items were : cookies , sandwiches with mayonnaise and ketchup , soft drinks and chocolate milk . most of the students were interested in having roles in their cafeterias.conclusions : schools cafeteria are significant sources of supplying adolescents snacks , so developing hygienic stores containing healthy and nutritious food items is a key element to affect their snack selection positively . reaching this goal requires a multi disciplinary approach through participation of students , school staff , parents , and the support of community and media .

very soon after the initial reports of its selective antipsychotic action , it was tested and applied around the world in psychotic patients . today 's clinicians may underappreciate the potency of chlorpromazine in those neurolepticnaive individuals : the average symptom diminution was 80% or more . although a potent antipsychotic , the drug has significant motor , sedative , and cardiovascular side effects ; consequently , its use in schizophrenia has gradually diminished over the years . all were characterized by dopamine receptor blockade and catalepsy ( in rats ) or parkinsonism ( in humans ) . gradually , the compounds became purer dopamine receptor antagonists , without other monoaminergic , cholinergic , or histaminergic blockade . haloperidol is a typical example of these newer agents , which still acted predominantly via d2 dopamine receptor blockade . it was introduced in the 1960s , and soon became the most widely used antipsychotic drug . haloperidol had the same antipsychotic potency as chlorpromazine , but lacked several of its more significant side effects , including cardiovascular side effects , and much of its sedative effect . the efficacy of haloperidol was established in controlled trials in the 1960s , and it was used by clinicians thereafter over a wide dose range , often up to hundreds of milligrams per day . pharmacokinetic studies suggested that its active antipsychotic dose range was 4 to 16 mg / day/ however , a random assignment dose - response trial with haloperidol was not carried out until the early 1990s . this dose - response study compared doses of 0 , 4 , 8 , and 16 mg / day . the results showed a significant difference only between placebo and the 8 mg / day and 16 mg / day doses , but no differences between any of the doses either statistically or in overall magnitude of response . none of the items of the brief psychiatric rating scale ( bprs ) had a linear dose - response relationship , not even the positive symptom scores . moreover , parkinsonism and akafhisia were significantly present with the 4 mg / day dose , and remained at a maximal score at all higher dose levels . these results demonstrate that haloperidol is a potent antipsychotic and has significant motor side effects , even at its lowest threshold of antipsychotic dose ( 4 mg / day ) . clozapine was the first of the new antipsychotics , even though it was not new at all at the time of its introduction to the us market . it was marketed in europe in the 1970s , and its widespread european inpatient use allowed the detection of its most serious side effect , agranulocytosis . the clinical use of clozapine led to the hypothesis that it was a superior antipsychotic , which was tested by kane et al in a controlled trial . their initial study , carried out in fully treatment - resistant schizophrenic individuals , was followed up by a clozapine vs haloperidol comparison in schizophrenic partial responders . in both studies , the data show that clozapine has a significantly greater antipsychotic action than chlorpromazine or haloperidol in schizophrenic individuals . clozapine remains the only antipsychotic whose efficacy has been demonstrated to be superior to other agents in the antipsychotic class . unfortunately , in addition to the serious side effect of agranulocytosis ( which can be successfully managed by weekly plasma monitoring ) , clozapine also has a diverse array of additional side effects , some of which are serious , others merely bothersome . these include tachycardia , hypotension , sedation , seizures , akathisia , drooling , and significant weight gain . the disincentives to clinical use produced by these many side effects are significant , but the drug is still used around the world , indicating its superior efficacy . most psychiatrists would agree that clozapine is underutilized in the us , given its superior antipsychotic efficacy . four new antipsychotics have since followed clozapine to market . with these , there has been an attempt to reduce motor side effects and increase treatment efficacy . to some extent , this has been achieved with the new antipsychotics ; most prominently , they lack motor side effects . the approval of the new compounds by the us food and drug administration ( fda ) ( first risperidone , then olanzapine , quetiapine , and finally ziprasidone ) fails to recognize the significant number of drugs that nearly reached general approval , but failed for safety or efficacy reasons . this list includes drugs like remoxipridc , which caused aplastic anemia ; sertindole , which prolongs the qt interval on the electrocardiogram ; and ml 00907 , which failed because of reduced efficacy . the difficulty in the development of drugs for schizophrenia is primarily due to the lack of a pathophysiologic understanding of the illness and , consequently , the lack of a known drug target . animal testing to help focus drug candidate choices is not usually helpful because of the obvious difficulties in modeling psychosis . nonetheless , it is an area of the highest medical need and , for that reason , pharmaceutical companies continue to invest in antipsychotic drug development . it is fortunate that each new drug candidate introduced to the market to date has provided additional advances in patient response and has been widely used . risperidone , the first drug to market after clozapine , is predominantly a d2 dopamine receptor antagonist and a 5-ht2 receptor antagonist at clinical doses . it was shown to be effective against placebo with an antipsychotic response comparable to that of haloperidol . in several studies , greater efficacy is apparent at a lower dose ( < 6 mg / day ) . this initial observation by marder et al preceded several later reports showing the same phenomenon in naturalistic studies , that low daily dose averages are 4 to 4.5 mg / day . at these dose levels , risperidone 's motor side effects are minimal , although they do increase at daily doses above 6 mg / day . galactorrhea secondary to elevated prolactin levels is one of its major side effects and a moderate weight gain is apparent . risperidone has been studied in psychosis of dementia and found to be therapeutic at the lower dose range of 1 to 2 mg / day . the drug has a broad receptor affinity profile , similar to that of clozapine , except for a generally higher receptor affinity at each site . its antipsychotic action tested against haloperidol is at least comparable , with both drugs showing significantly better effects than placebo . with respect to its therapeutic action , olanzapine has broader effects than a traditional compound like haloperidol , with some antianxiety , antidepressant , and arguably antinegative symptom actions as well . olanzapine has been tested in the psychosis of bipolar illness and found to be therapeutic . olanzapine 's side effects are mostly benign , with no parkinsonism , mild akathisia , and no blood dyscrasias or prolactin elevations . significant weight gain and its consequences , including adult - onset diabetes and hyperlipidemia , are its most significant side effects . this low affinity but broad spectrum compound ( like clozapine ) is an effective antipsychotic . worldwide use has been relatively low , despite its efficacy and attractive side - effect profile : placebo - level parkinsonism and akathisia with no prolactin elevation but moderate weight gain . moreover , quetiapine has been studied in the psychosis of dementia with oral reports of good activity . amisulpride is an antipsychotic available in several european countries , but not yet in the us . its antipsychotic efficacy has been demonstrated , together with a low , but not placebo level , motor side - effect profile . several studies have suggested an antinegative symptom profile for this drug . such a unique characteristic has not yet been rigorously demonstrated , but repeatedly suggested in the literature . even beyond these compounds , the industrial pipelines for new antipsychotics are not dry . newer drugs are being tested in all stages of trials : phases 1 through 4 . currently , all the compounds under study block d2 dopamine receptors , but additional novel strategies are also being evaluated , like partial dopamine agonists and indirect - acting n - methyl - d - aspartate ( nmda)-sensitive glutamate agonists . the discovery of a disease mechanism will catapult the discovery process ahead . currently , discovery efforts are tending to focus on the different mechanisms for the positive , negative , or cognitive manifestations of schizophrenia . basic to a full understanding of the biology of the mental dysfunction in psychosis and the therapeutic action of these drugs in psychosis is the concept of the brain as a set of overlapping distributed neural systems , each of which utilizes particular brain regions as needed to fulfill their circuit function . the best understood set of these distributed systems has been identified using motor out - puts , since motor end points can be most easily measured in experimental situations . one set of distributed systems governing aspects of motor function is made up of parallel , segregated neuronal circuits that project from the frontal cortex , supplementary motor area ( sma ) , to the basal ganglia , and then on to the thalamus , and thereafter return to the sma . the frontal projections begin in the neocortex , synapse in the caudate / putamen , and then split into two pathways , an indirect pathway through the globus pallidus ( ultimately inhibitor } ' ) and a direct pathway to the substantia nigra ( ultimately facilitatory ) , which both pass into the substantia nigra pars reticulata and from there to the specific nuclei of the thalamus . the segregated pathways project from the thalamus back to their specific regions of the frontal cortex , presumably carrying a subcortically modified neuronal signal back from the basal ganglia to the frontal cortex . d2 dopamine receptors , the putative site of action of antipsychotic drugs , reside in very high concentrations in the caudate and the putamen . antipsychotic drugs are believed to exert their primary therapeutic action here in the basal ganglia . yet , a mechanism to transmit this action in the basal ganglia back to the neocortex , particularly the frontal cortex , would deliver such a basal ganglia action to the neocortical brain areas , those presumably affected by schizophrenia . the transmission of this d2-receptor - modified message through the basal ganglia and thalamus , then back to frontal cortex , is the mechanism that we have proposed to mediate the therapeutic action of these drugs in humans . indeed , we have tested this hypothesis in our clinical laboratory with patient volunteers using regional functional imaging techniques with fluorodeoxyglucose ( fdg ) and positron emission tomography ( pet ) or regional cerebral blood ( rcbf ) flow with and without antipsychotic drugs . simply stated , schizophrenic volunteers received a fixed dose of haloperidol ( 0.3 mg / kg / day ) for 30 days ; and then an fdg / pet scan was done . the volunteers then received the same dose of placebo ( matched pill number ) with a repeat fdg / pet after the second period of 30 days . computer subtractions were done between the group - average on drug scan and its off drug counterpart , showing the regions where haloperidol increased and decreased neuronal activity . haloperidol had the following actions in these volunteers : ( i ) it increased neuronal activity in the caudate / putamen ( presumably a disinhibition ) ; ( ii ) it increased neuronal activity in the thalamus ( presumably associated with a diminished inhibitory reticulothalamic signal ) ; ( iii ) it decreased anterior cingulate neuronal activity ( presumably secondary to reduced activity in the thalamocortical excitatory afferent pathway ) ; and ( iv ) it decreased middle frontal cortical activity ( ie , the same explanation as for [ iii ] ) . the explanations ( given in parentheses above ) represent the interpretation we have made of the functional data to shed light on the question of the neural mechanism of antipsychotic drug action . wc propose that the disinhibition that haloperidol ( or any d2 dopamine receptor antagonist ) produces in the caudate / putamen is transmitted through the basal ganglia and thalamus to ultimately inhibit key areas of the neocortex . these pet findings have been replicated in our laboratory using rcbf , and the data are entirely consistent . these results are consistent with many of the functional imaging results from other laboratories doing similar kinds of studies . experiments in our laboratory over the last few years have involved the administration of traditional and new antipsychotic drugs to laboratory rats for subchronic time periods ( 6 months ) for the purpose of examining critical neurotransmitter systems in the central nervous system ( cns ) regions ( the basal ganglia - thalamocortical neural circuit ) and their alteration with chronic drug treatment . we postulated that the neurochemical marker for d2 dopamine receptor blockade ( d2 upregulation ) and the transmitted signals through this system would both vary between the traditional and new drugs . we measured d2 dopamine receptor density in rat caudate , gabaa ( gaba : gamma - aminobutyric acid ) receptor density and dj dopamine receptor density in rat substantia nigra , and gabaa receptor density and glutamic acid decarboxylase ( gad ) mrna expression in rat thalamus . with haloperidol , all these markers significantly changed in each region , implying a potent drug action in the caudate / putamen and a strong transmitted signal through the rest of the basal ganglia to the thalamus and thereafter to the cortex . these data are direct evidence from the experimental animal of the idea of a transmitted antipsychotic action through the basal ganglia and the thalamus to the cortex . with the new antipsychotics , these neurochemical changes were milder and not as broad , but always involved the basal ganglia and the thalamus . while the dopaminergic component of antipsy - chotic drug action is putativcly mediated through these defined neural circuits , other transmitter - specific components of drug action ( eg , antiserotonergic or anti- adrenergic ) are likely produced directly in the neocortex . basic to a full understanding of the biology of the mental dysfunction in psychosis and the therapeutic action of these drugs in psychosis is the concept of the brain as a set of overlapping distributed neural systems , each of which utilizes particular brain regions as needed to fulfill their circuit function . the best understood set of these distributed systems has been identified using motor out - puts , since motor end points can be most easily measured in experimental situations . one set of distributed systems governing aspects of motor function is made up of parallel , segregated neuronal circuits that project from the frontal cortex , supplementary motor area ( sma ) , to the basal ganglia , and then on to the thalamus , and thereafter return to the sma . the frontal projections begin in the neocortex , synapse in the caudate / putamen , and then split into two pathways , an indirect pathway through the globus pallidus ( ultimately inhibitor } ' ) and a direct pathway to the substantia nigra ( ultimately facilitatory ) , which both pass into the substantia nigra pars reticulata and from there to the specific nuclei of the thalamus . the segregated pathways project from the thalamus back to their specific regions of the frontal cortex , presumably carrying a subcortically modified neuronal signal back from the basal ganglia to the frontal cortex . d2 dopamine receptors , the putative site of action of antipsychotic drugs , reside in very high concentrations in the caudate and the putamen . antipsychotic drugs are believed to exert their primary therapeutic action here in the basal ganglia . yet , a mechanism to transmit this action in the basal ganglia back to the neocortex , particularly the frontal cortex , would deliver such a basal ganglia action to the neocortical brain areas , those presumably affected by schizophrenia . the transmission of this d2-receptor - modified message through the basal ganglia and thalamus , then back to frontal cortex , is the mechanism that we have proposed to mediate the therapeutic action of these drugs in humans . indeed , we have tested this hypothesis in our clinical laboratory with patient volunteers using regional functional imaging techniques with fluorodeoxyglucose ( fdg ) and positron emission tomography ( pet ) or regional cerebral blood ( rcbf ) flow with and without antipsychotic drugs . simply stated , schizophrenic volunteers received a fixed dose of haloperidol ( 0.3 mg / kg / day ) for 30 days ; and then an fdg / pet scan was done . the volunteers then received the same dose of placebo ( matched pill number ) with a repeat fdg / pet after the second period of 30 days . computer subtractions were done between the group - average on drug scan and its off drug counterpart , showing the regions where haloperidol increased and decreased neuronal activity . haloperidol had the following actions in these volunteers : ( i ) it increased neuronal activity in the caudate / putamen ( presumably a disinhibition ) ; ( ii ) it increased neuronal activity in the thalamus ( presumably associated with a diminished inhibitory reticulothalamic signal ) ; ( iii ) it decreased anterior cingulate neuronal activity ( presumably secondary to reduced activity in the thalamocortical excitatory afferent pathway ) ; and ( iv ) it decreased middle frontal cortical activity ( ie , the same explanation as for [ iii ] ) . the explanations ( given in parentheses above ) represent the interpretation we have made of the functional data to shed light on the question of the neural mechanism of antipsychotic drug action . wc propose that the disinhibition that haloperidol ( or any d2 dopamine receptor antagonist ) produces in the caudate / putamen is transmitted through the basal ganglia and thalamus to ultimately inhibit key areas of the neocortex . these pet findings have been replicated in our laboratory using rcbf , and the data are entirely consistent . these results are consistent with many of the functional imaging results from other laboratories doing similar kinds of studies . experiments in our laboratory over the last few years have involved the administration of traditional and new antipsychotic drugs to laboratory rats for subchronic time periods ( 6 months ) for the purpose of examining critical neurotransmitter systems in the central nervous system ( cns ) regions ( the basal ganglia - thalamocortical neural circuit ) and their alteration with chronic drug treatment . we postulated that the neurochemical marker for d2 dopamine receptor blockade ( d2 upregulation ) and the transmitted signals through this system would both vary between the traditional and new drugs . we measured d2 dopamine receptor density in rat caudate , gabaa ( gaba : gamma - aminobutyric acid ) receptor density and dj dopamine receptor density in rat substantia nigra , and gabaa receptor density and glutamic acid decarboxylase ( gad ) mrna expression in rat thalamus . with haloperidol , all these markers significantly changed in each region , implying a potent drug action in the caudate / putamen and a strong transmitted signal through the rest of the basal ganglia to the thalamus and thereafter to the cortex . these data are direct evidence from the experimental animal of the idea of a transmitted antipsychotic action through the basal ganglia and the thalamus to the cortex . with the new antipsychotics , these neurochemical changes were milder and not as broad , but always involved the basal ganglia and the thalamus . while the dopaminergic component of antipsy - chotic drug action is putativcly mediated through these defined neural circuits , other transmitter - specific components of drug action ( eg , antiserotonergic or anti- adrenergic ) are likely produced directly in the neocortex . it is our current hypothesis that both traditional and new antipsychotic drugs exert the dopaminergic component of their action in the caudate / putamen and that this action is transmitted through the basal ganglia - thalamo - cortical system to the frontal cortex where the pivotal therapeutic action is delivered . other neurotransmitter influences are most likely exerted in all parts of this circuit , in both the basal ganglia and the cortex . given this hypothesis , the obvious proposition is to modulate the circuit 's activity at other neurochemical sites in the circuit . this proposition may underlie the putative therapeutic actions of glutamatergic and gabaergic compounds in schizophrenia .

the first generation of antipsychotic drugs was discovered in the 1960s and 1970s , these agents were effective in treating psychosis , but were accompanied by significant side effects , including severe parkinsonism and akathisia . second - generation antipsychotics were introduced in the 1990s , these drugs have at least equal efficacy to their predecessors , but far fewer side effects . some data suggest a broader efficacy profile . clozapine remains the only superior antipsychotic in terms of the magnitude of psychotic symptom reduction . clinical and animal studies are consistent in suggesting that the antipsychotic component of antidopaminergic treatments is initiated by dopamine receptor blockade in the striatum and that the signal is transmitted to the neocortex through the established basal ganglia - thalamo - cortical neuronal circuits . other neurotransmitter actions ( eg , antiserotonergic ) can be exerted locally , in the neocortex . defining tissue targets of drug action may suggest additional strategies for developing new antipsychotic drugs .

systemic lupus erythematosus ( sle ) ( omim 152700 ) is a debilitating autoimmune disease which affects multiple organs and is characterized by a loss of tolerance to self - antigens , inflammation , and dysregulated immune responses , resulting in significant morbidity and mortality . although many new loci which contribute to the pathogenesis of sle have been identified by genome wide association studies ( gwas ) and other association studies , they collectively do not explain all the risk contributed by heritable factors , indicating that other , as - of - yet unidentified genes are likely to be involv1ed . the power of gwas is in their agnostic approach , which does not require prior knowledge of any of the genetics or cellular mechanisms underlying a phenotypic trait . this comes at the cost of testing a large number of single nucleotide polymorphisms ( snps ) , thereby increasing the multiple - testing correction , and ignoring ( often decades of ) prior research into a phenotypic trait which may increase the power of a genetic study . in the present study we report the discovery of 13 novel genes outside of the human leukocyte antigen ( hla ) region ( 4 family - wise error rate ( fwer ) significant ) and confirmation of 4 genes ( 4 fwer significant ) which were reported as significant in previously published studies ( table 1 ) . furthermore , we have imputed all associated regions using the largest panels publicly available and used bioinformatics tools to test for deleterious effects of non - synonymous variants . imputation can be used to deconvolute multiple signals in regions which have complex interdependent signals , such as the hla region . imputation ( hla*imp ) has also been used in this study to assign samples to hla alleles and then to hla serotypes , connecting modern gwas based association results to earlier genetic and serotypic association results . many snps are associated with a disease because they are in linkage disequilibrium ( ld ) with another snp which is the primary signal in that region . to disambiguate between independent and dependent signals , we used conditional regression analysis and lasso regularization to eliminate signals whose association was due to correlation with another primary signal . this enabled us to identify the minimum number of signals required to explain the association results present in every associated region , an approach pioneered by raychaudhuri et al . , and adapted here for sle . this study has also assigned hla alleles and serotypes to each subject and has refined the association signals within the hla and other regions . we detected 430 significant snps ( false discovery rate ( fdr ) 0.05 ) in 160 genes . 405 snps were in the hla region ( supplemental table s1 and s3 ) , and 25 snps were in 17 genes in other regions ( table 1 , supplemental table s3 ) . an additional 7 genes were found to be consistent with previously published results , although they did not meet the non - hla fdr level of this study ( supplemental table s2 ) . figure 1 shows a manhattan plot of the genome wide association data showing the p values of all tested snps . as this figure shows , because of these results , coupled with the well - known association of the hla region with sle and the extensive ld in the hla region , we have separated the analysis and discussion of hla genes from non - hla genes . 25 significant snps within 16 regions corresponding to 17 genes were detected in non - hla regions . of these genes , 13 have not been previously associated with sle ( previous association columns in table 1 ) . two of the 17 genes are transcription factors ( ehf and med1 ) , and two are involved in nfb signaling ( rassf2 and rnf114 ) . two genes are involved in antigen presentation ( bin1 and sec61 g ) , and one in cell adhesion / tissue remodeling cntn6 . all of the significant genes which have mrna expression information available are expressed in at least one relevant immune cell type ( figure s2 ) . the presence of multiple significant snps in a region can be due to ld or multiple independent signals . to distinguish between these possibilities , we performed multiple regression analysis on the significant snps in each of the 4 regions which contained multiple significant snps . we found snps in 1 gene which were dependent on other genes or interdependent on each other where significance of either gene could be explained by the other gene ( table 1 dependent on column ) . in the region including and surrounding irf5 and tnpo3 ( 7q32.1 ) there are 8 significant snps ( figure 2 panel a , supplemental table s3 ) . after accounting for rs10488631 in tnpo3 ( figure 2 panel b ) and rs4728142 in irf5 ( figure 2 panel c ) , the next most - significant snp is rs1665105 , whose p value ( 3.2910 ) is greater than the irf5/tnpo3 region multiple - family fdr ( 2.2210 ) , and furthermore was not found to be significant in this study . this suggests that there are at least two causal variants in the irf5 and tnpo3 region , and that irf5 and tnpo3 are independently associated with sle . in the 15q14 region , the significant snp in fam98b , rs11073328 , accounts for the original significance of the snp in tyro3 , rs12259 ( pmult = 2.6110 , pmult = 8.3010 in the multiple regression , respectively ) this indicates that the association of tyro3 is due to fam98b ; there is evidence for only one signal in the 15q14 region . the final two regions which have multiple snps are sec61 g and ehf ; each of these regions has two significantly associated snps , and in both regions , the significantly associated snps are in complete ld with each other . thus , there is only evidence for a single signal in sec61 g and another single signal in ehf . imputation was performed on all 16 non - hla regions comprising over 2.9 million snps . 840 additional significant snps were found in these regions , 3 of these snps were non - synonymous . however , bioinformatic analyses of these non - synonymous snps using polyphen , sift , and provean do not predict deleterious effects as a consequence of these mutations . thus , it is unlikely that we have identified the causal mutations responsible for the association of these regions with sle , and deep sequencing or other follow up studies of these regions are required . we have previously established that rs17849502 in ncf2 ( 1q25 ) is a causal mutation associated with sle which produces a two - fold reduction in fc receptor - mediated vav1 nadph oxidase response . in this region there is are multiple significant and suggestive ( fdr 0.1 ) snps by genotyping and imputation in each of edem3 , lamc1 , and ncf2 genes as shown in figure 3 . multiple regression indicates that the signals in edem3 and lamc1 are independent of each other ( data not shown ) . to determine if the significant association of edem3 and the suggestive association of lamc1 was due to the presence of rs17849502 in ncf2 , we examined the imputation results in this region . while rs17849502 was present in the imputation panels , it is rare , so the ability of the imputation panels to accurately impute this snp is greatly diminished , leading to its non - significance upon imputation ( impute v2 reports an metric of 0.05 for this snp ; accurately imputed snps should have an info however , rs17849502 and rs12565776 are in strong linkage disequilibrium ( d = 0.96 , figure 3 panel b ) , so it remains possible that the significance of rs12565776 is due to rs1789502 . the hla region has by far the largest number and most highly significant snps , reaching a minimum p value of 7.7610 ( fdr=2.5610 ) ( figure 1 , supplemental table s1 ) . the 6p21 - 22 region , which includes the classical hla genes , contains many alleles which are in linkage disequilibrium with each other . thus , many of the significant results seen could be due to a smaller number of primary signals . to determine the minimum number of signals capable of accounting for the significance seen in the 6p21 - 22 region we performed a step - wise multiple regression analysis , accounting for the most significant snp in the model at each step , and repeating until no additional terms had p values less than the p value corresponding to an fdr of 0.05 in this study . as shown in figure 4 , the most significant snp ( from 405 significant snps out of 4,748 genotyped snps in this region ) was rs558702 ( p = 7.76 10 ) ( figure 4 panel a ) . after accounting for rs558702 , the most significant unaccounted snp is rs9275572 ( p = 1.94 10 , figure 4 panel b ) . subsequently , after accounting for both of these snps , rs2764208 was the most significant snp unaccounted for ( p = 3.6110 , figure 4 panel c ) . finally , rs10946940 was most significant snp after accounting for all three of these snps ( p = 2.3710 , figure 4 panel d ) . these four snps accounted for the vast majority of the significant snps seen in the 6p21 - 22 region , although 6 additional weak signals remained ( figure 4 panel e ) . using lasso regularization across all significant snps in this region , both rs558702 and rs9275572 were the components in the model with the largest coefficients , further indicating their importance ( data not shown ) . thus , two signals in the classical hla are able to account for the vast majority of the significant associations seen in hla , and two additional signals in znf184 and snrpc explain most significance seen in the periphery of the hla region ( table 2 and figure 4 ) . the other signal is in snrpc which is involved in the formation of the spliceosome which is often a target of autoantibodies in sle . thus , the majority of the association of classical hla with sle in this study can be explained by two primary signals . to reconcile our snp findings with the established hla allele and serotype association with sle , we imputed hla alleles using hla*imp [ 1214 ] which assigned an hla haplotype to each sample on the basis of genotyped snps in the hla region . the alleles of hla - a , b , c , dpb1 , dqa1 , dqb1 , and drb1 were imputed at four digit accuracy and hla - drb3 , drb4 , and drb5 were imputed at two digit accuracy . notably , hla - dqa1 * 05:01 and hladqb1 * 02:01 , which have previously been shown to be significantly associated with sle and are part of the dr17/dq2 extended haplotype , were found to be highly associated in this study . we did not detect any significant associations with any of the alleles of hla - a , c , dpb1 , drb4 , or drb5 . finally , multiple regression with hla - dqb1 and hla - drb1 indicated that hla - dqb1 and hla - drb1 have an independent association with sle , and the association of one is not merely due to linkage with the other ( table s5 ) . furthermore , the association of hla - b is largely explained by hla - drb1 , but not hla - dqb1 ( table s5 ) . a regression analysis with the significant alleles of hla - dqb1 and hla - drb1 with the two significant primary snps signals in classical hla ( rs558702 and rs9275572 ) indicates that hla - dqb1 and hla - drb1 are strongly correlated with these snps . the snps outside of the hla region ( rs2764208 and rs10946940 ) were not accounted for by hla - dqb1 or hla - drb1 , indicating that their association is independent of the classical hla signal . dq7 ( broad antigen dq3 ) had a protective effect , whereas dr17 and b8 were associated with disease . we did not see any significant associations with the hla class i a or c antigens , nor did we see any significant associations with alleles of the extended dr / dq haplotype . 25 significant snps within 16 regions corresponding to 17 genes were detected in non - hla regions . of these genes , 13 have not been previously associated with sle ( previous association columns in table 1 ) . two of the 17 genes are transcription factors ( ehf and med1 ) , and two are involved in nfb signaling ( rassf2 and rnf114 ) . two genes are involved in antigen presentation ( bin1 and sec61 g ) , and one in cell adhesion / tissue remodeling cntn6 . all of the significant genes which have mrna expression information available are expressed in at least one relevant immune cell type ( figure s2 ) . the presence of multiple significant snps in a region can be due to ld or multiple independent signals . to distinguish between these possibilities , we performed multiple regression analysis on the significant snps in each of the 4 regions which contained multiple significant snps . we found snps in 1 gene which were dependent on other genes or interdependent on each other where significance of either gene could be explained by the other gene ( table 1 dependent on column ) . in the region including and surrounding irf5 and tnpo3 ( 7q32.1 ) there are 8 significant snps ( figure 2 panel a , supplemental table s3 ) . however , after accounting for rs10488631 in tnpo3 ( figure 2 panel b ) and rs4728142 in irf5 ( figure 2 panel c ) , the next most - significant snp is rs1665105 , whose p value ( 3.2910 ) is greater than the irf5/tnpo3 region multiple - family fdr ( 2.2210 ) , and furthermore was not found to be significant in this study . this suggests that there are at least two causal variants in the irf5 and tnpo3 region , and that irf5 and tnpo3 are independently associated with sle . in the 15q14 region , the significant snp in fam98b , rs11073328 , accounts for the original significance of the snp in tyro3 , rs12259 ( pmult = 2.6110 , pmult = 8.3010 in the multiple regression , respectively ) this indicates that the association of tyro3 is due to fam98b ; there is evidence for only one signal in the 15q14 region . the final two regions which have multiple snps are sec61 g and ehf ; each of these regions has two significantly associated snps , and in both regions , the significantly associated snps are in complete ld with each other . thus , there is only evidence for a single signal in sec61 g and another single signal in ehf . imputation was performed on all 16 non - hla regions comprising over 2.9 million snps . 840 additional significant snps were found in these regions , 3 of these snps were non - synonymous . however , bioinformatic analyses of these non - synonymous snps using polyphen , sift , and provean do not predict deleterious effects as a consequence of these mutations . thus , it is unlikely that we have identified the causal mutations responsible for the association of these regions with sle , and deep sequencing or other follow up studies of these regions are required . we have previously established that rs17849502 in ncf2 ( 1q25 ) is a causal mutation associated with sle which produces a two - fold reduction in fc receptor - mediated vav1 nadph oxidase response . in this region there is are multiple significant and suggestive ( fdr 0.1 ) snps by genotyping and imputation in each of edem3 , lamc1 , and ncf2 genes as shown in figure 3 . multiple regression indicates that the signals in edem3 and lamc1 are independent of each other ( data not shown ) . to determine if the significant association of edem3 and the suggestive association of lamc1 was due to the presence of rs17849502 in ncf2 , we examined the imputation results in this region . while rs17849502 was present in the imputation panels , it is rare , so the ability of the imputation panels to accurately impute this snp is greatly diminished , leading to its non - significance upon imputation ( impute v2 reports an info metric of 0.05 for this snp ; accurately imputed snps should have an info however , rs17849502 and rs12565776 are in strong linkage disequilibrium ( d = 0.96 , figure 3 panel b ) , so it remains possible that the significance of rs12565776 is due to rs1789502 . the hla region has by far the largest number and most highly significant snps , reaching a minimum p value of 7.7610 ( fdr=2.5610 ) ( figure 1 , supplemental table s1 ) . the 6p21 - 22 region , which includes the classical hla genes , contains many alleles which are in linkage disequilibrium with each other . thus , many of the significant results seen could be due to a smaller number of primary signals . to determine the minimum number of signals capable of accounting for the significance seen in the 6p21 - 22 region we performed a step - wise multiple regression analysis , accounting for the most significant snp in the model at each step , and repeating until no additional terms had p values less than the p value corresponding to an fdr of 0.05 in this study . as shown in figure 4 , the most significant snp ( from 405 significant snps out of 4,748 genotyped snps in this region ) was rs558702 ( p = 7.76 10 ) ( figure 4 panel a ) . after accounting for rs558702 , the most significant unaccounted snp is rs9275572 ( p = 1.94 10 , figure 4 panel b ) . subsequently , after accounting for both of these snps , rs2764208 was the most significant snp unaccounted for ( p = 3.6110 , figure 4 panel c ) . finally , rs10946940 was most significant snp after accounting for all three of these snps ( p = 2.3710 , figure 4 panel d ) . these four snps accounted for the vast majority of the significant snps seen in the 6p21 - 22 region , although 6 additional weak signals remained ( figure 4 panel e ) . using lasso regularization across all significant snps in this region , both rs558702 and rs9275572 were the components in the model with the largest coefficients , further indicating their importance ( data not shown ) . thus , two signals in the classical hla are able to account for the vast majority of the significant associations seen in hla , and two additional signals in znf184 and snrpc explain most significance seen in the periphery of the hla region ( table 2 and figure 4 ) . the other signal is in snrpc which is involved in the formation of the spliceosome which is often a target of autoantibodies in sle . thus , the majority of the association of classical hla with sle in this study can be explained by two primary signals . to reconcile our snp findings with the established hla allele and serotype association with sle , we imputed hla alleles using hla*imp [ 1214 ] which assigned an hla haplotype to each sample on the basis of genotyped snps in the hla region . the alleles of hla - a , b , c , dpb1 , dqa1 , dqb1 , and drb1 were imputed at four digit accuracy and hla - drb3 , drb4 , and drb5 were imputed at two digit accuracy . notably , hla - dqa1 * 05:01 and hladqb1 * 02:01 , which have previously been shown to be significantly associated with sle and are part of the dr17/dq2 extended haplotype , were found to be highly associated in this study . we did not detect any significant associations with any of the alleles of hla - a , c , dpb1 , drb4 , or drb5 . finally , multiple regression with hla - dqb1 and hla - drb1 indicated that hla - dqb1 and hla - drb1 have an independent association with sle , and the association of one is not merely due to linkage with the other ( table s5 ) . furthermore , the association of hla - b is largely explained by hla - drb1 , but not hla - dqb1 ( table s5 ) . a regression analysis with the significant alleles of hla - dqb1 and hla - drb1 with the two significant primary snps signals in classical hla ( rs558702 and rs9275572 ) indicates that hla - dqb1 and hla - drb1 are strongly correlated with these snps . the snps outside of the hla region ( rs2764208 and rs10946940 ) were not accounted for by hla - dqb1 or hla - drb1 , indicating that their association is independent of the classical hla signal . dq7 ( broad antigen dq3 ) had a protective effect , whereas dr17 and b8 were associated with disease . we did not see any significant associations with the hla class i a or c antigens , nor did we see any significant associations with alleles of the extended dr / dq haplotype . in the present study we report the discovery of novel non - hla genes not previously associated with sle and the confirmation of genes which were reported as significant in previously published studies ( table 1 ) . the novel discoveries were made in part due to the increase in statistical power of the study through the use of a fdr multiple testing correction instead of a fwer multiple testing correction . it is therefore expected that genes involved in regulation of various immune cells and their functions would be an important part of the genetic susceptibility architecture of sle . indeed , among the novel genes associated with sle susceptibility found in the present study , two genes code for transcription factors ( ehf and med1 ) , two are components of the nfb pathway ( rassf2 and rnf114 ) , two are involved in antigen presentation ( bin1 and sec61 g ) , and one is involved in adhesion and endothelial migration ( cntn6 ) . some of the proteins encoded by these susceptibility genes have been previously considered to be involved in the pathogenesis of the disease , but this is the first study to demonstrate that they are actually associated with genetic susceptibility to sle . the ets family of transcription factors and especially ets-1 , have been previously associated with sle . here we identified ehf , another member of the ets family as associated with sle . ehf is particularly important in the differentiation of dendritic cells ( dc ) , an important antigen presenting cell and producer of cytokines , including type i ifns that are essential for disease pathogenesis . interestingly , the cytokine tgfa , specifically involved in dc differentiation , is suggestively associated with sle ( p = 2.62 10 , fdr = 1.6010 ) . furthermore , mafb , a repressor of ets - mediated transcription , is also suggestively associated with sle ( p = 9.8810 , fdr = 8.8310 ) . the ets pathway exemplifies the recurring motif of genes at multiple steps of a disease - relevant pathway being found to be associated with that disease . the transcription factor med1 is essential for the intrathymic development of inkt cells , a subgroup of immune cells found at abnormal levels in sle subjects . components of the nfb pathway , the master transcriptional regulator of inflammation , have been associated with increased sle susceptibility ( irak1 , tnfaip3 , ube2l3 , prkcb ) . the present study adds two new genes involved in nfb signaling ( rassf2 and rnf114 ) , highlighting the importance of this pathway for sle genetic susceptibility and pathogenesis . rnf114 , an ubiquitin binding protein , regulates a positive feedback loop that enhances dsrna - induced production of type i ifn through the activation of the irf3 and nfb transcription factors , previously shown to be associated with the immune - mediated skin disease psoriasis . genetic variants related to adhesion and endothelial migration of the various immune cell types have been associated with sle susceptibility ( itgam , icam1 , selp)[1 , 20 ] . in the present study we have identified an additional gene involved in cell adhesion and tissue remodeling ( cntn6 ) . although the primary auto - antigens driving sle pathogenesis are quite elusive , it is clear that abnormal antigen processing and presentation play an important part . in the current study we identified two new susceptibility genes involved in antigen processing and presentation ( bin1 and sec61 g ) . classical mhc class i and class ii genes on the surface of antigen presenting cells are responsible for antigen presentation to the t cell receptor on t lymphocytes . mhc loci were the first reported genetic association with sle and remain the strongest . understanding the genetic risk of this region is therefore critical to the understanding of the pathogenesis of the disease however , the region is extremely gene - dense with long range ld and hundreds of immunologically active genes , which makes identification of the true causal loci very difficult . following the approach of raychaudhuri et al . using conditional logistic regression , we showed that two signals inside of the classical hla and two additional in the periphery can account for the vast majority of the significant snps in the entire hla region . we confirm the findings of morris et al . by showing the involvement of hla - drb1 * 03:01 and the involvement of snps in addition to hla alleles . we extend their findings by showing the association of sle with serotypes , confirm the number of primary signals using lasso regularization , and provide evidence for the requirement of hla - dqb1 * 02:01 to explain association of hla with sle . our use of hla imputation has also enabled us to deconvolute the large number of associated snps in the hla region and reconcile these findings with decades - old work showing the association of serotypes and hla alleles with sle . the new sle - associated genes and genetic regions discovered in this study expand our understanding of sle and provide new putative targets for diagnostics and treatment of this important autoimmune disease . this study was approved by the irb of university of southern california school of medicine , and by the irb of the institutions participating in the study , namely , university of california los angeles , university of california san francisco , oklahoma medical research foundation , university of oklahoma health sciences center , university of alabama at birmingham , cincinnati children s hospital medical center , and the irb of king s college , london . 735 unrelated self - identified females of european ancestry with sle ( satisfying the revised criterion for sle from the american college of rheumatology [ 22 , 23 ] ) were collected ( along with 480 unrelated female controls ) by slegen members . in addition , 2057 female controls were obtained from the illumina icontroldb resource giving 2537 total controls . genotyping was performed using the illumina infinium humanhap300 genotyping bead chip as previously described . in order to avoid including samples with genetic background not representative of the overall study , a principle component analysis ( pca ) analysis was performed which identified 109 non - representative samples which were removed from further analysis . to eliminate duplicate and cryptic relationships , a set of 500 snps with minor allele frequency ( maf ) 0.4 were selected . 98 sets of duplicates ( three cases , two slegen controls , 93 illumina controls ) which matched at 99.9% of the snps and 44 sets of related samples ( 16 cases , three slegen controls and 25 illumina controls ) had all but one duplicate / related removed . in order to address population structure which can be a source of confounders [ 25 , 26 ] , we performed pca on a subset of snps which are informative for ancestry ( ancestry informative markers ( aims ) ) . in figure s1 , a pca biplot of the samples collected for this study is shown . the vast majority of samples are present in a homogeneous grouping , with no clear delineations between case and control samples . a prominent exception to this are the samples in orange which are found in the lower left of panel a and lower right of panel b. these 109 samples ( 10 cases , 6 slegen controls , and 93 illumina controls ) are not representative of the ancestral background of the other samples , and therefore have been excluded from further analysis . to control for any remaining population structure , the same first three pca rotation axes were used as cofactors in the association analysis . after correcting for these three factors and eliminating the outlier samples , the remaining dispersion of cases and controls is even and symmetrical ( figure s1 panels c and d ) . the significance of association was calculated using logistic regression with sle status as the dependent variable and the additive dosage of the snp and the three first pca axes as independent variables with custom routines written in r . p values obtained were corrected for multiple testing by estimating the fdr using the benjamini and hochberg method . since the vast majority of significant snps are located in the hla region , calculating the fdr study - wide would result in an under - estimation of the fdr in the non - hla region . thus , the fdr was calculated separately for the hla regions and non hla regions . because each of these regions contains significant results imputation on regions containing significantly associated snps was performed using impute ( v2.3.0 ) with the 1k genomes and hapmap panels , covering over 3.1 million snps . significance of association of imputed snps was calculated in the same manner as for genotyped snps , with multiple - testing corrections being applied within the region instead of study - wide . multiple logistic regression was performed by adding the most significant snp in a region as covariates to the logistic regression model , and repeating the regression on the remaining snps in the region . this procedure was repeated until no snps had a multiple - testing corrected fdr less than the per - family fdr cutoff of 2.6810 ( q = 0.05 , m = 298 , r = 16 as defined in using the ( bh q , bh rq / m ) procedure ) . the best guess haplotype was used in logistic regression with three pca axes as was done for genotyped values . hla haplotypes were assigned to serotypes using the wmda directory release 3.12.0 , and logistic regression with three pca axes was performed on the resultant serotypes .

in a genome wide association study ( gwas ) of individuals of european ancestry afflicted with systemic lupus erythematosus ( sle ) the extensive utilization of imputation , stepwise multiple regression , lasso regularization , and increasing study power by utilizing false discovery rate ( fdr ) instead of a bonferroni multiple test correction enabled us to identify 13 novel non - human leukocyte antigen ( hla ) genes and confirmed the association of 4 genes previously reported to be associated . novel genes associated with sle susceptibility included two transcription factors ( ehf , and med1 ) , two components of the nfb pathway ( rassf2 and rnf114 ) , one gene involved in adhesion and endothelial migration ( cntn6 ) , and two genes involved in antigen presentation ( bin1 and sec61 g ) . in addition , the strongly significant association of multiple single nucleotide polymorphisms ( snps ) in the hla region was assigned to hla alleles and serotypes and deconvoluted into four primary signals . the novel sle - associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease .

methamphetamine ( n - methyl-1-phenyl - propan-2-amine ) , propranolol ( rs)-1-(isopropylamino)-3-(1-naphthyloxy)propan-2-ol ) and pyrrolidine ( tetrahydropyrrole ) were purchased from sigma - aldrich ( uk ) . ultra - purified ( deionised ) water was prepared in - house using a milli - q water system ( millipore , uk ) . methamphetamine and propranolol tablets were obtained from local pharmacy ( oxford , uk ) . a knauer hplc system ( germany ) equipped with a model 1000 lc pump , an online degasser , model 3950 autosampler , and model 2600 photodiode - array detector was used . xterra rp18 ( 1504.6 mm , 5 m particle size ) column ( waters , uk ) was used . the mobile phase consisted of a mixture of 50 mm pyrrolidine ( ph 11.5 ) acetonitrile ( 50:50 , v / v ) the injection volume was 10 l and the uv detection wavelength was set at 214 nm . reversed - phase hplc analysis was performed isocratically at 30. stock solutions of met and pro were prepared in mobile phase at concentrations of 1 mg / ml ( s1 ) . series of standards for each of the substance was prepared by progressive dilution of the stock solution for calibration study . fifteen millilitre aliquot of s1 was transferred to another 50 ml volumetric flask and diluted in mobile phase yielding a final concentration of 0.3 mg / ml . the mean weight of finally powdered pro tablets containing 80 mg of pro was accurately transferred into 50 ml volumetric flask and about 30 ml of mobile phase was added ; the mixture was extracted in the ultrasonic bath for 10 min at room temperature and diluted with mobile phase to the mark . two millilitre of this solution was transferred to the 10 ml volumetric flask and diluted with mobile phase to the mark and 10 l was injected into the chromatographic system . the ability of an analytical method to unequivocally assess the analyte in the presence of other component in the formulation ( impurities , degradations , excipients ) can be demonstrated by evaluating specificity . the specificity of the method was determined by injecting placebo solution having the same concentration as that of the tablet solution . forced degradation studies of the tablet sample tablet samples were prepared and degraded under stress conditions like acidic hydrolysis , basic hydrolysis , oxidative degradation , photo degradation and thermal degradation for hplc method . for acid , base and oxidative degradation , samples were individually placed into three volumetric flasks and then 0.1 m hcl , 0.1 m naoh and 3% h2o2 were added separately into the flasks . all the three flasks were then heated in a water bath at 80 for 4 h. acid and base treated sample were neutralised and all the three samples were then diluted to a concentration of 0.3 mg / ml with the mobile phase . for thermal degradation sample was exposed to heat at 60 for 4 h and for photo degradation , the drug sample was exposed under a uv lamp for 24 h. the samples were withdrawn and analysed using hplc / dad uv . stock solutions of met and pro were prepared in mobile phase at concentrations of 1 mg / ml ( s1 ) . series of standards for each of the substance was prepared by progressive dilution of the stock solution for calibration study . fifteen millilitre aliquot of s1 was transferred to another 50 ml volumetric flask and diluted in mobile phase yielding a final concentration of 0.3 mg / ml . the mean weight of finally powdered pro tablets containing 80 mg of pro was accurately transferred into 50 ml volumetric flask and about 30 ml of mobile phase was added ; the mixture was extracted in the ultrasonic bath for 10 min at room temperature and diluted with mobile phase to the mark . two millilitre of this solution was transferred to the 10 ml volumetric flask and diluted with mobile phase to the mark and 10 l was injected into the chromatographic system . the ability of an analytical method to unequivocally assess the analyte in the presence of other component in the formulation ( impurities , degradations , excipients ) can be demonstrated by evaluating specificity . the specificity of the method was determined by injecting placebo solution having the same concentration as that of the tablet solution . tablet samples were prepared and degraded under stress conditions like acidic hydrolysis , basic hydrolysis , oxidative degradation , photo degradation and thermal degradation for hplc method . for acid , base and oxidative degradation , samples were individually placed into three volumetric flasks and then 0.1 m hcl , 0.1 m naoh and 3% h2o2 were added separately into the flasks . all the three flasks were then heated in a water bath at 80 for 4 h. acid and base treated sample were neutralised and all the three samples were then diluted to a concentration of 0.3 mg / ml with the mobile phase . for thermal degradation sample was exposed to heat at 60 for 4 h and for photo degradation , the drug sample was exposed under a uv lamp for 24 h. the samples were withdrawn and analysed using hplc / dad uv . the procedure for the simultaneous analysis of met and pro using isocratic hplc / dad uv method is reported . the mobile phase was chosen after several trials with methanol , acetonitrile , water and buffer solutions in various compositions and at different ph values . the best separation was obtained using the mobile phase consisted of a mixture of 50 mm pyrrolidine ( ph 11.5 ) and acetonitrile ( acn ) in ratio of ( 50:50 , v / v ) . in reversed - phase hplc , buffers are required when the sample contains ionic or ionisable analytes . without a buffer , poor peak shape and variable retention the organic buffer pyrrolidine ( pka 11.1 ) was chosen for optimum column life time as phosphate buffers accelerate the dissolution of silica at ph>7 . a flow rate of 1 ml / min gave an optimal signal to noise ratio with excellent separation time . the diode - array uv absorbance detector was set at 200 to 400 nm and met and pro drug components were extracted at maximum absorption at 214 nm and this optimal wavelength was chosen for the assay method . the separation of basic compounds requires special reversed - phase ( rp ) chromatographic sorbents . retention , selectivity and peak symmetry of basic compounds are strongly been influenced by the silica matrix . strongly distorted peaks of the basic compounds are often been observed when unsuitable rp sorbents are used , due to the interaction of the basic compounds with un - reacted silica gel ( sioh ) groups on the silica matrix . xterra rp18 is a spherical porous silica carrier , in which the starting silica material optimised in order to prevent any secondary interactions with basic compounds . the usage of this type of column allows separation of basic compounds such as met and pro with dissociation constants values ( pka ) of 9.9 and 9 , respectively without the need of ion pair reagents . using xterra rp18 , the retention times for met and pro were found to be 2.30 and 2.86 min , respectively . total time of analysis was < 4 min . using these optimised conditions , typical chromatogram obtained the robustness of the analytical method is defined as the measure of its capacity to remain unaffected by small but deliberate variations in the method parameters and provides an indication of its reliability during normal usage . one way to gauge robustness is to examine some relevant factors , which might influence the reliability of the developed method . selected factors , namely the mobile phase composition ( 2 ml ) , flow rate ( 0.1 unit ) , temperature ( 2 ) , wavelength ( 5 nm ) and column from different lots were investigated . in all cases , good separations of both drug components were always achieved , indicating that the analytical method remained selective and robust under the optimised conditions . hplc chromatogram obtained from sample : ( a ) met ( tr=2.30 min ) and pro ( tr=2.86 min ) and ( b ) placebo system suitability testing verifies that the hplc system is working as expected . it is based on the concept that the equipment , electronics , analytical operations and samples to be analysed constitute an integral system . system suitability was evaluated by injecting a solution of met and pro drugs at 100% test concentration ( 0.3 mg / ml ) in six replicates at the beginning of the validation run . 2a ) , such as peak capacity factor ( k ) , tailing factor ( t ) , resolution factor ( rs ) , theoretical plate numbers ( n , column efficiency ) and percent relative standard deviation ( rsd ) of peak areas are given in table 1 . the obtained values of these parameters ( 1<k<10 , t2 , rs>2 , n>2000 ) show that the proposed chromatographic conditions are suitable for separation of the analysed drug components . the values for the injection repeatability ( rsd < 2% , n=6 ) show that the system is precise . system suitability test parameter linearity test solutions were prepared by diluting stock solution ( 1 mg / ml ) at six concentration levels from 25 to 200% of analytes concentration ( 0.075 - 0.6 mg / ml ) for both drugs . the solution was injected in triplicate and curves were obtained by plotting the peak area against concentration of the drugs . the mean of two different calibration graphs yielded the following equations : y=203.17x-0.2552 ( r=0.9998 ) for met and y=133.99x+5.0261 ( r=0.9999 ) for pro ( table 2 ) . an excellent correlation exists between the peak areas and concentration of met and pro drugs . method validation results the precision of the test method was demonstrated by intra - day and inter - day variation studies . the intra - day ( repeatability ) studies were carried out by injecting six repeated injections of standard solution of 0.3 mg / ml on the same day , by one analyst under the same experimental conditions . the rsd values for peak areas were found to be 0.27% and 0.19% , respectively ( table 2 ) . the intermediate precision ( inter - day variation ) was studied over three consecutive days at three different concentration levels 0.15 , 0.3 and 0.45 mg / ml for met and pro that cover the assay range ( 80 - 120% ) . the mean and rsd across the system were calculated from the individual peak area % purity mean values at the 50 , 100 and 150% testing amounts . 3 ) , and met the intermediate precision criteria ( rsd < 2% ) which illustrated the good precision of this analytical method . comparison of intermediate precision evaluated over three days at three different concentration levels of drug analytes ( n=3 ) day 1 , day 2 , day 3 the accuracy of an analytical method is determined by how close the test results obtained by that method come to the true value . it can be determined by application of the analytical procedure to an analyte of known purity ( for the drug substance ) or by recovery studies , where a known amount of standard is spiked in the placebo ( for drug product ) . in the present study , a number of different solutions were prepared with a known added amount of 50 , 100 and 150% for met and pro drug components and injected in triplicate ( n=3 ) . percent recoveries of response factor ( area and concentration ) were calculated ranged from 99.62 - 101.07% ( table 2 ) which indicated the accuracy of the method was accurate within the desired range . injections of the placebo were performed to demonstrate the absence of interference with the elution of the met and pro drugs . 2b ) demonstrate that there was no interference from the other compounds and , therefore , confirm the specificity of the method . for the further evaluation of the selectivity of the hplc method , the forcibly degraded tablet sample solutions prepared by subjecting the tablet samples to such stress conditions as acid , base , heat , light and oxidative agent were determined under the proposed optimised hplc conditions . a summary data of the stress results is shown in table 3 , which showed no changes in retention times of drug components and no degradation peaks were observed . this was further confirmed by peak purity analysis on a hplc / dad uv and , therefore , confirms the specificity of the method . results of the stress conditions experiments the limit of detection ( lod ) and limit of quantitation ( loq ) were determined by the calibration plot method . a specific calibration plot was constructed using samples containing amounts of analytes in the range of lod and loq . the values of lod were 0.85 g / ml ( s / n=3.2 ) and 0.95 g / ml ( s / n=3.3 ) and loq were 2 g / ml ( s / n=10.3 ) and 2.5 g / ml ( s / n=10.2 ) for met and pro , respectively . lod and loq were calculated by using the equations : lod = cdsyx / b and loq = cqsyx / b where cd and cq are the coefficients for lod and loq , syx is the residual variance of the regression , and b is the slope . precision at the limits of quantitation and detection was checked by analysis of six test solutions prepared at three levels . the rsd values for peak area were < 5% for lod and < 2% for loq solutions the stability of analyte stock solutions with mobile phase as solvent , stored at room temperature , was studied for 48 h. no analyte ( met or pro ) degradation was detected during this time period ( < 2% ) . to demonstrate the applicability of the present method , commercially available three batches of tablets containing 5 mg met and assay results for three samples of tablets expressed as the percentage of the label claim , were found 98.40 - 101.02 for met and 98.66 to 102.11% for pro . results showed ( table 4 ) that the content of met and pro in tablet formulation were to the counter requirements ( 90 - 110% of the label claim ) . the above results demonstrated that the developed method achieved rapid and accurate determination of compound studied and can be used for the simultaneous determination of met and pro in drug substances and pharmaceutical formulations . recovery studies of methamphetamine and propranolol content in tablets a new isocratic rp - hplc / dad uv method has been developed for the determination of met and pro . low cost , environment friendly , faster speed analysis , and satisfactory precision and accuracy are the main features of this method . the method was critically validated and statistical analysis of generated high quality data proves that the method is sensitive , specific and robust . the method is stability - indicating and can be conveniently applied for the testing of studied components raw materials , in tablet formulations and batch release by industry .

a new isocratic reversed - phase hplc method with diode - array uv detection was developed and validated for the determination of methamphetamine and propranolol in tablet dosage forms . chromatography was carried out on an xterra rp18 ( 1504.6 mm , 5 m ) column using 50 mm pyrrolidine ( ph 11.5 ) acetonitrile ( 50:50 , v / v ) as mobile phase at a flow rate of 1 ml / min . spectrophotometric detection was performed at a wavelength of 214 nm . the linearity was established over the concentration range of 0.075 - 0.60 mg / ml for both drugs . the correlation coefficients ( r2 ) were 0.9998 in each case . the relative standard deviation values for intermediate precision studies were < 1% . statistical analysis of the data showed that the method was precise , accurate , reproducible and selective for the analysis of methamphetamine and propranolol drugs . the method was successfully employed for the determination of propranolol and methamphetamine in commercially available tablet dosage form .

molecular dynamics ( mds ) simulation is a powerful tool to study biomolecular processes an atomistic detail [ 1 , 2 ] . processes like molecular encounter , that have been traditionally studied by brownian dynamics simulations [ 35 ] have also been addressed by md simulation in the last decade [ 614 ] . the time required for assembly processes may be exceedingly large , and for this reason implicit solvent or coarse - grained models have been used [ 9 , 1520 ] . for these models should catch , through relevant energy terms , the essence of the phenomena to be simulated and therefore provide a likely representation of the simulated process . if the interest is in the encounter of proteins or of a protein and a ligand , much shorter times are required , and if the conformational changes in the molecules are not large , translational and rotational diffusion of the systems ( possibly at high concentration , in order to increase sampling ) provides a large number of encounters possibly representative of different frequencies and lifetimes . in order to address recognition at the atomic level , we have performed recently all - atom explicit solvent simulations using a high concentration of interacting molecules [ 10 , 12 , 13 ] . the aim of this paper is not to provide an extensive review of methods and results , but rather to consider the practical problems for setting up the simulation and for analysing the results of the simulation . in setting up simulations aimed at observing molecular encounters , the spacing of the molecules and total simulation time should be considered carefully in order to allow translational and rotational diffusion to take place effectively during the simulation . an excellent review about biomolecular hydrodynamics and diffusion has been given by bloomfield in an online review , which is unfortunately not available anymore , and by cantor and schimmel in their textbook . the topic is also discussed by hunter in his treatise on colloid science . for translational diffusion the einstein - smoluchowski equation relates the average quadratic displacement to time ( 1)||x||2=2ddtt , with a standard deviation equal to 2||x||2 , where dt is the diffusion coefficient and d is the dimensionality ( 3 in standard molecular dynamics simulations ) . the translational diffusion constant dt depends on the shape of the molecule and must be computed numerically under proper assumptions . when the molecule may be considered as a sphere the stokes - einstein equation relates the diffusion coefficient dt to the solvent and solute properties : ( 2)dtsphere = kt6r , where k is the boltzmann constant , t is the temperature , r is the hydrodynamic radius of the sphere , and is the medium viscosity . sometimes proteins do not resemble spheres , but they rather have an elongated shape that can be approximated by a prolate spheroid , that is , an ellipsoid with the two short axes of equal length b and a long axis of length a , with q = b / a . in this case , the above relations are generalized with respect to an equivalent sphere of radius r = ( ab ) [ 2224 ] . for the translational diffusion coefficient we have ( 3)dtps = dtsphereq2/3log ( ( 1+(1q2))/q)(1q2 ) . for the simulation to be able to sample reciprocal orientations , the simulation time must be long enough as to allow reorientation of molecules . for rotational diffusion , the debye relaxation equation holds . for a set of molecules with one axis aligned with the z - axis at time 0 : ( 4)cos()=exp(2drt ) , where is the angle of the same axis with the z - axis at time t. based on ( 4 ) reported in the work by smith and van gunsteren , the standard deviation is ( 5)13e4drt+23e6drt . for the rotational diffusion coefficient of a sphere , an equation similar to the stokes - einstein equation , the stokes - einstein - debye relation , holds : ( 6)drsphere = kt8r3. for elongated molecules , frictional coefficients for rotations about the long and short axis are computed first as ( 7)frps(l)=frsphere 2(1q2)3(1q2(1q2)1/2 log((1+(1q2))/q)),frps(s)=frsphere 2(1q4)3q2(2q2)(1q2)1/2log((1+(1q2))/(q3q2 ) ) . then rotational diffusion coefficients are computed based on the frictional coefficients as ( 8)drps(l)=drsphere(frps(s)frsphere)1,drps(s)=drsphere2((frps(s)frsphere)1+(frps(l)frsphere)1 ) , and where dr(l ) and dr(s ) are the diffusion constants that enter the debye relaxation equation for reorientation of vectors along the long and short axes , respectively . for water , the viscosity has a sharp dependence on temperature which can be described by the following equation reported in ( and corrected here ) : ( 9)log10=1301998.333 + 8.1855(t20)+0.00585(t20)2 4.30233 pa s , in the range 020 c and ( 10)log1020=1.3272(20t)0.001053(t20)2t+105 , in the range 20100 c , with the temperature t in celsius degrees . at 20 c , this results in = 1.0 10 pa s. for a globular protein with hydrodynamic radius of 20 ( corresponding to a molecular weight of ca . 28 kda ) , the rotational diffusion constant dr at 20 c is 2.0 10 s which corresponds to a rotational time constant of 25 ns . the rotational autocorrelation time scales linearly with the mass of the molecule and with the third power of the radius . note that the rotational time constants considered here are 3 times longer than those considered in nmr experiments , where a different function of ( 1/2(3cos( ) 1 ) is used for defining rotational diffusion . the dependence of the diffusion coefficients on the shape is not much pronounced . for long - to - short axis ratios that do not exceed 1.5 , the rotational time constant increases by less than 20% . in practice , the size of the system , but not its shape , for typical proteins , determines the choice of the simulation time . from the above considerations , it is seen that simulations in the range of tens to one hundred of nanoseconds should allow complete reorientation of typical proteins studied by molecular dynamics simulations . for convenience , considering an average protein molar volume 0.73 cm g , the radius ( in ) of a protein of molecular weight mw da is the hydrodynamic radius of the protein may be larger due to hydration water by up to ca . 3.0 . the rotational time constant ( in ns ) at 20 c is ( 12)0.0031r3 , or three times less when the decay of the quantity 1/2(3cos( ) 1 is considered . during one rotational time constant , under the same solvent conditions , the molecule will experience a root mean square displacement equal to its hydrodynamic diameter . for practical purposes , therefore , one may first estimate the rotational correlation time constant 1/2dr and setup the simulation time , to say , at least twice the rotational correlation time . during this time the spacing of the molecules ( i.e. , center - to - center distance ) can be chosen therefore equal to four - to - five times the hydrodynamic radius , so that the molecules will get in contact on average after one - to - two rotational correlation times . the spacing will most likely be reduced in order to compromise with the need for a limited - size system . molecules are arranged in a regular way in order to maximize the chances of encounters , and also they are randomly oriented in order to maximize the number of relative orientations sampled . for arrangement on a cubic periodic lattice n should be larger than 2 to ensure that the 26 nearest neighbours are truly different molecules and not just molecules and their periodic images . the spacing of the lattice should be of the order of few molecular radii in such a way that reoriention may take place before encounter , as discussed above . for protein - protein association , these prescriptions may lead to a very large system size with consequent large number of atoms , which in turn may limit the simulation time . for protein - small molecule association , the above requirements are easily met . it is often reported that molecules may be randomly rotated by choosing a random rotation axis and a random rotation angle . a simple way to do this is to choose a random axis in space and perform a rotation by a random angle about that axis . although this procedure is reasonable , and a fast algorithm implementing this idea is available , the resulting distribution of rotations is not uniform . a truly uniform distribution is obtained by taking random unit quaternions , which can be generated in a simple way by taking four random quantities ( w , x , y , and z ) between 1 and 1 subject to the condition w + x + y + z 1.0 , and normalizing them to 1.0 . the four normalized values are related to the rotation axis v and the rotation angle as follows : ( 13)(w , x , y , z)=(cos(2),vsin(2 ) ) , and the corresponding rotation matrix is ( 14)r(w , x , y , z ) = ( 12(y2+z2)2xy2wz2xz+2wy2xy+2wz12(x2+z2)2yz2wx2xz2wy2yz+2wx12(x2+y2 ) ) . besides traditional analyses that are typically performed on molecular dynamics trajectories ( see e.g. , those provided with the package gromacs ) , it is obvious to check that the molecule 's translational and rotational diffusion during the simulation is taking place as expected . for translation , it is sufficient to monitor the center of mass of each molecule as a function of time and apply due corrections for periodic boundary conditions . as long as the particles do not travel an entire periodic box length , the correction for periodic boundary crossing is very easily applied . the average square of the distance from the starting position can be fitted as a linear function of time , the proportionality constant being six times the translational diffusion constant dt . for the rotational diffusion constant , each molecule may be superimposed to the starting structure using programs like profit . the rotation matrix may then be used to obtain the dot product between any given unit vector before and after rotation , that is , v rv , which is in turn equal to the cosine of the angle between the two vectors . averaging overing all possible unit vectors leads to the average cosine value ( cos() ) , entering the rotational diffusion equation . the average is equal to the trace of the rotation matrix divided by 3 , that is , v rvv = ( rxx + ryy + rzz)/3 . in turn , the average of the latter quantity over all molecules can be fitted to an exponential whose time decay constant is twice the rotational diffusion constant dr . except for long - range electrostatic effects , the use of a large number of identical molecules increases the number of both random and specific contacts . the affinity of molecular regions between themselves and with small ligands may be measured by the count of contacts . contacts are typically defined using a cutoff on the distance between heavy atoms . a typical value , when one considers all heavy atoms , is 4.5 ( e.g. , ) . a different definition was used by berrera et al . to take into account the different size of atoms . in this definition , two atoms are in contact when their van der waals surfaces are closer than 1.0 . only heavy atoms are considered and the radii are 1.4 for oxygen , 1.5 for nitrogen , 1.9 for carbon , and 1.85 for sulphur . we found convenient to map all atoms at nodes of a periodic cubic lattice and then list contacts within the atoms assigned to one node and with the atoms assigned to the 26 neighbouring nodes . the lists of contacts corresponding to different snapshots are grouped together , and the number of occurrences of each atomic contact is counted . the cumulative counts of atomics contacts , independent of the specific molecules interacting , provide the affinity of a contact . for instance , the same hydrophobic part of different copies of a protein may be contacted by the same hydrophobic part of different copies of a ligand . this will result in a large cumulative number of the same atomic contacts , although due to different interacting molecules . the frequency of atomic contacts ( independent of the specific molecular copies involved ) is a measure of the affinity , but it does not tell how long a single ligand resides at a given location . in this respect , we can take advantage of the fact that , if the number of interacting molecules is large , the probability of observing the same long - lived contacts for the same molecular copies is very low . in such situation , the count of atomic contacts for specific molecules is proportional to the time the two atoms are close to each other continuously . the analysis of most frequent contacts just requires a sufficient number of snapshots spread over an interval of time sufficient to sample all possible contacts . the analysis of longest - lived contacts requires snapshots to be taken at time intervals which are fractions of the time used for defining a long - lived contact . for instance , if 0.5 ns is considered as a long - residence time than snapshots at 100 ps guarantees that a long - lived contact will count at least five times more than a short - lived contact . beta 2-microglobulin is a 99-residue protein which is responsible of dialysis - related amyloidosis [ 33 , 34 ] . due to its small size and its solubility it has been extensively studied in recent years , and much knowledge has been gained on its structural and dynamical properties in relation with the onset of aggregation [ 35 , 36 ] . the early steps of protein - protein association have been studied by simulating 27 copies of the protein in explicit solvent . all the details and general context of the simulation are reported in the original paper . here , we will address only the issue of how the system was set up . with a molecular weight of 11.7 kda , the hydrodynamic radius estimated from ( 11 ) the simulation was run at 300 k. at this temperature , the viscosity of water is 0.85 10 pa s. the predicted rotational diffusion time is therefore 8.7 ns , according to the rotational diffusion constant estimated using ( 6 ) . this choice was dictated by the computational time available at the time and by the large size of the simulated system ( ca . the large number of reciprocal orientations between pairs of molecules should , however , compensate for the short simulation time . note also that the standard deviation of cos() is fastly increasing towards the limiting value 1/3 . during the simulation time , the molecules should travel on average 22.7 based on the translational diffusional coefficient estimated using ( 2 ) . for this reason , the spacing of molecules was chosen 50 in such a way that the average van der waals surface to van der waals surface distance between closest molecules was slightly less ( 20 ) than the average displacement of the molecules . the intermolecular distance was not reduced further in order to avoid close contacts of the randomly oriented molecules which have an elongated shape . this system illustrates well the kind of considerations and compromises one is to face when dealing with an ensemble of large molecules . notwithstanding the nonoptimal simulation parameters , the contact analysis of the trajectory suggested a predominant role of trp 60 in the early association of beta 2-microglobulin . following that study , experiments with the w60 g mutant showed that indeed the mutation resulted in absence of formation of fibrils by the protein in nonextreme conditions , as a combined result of increased stability and removal of the aromatic chain of trp 60 prone to intramolecular contacts . glutathione peroxidases were the first seleno - enzymes that were discovered in mammals and up to 8 distinct members have been detected so far . most of them are selenoproteins ( the classical gpx-1 then gpx-2 , gpx-3 , gpx-4 and , depending on species , gpx-6 ) , while the remaining 2 or 3 variants have a cysteine in the active site . they are included in the heme - free thiol peroxidase class together with peroxiredoxins and catalyze the reduction of h2o2 or organic hydroperoxides to water or corresponding alcohols , thus mitigating their toxicity . the catalytic mechanism is still a question of open debate and could be different for selenium versus sulphur - based enzymes . presently , the commonly accepted kinetic is an enzyme substitution mechanism , as revealed by ping - pong kinetics rather than classical michaelis - menten kinetics . this implies that the catalytic mechanisms do not involve any central complexes of the enzymes with all the substrates bound simultaneously . instead , the catalytic cycles are composed of sequences of bimolecular reactions between the enzymes and their substrates . the first one is the oxidation of the catalytic site to a selenenic or sulfenic acid derivative after collision with a hydroperoxide , and the second one is its reduction by a reducing substrate . following a previous study , the structure of drosophila melanogaster glutathione peroxidase was surrounded by 198 randomly oriented hydrogen peroxide molecules . for such small molecule the translational and rotational coefficients may be estimated , according to ( 2 ) and ( 6 ) . the hydrodynamic radius of hydrogen peroxide may be estimated in the range of 2.1 to 5.1 for the absence or presence of hydration , respectively , based on its elongated shape and the average hydrogen to hydrogen distance of 2.4 . the van der waals longest dimension would thus be 4.4 , whereas the shortest dimension of the molecule is the radius of the oxygen ( 1.4 ) . the upper limit has been taken 5.1 in order to account the possibility of hydration , increasing typically the minimum hydrodynamics radius by 3.0 . the range of estimates for the translational and diffusional coefficients for hydrogen peroxide are thus 12.3 to 5.0 10 m s and 21.0 to 1.5 10 s , respectively at 300 k. these figures mean that reorientation will take place in the ns timescale and that in half nanoseconds a molecule is expected to travel on average 12 . hydrogen peroxide molecules were arranged on a 6 6 6 grid with 12 spacing in each dimension ( figure 1 ) . after solvent was added and following relaxation and equilibration , the size of the system was 64.6 64.6 64.6 entailing 27300 atoms ( figure 1 ) . the concentration of hydrogen peroxide was unrealistically high ( 1.25 m ) in order to increase the number of molecular encounters . in practice , however , the consequences of this choice are not so important because water concentration is still dominant and no chemical reactions can take place in simulations . based on the above figures , the total simulation time was set to 10 ns or larger in order to let all molecules reach the target protein and to possibly probe multiple binding events . for all simulations , the forcefield used is charmm v.27 with the cmap correction except where mentioned . forcefield parameters for hydrogen peroxide were taken from the study of de gioia and fantucci . solute molecules , including ions and cosolutes , were fixed , and the system was energy - minimized by 300 conjugate gradients steps using periodic boundary conditions and the particle mesh ewald ( pme ) method for electrostatic interactions . for all simulations , pme employed a grid of 128 128 128 points corresponding to a spacing of ca the pme tolerance was set to 10 that , together with the cutoff of 12 , resulted in an ewald coefficient of 0.257952 . the minimized system was further relaxed , keeping the solute molecules ( including ions ) fixed , by molecular dynamics simulation . the system was heated to 300 k in 2 ps , and a further 18 ps simulation was run in order to let water molecules reorient , consistent with the average lifetime of a hydrogen bond in water . the system without restraints on solute molecules was energy - minimized by 300 conjugate gradients minimization steps . the system was then heated to 300 k in 2 ps , and a further 1.118 ns simulation was run in order to let the system equilibrate and finally production run could start . the temperature was kept constant through a simple velocity rescaling procedure , with a relaxation time of 1 ps , while the pressure was controlled through a berendsen bath using a relaxation time of 100 fs , the default value in the program namd . it is well known that a simple rescaling procedure does not provide a correct canonical ensemble and extended - system approaches have been proposed to simulate correct thermodynamic ensembles [ 4952 ] . recently a stochastic velocity rescaling has been proposed that could provide correct ensemble with limited overhead computation . however , the adopted protocol should not be detrimental for the kind of analysis presented here . for all simulations , the size of the box was fluctuating around its average value within fractions of . we have used high - concentration molecular dynamics simulations in previous works that addressed different problems [ 10 , 12 , 13 ] . many analyses have been already reported in those works . as illustrations of the protocols described in the methods section , we consider two applications : the simulation of the association of an amyloidogenic protein ( beta 2-microglobulin ) which illustrates the setup of the simulation system , detailed in the methods section , and the analysis of rotational and translational diffusion constants reported hereafter ; the simulation of the encounter of hydrogen peroxide with glutathione peroxidase which illustrates the analysis and the general usefulness of the approach . the value of cos() is computed as ( 1/3)tr(r ) where r is the matrix that describes the rotation of the molecule with respect to its orientation at the start of the simulation , and tr denotes the trace operation , as discussed in section 2 . the average cos() is further averaged over all 27 molecules , thus providing also standard deviation . the time constant ( 9.6 ns ) obtained fitting the decay with an exponential is larger than expected ( 8.7 ns ) , but still consistent with the size of the protein . analogously , the diffusion coefficient of the molecule is 20.9 ns , close to what expected ( 17.2 ns ) ( figure 3 ) . also here the square displacement is averaged over all 27 molecules . similar results are obtained by considering the average translation and rotation in a small interval of time and fitting the linearized exponential equations of diffusion reported above . for hydrogen peroxide , the translational diffusion constant was 443 ns , and orientation was randomized in less than 0.01 ns . two simulations were performed with different seed number , one for 10 ns and one for 30 ns . one simulation ( 20 ns long ) , was performed with the catalytic cys 36 ionized . in one simulation ( 10 ns long ) the backbone dihedral angle energy correction term cmap was not applied in order to test the effect of different backbone mobility . only few hydrogen peroxide molecules establish long - lived contacts with the peroxidase in each simulation . ideally , we would like to sample the same long - lived contact many times during each simulation or at least in different simulations . although in all simulations at least one hydrogen peroxide molecule reaches the catalytic region establishing contacts for more than 500 ps , different long - lived contacts are observed in different simulations . the different results obtained in the two simulations differing only for the seed number point out that a larger number of simulations should be performed for exploring all possible encounter events . for all simulations , snapshots were taken at 0.1 ns time intervals . as detailed in the methods section , this led to a list of the atoms of the protein contacted by hydrogen peroxide molecules together with the number of occurrences of such contacts . with the definition of contact adopted here , on average there are 386 hydrogen peroxide - protein contacts for each snapshot in the longest simulation . the number of different pairs of atoms involved in contacts in the 300 snapshots for the longest simulation is 71830 . of these , only 2000 are involved in contacts lasting more than 0.5 ns . most frequently contacted atoms overlap in some cases with the longest - lived contacts . as observed with other small molecules , it is not infrequent that some of the hydrogen peroxide molecules gets trapped in protein cavities and stay for times even longer than 20 ns , as observed in the longest simulation . one such cavity is defined by residues { k45 , l46 , l49 , k130 , t147 , d148 , p149 , i152}. other cavities are defined by other residues throughout the protein . in all simulations , there were contacts of hydrogen peroxide with residues in the catalytic site lasting at least 0.5 ns . the contacting residues in the catalytic site are { c36 , l38 , n42 , w126 , n127 , f128 , p145}. in general , however , these were not the longest - lived contacts found . the arrangement of the catalytic site is rather stable for the simulations using the cmap energy correction , with fluctuations at the backbone of the residues { c36 , q71 , w126 , n127 } on average 0.45 0.15 , much lower than the fluctuations displayed by loops . in the same simulations also , hydrogen peroxide is able to reach the catalytic site with no aid from other sidechains as can be seen following the trajectories of the molecules before reaching glutathione peroxidase . however , in several cases the hydrogen peroxide reaches the catalytic site by shifting between the c - terminal residues and the helix following c36 or approaching the catalytic site from the side of c36 and w126 . when the cmap correction energy term is not included in the forcefield , the region entailing c36 moves apart from the other residues and a hydrogen peroxide molecule can enter the cavity entailing residues i32 , a33 , c36 , and f128 . in view of the low stability of the catalytic region the distances of atoms w126 he1 , n127 hd2 , and q71 he2 with the peroxidatic cysteine c36 sg are overall short . as evidenced by other works , these interactions lead to a stabilization of the developing negative charge at the sulphur atom upon deprotonation . during the simulation , an unexpected interaction is conserved between the hydrogen of hydrogen peroxide and the carbonyl oxygen of residue n127 . besides these hydrogen bonds , the sidechain of n127 establishes a hydrogen bond with the thiol group of c65 . a representative snapshot of the sidechains involved in this fluctuating network of hydrogen bonds is shown in figure 4 . residues whose sidechains are shown as sticks are q71 , w126 , n127 , c36 , and c65 . once a hydrogen peroxide molecule exhibiting a long - lived contact has been found , its interactions with the protein may be obtained throughout the simulation ( not only at 0.1 ns - spaced snapshots ) by using efficient programs like vmd . in figure 5 , as said above , the different results obtained in the different simulations point out the need to run more and longer simulations in order to acquire statistics . on the other hand , the results and the other examples cited prove how effective can be high - concentration molecular dynamics simulations .

molecular dynamics simulations have been used to study molecular encounters and recognition . in recent works , simulations using high concentration of interacting molecules have been performed . in this paper , we consider the practical problems for setting up the simulation and to analyse the results of the simulation . the simulation of beta 2-microglobulin association and the simulation of the binding of hydrogen peroxide by glutathione peroxidase are provided as examples .

type vii total laparoscopic hysterectomy ( tlh ) is a laparoscopic hysterectomy in which all of the surgical dissections , ligations , and sutures are completed entirely through the trocars , including the closure of the vagina . when compared with abdominal hysterectomy ( ah ) and laparoscopic - assisted vaginal hysterectomies ( lavh ) , tlh has been reported to result in shorter procedure durations , lower blood losses , and shorter hospital stays . typically , vaginal hysterectomy ( vh ) and lavh are performed in patients with at least moderate prolapse usually associated with parity , but some of these patients may later develop vaginal prolapse or incontinence . one recent report describes recurrence of prolapse in 12% of patients having a vaginal hysterectomy with vaginal vault suspension compared with 4% having a laparoscopic uterosacral ligament uterine suspension . among nulliparous patients , the vaginal dissection may be difficult due to absence of prolapse and small vaginal capacity and may result in more complications . tlh may offer a minimal blood loss , short hospital stay , and be practicable in most women with minimal risk of complications , but randomized trials are lacking . a recent cochrane meta - analysis of hysterectomy approaches reviewed 24 randomized trials of 3643 women , but only 2 trials with 71 women were identified for type vii tlh . very few contemporary newly trained gynecologists will have sufficient expertise and confidence to tackle tlh , which requires the highest level of surgical skill . they concluded that [ t]he newest approach to hysterectomy ( tlh ) should be further evaluated . it is speculated that if a tlh technique used familiar open technique standards and was observed to be safe in a large retrospective series , potentially more surgeons would learn and safely perform the technique , and randomized controlled trials could be designed to compare the various routes . in this article , technique and complications are reviewed and analyzed in great detail so that surgeons learning and newly performing tlh may avoid complications , and subsequent randomized trials could be planned . all tlh cases were identified from computerized billing lists by using the current procedure terminology codes for all types of hysterectomy . approval by the investigation review board at sequoia hospital in redwood city , california , has been maintained with yearly updates . all consecutive , eligible patients were offered simple tlh for every benign gynecologic indication , stage ia1 ( < 37 mm invasion ) cervical cancer , occult ovarian cancer , and clinical stage iia or less endometrial cancer . patients were ineligible if they had documented severe abdominal adhesions from a prior operative report , or endometrial cancer in a uterus too large for intact removal through the vagina . every surgery was performed by the author ( kao'h ) from september 5 , 1996 to april 1 , 2006 , at 4 california hospitals , assisted by a categorical obstetrics and gynecology resident , a gynecologist , or a general surgeon . for clarification in this article , the technique used for this type vii tlh the patient is in modified lithotomy position with hips extended , in a 40-degree angle trendelenburg position . shoulder bolsters prevent slippage up the table , and the arms are padded and tucked by the side . after the examination with the patient anesthetized , a humi uterine manipulator ( cooper surgical , inc . trumbull , ct , usa ) is inserted , unless the patient has endometrial cancer , in which case the manipulator is inserted after laparoscopic washings and bipolar cautery occlusion of the proximal fallopian tubes . after visual inspection of the ureter at the pelvic brim , the infundibulopelvic ligament , or , utero - ovarian ligament is coagulated and incised with an ultrasonic scalpel ( ethicon endo - surgery , cincinnati , oh , usa ) . immediately after the round ligament is incised , the uterus , on the uterine manipulator , is pushed cephalad to recreate the traction - counter - traction a bladder flap is incised and the anterior cervical fascia is exposed with blunt dissection off of the cervix broadly below the cervicovaginal margin . the uterine arteries are coagulated with a bipolar cautery at the mid lower cervical length , and then incised with the ultrasonic scalpel . the uterine arteries are pushed downward to expose the cardinal ligament fibers attaching the arteries to the cylindrical cervical fascia ; then the cardinal fibers are incised posteriorly to the uterosacral ligaments , and inferiorly , identifying the cervicovaginal margin as the lowest limit of dissection . the cervicovaginal margin is laparoscopically palpated with the laparoscopic instruments by using visual and manual cues to delineate the posterior margin , the lateral edges , and the anterior margin of the cervical stroma ( figure 1 ) . typically , the cervical stroma is firm and moves as a solid object , and the vaginal wall is pliant and dimples with pressure . if the cervicovaginal margin is not obvious , a thin malleable ribbon is inserted into the anterior vagina and elevated , identifying the anterior - most cervicovaginal margin . then the ultrasonic scalpel incises into the vagina at the precise margin of the cervix and vagina ( figure 2 ) . when the pneumoperitoneum is lost , the uterine manipulator is removed and a surgical glove containing 2 fluffed 44 gauze sponges is inserted to obturate the lower vagina . two semb toothed cupped graspers ( # a5261 , olympus surgical , orangeburg , ny , usa ) are used to expose the cervicovaginal margin for ultrasonic incision around the cervix . a tenaculum is inserted through the vagina ( under or beside the glove ) to grasp the cervix and remove the uterus . when the uterus is large , but only in the absence of endometrial cancer , it is morcellated transvaginally . any pelvic mass is placed intact in a lapsac surgical tissue pouch ( # 054100 , cook urological , spencer , in , usa ) for frozen section . the edges of the pouch are brought out through the introitus , and a speculum is inserted into the pouch , allowing puncture or morcellation and removal of the mass with no intraperitoneal spillage . the vaginal apex is closed with # 1 polygalactic acid jk10 ( ethicon endo - surgery , cincinnati , oh , usa ) suture in 3 or more figure - of - eight ( technically spiral ) sutures with semb graspers and a wolfe needle driver ( # 8393.502 , richard wolf surgical instruments , vernon hills , il , usa ) , fixing the vaginal angle to the uterosacral ligaments for suspension . anova , chi - square , and spearman and pearson correlation techniques were used with significance set at p<0.05 . the right lower uterine segment is seen with suction irrigator delineating the posterior cervicovaginal margin . after incising the cardinal ligament , the pliant vaginal wall is palpated laterally , anteriorly , and posteriorly to confirm the location of the cervicovaginal margin . three had massive myomata in the lower uterine segment obstructing laparoscopic ligation of the uterine arteries . the fourth patient was converted to laparotomy to explore a limited retroperitoneal bleeding site that was identified as a likely trocar injury . the fifth patient had persistent bleeding of the left infundibulopelvic vessels that had retracted into the retroperitoneum . preoperative diagnoses additional procedures performed with total laparoscopic hysterectomy * some patients had more than one procedure . all cases of malignancy were managed with strict standards of oncological principles : pelvic masses were removed intact in a ripstop nylon bag . the typical patient in this series had a mean age of 50.1 years ( sd=11.0 ; range , 21 to 90 ) . the average parity was 1.3 ( sd=1.3 ; range , 0 to 9 ) with 38.2% ( n=315 ) of these women being nulliparous . their mean body mass index ( bmi ) was 27.8 kg / m ( sd=7 ; range , 16 to 71 ) with 57% ( n=468 ) of the women being in the overweight or higher bmi range ( > 24.9kg / m ) . surgical duration was abstracted from operating room records and included all gynecologic procedures and incidental appendectomy . patients requiring staging lymphadenectomy and omentectomy for malignancy , or other general surgical procedure such as cholecystectomy were not included in the duration analysis . the mean surgery duration was 130 minutes ( sd=56 ; range , 28 to 355 ) . over one fifth ( 23.7% ) of patients ' surgeries were completed in 90 minutes or less . neither age ( r=0.025 , p=0.513 ) , body mass index ( r=0.060 , p=0.115 ) , nor parity ( t=1.896 , p=0.058 ) impacted duration , but the increasing number of cases performed by the surgeon reduced duration ( r=0.448 , p<0.001 ) . blood loss was estimated by examining the contents in the canister before irrigation , or by surgeon and anesthesiologist agreement . the average blood loss was 130 ml ( sd=178 ; range , 0 to 1200 , median 75 ) with 16% of patients ( n=132 ) losing less than an estimated 10ml of blood . this estimate is made when there is virtually no observable blood lost during the procedure , little or none observed in the pelvic cul de sac at the end of the procedure , and little or none lost from the laparoscopic incisions . more than half of the patients lost less than 50 ml of blood . while age ( r = 0.018 , p=0.633 ) and bmi ( r=0.050 , p=0.184 ) did not correlate with blood loss , lower parity ( t = 2.91 , p=0.004 ) and increasing experience ( r=0.247 , p<0.001 ) correlated with reduced blood loss . transfusions , mostly related to complications , were needed in 3.0% of women ( 11.0 vs. 1.7% , p=0.0001 ) , especially a reoperative complication ( 13 vs. 2.4% , age was not significantly related to having a transfusion ( t=0.09 , p=0.93 ) , nor was bmi ( t=0.7109 , p=0.48 ) . however , those requiring transfusions had longer surgery ( t=3.50 , p=0.000 ) and more days in the hospital ( t=2.35 , p=0.028 ) . the median uterine weight was 160 , and the mean was 239 g , ( sd = 291 ; range , 50 to 3131 ) . of the 632 with recorded uterine weights , 16% were between 250 grams and 499 g , and 12% were between 500 grams and 3131 g. bmi did not impact uterine weight ( r=0.056 , p=0.156 ) ; however , increasing age correlated with smaller uteri ( r = 0.113 , p=0.004 ) . having a larger uterus correlated with increased surgical time ( r=0.268 , p<0.001 ) and more blood loss ( r=0.347 , p<0.001 ) . seven nulliparous , obese patients with bmi ranging from 31 to 46 , and massive uteri , had a tlh followed by a planned 6-cm pfannenstiel incision used only for evacuation of the disconnected uterine tissue , weighing 1047 g to 3131 g. only evacuation of tissue was performed through the pfannenstiel incisions . the pfannenstiel incisions were immediately closed , and the remainder of the case , including suture of the vaginotomy , was completed laparoscopically . had any portion of these cases required laparotomy for completion of the hysterectomy / oophorectomy dissection , these cases would have been classified as converted or as a laparoscopic - assisted abdominal hysterectomy . the average hospital stay was 1.37 days ( sd=0.97 ; range , 0 to 13 , median , 1 day ) . duration of hospital stay was recorded only by days , not hours ; however , 73% of patients left the hospital by lunchtime the next day . longer stays were not associated with age , bmi , parity , or uterine size , but did correlate with longer surgical times ( r=0.347 , p<0.001 ) and more blood loss ( r=0.346 , p<0.001 ) . the length of hospital stay decreased with the increasing number of cases performed by the surgeon ( r=0.452 , p<0.001 ) . complications occurred in 83 patients ( 10% ) , and of those complications about half required reoperation ( 39 patients , 4.7% ; table 3 ) . urologic complications occurred in 23 ( 2.8% ) patients and resulted in reoperation in about half ( 1.4% ) . complications were more likely to occur in women with a lower bmi ( or 0.95 ; 95% ci=0.90 to 0.99 ) , with a high surgical blood loss ( or 1.002 ; 95% ci=1.001 to 1.003 ) resulting in longer operating times ( or 1.007 ; 95% ci=1.003 to 1.012 ) and a longer length of stay in the hospital ( or 1.78 ; 95% ci=1.39 to 2.28 ) . the demographics , diagnoses , and surgicopathological data reflect a broad diversity of consecutive patients and demonstrate a broad utility for tlh . senior age and high bmi are not contraindications to tlh . because type vii tlh does not depend on vaginal descensus , capacity , or laxity , minimally invasive hysterectomy is available to more women , including the nulliparous and obese . with a 38% nulliparity rate , few of these patients would have qualified for a vh or an lavh . agostini et al report a high rate of successful vaginal hysterectomy in their 52 nulliparous patients , but their complication rate was 13.46% , higher than our total complication rate for this subset ( 10.82% ) , and much higher than our reoperative rate ( 4.5% ) in this subset . their hemorrhage rate was 7.69% , which was much higher than our transfusion rate of 3.2% . this report responds to their conclusion that laparoscopic assistance for nulliparous women having hysterectomy needed further investigation . their findings underscore the benefit of performing the entire surgery laparoscopically rather than struggling through a narrow vagina to reach a cervix with no descensus . it is unfortunate that operating time for tlh only was not recorded in the operating room records . the mean operating time of 132 minutes is thus an overestimate of the time needed for tlh only . the duration decreased with surgeon 's experience , with the last 100 cases taking 99 minutes or less . in controlled trials , type vii tlh has been observed to take the same or slightly more time than ah , similar or shorter time than lavh , and 53 minutes longer than vh . it is a weakness of this study that blood loss was not measured in a more precise fashion . in randomized trials , type vii tlh confers less blood loss than ah and lavh do , and confers a similar blood loss as that of vh . precise assessment of patient hospital stays would have required calculation of the hours from the end of surgery to the hour of discharge . patients stayed in the hospital 2 days ; however , in the second half of the series , it became routine to discharge patients on postoperative day one . this has caused no problems , and no readmissions have been observed for an extra day of observation . randomized trials reveal that type vii tlh confers a shorter hospital stay than do ah and lavh , and a similar stay as that of vh . uterine size did not correlate with increasing likelihood of complication ( p=0.6705 ) , urologic complication ( p=0.5782 ) , or conversion to laparotomy , as others have seen . lower uterine segment fibroids in 3 cases precluded access to the uterine arteries necessitating conversion to laparotomy for bleeding control . preoperative evaluation of the parametrium of patients with lower uterine segment fibroids can identify patients with an increased risk of laparotomy . laparoscopic myomectomy for optimal visualization of the cervical anatomy is used when lower uterine fibroids are large . the total complication rate of 9.8% and major complication rate of 4.5% in this series are comparable to rates in other tlh series ( table 3 ) . hoffman et al had a total and major complication rate of 10% and 5.6% , while heinberg et al had a major complication rate of 14.4% , and chapron reported complications in 10% . a complication rate of 13.4% has been observed for vh performed on nulliparous women , and 18% for vh after cesarean delivery . in randomized , controlled trials , type vii tlh conferred fewer complications , fewer wound infections , similar urologic complication rates , and was more cost effective than was ah , and resulted in comparable complications as lavh and vh . six categories of complications are analyzed for possible prevention strategies : urologic injuries , intestinal injuries , hemorrhagic events , infections , wound healing problems , and retained device complications . nineteen ( 2.3% ) patients had a urological injury , with half requiring reoperation : 3 cystoscopic ureteral stent placements , 4 ureteral reimplantations , and 2 laparotomies for closure of bladder fistula . tlh urological injury rates are reported at 2.5% to 3.4% and 2.2% in the first half of one series , decreasing to 0.9% in the second half . six injuries occurred during bladder flap dissections in patients with large lower uterine fibroids ( uterine weights all > 510 g ) and 3 with prior cesarean deliveries . one injury occurred during dissection of the anterior abdominal wall peritoneum to access the space of retzius for a burch procedure . to repair these , a running 3- 0 polygalactic acid suture in 2 layers it was successful in all but 2 patients who required laparotomy for successful repair after developing delayed breakdown of the laparoscopic cystotomy repair . when the margins of the bladder are not obvious before any dissection , inflating the bladder with a small amount of carbon dioxide can effectively delineate the edges . by clamping a hemostat on the foley catheter , attaching the insufflator tubing to it , and slowly opening the clamp while laparoscopically watching the bladder inflate with a small amount of carbon dioxide , patients typically have scarring on the upper portion of the anterior cervix and lower uterine segment , extending as low as the middle level of the cervix , but rarely ever lower . to dissect below this scar to unoperated vesicocervical fascia , the broad ligament adventitia is opened widely along the anterolateral aspect of the cervix until below the scar , where the smooth anterior white fascia of the cervix is easily separated from the bladder . then cutting ultrasonic dissection of the scar ureterotomy or ureteral transsection with immediate and successful repair occurred in 2 patients . in one case very early in the series ( case # 31 ) , in the other , redundant peritoneum was being trimmed after the completed hysterectomy for a massive , predominantly retroperitoneal fibroid uterus , without reconfirmation of the ureteral location . in both cases , a stent was inserted cystoscopically , and laparoscopically placed in the proximal portion of the ureter , with ureteroureterostomy by using 4 - 0 vicryl suture on the spatulated ends , and peritoneal closure without a drain . constant attention to the ureteral location would have prevented both injuries . eight ureteral fistulae developed postoperatively , most in the first third of the series , due to 2 sites of potential injury.it is well known that lack of recognition of the ureter in the lateral parametrial canal when ligating the uterine artery can result in ureteral injury 4 cm to 5 cm from the ureterovesical junction . an additional site of ureteral injury has come about solely through tlh due to the lack of precise recognition of the cervicovaginal margin and dissection too far down the vagina . dissection below the cervicovaginal margin can injure the ureter 2 cm to 3 cm from the ureterovesical junction . in open cases , surgeons have easily avoided this injury by carefully palpating the cervicovaginal margin between thumb and forefinger . surgeons keep the uterus on traction - counter - traction during laparotomy and feel the cervicovaginal margin repeatedly before incising into the vagina precisely at the cervicovaginal margin . the standard for laparoscopic surgeons must be the same : always know the exact site of the cervicovaginal margin by palpating the cervicovaginal margin with laparoscopic instruments to feel the precise change from the firmness of the cervical body to the pliancy of the vaginal wall , anteriorly , posteriorly , and laterally . when any of these landmarks are in question , a long right - angled heaney retractor or 1-inch ribbon is inserted to the anterior vagina and elevated , and the vaginotomy is performed safely onto the retractor 's edge at the anterior central cervicovaginal margin . cystoscopy after tlh should be performed in any case in which bladder or ureteral integrity is in question , to confirm closure of any repaired defect or to confirm patency of ureters after procedures , such as uterosacral ligament plications or burch colposuspensions . a 5-mm laparoscope can be used after instilling saline in the bladder with the suction irrigator . no indigo carmine is required to see the ureteral jets . using the irrigator and laparoscope gently as described three intestinal injuries occurred in the series ; only one was recognized immediately and repaired . one patient had slippage of the trocar outside of the abdominal cavity , with reinsertion performed without direct observation , through the bowel wall , requiring a figure - of - eight imbrication of both sites with 3- 0 polygalactic acid suture . the other 2 were occult injuries , presenting on days 7 and 29 , possibly from a lighted scope left in the abdomen touching the bowel , or inadvertent injury from ultrasonic scalpel dissection . three patients developed obstructive small - bowel adhesions to the vaginal apex , as evidenced by prolonged ileus and radiological demonstration of small - bowel obstruction at the vaginal apex . vaginal hemorrhage , retroperitoneal hemorrhage , and intraabdominal hemorrhage occurred in 11 ( 1.3% ) , 6 ( .8% ) , and 5 ( .7% ) patients , respectively , as observed in other series . of the 11 patients with vaginal hemorrhage , 5 occurred in the recovery room with immediate return to the operating room for additional suture placement vaginally . using at least 3 figure - of - eight ( technically spiral ) sutures along the vaginal apex seems essential , even when there is good hemostasis of the cuff edge . six patients developed brisk arteriolar cuff bleeding on days 7 through 10 , and 4 required sutures in the office . none of these 4 patients were diagnosed with a coagulopathy ; however , the suture material appeared dissolved in each . two patients had bleeding remote from the hospital and were observed in an emergency room without suture . of the 11 patients with abdominal or retroperitoneal hemorrhage , 6 were managed expectantly because they maintained normal vital signs or presented long after discharge with hematoma , which resolved . two had unexpectedly intense pain in the first 12 hours after surgery , which might have been an early warning symptom . no bleeding sites were identified in any of the reoperative cases , and none required laparotomy . nine patients ( 1% ) were diagnosed with pelvic cellulitis and received oral antibiotics with resolution of symptoms . five ( 0.4% ) patients were suspected of having pelvic abscess : 2 had 3.0-cm fluid collections with ct aspirate of clear sterile fluid . three had abscess : 2 were laparoscopically irrigated and drained , while one patient required laparotomy . four patients developed small - bowel herniation into a 5-mm trocar site , with 3 diagnosed after discharge from the hospital . each patient developed nausea and abdominal distension ; and ct was confirmatory in all cases . in each case , laparoscopic release resulted in recovery of bowel function after 3 days . herniation into a 5-mm trocar may occur with more degrees of instrument manipulation stretching the fascial incisions . five patients have had partial vaginal dehiscence , most after sexual penetration after the 6-week postoperative vaginal examination . one patient developed complete dehiscence of the vaginal apex 18 days after her surgery , and required operative repair . none of the other 4 patients required surgical repair , but all patients were advised to use a 1-cm thick foam ring around their partner 's penis during sexual activity to prevent their deepest penetration for the next 3 months . there have been no further incidents since warning all patients of the 1% chance of rupture with deep penetration after 6 weeks . an 8-mm segment of metal element from the bipolar cautery was retrieved laparoscopically from 1 patient in postoperative week 4 . most of the gynecological , hemorrhagic , infectious , and general surgical complications were addressed laparoscopically , vaginally , or locally . while all of the urological complications were treated by laparotomy except for cystoscopic stenting , this statistic will likely shift in time to laparoscopic repair . nineteen ( 2.3% ) patients had a urological injury , with half requiring reoperation : 3 cystoscopic ureteral stent placements , 4 ureteral reimplantations , and 2 laparotomies for closure of bladder fistula . tlh urological injury rates are reported at 2.5% to 3.4% and 2.2% in the first half of one series , decreasing to 0.9% in the second half . six injuries occurred during bladder flap dissections in patients with large lower uterine fibroids ( uterine weights all > 510 g ) and 3 with prior cesarean deliveries . one injury occurred during dissection of the anterior abdominal wall peritoneum to access the space of retzius for a burch procedure . to repair these , a running 3- 0 polygalactic acid suture in 2 layers it was successful in all but 2 patients who required laparotomy for successful repair after developing delayed breakdown of the laparoscopic cystotomy repair . when the margins of the bladder are not obvious before any dissection , inflating the bladder with a small amount of carbon dioxide can effectively delineate the edges . by clamping a hemostat on the foley catheter , attaching the insufflator tubing to it , and slowly opening the clamp while laparoscopically watching the bladder inflate with a small amount of carbon dioxide , , patients typically have scarring on the upper portion of the anterior cervix and lower uterine segment , extending as low as the middle level of the cervix , but rarely ever lower . to dissect below this scar to unoperated vesicocervical fascia , the broad ligament adventitia is opened widely along the anterolateral aspect of the cervix until below the scar , where the smooth anterior white fascia of the cervix is easily separated from the bladder . then cutting ultrasonic dissection of the scar ureterotomy or ureteral transsection with immediate and successful repair occurred in 2 patients . in one case very early in the series ( case # 31 ) , parametrial dissection landmarks were distorted by large lower uterine segment fibroids . in the other , redundant peritoneum was being trimmed after the completed hysterectomy for a massive , predominantly retroperitoneal fibroid uterus , without reconfirmation of the ureteral location . in both cases , a stent was inserted cystoscopically , and laparoscopically placed in the proximal portion of the ureter , with ureteroureterostomy by using 4 - 0 vicryl suture on the spatulated ends , and peritoneal closure without a drain . eight ureteral fistulae developed postoperatively , most in the first third of the series , due to 2 sites of potential injury.it is well known that lack of recognition of the ureter in the lateral parametrial canal when ligating the uterine artery can result in ureteral injury 4 cm to 5 cm from the ureterovesical junction . an additional site of ureteral injury has come about solely through tlh due to the lack of precise recognition of the cervicovaginal margin and dissection too far down the vagina . dissection below the cervicovaginal margin can injure the ureter 2 cm to 3 cm from the ureterovesical junction . in open cases , surgeons have easily avoided this injury by carefully palpating the cervicovaginal margin between thumb and forefinger . surgeons keep the uterus on traction - counter - traction during laparotomy and feel the cervicovaginal margin repeatedly before incising into the vagina precisely at the cervicovaginal margin . the standard for laparoscopic surgeons must be the same : always know the exact site of the cervicovaginal margin by palpating the cervicovaginal margin with laparoscopic instruments to feel the precise change from the firmness of the cervical body to the pliancy of the vaginal wall , anteriorly , posteriorly , and laterally . when any of these landmarks are in question , a long right - angled heaney retractor or 1-inch ribbon is inserted to the anterior vagina and elevated , and the vaginotomy is performed safely onto the retractor 's edge at the anterior central cervicovaginal margin . cystoscopy after tlh should be performed in any case in which bladder or ureteral integrity is in question , to confirm closure of any repaired defect or to confirm patency of ureters after procedures , such as uterosacral ligament plications or burch colposuspensions . a 5-mm laparoscope can be used after instilling saline in the bladder with the suction irrigator . no indigo carmine is required to see the ureteral jets . using the irrigator and laparoscope gently as described three intestinal injuries occurred in the series ; only one was recognized immediately and repaired . one patient had slippage of the trocar outside of the abdominal cavity , with reinsertion performed without direct observation , through the bowel wall , requiring a figure - of - eight imbrication of both sites with 3- 0 polygalactic acid suture . the other 2 were occult injuries , presenting on days 7 and 29 , possibly from a lighted scope left in the abdomen touching the bowel , or inadvertent injury from ultrasonic scalpel dissection . three patients developed obstructive small - bowel adhesions to the vaginal apex , as evidenced by prolonged ileus and radiological demonstration of small - bowel obstruction at the vaginal apex . vaginal hemorrhage , retroperitoneal hemorrhage , and intraabdominal hemorrhage occurred in 11 ( 1.3% ) , 6 ( .8% ) , and 5 ( .7% ) patients , respectively , as observed in other series . of the 11 patients with vaginal hemorrhage , 5 occurred in the recovery room with immediate return to the operating room for additional suture placement vaginally . using at least 3 figure - of - eight ( technically spiral ) sutures along the vaginal apex seems essential , even when there is good hemostasis of the cuff edge . six patients developed brisk arteriolar cuff bleeding on days 7 through 10 , and 4 required sutures in the office . none of these 4 patients were diagnosed with a coagulopathy ; however , the suture material appeared dissolved in each . two patients had bleeding remote from the hospital and were observed in an emergency room without suture . of the 11 patients with abdominal or retroperitoneal hemorrhage , 6 were managed expectantly because they maintained normal vital signs or presented long after discharge with hematoma , which resolved . two had unexpectedly intense pain in the first 12 hours after surgery , which might have been an early warning symptom . no bleeding sites were identified in any of the reoperative cases , and none required laparotomy . nine patients ( 1% ) were diagnosed with pelvic cellulitis and received oral antibiotics with resolution of symptoms . five ( 0.4% ) patients were suspected of having pelvic abscess : 2 had 3.0-cm fluid collections with ct aspirate of clear sterile fluid . three had abscess : 2 were laparoscopically irrigated and drained , while one patient required laparotomy . four patients developed small - bowel herniation into a 5-mm trocar site , with 3 diagnosed after discharge from the hospital . each patient developed nausea and abdominal distension ; and ct was confirmatory in all cases . in each case , laparoscopic release resulted in recovery of bowel function after 3 days . herniation into a 5-mm trocar may occur with more degrees of instrument manipulation stretching the fascial incisions . five patients have had partial vaginal dehiscence , most after sexual penetration after the 6-week postoperative vaginal examination . one patient developed complete dehiscence of the vaginal apex 18 days after her surgery , and required operative repair . none of the other 4 patients required surgical repair , but all patients were advised to use a 1-cm thick foam ring around their partner 's penis during sexual activity to prevent their deepest penetration for the next 3 months . there have been no further incidents since warning all patients of the 1% chance of rupture with deep penetration after 6 weeks . an 8-mm segment of metal element from the bipolar cautery was retrieved laparoscopically from 1 patient in postoperative week 4 . most of the gynecological , hemorrhagic , infectious , and general surgical complications were addressed laparoscopically , vaginally , or locally . while all of the urological complications were treated by laparotomy except for cystoscopic stenting , this statistic will likely shift in time to laparoscopic repair . this technique for type vii tlh appears safe and allows excellent access to the entire abdomen as needed in cancer surgeries , for patients with pelvic mass , endometriosis , pain , or adhesions with minimal morbidity . a total laparoscopic capability makes the benefits of a minimally invasive approach available to more women , including obese and nulliparous women . with an understanding of the complications from this technique , it is hoped that complications can be avoided and that more surgeons will safely learn tlh . experienced laparoscopic surgeons are urged to initiate needed randomized clinical trials of type vii tlh .

objective : this study analyses the technique and complications from total laparoscopic hysterectomy.methods:retrospective chart abstraction was performed on 830 consecutive patients operated on between 1996 and 2006 . demographic and surgical data were analyzed by anova , chi - square , and spearman and pearson correlation techniques were used with significance set at p<0.05.results : of 830 consecutive patients , 5 ( 0.6% ) were converted to laparotomy . patients had a mean age of 50 ( 11 ) years , a mean of 1.3 ( 1.3 ) pregnancies , and a mean bmi of 27.6 ( 6.8 ) kg / m2 . the mean surgical duration was 132 ( 55 ) minutes , with mean blood loss of 130 ( 189 ) ml and average hospital stay of 1.4 ( 0.9 ) days . duration of surgery , blood loss , and hospital stay all decreased with the surgeon 's increasing experience . reoperative complications occurred in 38 patients ( 4.7% ) . urologic injuries were observed in 23 patients ( 2.6% ) , with 9 ( 1.1% ) requiring reoperation.conclusions:this technique for tlh offers the benefits of minimally invasive surgery for patients needing hysterectomy , even those without vaginal capacity and uterine prolapse .

in general terms , epigenetic mechanisms include dna methylation , covalent modification of histones , chromatin remodelling and histone replacement through incorporation of the so - called histone variants ( fig . histone marks have emerged as one of the main players involved in epigenetic mechanisms ( kouzarides , 2007 ) . although it is still unclear whether they are the actors of the epigenetic information or the epigenetic information itself , it is evident that covalent modifications of histones are essential components of the epigenome . histones can be modified by a number of enzymes that mediate methylation , acetylation , phosphorylation , ubiquitynation and adp - ribosylation of specific amino acid residues ( reviewed in kouzarides , 2007 ) . by and large , the highest density of modifications so far described occurs in histone h3 , particularly on its tail . the effects of these modifications on the chromatin and on cellular processes are very diverse , and a modification of the same residue can even have opposite effects depending on the type of modification . for example , trimethylation of h3k9 is considered as a repressive mark , whereas acetylation of the same lysine has a positive effect on transcription ( bannister and kouzarides , 2005 ) . likewise , methylation of arginine residues can have a positive effect on transcription ( chen et al . , 1999 ) or a repressive effect ( pal et al . , 2004 ) , depending both on the targeted residue and on whether the methylation is symmetric or asymmetric . for some of the modified residues , there is a very clear view of the outcome of an eventual modification : h3k9me3 creates a specific docking site for the heterochromatin protein 1 ( hp1 ) , which subsequently recruits the h3k9 methyltransferase su(var)3 - 9 and reinforces an autoregulatory loop for heterochromatin formation and maintenance ( bannister et al . , 2001 ; lachner et al . , 2001 ; nakayama et al . , they include the regulation of the subcellular localization of dna methyltransferase activity , highlighted by the cytoplasmic retention of dnmt1o ( carlson et al . , 1992 ) ; the exclusion of a particular histone modification from the chromatin , which is exemplified by the lack of detection of h3k9me3 in the paternal pronucleus after fertilization resulting in an asymmetry of histone marks between the two pronuclei ( arney et al . , 2002 ; santos et al . , 2005 ) , the differential incorporation of chaperons and histone variants in the parental chromatin ( van der heijden et al . , 2005 ; torres - padilla et al . , 2006 ) and , 1992 ; tanaka et al . , 2001 ; govin et al . , 2007 ) . further , the maternal and paternal pronuclei exhibit different patterns of global dna methylation : while the paternal pronucleus is rapidly demethylated presumably through an active mechanism right after fertilization , the maternal pronucleus is only passively demethylated through the subsequent rounds of replication and cell division that follow the first mitosis of the embryo ( mayer et al . , 2000 ) . moreover , while the centromeric and pericentric paternal chromatin remain dna methylated , the maternal dna loses methylation in such regions ( rougier et al . , 1998 ) . the changes in the levels of dna methylation as development proceeds in the preimplantation embryo are dynamic . global levels of dna methylation have been analysed by immunofluorescence , bisulphate sequencing and restriction digestion ( rougier et al . , 1998 ; mayer et al . , 2000 ; santos et al . , 2002 ; aranyi and paldi , 2006 ) . bisulphate sequencing and restriction digestion have also been used to analyse the methylation status of repeat sequences ( such as l1 and iap repeats ) and some single - copy sequences ( such as actin ) ( howlett and reik , 1991 ; oswald et al . , 2000 ) . these studies have revealed that although global levels of dna methylation decrease until the blastocyst stage , changes in dna methylation do not occur to the same extent on all genes . remarkably , imprinted genes ( such as h19 ) and some repeat sequences ( such as iaps ) do not undergo demethylation ( tremblay et al . , 1997 ) . during early stages of development , decisions involved in cell fate determination and pluripotency have to be assumed . this implies that the mechanisms involved in regulation of chromatin structure need to ensure stability across generations and cell division , but they also need to be flexible ( reik , 2007 ) . the double nature of a covalent modification either on histones and/or on the dna as dynamic ( because in principle it can be added and removed ) and at the same time its potential ability to propagate a memory , fits well with these aforementioned needs . moreover , in keeping with the importance of epigenetic mechanisms during early development , the possibility for an epigenetic mark(s ) underlying these phenomena appears very attractive . concerning the epigenetic asymmetries of the icm and the trophectoderm , the icm displays , in global terms , higher levels of dna methylation compared with the trophectoderm ( dean et al . specific histone marks such as trimethylation of lysines 9 and 27 of histone h3 ( h3k9me3 and h3k27me3 , respectively ) are enriched in the icm compared with the trophectoderm ( erhardt et al . , 2003 ) . likewise , the trophectoderm retains an imprinted form of x inactivation , where the paternal x chromosome is silenced ( heard and disteche , 2006 ) . this is in contrast to the icm , where there is reactivation of the inactive x chromosome and a subsequent round of inactivation occurs at random in which either the maternal or the paternal chromosome is inactivated ( mak et al . , 2004 ; okamoto et al . , the aforementioned epigenetic asymmetries of the two lineages of the blastocyst are evident once lineage allocation has taken place and might reinforce their molecular identity . however , epigenetic asymmetry could also act as a driver for lineage allocation , in which case , the former would precede the latter ( fig . from experimental embryology , we have learnt from pioneer experiments performed in the 50 s that after mechanical separation of the blastomeres of a 2-cell stage embryo and transfer into foster mothers , each of these two cells gives rise to an adult mouse ( tarkowski , 1959 ) . this indicates that mouse embryos are very flexible in what people have referred to as developmental potential . derivation of twins from mouse blastomeres at later stages of development ( e.g. 4-cell stage or later ) has not been possible . although this has been linked to the low number of cells present in the resulting embryos and hence their inability to form an icm with a normal cell number , rather than to their developmental potential or identity ( tarkowski and wroblewska , 1967 ; rossant , 1976 ) . indeed , when random single 4- and 8-cell stage blastomeres are aggregated with carrier blastomeres , their progeny is able to contribute to all the tissues of the embryo ( kelly , 1977 ; tarkowski et al . , 2005 ) , and in this sense , the blastomeres were considered to be totipotent . it is also possible however that some subtle intrinsic differences of these blastomeres might be masked by the limitations of the outcome of transplantation procedures , given that the results of these studies are very often far from 100% and the transplantation efficiency is never absolute . whether this is solely related to technical difficulties linked to these challenging manipulations or to an intrinsic property of specific blastomeres of the embryo is impossible to ascertain . despite a controversial viewpoint on whether there is any polarity in the early mouse embryo or not , most reports coincide with the interpretation that a blastomere at the 2-cell stage contributes to both the icm and the trophectoderm ( reviewed in edwards and beard , 1997 ; zernicka - goetz , 2006 ) . the suggestion of a given blastomere at the 2-cell stage having a preferential fate towards either of the blastocyst lineage is not resolved and some researchers have suggested that a slight , but distinct difference in the fate of 2-cell stage blastomeres might be disturbed by experimental manipulations ( alarcon and marikawa , 2005 ; hiiragi et al . , 2006 ) . the role of extrinsic factors to the embryo , such as the shape of the zona pellucida , in axis specification of the blastocyst is also a matter of controversy ( gardner , 2007 ; kurotaki et al . , 2007 ) . however , there are some indications that a bias for a blastomere to contribute to a given region of the embryo in the blastocyst could already exist at the 4-cell stage ( fujimori et al . , 2003 ; piotrowska - nitsche and zernicka - goetz , 2005 ) . these conclusions are mostly based on lineage tracing experiments of labelled blastomeres , and their degree of invasiveness is debatable . some groups have used the plane of division in relation to the animal vegetal axis of the embryo as a sort of guideline to distinguish and characterize blastomeres according to their cleavage plane . by convention , the animal pole is demarcated by the position of the second polar body ( which is extruded after resumption of meiosis ii upon fertilization ) and hence the vegetal pole lies on the opposite side ( fig . the division from the 2- to the 4-cell stage would segregate for the first time the animal and the vegetal components of the zygote if it occurs equatorially , that is , perpendicular to the animal vegetal axis of the conceptus ( gardner , 2002 ) . thus , whereas a cell that derives from a meridional division ( parallel to the animal vegetal axis ) inherits both components , an equatorial division gives rise to an by looking into the plane of division and the order at which this division occurs from the 2- to the 4-cell stage and subsequent lineage tracing , a subgroup of embryos was identified where it is possible , with a relatively high probability , to predict the future position of the blastomeres in the blastocyst ( piotrowska - nitsche et al . , 2005 ) . this group of embryos are referred to as me embryos ( for meridional equatorial , reflecting the type of cleavage plane and order of division that generated them ( fig . 2b ) ( piotrowska - nitsche et al . , 2005 ; piotrowska - nitsche and zernicka - goetz , 2005 ) . although the me type embryos constitute only a small part ( 20% ) of a complete litter , they provide a very nice system where it is possible to explore the foundations of differentiation in the embryo . indeed , the vegetal blastomere would most often populate the abembryonic region of the blastocyst , which contains mainly mural trophectoderm . blastomere division planes according to the animal - vegetal axis in the embryo ( a ) the animal vegetal ( a v ) axis of the preimplantation embryo is demarcated , by convention , by the position of the second polar body , which marks the animal pole . the opposite side to the site of extrusion of the polar body is , by default , the vegetal pole of the embryo . ( b ) the division pattern from 2-to- 4-cell stage of a typical embryo that undergoes one meridional ( m ) and one equatorial ( e ) division ( me embryo ) is represented . the cleavage plane is depicted by a red dashed line ( embryo on the left ) . vegetal components ( two cells with pink and yellow motifs on the embryo depicted on the right ) . in contrast , when a 2-cell stage blastomere divides equatorially , a segregation of the animal and animal ( pink ) and a vegetal ( yellow ) blastomere in looking for epigenetic marks that could be involved in an eventual cell fate decision of the blastomeres of these me embryos , it was found that the vegetal blastomere displays the lowest levels of dimethylated arginine 26 of h3 ( h3r26me2 ) . if h3r26me2 participates in lineage allocation , one might predict that modulating the levels of histone arginine methylation , would have an effect over cell fate . overexpression of the histone methyltransferase that methylates this residue on h3 , prmt4/carm1 , into individual blastomeres not only induced upregulation of nanog and sox2 , but also resulted in an almost complete allocation of these blastomeres into the icm compartment ( torres - padilla et al . , 2007 ) . it is interesting to note that in the mouse , the differences described in histone h3 arginine methylation appear at the 4-cell stage ( torres - padilla et al . , 2007 ) , that is . as early as one cell cycle after the major wave of embryonic genome activation occurs ( schultz , 2002 ; hamatani et al . , 2004 ) , which suggests that these events might be , at least in part , linked to the transcriptional programme of the embryo . the developmental stage at which genome activation occurs in other mammalian species varies considerably : it takes place at the 1-to- 2-cell stage in mice , the 4-to- 8-cell stage in cows and humans , and the 8-to- 16-cell stage in sheep and rabbits ( schultz and heyner , 1992 ) . would this anticipate a different timing for an eventual cell fate path for other species ? normal fertile adults can be derived from single blastomeres from 2- , 4- and 8-cell stage embryos in the rabbit , sheep and cattle ( moore et al . , 1968 ; willadsen , 1981 ; willadsen and polge , 1981 ) . thus , these species indeed support blastomere isolation and further development at later stages than the mouse does . transplantation of isolated 4-cell blastomeres into morula stage embryos has demonstrated that the blastomeres at the 4-cell stage are totipotent ( kelly , 1977 ) . in this context , it is important to note that aggregating the vegetal cell from me embryos to form chimeric embryos , showed that this cell is able to contribute to all tissues in the embryo . however , aggregating the same blastomere with other vegetal blastomeres from me embryos exclusively , results in a failure to proceed through development ( piotrowska - nitsche et al . , 2005 ) . thus , the environment where the blastomeres develop is crucial for the success of the embryo throughout development , and in a normal situation , where the embryo has not been perturbed and a given cell develops in its niche , some differences appear to develop , which are , at least partially , related to the epigenetic information of each of them ( torres - padilla et al . , 2007 ) . if epigenetic asymmetries of the early embryo are related to lineage allocation , it is still uncertain whether they are a cause or a consequence for lineage choice . also , it remains unknown whether such epigenetic asymmetries would affect only particular regions of the genome . for example , whether genes involved in specification of the icm such as sox2 and nanog would all be targeted by the same epigenetic marks in the same blastomere or whether such marks would vary among genes and/or among blastomeres . inner and outer cells at the 16-cell stage distinguishable in terms of their chromatin landscapes ? the experiments showing that blastomeres are able to respond to the overexpression of a histone modifier and change their fate , indicate that these cells have not yet acquired a fix destiny , but that they can still be responsive to some kind of signals . these experiments have an important impact on showing that manipulating the epigenetic information can affect cell fate in the preimplantation embryo , in line with the importance of epigenetic mechanisms being crucial for early development . moreover , these results do illustrate that such cells can still be flexible and accommodate themselves after a perturbing event ( in this case , overexpression of a histone methyltransferase and the downstream effects on the information that is these studies have originated some interest from the part of the medical community , particularly , in the context of preimplantation genetic diagnosis ( pgd ) ( goldman , 2007 ) . does it matter to the embryo which cell is used for genetic diagnosis ? might the death of one of these cells have an effect on subsequent development ? this question is equally valid on the impact of cell loss upon cryopreservation ( cohen et al . , 2007 ) . although these are very delicate questions with very likely no easy answer , from the perspective of the mouse embryo , at least four things are to be considered . a tendency for a blastomere of some 4-cell stage embryos to contribute to a given region of the embryo has been documented . second , the blastomeres in the 4-cell stage show clear differences not only in the levels of histone methylation , but also in their transcriptional activiy when they develop without being perturbed . however ( third ) , the cells undergo a redirection of cell fate when a histone methyltransferase is overexpressed , indicating that they can readapt . finally , the environment in which cells develop seems to be crucial for completing development and somehow the remaining cells in the embryo could compensate provided they are somehow different from each other . it is also important to note that a 4-cell stage mouse embryo might correspond to a very different developmental stage than a 4-cell embryo in other mammalian species , as illustrated by the differences in the onset of genome activation between them . indeed , pgd is most often performed at the 8-cell stage and some reports document a better rate of development when 6-to- 9-cell stage embryos are diagnosed , as opposed to 3-to- 4-cell stage embryos ( wang et al . , 2007 ) . moreover , the effects of in vitro fertilization procedures and culture on embryonic development are also extremely important , as they have been shown to alter epigenetic information in the mouse ( li et al . , 2005 ) . as a final consideration , i would like to leave the reader with an open perspective of some ongoing questions in the field . although there are strong indications that epigenetic mechanisms are involved in cell fate determination , we are still far from establishing a direct link between an epigenetic mark(s ) and the derivation of a particular cell lineage in the embryo . much work is still to be done to determine how these mechanisms are set in play upon fertilization and how they are transmitted during subsequent cleavage stages . also , what other epigenetic marks contribute to the inheritability of cell fate decisions ? how do these marks relate to and influence each other ? are different lineage - specific genes marked by a different combination(s ) of epigenetic marks ? it is also tempting to expand these notions into the stem cell field and question whether these mechanisms would also underlie the intrinsic self renewal ability of adult stem cells and their potential to differentiate into other cell types . the exquisite complexity and richness of chromatin - regulated events in the early embryo will certainly be the subject of exciting research in the future .

the early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation . following fertilization , a single cell , the zygote , will give rise to all tissues of the organism . the first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm , a differentiated tissue that envelopes the inner cell mass . the question of when and how the cells start to be different from each other in the embryo is central to developmental biology : as cell fate decisions are undertaken , loss of totipotency comes about . although the blastomeres of the preimplantation embryo are totipotent , as the embryo develops some differences appear to develop between them which are , at least partially , related to the epigenetic information of each of these cells . the hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented . although there are now some indications that epigenetic mechanisms are involved in cell fate determination , much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division . these considerations are further discussed in the context of preimplantation genetic diagnosis : does it matter to the embryo which cell is used for genetic diagnosis ? the exquisite complexity and richness of chromatin - regulated events in the early embryo will certainly be the subject of exciting research in the future .

total knee arthroplasty ( tka ) is widely accepted treatment for moderate or severe osteoarthritis and rheumatoid arthritis . significant blood loss can be seen during the early postoperative period where a blood transfusion may be necessary . however , it has been known that the donor blood may lead to an immunological reaction and the transmission of viral infections ( e.g. , hepatitis and aids).1 various methods have been suggested to reduce blood loss following tka , such as preoperative erythropoietin and iron supplementation , autotransfusion,23 postoperative blood salvage,4 hypotensive anesthesia,5 tranexamic acid administration,6 intraarticular epinephrine injection,7 thrombin based hemostatic agent,8 and temporary drain clamping.910 closed suction drainage is known to prevent the formation of hematomas in the operative field , decrease tension on incisions , diminish delayed wound healing and reduce the risk of infection.11 however , some studies have demonstrated that closed drainage leads to increased blood loss after tka because it eliminates the tamponade effect and may cause a retrograde infection.12 the role of wound drainage following knee arthroplasty is still controversial.1314 despite these disagreements , wound drainage is a procedure widely used by orthopedic surgeons and it is still assumed to be a standard of care and suggested for use in tka . subcutaneous indwelling closed suction drainage method has been known to be beneficial and an alternative to the intraarticular indwelling method.15 this method provides tamponade effect as the joint is not drained and in addition has subcutaneously drainage effect preventing the soft tissue hematoma and wound problems . the purpose of this prospective randomized study was to compare the visible , hidden , total blood loss and postoperative hemodynamic change of subcutaneous and intraarticular indwelling closed suction drainage method after tka . the hypothesis was that subcutaneous indwelling method would decrease the visible , total blood loss and reduction of postoperative hemoglobin ( hb ) or hematocrit ( hct ) change after tka . 160 patients with primary osteoarthritis , who underwent unilateral tka in our institute between january 2011 and september 2011 , were enrolled in this trial . all patients were randomly assigned into two groups ( 80 per group ) using computer - generated numbers . the sample size was calculated to detect a significant difference in the postoperative drainage and laboratory change between groups with a power of 80% with an value of 0.05 . group a and b were patients with subcutaneous and intraarticular indwelling closed suction drainage method , respectively . this left 157 tkas ( 78 patients for group a and 79 patients for group b ) who form the basis of the current report [ figure 1 ] . exclusion criteria include other diagnosis than primary osteoarthritis , preoperative valgus alignment , received a revision or bilateral tka , hematological disease , previous open knee surgery or vascular surgery . there were 53 males and 104 females with an average age of 69.5 5.8 years ( range 61 - 82 years ) , and the average body mass index was 24.9 4.7 kg / m [ table 1 ] . this study was conducted after the approval of the institutional review board and informed consent was obtained from each patient before enrollment in the study . flow char showing consolidated standards of reporting trials patient demographics , preoperative deformities and functional status all surgeries were performed by a single surgeon ( jhy ) using the navigation system ( orthopilot , version 4.0 ; b. braun aesculap , tuttlingen , germany ) under spinal anesthesia . all 160 knees underwent the same surgical approach consisting of a midline skin incision and a medial parapatellar approach under tourniquet . all patients were implanted with cemented type ultra - congruent fixed bearing design ( columbus uc , b. braun aesculap , tuttlingen , germany ) . none of the patients underwent lateral retinacular release or patellar resurfacing . in the subcutaneous indwelling group ( group a ) , a vacuum drainage system was placed in the subcutaneous space ( below the medial skin flap ) after joint capsule closure [ figure 2].15 in the intraarticular indwelling group ( group b ) , a vacuum drainage system was placed in the intraarticular space ( the medial gutter ) before joint capsule closure . the amount of drained visible blood was recorded at 24 and 48 h using the measuring cylinder with total 500 ml and 1.0 ml unit . using gross 's formula,16 hidden blood loss was calculated using patients height , weight , and preoperative and postoperative hct . the hb and hct levels were determined preoperatively and 12 h ( day 1 ) , 5 and 10 day postoperatively . the patients received packed red blood cells if their hb levels decreased to < 8 g / dl and presents compromised clinical criteria ( e.g. , tachycardia , hypotension , or symptoms of anemia that were relative to the preoperative medical condition of the patient ) necessitated transfusion . the postoperative timing of transfusion and the number of units of transfused red blood cell concentrates were recorded . all patients were given low molecular weight heparin ( enoxaparin 40 mg ) subcutaneously daily for 7 days postoperatively . note that the indwelling drains ( white arrows ) are placed subcutaneously superficial to the joint capsule closure after the operation , patients of both groups were encouraged to perform a mechanical ankle pumping exercise to prevent deep vein thrombosis as soon as possible after surgery . the bandage and foley 's catheter were removed on the second postoperative day . on the same day , the range motion ( rom ) exercise using continuous passive motion devices , an isometric / isotonic quadriceps exercise , a straight leg rising exercise and a walking exercise were initiated under the control of a physiotherapist . all the observations were recorded by clinical research orthopedic nurse who was unaware about the allotted groups . oozing persisting beyond 2 days after surgery , subcutaneous hematoma ( requiring aspiration or surgical drainage ) , hemarthrosis ( requiring aspiration or surgical drainage ) , ecchymosis ( larger than 3 cm in diameter ) and wound infection ( requiring additional treatments such as antibiotics coverage or surgical debridement ) were recorded . thigh and calf circumference were checked at day 7 after tka . the level for thigh circumference was set at 10 cm proximal from the patella upper pole and largest diameter for calf . symptom severity was assessed at 24 months using the knee society score ( kss)17 and western ontario and mcmaster universities osteoarthritis index ( womac ) score.18 passive maximum knee rom was measured using a goniometer . at these evaluations , a kss of 90 points was considered an excellent outcome , a score between 80 and 89 points was considered a good outcome , a score between 70 and 79 points was considered a fair outcome and a score of < 70 points was considered a poor outcome . the womac system involves the completion of a 24-item questionnaire with three sections : namely ; pain , stiffness and function.18 five response options are possible ( none , mild , moderate , severe and extreme ) , which are scored from 0 to 4 to yield subtotal scores for pain ( 5-item ; possible total score range 020 ) , stiffness ( 2-item ; possible score range 08 ) , and function ( 17-item ; possible score range 068 ) . the two study groups were compared with respect to blood volumes collected via indwelled drains , changes in hb / hct levels postoperatively , requirements for allogeneic blood transfusion , bleeding - related wound problems , and postoperative hospital stay . the kolmogorov fisher 's exact test or the yates chi - square test was used to analyze categorical variables . the complication rate between the two groups was determined by the chi - square test with fisher 's exact test . the statistical package for social science version 10.1 ( spss inc . , chicago , il , usa ) was used for all analysis and p < 0.05 was considered statistically significant . there were no significant differences between the subcutaneous drainage group ( group a ) and intraarticular drainage group ( group b ) regarding the preoperative demographs [ table 1 ] . the total blood loss after tka was significantly different between two groups ( p = 0.04 ) . the total blood loss ( visible drained blood + hidden loss ) was less in subcutaneous drainage group . the mean visible blood drainage in subcutaneous drainage group was significantly less than intraarticular drainage group , both during the first 24 h and between 24 and 48 h ( p < 0.001 ) . the hidden blood loss after tka was significantly different between two groups ( p < 0.001 ) [ table 2 ] . comparison of hematologic data there were no significant differences in hb or hct level in 2 groups before the surgery . however , significant differences of change of hb were demonstrated between groups at 12 h ( day 1 ) , 5 and 10 day postoperatively [ table 2 ] . allogeneic transfusion requirements between patients in the subcutaneous drainage and intraarticular drainage groups ( 6.4% versus 24.1% ) were significantly different ( p = 0.002 , table 3 ) . comparison of transfusion rate and the units in terms of wound problems [ table 4 ] , none of the patients developed delayed wound healing with skin edge necrosis , wound infection , deep vein thrombosis in this study . subcutaneous drainage group ( group a ) demonstrated a higher rate of minor complications such as the bullae formation and ecchymosis compared to intraarticular drainage group . mean postoperative hospital stay in group a and group b was 13.8 and 14.1 days , respectively ( p > 0.05 ) . comparison of bleeding - related wound problems , change of thigh - calf circumferences and postoperative hospital stay there were no significant differences between groups in functional outcomes at 2 year after surgery [ table 5 ] . the most important finding of this study was that subcutaneous indwelling closed suction drainage method reduces both the visible blood loss and total blood loss ( hemovac drainage + internal blood loss ) thus decreases the rate of allogeneic transfusion . although the incidence of minor complications such as the bullae formation and the ecchymosis were significantly higher in the subcutaneous indwelling group , the functional outcome at postoperative 2 years did not demonstrate the difference from intraarticular drainage group . a few studies have shown that closed negative pressure drainage is beneficial to early knee function recovery by reducing postoperative hematoma formation in the wound , relieving wound tension , preventing complications including pain and poor healing and lowering the incidence of deep infections.19 however , a number of other studies have not supported the advantages of closed negative pressure drainage and indicated that closed negative pressure drainage causes complications such as postoperative hemorrhage , increased blood transfusion and infection and affects the ability of postoperative functional training.122021 the use of vacuum drainage in tka to manage bleeding continues to be debated . because most of the blood loss in tka occurs during the first few postoperative hours ( 37% in 2 h and 55% in 4 h ) , it seems reasonable to clamp the drain tube in the first few hours after tka to temporarily create a tamponade effect for bleeding control . recommendations included no clamping,22 clamping for 1 h,23 10-min clamp releases every 2 h,24 and clamping for 4 h.10 the effect of clamping method is also in controversy . the method of subcutaneous indwelling closed suction drainage had been proposed as a potentially efficacious alternative to either intraarticular closed suction drainage or no drainage system.15 this method was suggested to exploit the advantages of both methods ; the subcutaneous blood drainage and the joint tamponade effect . seo et al.15 have shown that subcutaneous closed suction drainage involves equivalent blood loss with comparable wound problems and functional outcomes compared with intraarticular closed suction drainage . however , only exogenous blood loss was analyzed in their study while internal blood loss ( i.e. , total blood loss ) was calculated in this study . in addition , detailed postoperative minor complications were identified using the parameters such as the change of thigh and calf circumference . the occurrence of bullae formation and ecchymosis were significantly higher in subcutaneous indwelling closed suction drainage group in this series . although some studies have demonstrated no statistically significant difference in hematoma formation with or without postoperative drainage , tka patients without drainage system theoretically have a higher chance of hematoma formation . while pressure dressing reduces superficial bleeding at the incision , it is less effective on bleeding in deep locations such as the marrow and periprosthesis space , which can result in invisible blood loss and hematoma formation due to blood accumulation.25 after tka , because the capsule of the knee joint is damaged and the surrounding soft tissue has been incised , the blood infiltration can extend to the fascia and intramuscular space of the thigh and calf , which results in increase in thigh calf circumference and ecchymosis . subcutaneous indwelling closed suction drainage method may not be a perfect solution to prevent soft tissue accumulation from excessive blood infiltration . despite significantly increased minor wound problems and increased thigh - calf circumference compared to intraarticular drainage group , two groups had similar functional outcome at 2 years postoperatively . soft tissue swelling around the knee joint in subcutaneous indwelling closed suction drainage group might have reduced the rom shortly after the operation , but it did not influence the motion arc in the long run . limitation of this study are methods used regarding the drainage system in tka , subcutaneous indwelling closed suction method was compared with only the intraarticular indwelling drainage method . a group without closed suction drainage system or groups of various clamping methods have not been compared . therefore , conclusions can not be made whether subcutaneous indwelling closed suction drainage method has advantages or disadvantages from other methods . subcutaneous indwelling closed suction drainage method in tka reduces total blood loss and thus lowers the rate of allogeneic blood transfusion compared to the intraarticular indwelling drainage method . although the incidence of minor wound problems such as bullae formation and ecchymosis is higher than the intraarticular indwelling drainage method , eventual motion arc and functional outcome are similar . based on these results ,

background : total knee arthroplasty ( tka ) is widely accepted treatment for moderate or severe osteoarthritis and rheumatoid arthritis . significant blood loss can be seen during the early postoperative period where a blood transfusion may be necessary . closed suction drainage is known to prevent the formation of hematomas in the operative field , decrease tension on incisions , diminish delayed wound healing and reduce the risk of infection . subcutaneous indwelling closed suction drainage method has been known to be beneficial and an alternative to the intraarticular indwelling method . this prospective randomized study was to compare the visible , hidden , total blood loss and postoperative hemodynamic change of subcutaneous and intraarticular indwelling closed suction drainage method after tka.materials and methods : one hundred and sixty patients with primary osteoarthritis who underwent unilateral tka were enrolled ; group a with subcutaneous ( n = 78 ) and group b with intraarticular ( n = 79 ) indwelling closed suction drainage method . total blood loss , visible blood loss , internal blood loss , postoperative day 1 , 5th , 10th day hemoglobin , hematocrit levels were compared . allogeneic blood transfusion rate and complications related to soft tissue hematoma formation were additionally compared.results:allogenic transfusion requirements between subcutaneous drainage group and intraarticular drainage groups ( 6.4% vs. 24.1% ) were significantly different ( p = 0.002 ) . although the minor complications such as the incidence of bullae formation and the ecchymosis were higher in the subcutaneous indwelling group , the functional outcome at postoperative 2 year did not demonstrate the difference from intraarticular drainage group.conclusion:subcutaneous indwelling closed suction drainage method is a reasonable option after tka for reduction of postoperative bleeding and transfusion rate .

gestational diabetes mellitus ( gdm ) is defined as carbohydrate intolerance of any severity identified for the first time during pregnancy . though this definition uniformly detected and classified gdm , its limitations have been known for many years . in 2008 - 2009 , the international association of diabetes and pregnancy study groups ( iadpsg ) , an international consensus group with representatives from multiple obstetrical and diabetes organizations , including the american diabetes association ( ada ) , recommended that high - risk women found to have diabetes at their initial prenatal visit , using standard criteria , receive a diagnosis of overt , and not gestational diabetes . india has the second largest number of people with diabetes in the world ( 62.4 million ) and this number is expected to reach 100 million by the year 2030 . rapid changes in lifestyle including unhealthy diet and physical inactivity may have contributed to this rise in prevalence of diabetes in general with a parallel increase in the rates of gdm . the prevalence of gdm is reported to vary widely from 3.8 to 21% in different parts of india depending on the geographical location and on the diagnostic criteria used . gdm has been associated with neonatal morbidity and mortality , including macrosomia , shoulder dystocia , other birth injuries , and neonatal hypoglycemia , in addition to congenital anomalies and still births . further , the offspring are potentially at a higher risk of developing childhood obesity later in life . evidence suggests that a large percentage of these women are at a higher risk of developing type 2 diabetes mellitus ( t2 dm ) in the future . despite high rates of morbidity , most women remain asymptomatic for the duration of their pregnancy and gdm goes unnoticed until a routine screening of blood sugar is carried out . hence , it is recommended that all pregnant women undergo screening for gdm at 24 - 28 weeks of gestation . there are several reports of gdm from india , but few reports on outcomes or on development of diabetes postpartum . the objectives of this study were to describe the clinical profile , maternal and fetal outcomes , and conversion rates to diabetes in women with gdm seen at a tertiary care diabetes center in urban south india . in this retrospective clinic - based study , clinical case records of 1,003 pregnant women seen between the year 1991 and 2011 at dr . mohan 's diabetes specialties centre ( dmdsc ) , a tertiary diabetes center at chennai in south india , were extracted from the diabetes electronic medical records ( demr ) . the demr database at dmdsc connects data of 15 centers / clinics of dmdsc in different areas in southern india and has been used as an effective tool to improve diabetes care and research . study variables collated from the demr for this study included demographic profile ( name , age , and contact information ) , clinical profile ( family history , age of onset of diabetes , last menstrual period , and expected date of delivery ) , anthropometric measurements ( height , weight , body mass index ( bmi ) ) , biochemical investigations ( oral glucose tolerance test ( ogtt ) and glycated hemoglobin ( hba1c ) ) , and the treatment regimen ( diet / exercise / oral hypoglycemic agents / insulin ) . after excluding preexisting diabetes and impaired glucose tolerance cases , normal glucose tolerance and cases with incomplete data , 898 women with gdm were included in our study . unfortunately only 174 women ( 19.3% ) could be followed - up to determine maternal and neonatal outcomes [ figure 1 ] . one possible explanation for this could be that as ours being a tertiary referral center for diabetes with no obstetric unit , women tend not to follow - up with us postpartum . another common problem in india is that women generally go back to their maternal homes for delivery which maybe in another city and hence determining pregnancy outcomes becomes difficult . yet , we have attempted to report pregnancy outcomes in a small cohort of women , whom we were able to trace and collect , follow - up details from . flow chart of patient selection during the first visit , a complete medical history was elicited , including history of any past or current illnesses , dietary patterns , and family history of diabetes as well as current medications . height was measured in centimeters using a stadiometer and weight in kilograms using an electronic scale . bmi was calculated as weight ( kg ) divided by height ( m ) squared . a fasting blood sample was obtained after an overnight fast of at least 8 h. participants whose diabetes status had previously been confirmed were given a standard south indian breakfast and a venous blood sample was drawn after 90 min for the postprandial glucose sample . in all others an ogtt using 100 g glucose load were done and were diagnosed using the earlier carpenter and coustan criteria adopted by the ada . in a few cases , who were already on oral drugs ( metformin ) and were doing well this treatment , it was continued . the follow - up periods varied from 6 weeks to several years and the mean follow - up duration was 4.5 years . in women who could be followed after delivery to determine progression to diabetes , an ogtt was done using 75 g glucose load . plasma glucose ( glucose - oxidase peroxidase method ) was estimated using a hitachi912 autoanalyzer ( hitachi , mannheim , germany ) . hba1c was measured by high performance liquid chromatography ( hplc ) using variant machine ( bio - rad , hercules , ca ) . the intra- and interassay coefficients of variation ( cv ) for the biochemical assays ranged from 3.1 to 7.6% . hba1c was measured by hplc ( bio - rad variant , bio - rad , hercules , california , usa ) . all biochemical investigations were done at dmdsc laboratory , which is certified by the college of american pathologists ( cap ) and the national accreditation board for testing and calibration of laboratories ( nabl ) . kg / m and overweight as those who had a bmi 23 kg / m based on world health organization ( who ) asia pacific guidelines . this classification was used for nonpregnant adults , that is , during the follow - up period . for assessing obesity during pregnancy , in 2013 , the institute of medicine ( iom ) published revised pregnancy weight gain guidelines that are based on pre - pregnancy bmi ranges recommended by the who . these ranges are independent of age , parity , smoking history , race , and ethnic background . the revised iom recommendations define normal weight as a bmi of 18.5 - 24.9 , overweight as a bmi of 25 - 29.9 , and obesity as a bmi of 30 kg / m or greater . since the study was based between 1991 and 2011 , the older ada criteria were used to diagnose diabetes in which samples were drawn at 1 h ( 1 h pg ) , 2 h ( 2 h pg ) , and 3 h ( 3 h pg ) after ingestion of the glucose load of 100 g using cut points recommended by carpenter and coustan and adopted by ada . the who criteria for nonpregnant adults was used for diagnosing diabetes and prediabetes , that is , for diabetes ; fasting plasma glucose ( fpg ) > 126 mg / dl or 2 h pg > 200 mg / dl and for prediabetes , impaired fasting glucose ( ifg ) ; fpg between 110 and 125 mg / dl and/or impaired glucose tolerance ( igt ) ; and 2 h pg between 140 and 199 mg / dl . the indian consensus is that a new born weighing > 3.5 kg should be considered as macrosomia . statistical analysis was done in statistical package for the social sciences ( spss ) software ( version 15 ) and microsoft excel 2007 . percent frequencies , means , and standard deviations for quantitative variables were used wherever necessary . during the first visit , a complete medical history was elicited , including history of any past or current illnesses , dietary patterns , and family history of diabetes as well as current medications . height was measured in centimeters using a stadiometer and weight in kilograms using an electronic scale . bmi was calculated as weight ( kg ) divided by height ( m ) squared . a fasting blood sample was obtained after an overnight fast of at least 8 h. participants whose diabetes status had previously been confirmed were given a standard south indian breakfast and a venous blood sample was drawn after 90 min for the postprandial glucose sample . in all others an ogtt using 100 g glucose load were done and were diagnosed using the earlier carpenter and coustan criteria adopted by the ada . the vast majority of those not controlled with diet and exercise , received insulin . in a few cases , who were already on oral drugs ( metformin ) and were doing well this treatment , the follow - up periods varied from 6 weeks to several years and the mean follow - up duration was 4.5 years . in women who could be followed after delivery to determine progression to diabetes , an ogtt was done using 75 g glucose load . plasma glucose ( glucose - oxidase peroxidase method ) was estimated using a hitachi912 autoanalyzer ( hitachi , mannheim , germany ) . hba1c was measured by high performance liquid chromatography ( hplc ) using variant machine ( bio - rad , hercules , ca ) . the intra- and interassay coefficients of variation ( cv ) for the biochemical assays ranged from 3.1 to 7.6% . hba1c was measured by hplc ( bio - rad variant , bio - rad , hercules , california , usa ) . all biochemical investigations were done at dmdsc laboratory , which is certified by the college of american pathologists ( cap ) and the national accreditation board for testing and calibration of laboratories ( nabl ) . obesity was defined as those who had a bmi 25 kg / m and overweight as those who had a bmi 23 kg / m based on world health organization ( who ) asia pacific guidelines . this classification was used for nonpregnant adults , that is , during the follow - up period . for assessing obesity during pregnancy , in 2013 , the institute of medicine ( iom ) published revised pregnancy weight gain guidelines that are based on pre - pregnancy bmi ranges recommended by the who . these ranges are independent of age , parity , smoking history , race , and ethnic background . the revised iom recommendations define normal weight as a bmi of 18.5 - 24.9 , overweight as a bmi of 25 - 29.9 , and obesity as a bmi of 30 kg / m or greater . since the study was based between 1991 and 2011 , the older ada criteria were used to diagnose diabetes in which samples were drawn at 1 h ( 1 h pg ) , 2 h ( 2 h pg ) , and 3 h ( 3 h pg ) after ingestion of the glucose load of 100 g using cut points recommended by carpenter and coustan and adopted by ada . the who criteria for nonpregnant adults was used for diagnosing diabetes and prediabetes , that is , for diabetes ; fasting plasma glucose ( fpg ) > 126 mg / dl or 2 h pg > 200 mg / dl and for prediabetes , impaired fasting glucose ( ifg ) ; fpg between 110 and 125 mg / dl and/or impaired glucose tolerance ( igt ) ; and 2 h pg between 140 and 199 mg / dl . the indian consensus is that a new born weighing > 3.5 kg should be considered as macrosomia . since the study was based between 1991 and 2011 , the older ada criteria were used to diagnose diabetes in which samples were drawn at 1 h ( 1 h pg ) , 2 h ( 2 h pg ) , and 3 h ( 3 h pg ) after ingestion of the glucose load of 100 g using cut points recommended by carpenter and coustan and adopted by ada . the who criteria for nonpregnant adults was used for diagnosing diabetes and prediabetes , that is , for diabetes ; fasting plasma glucose ( fpg ) > 126 mg / dl or 2 h pg > 200 mg / dl and for prediabetes , impaired fasting glucose ( ifg ) ; fpg between 110 and 125 mg / dl and/or impaired glucose tolerance ( igt ) ; and 2 h pg between 140 and 199 mg / dl . the indian consensus is that a new born weighing > 3.5 kg should be considered as macrosomia . statistical analysis was done in statistical package for the social sciences ( spss ) software ( version 15 ) and microsoft excel 2007 . percent frequencies , means , and standard deviations for quantitative variables were used wherever necessary . the mean maternal age was 29 4 years ( range 19 - 42 years ) , mean gestational age at first visit was 24 8.4 weeks , and mean bmi was 28.6 4 kg / m . while , 34.4% of the women were obese , 47.2% were overweight , and only 18.4% had ideal body weight as per the iom guidelines , seventy percent of gdm women had a family history of t2 dm , which included 49% with one parent ( either father or mother ) having diabetes and 21% with both parents having diabetes . mean pregestational weight and gestational weight in a subset of 120 women in whom it was available were 65.7 13 and 71.3 10.4 , respectively . baseline demographic and clinical profile of women with gdm ( n=898 ) table 2 shows the changes in hba1c and serum fructosamine values in a subset of the population in whom the hba1c and serum fructosamine values were available at their first and last antenatal checkup ( anc ) visit before delivery . mean hba1c of the 272 women was found to be 6.2% at first visit which improved to 6.0% by the last anc visit . the mean fructosamine at first visit was found to be 219 37 mol which improved to 197 25 mol at last anc visit . anc visits among 893 women with gdm , 585 ( 65.5% ) were treated with insulin therapy , 280 ( 31.3% ) were advised diet modification and exercise therapy , and 28 women ( 3.2% ) were given oral hypoglycemic agents ( metformin ) . seventy - two women ( 41.3% ) underwent elective caesarian delivery , 42 ( 24.1% ) had emergency cesarean delivery , 53 ( 30.5% ) had normal vaginal delivery , and seven ( 4.1% ) had induced vaginal delivery , which included assisted delivery . table 3 presents the neonatal outcomes and complications during delivery of the 174 women in whom outcome data was available . outcomes and neonatal complications ( n=174 ) follow up ogtt data could only be obtained in 174/898 women ( 19.3% ) . of these 174 women , 101 ( 58% ) developed t2 dm postpartum and nine ( 5% ) developed igt , while 64 ( 37% ) reverted back to normal glucose tolerance ( ngt ) [ table 4 ] . of the 101 women who developed diabetes , 58 women ( 56.3% ) developed diabetes within 5 years , 35 ( 33.9% ) within 5 - 10 years , and eight women ( 9% ) 10 years after delivery . thus , 90.2% of conversion to t2 dm took place within 10 years after the delivery . the mean maternal age was 29 4 years ( range 19 - 42 years ) , mean gestational age at first visit was 24 8.4 weeks , and mean bmi was 28.6 4 kg / m . while , 34.4% of the women were obese , 47.2% were overweight , and only 18.4% had ideal body weight as per the iom guidelines , seventy percent of gdm women had a family history of t2 dm , which included 49% with one parent ( either father or mother ) having diabetes and 21% with both parents having diabetes . mean pregestational weight and gestational weight in a subset of 120 women in whom it was available were 65.7 13 and 71.3 10.4 , respectively . baseline demographic and clinical profile of women with gdm ( n=898 ) table 2 shows the changes in hba1c and serum fructosamine values in a subset of the population in whom the hba1c and serum fructosamine values were available at their first and last antenatal checkup ( anc ) visit before delivery . mean hba1c of the 272 women was found to be 6.2% at first visit which improved to 6.0% by the last anc visit . the mean fructosamine at first visit was found to be 219 37 mol which improved to 197 25 mol at last anc visit . among 893 women with gdm , 585 ( 65.5% ) were treated with insulin therapy , 280 ( 31.3% ) were advised diet modification and exercise therapy , and 28 women ( 3.2% ) were given oral hypoglycemic agents ( metformin ) . seventy - two women ( 41.3% ) underwent elective caesarian delivery , 42 ( 24.1% ) had emergency cesarean delivery , 53 ( 30.5% ) had normal vaginal delivery , and seven ( 4.1% ) had induced vaginal delivery , which included assisted delivery . table 3 presents the neonatal outcomes and complications during delivery of the 174 women in whom outcome data was available . follow up ogtt data could only be obtained in 174/898 women ( 19.3% ) . of these 174 women , 101 ( 58% ) developed t2 dm postpartum and nine ( 5% ) developed igt , while 64 ( 37% ) reverted back to normal glucose tolerance ( ngt ) [ table 4 ] . of the 101 women who developed diabetes , 58 women ( 56.3% ) developed diabetes within 5 years , 35 ( 33.9% ) within 5 - 10 years , and eight women ( 9% ) 10 years after delivery . thus , 90.2% of conversion to t2 dm took place within 10 years after the delivery . this paper reports on the clinical profile , outcomes , and conversion rate to diabetes in a large sample of women with gdm seen at a tertiary diabetes care center in south india . a positive correlation between maternal weight and risk of gdm was observed in studies by seshiah et al . , and chu et al . in our study , nearly half of the pregnant women were over 30 years of age and 81% were obese and 12% were overweight . some studies have also attributed the risk of adverse outcomes associated with gdm to confounding characteristics such as obesity and advanced maternal age of women with gdm . the hyperglycemia and adverse pregnancy outcome ( hapo ) study showed that lifestyle factors and obesity contribute significantly to the increasing incidence of gdm . hence , targeting overweight women in the population with lifestyle intervention program may help prevent gdm . there is also an important association between family history of t2 dm and gdm . in our study , 70% of women with gdm had family history of t2 dm which is higher compared to earlier studies . goldman et al . , reported cesarean section rates of 35.3% in women with gdm in united states with similar reports from casey et al . along with the parallel increase in gdm , these rates have also increased over the years with 65.2% women in the present study undergoing a cesarean section . as reported in other studies , macrosomia and perinatal mortality the neonatal complications are more than that reported in other studies . higher maternal age ( mean maternal age was 29 5 ) could be hypothesized as the cause to gdm and subsequent conversion to diabetes , which is seen in our study . while macrosomic infants are at an increased risk of developing t2 dm , hypertension , and obesity later in adulthood ; low birth weight infants , when exposed to excess food and sedentary lifestyle later in life ( catch up growth ) also develop t2 dm . therefore , curtailing excess weight gain during childhood is equally important to prevent diabetes . due to its poor sensitivity in diagnosis of gdm , our study showed that the mean hba1c level in women with gdm at diagnosis during different trimesters was 6.2 1.0% , which decreased to 6.0 1% by the last anc visit . as many women visited the clinic only in their second and third trimester there might not have been enough time for a further reduction in hba1c to be demonstrated . finally , some patients initially diagnosed with gdm were subsequently deemed to have type 1 diabetes which could explain higher than expected hba1c 's values . apart from hba1c , we also monitored serum fructosamine values during pregnancy as it is an index of control of glucose over a 2 - 3 week 's period . , identified fructosamine as a more sensitive test to detect abnormal glucose tolerance in gdm than hba1c because it is an index of shorter term diabetes control . we found that the difference in mean hba1c between first visit and last anc visit was only 0.2% , but that of the mean fructosamine between the visits was 22 mol ( 10% decrease ) . during pregnancy when hormonal changes cause fluctuation in blood glucose concentrations , estimation of fructosamine levels could be a useful test to monitor diabetes control as it reflects average blood glucose levels of shorter duration ( 2 - 3 weeks ) . serum fructosamine , though not a useful screening test , could still be used to assess short - term maternal diabetes control during pregnancy . use of this test as an alternative for hba1c for managing gdm in our population where iron deficiency anemia is very common during pregnancy needs to be investigated further . we found that the conversion to t2 dm occurred even within 5 years post - delivery in many women , and by 10 years , most women who would convert to diabetes had already done so . it is possible that these rates are higher because those who were at higher risk only came for the follow - up checkup . other reports have also shown that women with gdm have a 17 - 63% risk of t2 dm within 5 - 16 years . studies have also shown that women with gdm as well as their children are at a greater risk of developing future t2 dm . first , it is a clinic - based study , from a tertiary care diabetes center . thus , being a referral diabetes center , most women do not come for follow - up regularly after the initial visit . for the same reason , we could not obtain complete maternal and fetal outcomes from all our gdm patients for the cohort . however , the strength of the study are the fairly large numbers of gdm women studied , the hba1c and fructosamine data and the follow - up data including conversion to diabetes . this study also emphasizes the need to strategize ways to follow up gdm women in india . in conclusion , among those who came for follow - up , almost 60% women in india with gdm seen at our center progress to t2 dm within 5 years and > 90% , within 10 years , post - delivery . this study emphasize the need for not just prevention , but also for developing evidence based cost - effective and accessible models of care for all women with gdm . such a model called women in india with gdm strategy ( wings ) is currently under evaluation at our center and is supported by the international diabetes federation ( idf ) . this model plans to focus on management of gdm right from diagnosis , treating complications , ensuring a safe delivery , and preventing or delaying progression to t2 dm .

aim : to describe the clinical profile , maternal and fetal outcomes , and the conversion rates to diabetes in women with gestational diabetes mellitus ( gdm ) seen at a tertiary care diabetes center in urban south india.materials and methods : clinical case records of 898 women with gdm seen between 1991 and 2011 were extracted from the diabetes electronic medical records ( demr ) of a tertiary care diabetes center in chennai , south india and their clinical profile was analyzed . follow - up data of 174 gdm women was available . to determine the conversion rates to diabetes , oral glucose tolerance test ( ogtt ) was done in these women . glucose tolerance status postpartum was classified based on world health organization ( who ) 2006 criteria.results:the mean maternal age of the women was 29 4 years and mean age of gestation at first visit were 24 8.4 weeks . seventy percent of the women had a family history of diabetes . seventy - eight percent of the women delivered full - term babies and 65% underwent a cesarean section . the average weight gain during pregnancy was 10.0 4.2 kg . macrosomia was present in 17.9% of the babies , hypoglycemia in 10.4% , congenital anomalies in 4.3% , and the neonatal mortality rate was 1.9% . mean follow - up duration of the 174 women of whom outcome data was available was 4.5 years . out of the 174 , 101 women who were followed - up developed diabetes , of whom half developed diabetes within 5 years and over 90% , within 10 years of the delivery.conclusions:progression to type 2 diabetes mellitus ( t2 dm ) in indian women with gdm is rapid . there is an urgent need to develop standardized protocols for gdm care in india that can improve the maternal and fetal outcomes and help prevent future diabetes in women with gdm .