Datasets:

ewhfef

/

mix_cpt

like 1

strokes of the left middle cerebral artery ( lmca ) territory often lead to impairments in language function that are collectively referred to as aphasias . language recovery after lmca stroke is highly variable , and many patients remain chronically aphasic despite optimal rehabilitative approaches [ 2 , 3 ] . aphasia following lmca stroke typically results from lesions affecting frontal and/or temporal language regions in the left hemisphere and also often involves damage to white matter pathways connecting these regions [ 411 ] . functional neuroimaging studies indicate that the recovery of language abilities after lmca stroke involves the restoration of language - related processing in the remaining tissues near affected language areas as well as the compensatory recruitment of unaffected areas for language - related processing [ 1214 ] . while downregulated responses in affected left - hemisphere language areas and upregulated responses in unaffected right - hemisphere homologues are commonly observed during language task performance in acute patients [ 12 , 13 , 15 ] , the restoration of typical language - related responses in residual left - hemisphere language areas ( which is thought to be marked by a restoration of left - hemisphere dominance for language - related processing ) is likely critical for the successful long - term recovery of language functions [ 12 , 1520 ] . thus , while the upregulation of right - hemisphere responses during language task performance might reflect a form of compensatory reorganization , it is likely less effective than the reinstatement of left - hemisphere processing for accomplishing language task performance [ 13 , 16 , 2023 ] . studies investigating how changes in cortical function relate to language recovery following stroke provide strong evidence indicating that the preservation and/or restoration of language - related processing in the residual left inferior frontal gyrus ( ifg ) , a region that has been strongly implicated in various language processes such as word processing and word generation [ 2426 ] , is strongly related to the recovery of language functions in both the acute and chronic stages of recovery . for example , adult patients with acute injury who show preserved dominance of the residual left ifg for language task performance have less severe language impairments than patients who depend on the compensatory recruitment of the right ifg to accomplish the same task , indicating that the preservation of language - related processing in this region after lmca stroke is an important factor in determining initial aphasia severity [ 21 , 22 ] . in addition , adult patients that receive early poststroke aphasia rehabilitation show enhanced language - related responses in the residual left ifg compared to patients that do not receive early rehabilitation , and the magnitude of treatment - related increases in left ifg responses during language task performance is correlated with improvements in language function after treatment . similarly , increases in the left - lateralization of ifg activity related to language task performance from early to chronic recovery phases correlate with improvements in naming ability in adult patients with poststroke aphasia , and the level of language - related activity in left frontal areas correlates with improvements in naming ability subsequent to behavioral treatments in chronic patients . the development of treatments that can facilitate the restoration of language - related processing in residual frontal language areas may , therefore , be an important step in improving both spontaneous and treatment - induced recovery in patients with poststroke aphasia . techniques such as transcranial magnetic stimulation ( tms ) enable the noninvasive manipulation of cortical excitability in specific parts of cortex and may provide a means for facilitating beneficial cortical plasticity in patients with poststroke aphasia [ 28 , 29 ] . experimental interventions utilizing these techniques typically attempt to induce changes in cortical function that mirror those observed in successfully recovered patients by transiently enhancing the excitability of residual left - hemisphere language areas or suppressing responses in their right - hemisphere homologues [ 2931 ] . high - frequency tms stimulation protocols ( e.g. , > 5 hz ) such as intermittent theta burst stimulation ( itbs ) are delivered in short intervals to produce a rapid facilitation of synaptic transmission in the stimulated cortex that can persist for over an hour after the initial stimulation session . in addition to facilitating changes in local synaptic transmission and evoked potential amplitude , itbs may also alter the temporal characteristics of ongoing oscillatory activity , suggesting that it may lead to changes in ongoing neural dynamics at larger spatial scales that reflect changes in the functional organization of distributed functional networks . excitatory stimulation protocols are typically applied to the residual left ifg in order to facilitate language - related processing [ 3436 ] . in contrast , low - frequency stimulation protocols ( e.g. , < 1 hz ) that are delivered in continuous trains for longer periods of time have predominantly inhibitory effects on synaptic transmission and are typically applied to the right ifg in order to reduce contralateral compensation and/or interference during language - related processing [ 3640 ] . studies investigating the efficacy of these paradigms for restoring language function after stroke have provided consistent evidence for improvements in language function subsequent to stimulation [ 34 , 3638 ] . studies assessing the general effects of excitatory and inhibitory stimulation paradigms on neuroimaging measures of language - related responses in aphasic patients suggest that improvements in language function are accompanied by changes in the responses of both the residual left - hemisphere language network and homologous areas in the right hemisphere , although research in this area remains limited . a previous behavioral and functional mri ( fmri ) study conducted by our laboratory found that after 10 sessions of itbs applied to residual language - responsive left frontal cortex identified with a semantic decision / tone decision task , patients with chronic poststroke aphasia showed significant improvements in word generation as well as changes in fmri responses during a semantic decision task that included a significant leftward shift in the lateralization of activity in the ifg . in addition , a previous analysis of concurrently collected diffusion tensor imaging ( dti ) data from the same patients found evidence for changes in white matter integrity in multiple regions including the left ifg following itbs treatment . however , a major limitation of our previous fmri study is that it was restricted to changes in activation associated with the same task that was used to define language - responsive cortex for targeting with itbs , and this limits inferences regarding whether or not similar effects might be observed for activation during other language tasks . our previous fmri analysis was also limited in that it only assessed changes in fmri measures of activation , and it is increasingly recognized that the characterization of changes in measures of interregional connectivity is important for developing a full understanding of how changes in interregional interactions relate to the recovery of function after stroke [ 42 , 43 ] . in addition , our previous structural analysis was restricted to investigating changes in white matter integrity after itbs , although changes in cortical grey matter morphology might also be expected since excitatory tms protocols have been found to result in measureable changes in cortical grey matter volume after as little as 5 days of treatment in individuals without stroke . here , we first analyzed the pre-/postintervention fmri data to assess whether or not itbs might have similar effects on fmri responses elicited by a covert verb generation ( vg ) paradigm that was not used to define itbs targets . notably , while both the semantic decision paradigm used in our previous study and the vg paradigm used in the current study reliably evoke strong responses in the left ifg in healthy individuals and in patients with poststroke aphasia , they target different functional domains ( word comprehension versus word generation ) and there is typically little overlap between the activations attained with these tasks beyond the left inferior frontal cortex . we hypothesized that if itbs has a general facilitatory effect on language - related processing in the residual left ifg ( i.e. , by modulating synaptic transmission to facilitate communication with other language - relevant areas or to suppress interference from language - irrelevant interactions with other areas ) , then patients should show increased responses in the left ifg during covert verb generation after treatment with itbs and increased left - lateralization of ifg activity associated with covert verb generation . because language lateralization in frontal cortex is associated with both higher levels of activity in the left hemisphere and lower levels of activity in the right hemisphere for language - related versus non - language - related tasks and because our previous study also found evidence for decreased activity in the right ifg related to semantic decisions following itbs , we expected that patients would also show reductions in right ifg activity during covert verb generation after itbs . in addition , because studies of healthy individuals indicate that the presence of left - lateralized ifg activation during language tasks may be in part due to a task - dependent reduction in connectivity between left and right ifg and because we are unaware of any studies that have investigated changes in functional mri measures of connectivity in patients with poststroke aphasia subsequent to itbs treatment , we also investigated whether or not interhemispheric connectivity between right and left ifg during covert verb generation was affected by itbs . finally , in order to fully characterize the structural effects of itbs in these patients , we also tested whether or not patients showed changes in regional grey matter volume after itbs treatment . eight prospectively identified patients ( 4 females ; mean age = 54.4 , sd = 12.7 ) with chronic aphasia resulting from lmca stroke were recruited as described previously [ 34 , 41 ] . the mean time since stroke for all patients included was 5.25 years ( sd = 3.62 ) . four patients presented with anomic aphasias ; of these subjects , two also presented with dysarthria and one also presented with conduction aphasia . none of the patients had contraindications to mri scanning , none had history of seizures , and all were right - handed prior to the stroke . the study was approved by the university of cincinnati , cincinnati children 's hospital medical center , and university of alabama at birmingham institutional review boards and adhered to the declaration of helsinki regarding human subject research . neuropsychological measures of language function were acquired before and after itbs treatment as described in previous publications [ 34 , 41 ] . briefly , naming and word - finding abilities were evaluated using the boston naming test ( bnt ) , receptive vocabulary was evaluated using the peabody picture vocabulary test ( ppvt ) , verbal fluency was evaluated using the semantic fluency test ( sft ) and controlled oral word association test ( cowat ) , and comprehension was evaluated using the complex ideation subtest of the boston diagnostic aphasia examination ( bdae compid ) . patients also completed the min - communicative abilities log in order to provide subjective measurements of progress in verbal communication . pre- and posttreatment testing used different versions of the assessments in order to reduce the potential for learning - related effects . detailed descriptions of all itbs and neuronavigation protocols performed on these patients can be found in our previous publication . briefly , all patients received itbs to residual language - responsive cortex in or near the left ifg as identified using an fmri semantic decision / tone decision language localizer task described in our previous publication . stimulation intensities used for each patient were set at 80% of the active motor threshold obtained from stimulation of the right motor cortex . stimulation sessions occurred each day for five consecutive weekdays over the course of two weeks , resulting in a total of 10 stimulation sessions . each session consisted of 600 total pulses , with three pulses at 50 hz given every 200 milliseconds in 2-second trains at 10-second intervals over a 200-second period . fmri - guided neuronavigation using brainsight2 ( rogue research inc . , montreal canada ) enabled the targeting of residual language - responsive cortex in the left frontal lobe near the ifg ( frontal targets were used for 7 patients ; language - responsive cortex in the left temporal lobe was targeted for one patient ; see figure 1 in ) that was identified using the fmri localizer task , and allowed for reliable and precise localization of the same location at each session . the functional and anatomical mri data presented in this study were acquired using a varian 4 tesla unity inova whole body mri / mrs scanner ( varian , inc . , palo alto , ca ) . for each patient , a high - resolution t1-weighted 3d - mdeft ( modified driven equilibrium fourier transform ) anatomical volume ( scan parameters : repetition time / echo time = 13.1/6 ms , field of view = 25.6 19.2 19.2 cm , flip angle = 22 , and voxel dimensions = 1 1 1 mm ) and t2-weighted blood oxygen - level dependent ( bold ) volumes ( scan parameters : repetition time / echo time = 3000/30 ms , fov = 25.6 25.6 cm , matrix = 64 64 pixels , number of slices = 30 , slice thickness = 4 mm , and flip angle = 75 ) were obtained at both pretreatment and posttreatment sessions . fmri data were collected while patients performed an alternating block - design covert verb generation ( vg ) task that consisted of alternating 30 s blocks of an active condition involving silent verb generation in response to binaurally presented nouns and a control condition involving bilateral sequential finger tapping ( ft ) in response to a frequency modulated tone centered on 400 hz that was modulated by 25% every 5 s. this task was chosen because previous studies indicate that it reliably produces left - lateralized activation patterns and because it has excellent test - retest ( across time points ) reliability for evoked activity patterns in patients with aphasia due to lmca stroke . the control condition served to control for the auditory stimulation during the noun presentation in the active condition and to distract patients from continuing to generate verbs outside of the active condition blocks while maintaining a task state . each patient 's understanding of and ability to perform the task were assessed prior to scanning by having the patients perform the task outside of the scanner . patients had to be able to generate at least one verb in response to each noun prior to proceeding to scanning . following each scan session , patients performed a forced - choice recognition test involving the nouns that were presented during the covert verb generation task , and the percentage of correctly remembered nouns was utilized as an indirect measurement of task performance . all mri data were preprocessed using matlab scripts implementing functions from the most recent release version of statistical parametric mapping ( spm12 , wellcome department of cognitive neurology , london , uk ) running in matlab r2014b ( the mathworks inc . , natick , ma ) . functional mri data from the baseline and follow - up scans were slice - time corrected , realigned and resliced , and coregistered to the structural image obtained during the same scan . deformation fields containing the deformation differences between across - session average anatomical volume and the anatomical scan from each session were used to warp the coregistered functional volumes to the across - session average anatomical volume . the average anatomical scan was then normalized to montreal neurological institute ( mni ) template using unified normalization - segmentation as implemented in the new segment tool in spm . the deformation parameters obtained from the warping of the anatomical volume were used to normalize the functional volumes to mni template space . the functional volumes were resampled to 2 2 2 millimeter isometric voxels and spatially smoothed with a 6-millimeter full - width half maximum ( fwhm ) gaussian kernel . functional volumes in which participants moved more than 0.5 mm in one frame ( 3 s ) were replaced with a volume interpolated from adjacent time points . volumes were to be rejected if they contained > 3 mm of motion , but no volumes met criteria for rejection . individual patient lesion delineations were created from the pre - itbs anatomical scans using an automated voxel - based bayesian classification algorithm developed by our lab and implemented in the lesion_gnb toolbox for spm12 . the resulting lesion delineations were used to create a group - level lesion frequency map . the group - level lesion frequency map is provided in figure 2 and illustrates the number of patients with lesioning at each voxel . the greatest across - patient lesion overlap was observed in the left insula , left putamen , and left precentral gyrus ( figure 2 ) . anatomical data utilized in the voxel - based morphometry ( vbm ) analysis were preprocessed according to a recently described longitudinal preprocessing pipeline for vbm analyses . first , probabilistic tissue segmentation implementing the new segment + dartel ( diffeomorphic automatic registration through exponentiated lie algebra ) approach with an additional tissue prior ( mean of white matter and csf tissue probability maps ) and medium bias regularization was used to obtain dartel - compatible tissue probabilistic maps ( tpms ) encoding the grey matter ( gm ) and white matter ( wm ) probabilities for each voxel . the additional tissue prior and medium bias regularization were used since this has been shown to improve template - space normalization using the new segment + dartel approach . next , patient - specific anatomical templates were created with dartel using the gm and wm tissue maps from the baseline and follow - up scans . for each patient , the baseline and follow - up tpms were then warped to the subject - specific templates and modulated using the jacobian determinant of the transformation to increase sensitivity to absolute differences in gm volume [ 58 , 59 ] . the creation of the patient - specific templates was performed in order to enable more precise between - session spatial alignment of tpms . a group template was then created by nonlinearly registering all of the patient - specific templates simultaneously using dartel . the modulated / warped gm and wm tpms obtained from each patient were then nonlinearly normalized to the population template and modulated with the jacobian determinant of the transformation . finally , the population template was then registered to mni space using an affine transformation , and each tpm was then coregistered to mni space using an identical transformation and smoothed using an 8 mm fwhm gaussian kernel . functional mri activity related to the covert verb generation task was quantified by contrasting the active condition blocks ( vg ) against the control condition blocks ( ft ) . for each patient , the fmri data were fit to a general linear model ( glm ) where each active block was modeled as a boxcar regressor convolved with a canonical hemodynamic response function . to account for temporal variability in the hemodynamic response , time and dispersion derivatives were modeled as basis functions in the first - level analyses . single - patient statistical maps containing contrast estimates quantifying differences between the active and control conditions were computed for both pre- and post - itbs scans . statistical comparisons of the contrast estimate maps were used to evaluate changes in activation between the pre- and post - itbs sessions . fmri data were first analyzed using a region of interest ( roi ) approach in order to directly test our hypotheses regarding changes in ifg activity and connectivity between pre- and post - itbs sessions . roi masks were created using the marsbar toolbox for spm ( http://marsbar.sourceforge.net/ ) , and rois in the left and right ifg were defined using peak activation coordinates obtained from an independent analysis of activation related to covert verb generation in healthy individuals . 8 mm radius spherical rois were centered on voxels in the left ifg ( mni coordinates : x = 50 , y = 16 , and z = 16 ) and on mirrored coordinates in the right ifg ( mni coordinates : x = 50 , y = 16 , and z = 16 ) . only two patients ( p1 and p4 ) had overlap between the left ifg roi and their lesion delineation . p1 's lesion encompassed nearly the entire left lateral prefrontal cortex and the left ifg roi fell directly onto the perilesional rim , resulting in 100% lesion overlap with the left ifg roi . p4 's lesion was primarily localized to the left ventral ifg and the roi overlapped by 60.2% with the perilesional rim . nonetheless , for both patients , the 1st principal component of the signal extracted from the roi showed phasic responses consistent with the design of the task . while anatomical overlap was noted , functional analyses indicated bold signal changes aligned with the box - car function of the fmri task design indicating that the fmri responses reflected the responses of perilesional cortex rather than csf . additionally , signal from the left ifg roi was more strongly correlated with signal from the right ifg roi than with the csf signal , and this was only marginally influenced by partialling out variability accounted for by the csf signal ( supplemental s1 ) ( see supplementary material available online at http://dx.doi.org/10.1155/2016/4796906 ) . nonetheless , control analyses indicated that excluding these patients did not substantially change the statistical significance of results for any analysis involving the left ifg roi , indicating that the presence of lesion - roi overlaps for these patients did not strongly influence the outcome of the roi analyses . to test our hypothesis about whether patients showed changes in fmri activity during covert verb generation in the left and right ifg rois after itbs , we extracted the mean parameter estimates for the active versus control condition contrast and compared the estimates obtained for each roi between the pre- and post - itbs sessions . this analysis provided information about the mean level of activity in the ifg rois for each session , with positive values indicating stronger activation during the active condition and negative values indicating stronger activation during the control condition . to test our hypothesis that patients would show more strongly left - lateralized ifg activation following itbs , laterality index ( li ) analyses were performed to quantify the lateralization of activity related to covert verb generation . for each patient li values range from 1 ( complete right - lateralization ) to 1 ( complete left - lateralization ) , and lis for each session were calculated according to the formula shown in(1)li=activationleft / mwfactivationrightactivationleft / mwf+activationright . using adaptive threshold determination , the term activation is defined as the values of voxels with intensities that are greater than the within - roi average intensity for the contrast of interest . this method was chosen because it has been shown to provide reliable li estimates that are more robust against interindividual variability in signal - to - noise ratio than approaches that employ arbitrary / fixed cut - off thresholds that are applied to all subjects ( e.g. , corrected p value thresholds ) ; this method does not substantially increase susceptibility to false positives . the mask weighting factor ( mwf term in ( 1 ) ) is used to adjust the li estimates to account for differences in the volume of each roi and is defined by the proportion of the volumes of the left- and right - hemisphere rois . to test our hypothesis regarding the effects of itbs on interhemispheric connectivity , we conducted a generalized psychophysiological interaction ( gppi ) analysis using the gppi toolbox for spm . gppi enables the modeling of context - specific changes in the relationship of activity in one brain region , referred to as a seed region , to activity in other brain regions by including a term specifying an interaction effect between the seed region time series and the task time series in each first - level glm . gppi effects are interpreted as changes in interregional connectivity which are driven by psychological states related to factors such as the task being performed [ 63 , 65 ] , making gppi an appropriate tool for testing our hypothesis that itbs would lead to changes in interhemispheric connectivity during covert verb generation . for each patient , the first principal component of the bold time series from each scan was extracted from the right ifg roi and entered as a seed time series for the gppi analysis . the right ifg roi , rather than the left ifg roi , was chosen in order to reduce potential confounds in the extracted time series related to lesion proximity since two patients showed substantial lesion overlap with the left ifg roi . cerebrospinal fluid ( csf ) and wm signals were also included as nuisance variables in the gppi model in order to reduce the influence of nonneural signals on estimates of task - dependent connectivity . for each patient , gppi estimates quantifying the level of condition - dependent connectivity from right to left ifg during each session were then extracted from the gppi model using the marsbar tool for spm and compared from pre- to post - itbs sessions . all between - session comparisons were tested for statistical significance using two - tailed dependent samples t - tests . correlations between changes in fmri measures of ifg function and behavioral measures were assessed using linear correlation analyses . multiple - comparisons correction for all roi - driven comparisons between pre - itbs and post - itbs scans was performed using the benjamini - hochberg procedure to control the false - discovery rate ( fdr ) at 0.05 [ 67 , 68 ] , and all associated p values presented are fdr - adjusted . exploratory whole - brain glm and gppi analyses were also performed in order to provide a more thorough characterization of functional mri measures of activity and right ifg connectivity related to the vg task at the pre - itbs and post - itbs sessions . exploratory and ad hoc partial correlational analyses were performed to further characterize the data and are presented with uncorrected p values . vbm is a technique that allows for the measurement of grey matter ( gm ) volume in t1-weighted mri data [ 58 , 69 ] . here , we used vbm to address the question of whether or not patients showed changes in gm volume after itbs treatment . the dartel - processed subject - level grey matter maps from baseline and follow - up scans were entered into a dependent samples t - contrast that also included each patient 's lesion volume as a nuisance covariate . changes in gm volume were assessed at the whole - brain level using dependent samples t - contrasts . analyses evaluating performance on the out - of - scanner forced - choice noun recognition task revealed good performance for both pre - itbs ( mean % correct = 94.13 ; sem = 1.63 ) and post - itbs ( mean % correct = 95.13 ; sem = 2.05 ) sessions . dependent samples t - test comparing pre - itbs and post - itbs evaluations did not reveal a significant change in noun recognition performance ( t7 = 0.415 , p = 0.69 ) . these results are consistent with previous studies that have reported good out - of - scanner noun recognition performance on this task in patients with poststroke aphasia [ 46 , 70 ] . the effects of itbs on neuropsychological measures of language function have been previously reported [ 34 , 41 ] and will briefly be described here to provide details relevant to the current study . our previous analysis revealed that patients showed a statistically significant ( at p < 0.05 ) improvement on the semantic fluency test , statistically nonsignificant ( at p < 0.05 ) improvements in performance on the boston naming test ( boston diagnostic aphasia examination , peabody picture vocabulary test , and the communicative abilities log ) , and a statistically nonsignificant ( at p < 0.05 ) decrease in performance on the controlled oral word association test ( see table 1 in ) . since statistically significant improvements were only observed for performance on the semantic fluency test , exploratory analyses investigating the relationship between changes in functional mri measures of ifg activation / connectivity and the behavioral effects of itbs were restricted to this test . while ideally behavioral correlations would have been performed on the out - of - scanner noun recognition task , the miniscule change between sessions and globally good performance precluded this approach . importantly , the semantic fluency test , like the covert verb generation task , required patients to generate words in response to a prompt . for the semantic fluency test , patients generated as many words as they could think of that were congruent with category prompt ( e.g. , animals ) with a 1-minute time limit , and performance on the semantic fluency test was measured by the number of congruent words produced within the 1-minute time limit . thus , whereas the covert verb generation task required patients to silently generate verbs in response to presented nouns , the semantic fluency test required patients to generate words in response to a given category . to test our hypotheses regarding the effects of itbs on the magnitudes of activity in left and right ifg during covert verb generation , we compared activation magnitudes at each roi between pre- and post - itbs sessions . it is worth noting that the rois used for these analyses were chosen a priori in order to avoid the introduction of bias by defining rois based on the glm results . our analyses revealed increased activation magnitudes in the left ifg ( t7 = 3.32 ; fdr p = 0.02 ; mean change = 0.54 , sem = 0.18 ) and decreased activation magnitudes in right ifg ( t7 = 2.3 ; fdr p = 0.05 ; mean change = 0.22 , sem = 0.09 ) related to covert verb generation after itbs treatment . left and right ifg activation magnitudes for each patient are shown in figures 3(b ) and 3(c ) . on average , patients showed lower levels of activity in left ifg and higher levels of activity in right ifg during covert verb generation compared to finger tapping pre - itbs . in contrast , patients showed higher levels of activity in left ifg and similar levels of activity in right ifg during covert verb generation compared to finger tapping post - itbs . accordingly , results from the li analysis indicated that overall ifg responses during covert verb generation were more strongly left - lateralized after itbs treatment ( t7 = 3.46 , fdr p = 0.02 ; mean change = 0.48 , sem = 0.14 ) . li estimates for each patient at pre - itbs and post - itbs sessions are shown in figure 3(d ) . on average , patients showed right - lateralized activation patterns in ifg pre - itbs . in contrast , patients showed left - lateralized activation patterns in ifg post - itbs . to allow for a more thorough characterization of the data , although no regions showed significant effects at a whole - brain fdr - corrected threshold of 0.05 , the uncorrected statistical maps provide evidence for increased responses related to covert verb generation in left - hemisphere frontal , temporal , and parietal regions after itbs ( figure 4(a ) ) . while no regions showed changes that were significant after multiple - comparisons correction , the most reliable ( voxelwise p < 0.001 , uncorrected ) increases in activity were observed in the left ifg pars opercularis ( peak mni coordinate : 40 , 14 , 10 ; 171 voxel clusters ) , the right thalamus ( peak mni coordinate : 10 , 14 , 20 ; 64 voxel clusters ) , and the right cerebellum vi ( peak mni coordinate : 30 , 62 , 30 ; 3 voxel clusters ) , and the most reliable ( voxelwise p < 0.001 , uncorrected ) decreases in activity were observed in the right cerebellum crus 2 ( peak mni coordinate : 48 , 52 , 42 ; 12 voxel clusters ) , the right cerebellum viii ( peak mni coordinate : 34 , 44 , 40 ; 6 voxel clusters ) , and the right inferior temporal gyrus ( peak mni coordinate : 54 , 6 , 30 ; 4 voxel clusters ) . to test our hypothesis regarding the effects of itbs on effective connectivity between the right and left ifg , we compared gppi estimates between the right ifg seed region and the left ifg target region between pre- and post - itbs sessions . it is important to note that gppi estimates reflect the magnitude of condition - dependent changes in the relationship between activity in the seed region and activity in the target region . thus , the connectivity estimates for each session quantify how the relationship between responses in the right ifg and responses in the left ifg differed between conditions . these analyses revealed that compared to the pre - itbs session , patients showed reductions in gppi estimates between the right ifg seed region and the left ifg target region for the active condition contrast at the post - itbs session ( t7 = 2.97 ; fdr p = 0.03 ; mean change = 0.24 , se = 0.09 ) . the gppi estimates for the active condition contrast for each patient are shown in figure 3(e ) . since the gppi measurement quantifies differences in the relationship between activity in the seed region ( r ifg ) and the target region ( l ifg ) that are moderated by task condition ( vg - ft ) , a reduction in the gppi estimate between right ifg and left ifg would indicate that the effect of right ifg activity on left ifg activity for covert verb generation relative to finger tapping was reduced after itbs . patients showed a small but positive mean effect of covert verb generation on connectivity between the right ifg and left ifg pre - itbs , indicating that right ifg activity was more positively associated with left ifg activity during covert verb generation than during finger tapping . in contrast , patients showed a negative mean effect of covert verb generation on connectivity between the right ifg and left ifg post - itbs , indicating that right ifg activity was more negatively associated with left ifg activity during covert verb generation than during finger tapping . thus , the direction of the effect of task condition on the relationship between right ifg activity and left ifg activity changed between pre - itbs and post - itbs sessions , with right ifg activity being more negatively associated with left ifg activity during covert verb generation than during finger tapping . to allow for a more thorough characterization of the data , whole - brain gppi analyses were also performed . although no regions showed significant effects at a whole - brain fdr - corrected threshold of 0.05 , the uncorrected statistical maps provide evidence for reduced connectivity between the right ifg and left - hemisphere frontal , temporal , and parietal regions after itbs ( figure 4(b ) ) . while no regions showed changes that were significant after multiple - comparisons correction , the most reliable ( voxelwise p < 0.001 , uncorrected ) reductions in right ifg connectivity associated with the vg task were observed in the right middle temporal gyrus ( peak mni coordinate : 36 , 74 , 6 ; 65 voxel clusters ) , the right superior frontal gyrus ( peak mni coordinate : 16 , 2 , 60 ; 32 voxel clusters ) , the left ifg pars opercularis ( peak mni coordinate : 50 , 8 , 22 ; 8 voxel clusters ) , the right postcentral gyrus ( peak mni coordinate : 36 , 32 , 56 ; 7 voxel clusters ) , the left lingual gyrus ( peak mni coordinate : 14 , 70 , 0 ; 6 voxel clusters ) , the right cerebellum vi ( peak mni coordinate : 24 , 60 , 24 ; 4 voxel clusters ) , the left caudate ( peak mni coordinate : 12 , 4 , 10 ; 4 voxel clusters ) , and the left temporal pole ( peak mni coordinate : 32 , 10 , 30 ; 1 voxel cluster ) . interestingly , comparable ( voxelwise p < 0.001 , uncorrected ) increases in right ifg connectivity associated with the vg task were not observed after itbs . prior to assessing behavioral correlations with fmri measures of ifg function , correlations between total lesion volume and each measure were assessed . this revealed moderate but nonsignificant correlations between total lesion volume and changes in l ifg activity ( r = 0.5 , p = 0.2 ) and changes in connectivity ( r = 0.33 , p = 0.43 ) and weak but nonsignificant correlations between total lesion volume and right ifg activity ( r = 0.11 , p = 0.79 ) . thus , partial linear correlations were computed to investigate the relationship between the changes in functional mri measurements of ifg function during covert verb generation and changes in performance on the semantic fluency test following itbs that were not attributable to total lesion volume . these analyses did not reveal significant correlations between overall changes in li and changes in performance ( partial r = 0.03 , p = 0.96 ) , changes in the magnitude of left ifg activity and changes in performance ( partial r = 0.16 , p = 0.74 ) , or changes in the effects of covert verb generation on interhemispheric connectivity and changes performance ( partial r = 0.15 , p = 0.76 ) . these analyses did reveal a strong negative correlation between changes in the magnitude of right ifg activity and changes in performance ( partial r = 0.82 , p = 0.01 ) , indicating that decreases in right ifg activity during covert verb generation between pre / post - itbs sessions were associated with concurrent improvements on the semantic fluency test . to further explore the effects of itbs on ifg function our a priori analyses indicated that itbs was associated with changes in the responses of both the left ifg and right ifg during covert verb generation and also indicated that itbs was associated with reduced connectivity from right ifg to left ifg during covert verb generation . the exploratory behavioral correlation analyses also revealed the somewhat surprising result that post - itbs improvements in performance on the semantic fluency test were most strongly related to reductions in the responses of right ifg during covert verb generation . these findings led us to question whether the relationship between post - itbs changes in the responses of left and right ifg during covert verb generation showed a consistent pattern across patients . they also led us to question whether the effects of itbs on the responses of left and right ifg during covert verb generation might relate to the pretreatment levels of effective connectivity from right ifg to left ifg during covert verb generation . it might be expected , for example , that preexisting interhemispheric dynamics might influence the effects of high - frequency itbs on the function of left and right ifg . these questions were addressed using additional exploratory partial correlation analyses that , while not related to our initial hypotheses , were included to more fully characterize the data . first , we addressed the question of whether or not changes in left ifg activity after itbs were correlated with changes in right ifg activity after itbs while controlling for total lesion volume . this revealed a nonsignificant negative correlation between post - itbs changes in left and right ifg activity ( partial r = 0.65 , p = 0.12 ) . second , we addressed the question of whether or not the effects of itbs on left and right ifg activation magnitudes were correlated with the effects of covert verb generation on interhemispheric connectivity prior to itbs treatment . this revealed a positive correlation between pre - itbs effects of verb generation on interhemispheric connectivity and changes in left ifg activation magnitude after itbs treatment ( partial r = 0.75 , p = 0.05 ) . a nonsignificant negative correlation was found between pre - itbs connectivity and changes in right ifg activation magnitude after itbs ( partial r = 0.60 , p = 0.15 ) . vbm was used to test our hypothesis that patients would show changes in gm volume between pre- and post - itbs sessions . an initial analysis using a voxelwise fdr threshold of 0.05 an evaluation of the unthresholded statistical maps ( figure 5 ) revealed that the most reliable ( p < 0.001 , uncorrected ) increases in gm volume occurred in the left medial orbital gyrus ( peak mni coordinates : 22 , 52 , 14 ; 123 voxel clusters ) and in the left lingual gyrus ( peak mni coordinates : 20 , 94 , 14 ) , and the most reliable ( p < 0.001 , uncorrected ) decreases in gm volume occurred in the right superior frontal gyrus ( peak mni coordinates : 18 , 66 , 12 ; 8 voxel clusters ) and in the right ifg pars opercularis ( peak mni coordinates : 36 , 6 , 30 ; 6 voxel clusters ) . growing evidence supports the use of techniques that utilize the noninvasive modulation of cortical excitability to improve language functions in patients poststroke aphasia [ 28 , 29 , 34 , 37 , 40 , 7173 ] . however , the development and optimization of future treatment protocols that harness the full potential of these techniques are limited by a rudimentary understanding of the changes in neural function that enable their therapeutic effects . the present study provides insights into this issue by characterizing changes in cortical function and structure following 10 sessions of itbs applied to residual language - responsive cortex in a group of chronic poststroke aphasia patients . our results show evidence for changes in language task - related responses in both the stimulated and unstimulated hemispheres following itbs treatment that are characterized by a general shift from right - lateralized to left - lateralized responses . moreover , we show evidence for changes in the language task - related connectivity of right - hemisphere homologues of residual left - hemisphere language areas subsequent to itbs treatment . these findings both replicate our previously reported observations of post - itbs changes in left- and right - hemisphere responses related to semantic decisions and extend them by demonstrating that similar changes in activation as well as additional changes in interhemispheric connectivity are observed during a covert verb generation task . importantly , the current study utilized a language task ( covert verb generation ) that targets different aspects of language function from our previous study , which focused on semantic decisions , and that is independent of the stimulation targeting paradigm . in addition , behavioral partial correlation analyses revealed that post - itbs changes in the function of contralesional cortex showed a strong relationship to improvements in neuropsychological measures of language function after treatment that could not be explained by interindividual differences in lesion extents . although we only found preliminary evidence for changes in regional grey matter volume following treatment with itbs , our results nonetheless provide evidence of structural and functional neuroplasticity subsequent to a short - duration itbs treatment in patients with poststroke aphasia . while preliminary , these results provide important insights into the changes in cortical function that enable improvements in language abilities following itbs treatment . the rerecruitment of residual left - hemisphere cortex for language processes is likely an important factor for the optimal long - term recovery of language functions following lmca stroke [ 12 , 1417 , 19 , 21 , 23 ] . noninvasive techniques such as itbs may facilitate the reintegration of residual cortex into cortical language networks by promoting beneficial neuroplasticity [ 29 , 40 ] . while the mechanisms underlying the neuroplastic effects of itbs in stroke patients are not fully understood , they likely involve multiple factors including modulations of gene expression , growth factor production , neurotransmitter release , and the facilitation of synaptic plasticity . our findings provide support for the conclusion that itbs can induce plastic changes in the function of both the stimulated and unstimulated hemispheres in patients with chronic poststroke aphasia . indeed , our roi results suggest that the application of itbs to residual language - responsive cortex in the left hemisphere has the potential to reduce contralesional compensation , increase residual left - hemisphere recruitment for language task performance , and alter task - dependent interhemispheric connectivity ( figure 3 ) . while exploratory in nature , the results from our whole - brain analyses support these conclusions . our glm analysis found evidence for distinct patterns of left versus right - hemisphere activity during pre - itbs and post - itbs sessions , with left - hemisphere frontotemporal areas showing increased activity related to covert verb generation post - itbs ( figure 4(a ) ) . similarly , our gppi analysis found evidence for large - scale changes in the connectivity of right ifg during covert verb generation between pre - itbs and post - itbs sessions , with widespread reductions in right ifg connectivity being observed after itbs ( figure 4(b ) ) . a speculative explanation for the observed effects is that the changes in left versus right ifg activity and connectivity during covert verb generation reflect the reinstatement of balanced inhibitory interactions between left and right ifc [ 13 , 75 ] . disproportionate influences of right - hemisphere homologues on left - hemisphere language areas have been previously documented in patients with aphasia resulting from lmca stroke , and it is possible that heightened right ifg activation during language tasks reflects the release of transcallosal inhibitory outputs from left to right ifg following left - hemisphere damage [ 14 , 75 ] . while not reaching our threshold for statistical significance , the results of our post hoc analyses suggested that increases in left ifg activation were related to decreases in right ifg activation regardless of lesion extent . this suggests that changes in right ifg activation depended in part on changes in the function of the left ifg , although this result is preliminary and should be interpreted as such . more highly powered analyses of larger samples are therefore necessary before conclusions about this relationship can be definitively drawn . additionally , results from our post hoc analyses indicated that the effects of itbs on left and right ifg function had opposite relationships with the pre - itbs strength of right ifg to left ifg connectivity during covert verb generation . while exploratory and requiring additional validation , these results have important implications , as they suggest that preexisting interhemispheric dynamics contribute to the effects of itbs on the function of both the stimulated and unstimulated hemispheres . this interpretation is consistent with results from a recent study that indicated that the preservation of frontal white matter tracts , specifically the left arcuate fasciculus , explains substantial interindividual variability in behavioral improvements following cathodal tdcs applied to right ifg in patients with left ifg lesions . our results indicate that patients that showed stronger right to left ifg connectivity during covert verb generation at the pre - itbs session also showed the most pronounced effects of itbs on both left and right ifg activation magnitudes at the post - itbs session . while future studies comparing connectivity between stroke patients in healthy controls are necessary to fully understand the implications of these findings , the general implications are that itbs treatment can lead to increased left ifg activity / reduced right ifg activity and reduced right ifg to left ifg connectivity during language task performance and that the effects of high - frequency itbs on both left and right ifg activation likely depend on the preexisting interhemispheric state prior to treatment . nevertheless , it is pertinent to address the question of how such large - scale changes in cortical function might result from the passive stimulation of residual left - hemisphere areas . after stroke , the loss of large - scale neural populations is thought to result in an acute breakdown of function in large - scale cortical networks that enable complex cognitive functions such as language and attention . this abrupt disruption of neural communication and regulation might be conceptualized as a large - scale perturbation of the brain 's functional state that alters the trajectory of ongoing neural signaling . during recovery , adaptive changes in the residual neural populations are thought to allow for the restoration of interregional communication and regulation , and it has been proposed that successful recovery of function after stroke may reflect the restoration of a near - normal functional state , whereas poor recovery may reflect ineffective reorganization that results in an aberrant functional state that is maladapted to generating normal cognitive / behavioral outputs . thus , it might be speculated that by passively stimulating the residual left - hemisphere cortex itbs might induce changes in the state of local neural populations that facilitate the restoration of a closer - to - normal functional brain state . an expected outcome in such a scenario would be that following itbs patients would show task / stimulus - evoked responses that more closely resemble those observed in healthy individuals . while preliminary , the observed changes in language - related responses and interhemispheric connectivity resemble the patterns of language - related responses [ 24 , 47 ] and task - dependent connectivity that are observed in healthy individuals with typical language function . while the combination of itbs with active language therapy might also be expected to lead to an enhancement of beneficial neuroplasticity , the expectation that passive stimulation can lead to changes in task - driven responses and connectivity is not unfounded . indeed , the cortical / subcortical networks that underlie cognitive and behavioral functions such as language and attention maintain ongoing interregional signaling even when tasks are not being performed [ 8083 ] . importantly , disruptions in resting state cortical networks are observed in stroke patients , and the structure of residual resting state networks is also altered by treatment . thus , it might be expected that passive high - frequency stimulation of a residual but dysfunctional language network node might lead to the strengthening of synaptic connections with other language network nodes and that this may facilitate the eventual reintegration of the stimulated node to the residual language network . such an effect would be consistent with the capabilities of itbs to induce ltp - like changes in synaptic transmission that persist beyond the stimulation period and with reports that these effects are paralleled by changes in the temporal coordination of large - scale , low - frequency oscillatory activity . the present study has limitations that must be acknowledged and considered in interpreting the results and in designing future studies . primarily , the lack of a sham - stimulation group precludes the ability to make definitive statements about whether the observed effects are specific to itbs treatment . it is worth noting that activation patterns during the covert verb generation task have been found to be remarkably consistent across time in patients with chronic poststroke aphasia , and the presence of reliable effects across chronic stroke patients in the present study indicates that the observed effects are not likely due to spontaneous changes in ifg function . nonetheless , future studies that employ a sham - stimulation control are necessary to make definitive statements regarding the neuroplastic effects of itbs in patients with chronic poststroke aphasia . while sham - controlled studies investigating the effects of neurostimulation on language function in patients with chronic poststroke aphasia have consistently reported behavioral improvements that are specific to real stimulation [ 36 , 71 , 73 , 85 ] , most of these have used stimulation protocols involving the application of low - frequency rtms to the unaffected right ifg . this does make our finding that behavioral improvements subsequent to high - frequency stimulation of left ifg negatively correlated with changes in right ifg activation particularly noteworthy , as this finding supports the use of paradigms such as these that aim to suppress dysfunctional activity in right ifg . while it is possible that both approaches may be manipulating similar mechanisms to achieve improvements in behavior , it is important for future studies to characterize the similarities and differences in the neuroplastic effects induced by each approach and to identify the mechanisms by which beneficial behavioral effects are achieved . however , since itbs increases local cortical excitability and potentiates cortical evoked responses [ 32 , 33 ] , it might potentiate language - related responses in residual ifg or lead to changes in li estimates even in the absence of beneficial neuroplastic effects . for example , in the absence of some preserved interactions between left and right ifg , high - frequency stimulation of left ifg ( or low - frequency stimulation of right ifg ) might lead to unilateral changes in activation during language tasks that could present as a transient overall shift in li estimates . this might provide an explanation for the absence of a relationship between overall changes in li and improvements in sft in the current study and for the absent or weak relationships between changes in li and behavioral improvements reported by other studies that have applied low - frequency rtms to right ifg . indeed , while measurements such as li provide useful summary statistics , they face intrinsic limitations that likely limit their utility in assessing the specific effects of itbs . thus , future studies investigating the effects of itbs in this population should consider independently the changes in left versus right - hemisphere function in addition to assessing changes in summary statistics such as li . although some would argue that this property makes the observed significant effects more compelling since the likelihood of finding significant - but - trivial effects increases with sample size , our limited sample likely also led to the obscuration of real effects due to relatively low power . indeed , in the current study , the detrimental effects of having a small sample size would most likely manifest as insufficient power to detect real effects , especially for the whole - brain glm , gppi , and vbm analyses at multiple - comparisons corrected thresholds . as we did not find any significant effects at corrected p value thresholds for the whole - brain analyses , the results from these analyses should be interpreted with caution . as such , we have refrained from drawing strong conclusions about the effects of itbs on gm volume or on activity / connectivity beyond those examined with our roi analyses . while the whole - brain results are exploratory and thus should not be used to draw strong conclusions about the effects of itbs , they do merit discussion . regarding the observed effects of itbs on regional gm volume , it would not be surprising if itbs did have an effect on gm morphology in these patients , as detectable changes following rtms have been reported after in as little as five days by studies investigating the effects of rtms on cortical morphology in healthy individuals . it is also worth noting that the previously reported effects of itbs on white matter integrity in these patients were obtained using similarly lenient thresholds , and given the large variability in lesion etiologies for the patients in this study , it is thus perhaps not surprising that stronger effects were not observed here . while the direction and locations of some of the most reliable vbm effects ( increased gm in left prefrontal areas / decreased gm in right ifg ) are in line with our expected results , the current results do not provide basis for strong conclusions but do provide support for future investigations into these effects . similarly , as noted earlier in the discussion , the whole - brain glm and gppi results do show effects consistent with larger - scale changes in the responses and interactions of the residual language network during covert verb generation . thus , future studies with larger sample sizes are necessary to provide a full characterization of the effects of itbs in this population . in conclusion , we investigated the effects of itbs applied to residual language - responsive cortex in the left hemisphere on mri measurements of cortical function and structure in eight patients with chronic poststroke aphasia . we found that itbs was associated with increased left - lateralization of ifg activity during covert verb generation . changes in lateralization were characterized by increases in left ifg activation magnitudes and decreases in right ifg activation magnitudes that presented as an overall shift in the lateralization of ifg activity during covert verb generation . itbs also led to reduced right to left ifg connectivity during covert verb generation , consistent with our interpretation that the effects of itbs are related in part to changes in context - dependent interhemispheric interactions . interestingly , our post hoc analyses suggest that the effects of itbs on left and right ifg function were negatively correlated across patients ( increased left ifg activity was associated with decreased right ifg activity ) , and the changes in left versus right ifg responses had opposite relationships to pre - itbs levels of right ifg to left ifg connectivity during covert verb generation . these data provide insights into the neuroplastic changes associated with itbs applied to residual left - hemisphere language areas in the treatment of chronic poststroke aphasia and provide support for future research in this area . randomized , blinded , and sham - controlled studies in a larger sample of patients are necessary and are currently in progress ( e.g. , nct01512264 ) in order to better clarify the neuroplastic effects of itbs in this population .

in a case of acidosis induced by the administration of a mineral acid , such as hcl or nh4cl , acidemia itself increases serum k concentration . it is well known that hyperkalemia is caused by the release of intracellular k into extracellular fluid . in contrast , in animal experiments involving the administration of nonmineral acids , such as lactic acid and methylmalonic acid there was no increase in serum k concetration by a nonmineral acid clinically except in diabetic ketoacidosis . we report 2 cases of hyperkalemia which developed after -fluoroethylacetate was ingested in which serum k was increased to 5.57 meq / l and 6.7 meq / l , respectively , with severe metabolic acidosis . a 59-year - old woman was admitted to the hospital via the emergency room in a semicoma with vomiting and intermittent general convulsions , following the ingestion of 6.0 gm of -fluoroethylacetate ( kilatol f ) in a suicide attempt . the laboratory findings on admission were as follows : hemoglobin 13.6 gm% , hematocrit 39.2% , white cell count 10,000 , platelet count 40,200 , total protein 6.2 gm , and albumin 3.9 gm% . she was in metabolic acidosis ( ph 7.26 , hco3 17 meq / l , pco2 38 mmhg ) . therefore , 24 meq / l of hco3 was given every 2 hours for 24 hours along with 10% glucose solution and 5% dextrose in saline . forty milligrams of lasix was also prescribed every 8 hours for 24 hours . on the second hospital day the ph was 7.34 , hco3 19 meq / l , pco236 mmhg and serum k 4.9 meq / l . bicarbonate and hypertonic glucose solution were given . on the 5th hospital day there was a marked improvement in the ecg , and lidocaine was withdrawn . on the 6th hospital day the results of the laboratory examinations were ph 7.36 , hco3 24 meq / l , pco2 43 mmhg and serum k 3.6 meq / l . a 38-year - old woman brought to the emergency room with convulsions , following the ingestion of 20 ml of -fluoroethylacetate solution 12 hours before in a suicide attempt . the laboratory findings on admission were as follows : hemoglobin 13.2 gm% , hematocrit 37.2% , white blood cell count 6,900 , platelet count 26,200 , total protein 6.5 gm% , and albumin 3.5 gm% . the findings on an arterial blood gas analysis were ph 6.83 , pco2 90 mmhg , hco3 15 mmol / l and po2 55 mmhg ( table 1 ) . in acidosis associated with mineral acids , acidemia , in which there is a predictable increase in serum k concentration occurs while in uncomplicated cases of acidosis associated with organic acids , the serum k concentration usually remains within the normal range . this difference is thought to be caused by the difference of membrane permeabilities between mineral acid and organic acid . normally , there exist some mechanisms to maintain the h balances between intracellular and extracellular fluid and some mechanisms to maintain the balanced k ratio . recently , alder et al . reported that there may be an undetermined relationship between intra - and extracellular h / k ratio , but the definite relationship between serum ph and k concentration remains unclarified . in 1958 he reported that there was a significant increase in serum k concentration in a hcl , nh4cl group after 2 hours and no change in that of the lactic acid and acetic acid groups . in 1980 they reported an increase in serum k concentration in the hcl and nh4cl group but a decrease in it in the initial period in the lactic acid group . mentioned in his review of a physiological phenomenon of lactate transport through cellular membrane , that lactic acid enters into cells in 2 forms , as ionized lactate and as unionized lactic acid . in 1983 oh et al . reported the development of hyperkalemia in dogs in a severe acidosis state ( ph 6.9 , hco3 6.0 he reported that the serum k concentration decreased below the baseline for the first 2 hours , before it started increasing . he suggested that hyperkalemia might develop if severe acidosis induced by nonmineral acid were to have been prolonged further . there have been a few reports to the effect that hyperkalemia has developed in humans with organic acidosis , especially diabetic ketoacidosis or lactic acidosis . the fluoroacetate which the patients had taken is a highly toxic , nonmineral acid which comes from the south african plant , dichapetalum cymosum . when administered to living subjects it interrupts the cellular metabolic process by blocking the citric acid cycle . fluoroacetate itself is nontoxic , but once transformed into fluorocitrate acid it is highly toxic , causing generalized convulsions and severe ventricular arrhythmia .

the potential for osteochondral defects to self - repair is limited in part due to the hypocellular and avascular nature of articular cartilage . osteochondral defects that fail to heal spontaneously may ultimately result in further joint destruction and osteoarthritis . numerous surgical treatment modalities have been developed , primarily focused on either repairing the damaged cartilage ( e.g. , bone marrow stimulation ) or replacing the lesion with a viable osteochondral graft ( e.g. , autologous osteochondral transplantation ) . regardless of whether the cartilage is repaired or replaced , the existing surgical treatment modalities are hindered by poor biology , leading to inferior quality repair tissue when compared to native hyaline cartilage . in an effort to improve the repair of osteochondral defects , biological products have seen increasing interest as an adjunct to surgical treatment and as a primary treatment modality . one such biological product is platelet - rich plasma ( prp ) , defined as an autologous blood product derived by the centrifugation of whole blood yielding a concentration of platelets that is increased from the baseline value . at the basic science level , prp has been shown to increase chondrocyte and mesenchymal stem cell proliferation , proteoglycan production , and type ii collagen deposition . few in vivo studies have been performed to evaluate the effect of prp alone on chondrogensis . therefore , the objective of this study was to assess the effect of prp on the repair of an osteochondral donor site ( i.e. , defect ) in a rabbit model . we hypothesized that prp would improve osteochondral healing at the donor site in the knee . a preliminary power analysis showed that a sample size of 12 rabbits had 88% power to detect a two point difference in the primary outcome of international cartilage repair society ( icrs ) histological score given a standard deviation of 2 points . therefore , 12 new zealand white rabbits were used with a mean weight of 4 kg ( range , 3.7 - 4.2 kg ) and a mean age of 23 weeks ( range , 22 - 24 weeks ) . a rabbit model was chosen for this study as the rabbit knee is large enough to accommodate the procedure and it is a well - established model that has been used previously for studies assessing cartilage repair procedures . the rabbits were held in individual cages and allowed unrestricted cage activity for 7 days preoperatively . additional experimental results derived from this animal model have been published previously . under anesthesia , 27 ml of blood was aspirated from the great aural artery of each rabbit and combined with 4 ml of anticoagulant citrate dextrose solution a ( acd - a ) . one milliliter of blood was then immediately dispensed into a separate syringe for cytological analysis . the remaining blood was then centrifuged using a standard , commercially available platelet concentration system ( magellan autologous platelet separator , arteriocyte , cleveland , oh ) to yield 3 ml of prp according to the manufacturer s protocol ( centrifuge force , 1200 g ; centrifuge time 17 minutes ) . from this sample , one ml was reserved for cytological analysis and the remainder was allocated for surgical use . the whole blood and prp samples underwent cytological analysis to measure platelet levels as well as red and white blood cell counts , as per previously published work . the anesthetic protocol used for the animals involved use of subcutaneous ketamine ( 40 mg / kg ) and acetylpromazine ( 0.5 mg / kg ) , and maintenance with isoflurane ( 2% ) inhalation through intubation . antibiotic prophylaxis ( ampicillin 25 mg / kg ) was given 30 minutes prior to surgery . following shaving and standard sterile preparation of both lower extremities , a 4 cm medial parapatellar arthrotomy was created in each knee exposing the medial and lateral femoral condyles . the joint was then carefully inspected to ascertain whether any preexisting cartilage pathology was present . using a trephine with an outer diameter measuring 3.0 mm and 5 mm in depth ( smith & nephew , memphis , tn ) an osteochondral graft was harvested on the medial femoral condyle of the left knee and lateral femoral condyle of the right knee , thereby creating a donor site . the graft harvested using the trephine was transplanted to the opposing femoral condyle for a separate study assessing autologous osteochondral transplantation that has been published previously . the use of an autologous osteochondral transplant donor site as a rabbit osteochondral defect model has been widely published in the literature . each wound was then closed in layers using simple interrupted sutures ( 4 - 0 vicryl ) , with final skin closure achieved using a running subcutaneous stitch technique ( 4 - 0 vicryl ) to obtain a water - tight seal . prior to wound closure , the knees were randomized to receive an injection of either 0.5 ml of prp or saline ( placebo ) . randomization was done through a computer - generated list , with results placed in a sealed envelope by an individual not involved with the surgical procedure . the envelopes were opened prior to wound closure so as to determine the laterality of the treatment . therefore , following wound closure , 0.5 ml of prp or saline was administered as an intra - articular injection . prior to concluding the surgical procedure , the knee was moved through a full range of motion to ensure that normal patellar tracking occurred . postoperatively , the animals were given fentanyl skin patches ( 12 g / h ) and allowed unrestricted cage activity . the rabbits were euthanized using pentobarbital ( 100 - 150 mg / kg , intravenously ) at 3 , 6 , and 12 weeks following the initial surgery , with 4 rabbits euthanized at each time interval . two reviewers who were blinded to the treatment groups performed all scoring independently . prior to assigning a final score for a given sample , the reviewers deliberated in order to reach a final consensus . the icrs macroscopic scoring system on a scale from 0 to 12 , with 12 being normal cartilage and 0 indicating severely abnormal , was used to assess the gross specimens ( table 1 ) . international cartilage repair society macroscopic score . at the time of euthanasia , both knee joints were resected en bloc and placed in 10% formalin for 7 days . following formalin fixation , the specimens were decalcified in a sodium citrate - formic acid solution for an additional 7 days prior to being embedded in paraffin wax . this is a previously published method of preparing osteochondral specimens in our institution s laboratory . the specimens were cut into 8 um sections at the posterior , middle , and anterior aspect of the osteochondral donor site , which provided a representative view of overall lesion healing . the osteochondral sections were stained with hematoxylin and eosin for icrs histological scoring , alcian blue to assess glycosaminoglycan presence , and processed for type ii collagen immunohistochemistry . the icrs histological score was graded on a scale of 0 to 18 ( table 2 ) . descriptive statistics were generated by treatment group for the overall cohort and for each time period . comparison of icrs macroscopic and histological scores between prp - treated and placebo defects for the overall cohort was performed using the wilcoxon sign - rank test . the mean difference in icrs scores with 95% confidence interval ( ci ) was also calculated for each time period separately . spearman s rank correlation coefficients were calculated to evaluate associations between cytological variables and histological scores . all analyses were performed using sas software version 9.2 ( sas institute , cary , nc ) . under anesthesia , 27 ml of blood was aspirated from the great aural artery of each rabbit and combined with 4 ml of anticoagulant citrate dextrose solution a ( acd - a ) . one milliliter of blood was then immediately dispensed into a separate syringe for cytological analysis . the remaining blood was then centrifuged using a standard , commercially available platelet concentration system ( magellan autologous platelet separator , arteriocyte , cleveland , oh ) to yield 3 ml of prp according to the manufacturer s protocol ( centrifuge force , 1200 g ; centrifuge time 17 minutes ) . from this sample , one ml was reserved for cytological analysis and the remainder was allocated for surgical use . the whole blood and prp samples underwent cytological analysis to measure platelet levels as well as red and white blood cell counts , as per previously published work . the anesthetic protocol used for the animals involved use of subcutaneous ketamine ( 40 mg / kg ) and acetylpromazine ( 0.5 mg / kg ) , and maintenance with isoflurane ( 2% ) inhalation through intubation . antibiotic prophylaxis ( ampicillin 25 mg / kg ) was given 30 minutes prior to surgery . following shaving and standard sterile preparation of both lower extremities , a 4 cm medial parapatellar arthrotomy was created in each knee exposing the medial and lateral femoral condyles . the joint was then carefully inspected to ascertain whether any preexisting cartilage pathology was present . using a trephine with an outer diameter measuring 3.0 mm and 5 mm in depth ( smith & nephew , memphis , tn ) an osteochondral graft was harvested on the medial femoral condyle of the left knee and lateral femoral condyle of the right knee , thereby creating a donor site . the graft harvested using the trephine was transplanted to the opposing femoral condyle for a separate study assessing autologous osteochondral transplantation that has been published previously . the use of an autologous osteochondral transplant donor site as a rabbit osteochondral defect model has been widely published in the literature . each wound was then closed in layers using simple interrupted sutures ( 4 - 0 vicryl ) , with final skin closure achieved using a running subcutaneous stitch technique ( 4 - 0 vicryl ) to obtain a water - tight seal . prior to wound closure , the knees were randomized to receive an injection of either 0.5 ml of prp or saline ( placebo ) . randomization was done through a computer - generated list , with results placed in a sealed envelope by an individual not involved with the surgical procedure . the envelopes were opened prior to wound closure so as to determine the laterality of the treatment . therefore , following wound closure , 0.5 ml of prp or saline was administered as an intra - articular injection . prior to concluding the surgical procedure , the knee was moved through a full range of motion to ensure that normal patellar tracking occurred . postoperatively , the animals were given fentanyl skin patches ( 12 g / h ) and allowed unrestricted cage activity . the rabbits were euthanized using pentobarbital ( 100 - 150 mg / kg , intravenously ) at 3 , 6 , and 12 weeks following the initial surgery , with 4 rabbits euthanized at each time interval . two reviewers who were blinded to the treatment groups performed all scoring independently . prior to assigning a final score for a given sample , the reviewers deliberated in order to reach a final consensus . the icrs macroscopic scoring system on a scale from 0 to 12 , with 12 being normal cartilage and 0 indicating severely abnormal , was used to assess the gross specimens ( table 1 ) . international cartilage repair society macroscopic score . at the time of euthanasia , both knee joints were resected en bloc and placed in 10% formalin for 7 days . following formalin fixation , the specimens were decalcified in a sodium citrate - formic acid solution for an additional 7 days prior to being embedded in paraffin wax . this is a previously published method of preparing osteochondral specimens in our institution s laboratory . the specimens were cut into 8 um sections at the posterior , middle , and anterior aspect of the osteochondral donor site , which provided a representative view of overall lesion healing . the osteochondral sections were stained with hematoxylin and eosin for icrs histological scoring , alcian blue to assess glycosaminoglycan presence , and processed for type ii collagen immunohistochemistry . the icrs histological score was graded on a scale of 0 to 18 ( table 2 ) . descriptive statistics were generated by treatment group for the overall cohort and for each time period . comparison of icrs macroscopic and histological scores between prp - treated and placebo defects for the overall cohort was performed using the wilcoxon sign - rank test . the mean difference in icrs scores with 95% confidence interval ( ci ) was also calculated for each time period separately . spearman s rank correlation coefficients were calculated to evaluate associations between cytological variables and histological scores . all analyses were performed using sas software version 9.2 ( sas institute , cary , nc ) . no rabbit exhibited any signs of lameness or developed an infection postoperatively at any time point . as previously reported , the mean values of the prp produced showed a 5.1-fold increased platelet count compared to whole blood ( 817.6 155.0 10/l vs. 161.5 42.6 10/l ; p < 0.001 ) . the prp showed a white blood cell count 1.9 times greater than the whole blood ( 10.0 3.2 10/l vs. 5.1 1.0 10/l ; p < 0.001 ) . the red blood cell count of the prp was 28% that of the whole blood ( 10.1 1.8 10/l vs. 35.6 2.4 10/l ; p < 0.001 ) . the rabbits were euthanized at 3 , 6 , and 12 weeks following surgery and their knee joints were resected , with 4 rabbits euthanized at each time point . the mean macroscopic review of the osteochondral donor sites across all time points showed a greater mean icrs macroscopic score in the prp treated knees compared with the placebo group ; however , this was not statistically significant ( 5.5 3.8 vs. 3.8 3.5 ; p = 0.13 ) . the mean icrs macroscopic scores for the prp - treated group at 3 , 6 , and 12 weeks postoperatively were 4.0 3.7 , 4.5 3.3 , and 8 4.1 , respectively . the mean icrs macroscopic scores for the placebo donor sites at the same time points were 3.5 3.1 , 1.5 1.3 , and 6.5 4.2 , at 3 , 6 , and 12 weeks , respectively . furthermore , the tissue in the prp - treated donor sites appeared smoother and more hyaline like , with fewer fissures , when compared with the placebos ( figs . 1 and 2 ) . photograph of osteochondral defect in control group at 12 weeks following surgery . photograph of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . the mean icrs histological score was higher overall and at each time point for the prp - treated osteochondral donor sites compared to the placebo group ( overall mean 9.8 2.0 vs. 7.8 1.8 ; p = 0.04 ) . the prp - treated donor sites showed a greater amount of reparative tissue , with increased cellularity . in addition , the reparative tissue was more hyaline - like in structure in the prp treated group when compared with the placebo . cell viability was also noted to be more apparent in the prp - treated group ( figs . 3 and 4 ) . hematoxylin and eosin was used to stain the sections for assessment using the international cartilage repair society scoring system . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . hematoxylin and eosin was used to stain the sections for assessment using the international cartilage repair society scoring system . image magnification 20. the mean icrs histological scores for the prp treated donor sites at 3 , 6 , and 12 weeks postoperatively were 10 0.8 , 9 2.9 , and 10.3 1.9 , respectively . these were all higher than the mean icrs histological score for the placebo donor sites at the same time points ( 8.0 2.2 , 7.5 0.6 , and 8.0 2.6 , at 3 , 6 , and 12 weeks , respectively ) . the mean difference in the icrs histological score comparing the prp - treated donor sites to the placebo donor sites at 3 weeks was 2.00 , 95% ci = 2.68 to 6.68 ; at 6 weeks was 1.50 , 95% ci = 2.71 to 5.71 ; and at 12 weeks was 2.25 , 95% ci = 3.01 to 7.51 . spearman s correlation coefficients showed no significant association between icrs histological score and any cytological variable ( white blood cells , = 0.29 ; platelets , = 0.15 , red blood cells , = 0.08 ; p < 0.05 for all analyses ) . the reparative tissue of the prp treated donor sites demonstrated greater alcian blue staining when compared with the placebo , representing an increase in glycosaminoglycan deposition . in addition , greater type ii collagen immunoreaction could be seen in the prp - treated group . the placebo group had sparse evidence of glycosaminoglycan and type ii collagen production , particularly at the center of the donor site . in comparison , the prp - treated group showed more diffuse staining for both glycosaminoglycan and type ii collagen throughout the reparative infill . . 5 -8 ) sagittal section of osteochondral defect in control group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in control group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . as previously reported , the mean values of the prp produced showed a 5.1-fold increased platelet count compared to whole blood ( 817.6 155.0 10/l vs. 161.5 42.6 10/l ; p < 0.001 ) . the prp showed a white blood cell count 1.9 times greater than the whole blood ( 10.0 3.2 10/l vs. 5.1 1.0 10/l ; p < 0.001 ) . the red blood cell count of the prp was 28% that of the whole blood ( 10.1 1.8 10/l vs. 35.6 2.4 10/l ; p < 0.001 ) . the rabbits were euthanized at 3 , 6 , and 12 weeks following surgery and their knee joints were resected , with 4 rabbits euthanized at each time point . the mean macroscopic review of the osteochondral donor sites across all time points showed a greater mean icrs macroscopic score in the prp treated knees compared with the placebo group ; however , this was not statistically significant ( 5.5 3.8 vs. 3.8 3.5 ; p = 0.13 ) . the mean icrs macroscopic scores for the prp - treated group at 3 , 6 , and 12 weeks postoperatively were 4.0 3.7 , 4.5 3.3 , and 8 4.1 , respectively . the mean icrs macroscopic scores for the placebo donor sites at the same time points were 3.5 3.1 , 1.5 1.3 , and 6.5 4.2 , at 3 , 6 , and 12 weeks , respectively . furthermore , the tissue in the prp - treated donor sites appeared smoother and more hyaline like , with fewer fissures , when compared with the placebos ( figs . 1 and 2 ) photograph of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . the mean icrs histological score was higher overall and at each time point for the prp - treated osteochondral donor sites compared to the placebo group ( overall mean 9.8 2.0 vs. 7.8 1.8 ; p = 0.04 ) . the prp - treated donor sites showed a greater amount of reparative tissue , with increased cellularity . in addition , the reparative tissue was more hyaline - like in structure in the prp treated group when compared with the placebo . cell viability was also noted to be more apparent in the prp - treated group ( figs . 3 and 4 ) . hematoxylin and eosin was used to stain the sections for assessment using the international cartilage repair society scoring system . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . hematoxylin and eosin was used to stain the sections for assessment using the international cartilage repair society scoring system . image magnification 20. the mean icrs histological scores for the prp treated donor sites at 3 , 6 , and 12 weeks postoperatively were 10 0.8 , 9 2.9 , and 10.3 1.9 , respectively . these were all higher than the mean icrs histological score for the placebo donor sites at the same time points ( 8.0 2.2 , 7.5 0.6 , and 8.0 2.6 , at 3 , 6 , and 12 weeks , respectively ) . the mean difference in the icrs histological score comparing the prp - treated donor sites to the placebo donor sites at 3 weeks was 2.00 , 95% ci = 2.68 to 6.68 ; at 6 weeks was 1.50 , 95% ci = 2.71 to 5.71 ; and at 12 weeks was 2.25 , 95% ci = 3.01 to 7.51 . spearman s correlation coefficients showed no significant association between icrs histological score and any cytological variable ( white blood cells , = 0.29 ; platelets , = 0.15 , red blood cells , = 0.08 ; p < 0.05 for all analyses ) . the reparative tissue of the prp treated donor sites demonstrated greater alcian blue staining when compared with the placebo , representing an increase in glycosaminoglycan deposition . in addition , greater type ii collagen immunoreaction could be seen in the prp - treated group . the placebo group had sparse evidence of glycosaminoglycan and type ii collagen production , particularly at the center of the donor site . in comparison , the prp - treated group showed more diffuse staining for both glycosaminoglycan and type ii collagen throughout the reparative infill . . 5 -8 ) sagittal section of osteochondral defect in control group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in control group at 12 weeks following surgery . image magnification 20. sagittal section of osteochondral defect in platelet - rich plasma treated group at 12 weeks following surgery . the most important finding of this study is evidence that prp may improve healing of an osteochondral donor site in vivo in the first 12 weeks postoperatively . while a statistically significant difference was not measured using the icrs macroscopic scoring system , a significant improvement was quantified at the histological level . in contrast to the current study , the use of prp in an empty donor defect resulted in lower icrs scores by comparison with those seen in autologous osteochondral transplantation . while these results were expected , the tissue regeneration seen in the prp group by comparison with the placebo is nonetheless encouraging and may prove beneficial clinically in patients requiring the harvest of an osteochondral graft from a non - weightbearing portion of the femoral condyle . the findings of the current study reflect the general trend seen in the current literature regarding the use of prp in cartilage pathology . multiple systematic reviews investigating the role of prp in cartilage pathology have been published recently with similar findings . zhu et al . concluded that the current basic science , preclinical , and clinical evidence for prp use in osteochondral defects shows promise . the evidence for this assertion , as well as the potential explanations for the results of the present study , are multifactorial . there is also evidence that prp upregulates the synthetic capacity of chondrocytes , with multiple studies citing a rise in glycosaminoglycan production and type ii collagen deposition . the effect of prp on mesenchymal stem cells ( mscs ) is also relevant to osteochondral defect healing as the lesions in this animal model have a bleeding base , leading to recruitment of subchondral - derived mscs . others , have shown that prp increases msc migration , chondrogenic differentiation , and subsequent proteoglycan production . in addition , prp may also have an effect on the joint as a whole by modifying the levels of catabolic cytokines that are seen in the presence of a cartilage lesion . these are known to include interleukin-1 ( il-1 ) and tumor necrosis factor alpha ( tnf- ) . in vitro evidence indicates that prp may inhibit the degradative effects of these cytokines , such as the downregulation of type ii collagen and aggrecan gene expression . recently , sundman et al . demonstrated that human synovium taken from knees with cartilage pathology expressed decreased tnf- levels when cultured with prp . these findings mirror the conclusions drawn by anitua et al . who determined that prp increased the secretion of hyaluronic acid by synovium cultured from osteoarthritic patients . as prp has been shown to exhibit an effect on the joint as whole , affecting not only cartilage but also the synovium and intra - articular cytokine levels , it was decided that prp would be administered as an intra - articular injection in this study . this contrasts with the aim of combining prp with fibrin glue or other biological product to limit it to the confines of the osteochondral lesion . there are discrepancies in the literature with respect to the fact that prp has not been found to positively influence cartilage repair in all instances . an in vivo study conducted using a goat model by van bergen et al . concluded that prp did not improve cartilage repair of an osteochondral lesion when combined with demineralized bone matrix . similarly , serra et al . conducted a rabbit osteochondral lesion study comparing 7 intra - articular prp injections with saline injections and found that the evolution of the healing tissue showed no difference between the 2 groups at 8 weeks postoperatively . the results of these studies , as well as other similar findings reported in the literature , may potentially be explained by the variance of adjuncts used in combination with prp , including demineralized bone matrix , biomimetic scaffolds , and stem cells . another potential explanation for the varying results seen in the literature may be the different formulations of prp used in each study . while the term platelet - rich plasma implies a pure mixture of platelet and acellular plasma , this may not actually reflect the true composition of the biological product used in clinical practice . prp may in fact be leukocyte - rich , leukocyte - poor , contain varying concentrations of growth factors , or be reduced to platelet - rich fibrin . to further compound this issue , the majority of studies fail to adequately characterize the cytological composition of the prp used , thus making cross - study comparisons difficult . while each rabbit in our study received a prp injection , no correlation was found between the cytological components of the prp ( platelets , white blood cells , red blood cells ) and the histological score . it is likely that a larger sample size may be required to determine optimum platelet , wbc and rbc concentration on cartilage repair . studies have also used a myriad of adjuncts in combination with prp , and it is likely that the interaction between the biologically active components of prp and the adjunct ( e.g. , synthetic scaffold ) cause different results than that of prp in isolation . last , there is a lack of uniformity in the literature regarding the optimum number of prp applications , further compounding comparison between studies . a potential limitation of the study is the model used for creation of an osteochondral donor site . while this is an established model in the rabbit , it is an acutely created defect . this differs from the classic clinical presentation of an osteochondral lesion , which is often more chronic in nature . therefore , the interaction of prp with the defect and the joint itself in the animal in vivo setting may differ from what would take place in the clinical setting with a chronic osteochondral lesion . however , prp may play a further role in healing acutely established osteochondral defects , such as those created at the osteochondral graft donor site in clinical practice . in this noted in an ovine model that osteochondral donor sites show failure of defect closure at 6 months post - operatively . donor site morbidity is a known complication of autologous osteochondral transplantation , and prp may have the potential to mitigate this risk in the clinical setting . while the initial findings assessing the effect of prp on patient reported outcome measures have been reported , there is little published literature quantifying the cartilage repair tissue found following prp treatment . ultimately , clinical trials assessing cartilage repair in the presence of prp , performed using second - look arthroscopy and/or advanced noninvasive imaging techniques , will determine whether the results found in the basic science literature translate to clinical practice . the results of this study indicate that prp used as an intra - articular injection may improve cartilaginous healing of an osteochondral donor site in a rabbit model .

during the fifth week of gestation , ectodermal thickening is differentiated to form milk - line or mammary ridges on the ventral side , which are extended bilaterally from the axilla to the groin . normally , these ridges persist in the pectoral region allowing for the development of breast tissue and regress in non - pectoral areas . any disruption in the embryonic regression of these ridges may result in the formation of ectopic breasts through this line . according to the prevalence rate of ectopic breast tissue among the general population ( 0.2 - 6% ) however , the prevalence of ectopic breast ( supernumerary and/or aberrant ) outside the mammary ridges is exceptionally uncommon . the face , chest wall , back , buttocks , and thighs are just some of the various places where ectopic breast tissue ( ebt ) have been found . to date , there are only three cases of ebt documented in internal organs . a 79-year - old caucasian female with a medical history significant for ischemic heart disease , hypertension , and diabetes mellitus , was found to have a pulmonary mass visualized on high resolution computed tomography ( hrct ) of the chest in 2002 during a workup for dyspnea . based on the hrct , the mass appeared cystic in nature but the patient was lost to follow - up over the last ten years . recently , she presented to the emergency department for worsening dyspnea on exertion and pleuritic chest pain over the last few days . initial chest radiography revealed a large cystic mass in her left pleural space [ figure 1 ] . chest radiography of the patient before thoracotomy on physical examination , vital signs were within normal limits and pertinent findings during pulmonary auscultation revealed decreased breath sounds on the left side along with left - sided dullness with percussion . the results of the initial laboratory tests at this time , which included complete blood count ( cbc ) , chemistry panel , and liver function , were normal . a thoracic ct scan was requested by the surgical team and it described the mass as a well - defined concentric thick wall cavity in the left upper lobe with smooth inner surface without any calcification and with a region of interest ( roi ) of -124 hu , suggesting a pulmonary lipoma [ figure 2 ] . chest ct before surgery ( note the large mass in the left lung ) due to size of the probable tumor and the patient 's symptoms , the surgical team recommended a thoracotomy under general anesthesia in the right lateral decubitus position . the left lower lobe of the lung was collapsed due to the large size of the tumor and its compression effect . after pneumolysis , the mass was observed to occupy the entire left upper lobe . upon visualization of numerous , enlarged lymph nodes in the mediastinum , lobectomy of the left upper lobe , lymphadenectomy , and contrapleural costal resection grossly , the mass appeared to have features of a benign soft tissue tumor ( i.e. lipoma ) measuring 240 130 100 mm [ figure 3 ] . gross appearance of the pulmonary mass histological examinations revealed papillary and tubular structures , similar to breast tissue . no mitosis , necrosis , or cells with atypical features were detected via histologic evaluation , suggesting the benign nature of the tumor [ figure 4 ] . histopathology of pulmonary ectopic breast ( h and e , 100 ) formalin - fixed , paraffin - embedded sections of the sample were stained for estrogen receptor ( er ) and progesterone receptor ( pr ) using immunohistochemistry ( ihc ) . for ihc investigation , we utilized flex monoclonal mouse anti - human antibodies : 1d5 at dilution 1:60 for er , and pgr636 at dilution of 1:300 for pr from dako - denmark ( a / s production svej 42 dk-2600 glostrup , denmark ) according to the manufacturer 's protocol . all slides which were stained on an automated system were negative for both er and pr . post - operatively , the patient was monitored for six days and was discharged to home without any surgical or hospital complications . during a 6-month follow - up appointment , the patient denied any symptoms of shortness of breath or dyspnea on exertion . re - expansion of the left lower lobe significantly improved the patient 's condition , and she is currently undergoing regular medical follow - up . ebt is residual tissue abnormally persisting during the embryological development against the gradual regression of mammary ridges in non - pectoral regions . ebt may be accompanied with symptoms like discomfort or pain during pregnancy , lactation , or in the pre - menopausal period . according to de - cholnocky , approximatly 4% of ebt cases therefore , the conversion of ebt to malignancy should be considered in all diagnosed cases . yeniay et al . , reported a 71-year - old female with an abdominal aberrant breast tissue found incidentally in a piece of mesenteric biopsy during operation for a recurrent endometrial malignant tumor . another case of developed heterotropic breast tissue found in internal organs was reported by sasaki et al . they reported a heterotopic breast epithelial inclusion of the heart incidentally found on a native heart of a 73-year - old man who received an orthotopic heart transplantation for ischemic cardiomyopathy . sundaram in 1969 reported several pseudo - tuberculosis conditions of the lung including silicosis , flurosis , etc . one of the reported cases was of a 20-year - old woman with throat irritation and chronic cough , bilateral axillary lymph nodes , and streaky shadows on chest x - ray localized to the left upper lobe . she was treated as a case of smear - negative tb laryngitis with anti - tb therapies that resulted in no appreciable changes . the histopathologic examination of the removed axillary glands yielded the diagnosis of mature lobule of breast . although the author proposed the diagnosis of accessory breast for the pulmonary findings in chest x - ray , no sampling was performed from the pulmonary inclusions in that case . our case is the only well - documented case of pulmonary ectopic breast worldwide . although the results of er and pr antibody studies did not add any confirmation to the diagnosis of ectopic breast , the obvious resemblance of the hisopathologic pattern of tumor to the breast tissue rejected any other possibility . unfortunately the concordance of er and pr expression with the existence of breast tissue in samples was not investigated before . there are some studies around the concordance of er and pr expression and existence of ectopic breast carcinoma . in the study of shabaik et al . , the results showed a sensitivity of 85.7% and specificity of 100% for er , and a sensitivity of 80% and specificity of 100% for pr . three cases ( 16.6% ) among the 18 different articles which investigated the er , pr antibodies in ectopic breast cancer cases , were reported to have negative er and pr results . as mentioned above , our patient did not recall any symptoms during pregnancy , lactation , or in the pre - menopausal period . this could be interpreted by the negative er and pr status of the ectopic breast tissue . this infers that many of the cells may not contain receptors that are influenced by the alterations in hormone levels . generally despite the rare nature of pulmonary ectopic breast tissue , the clinicians should take into consideration this disease in evaluating the differential diagnoses of pulmonary masses .

this disorder is highly prevalent around the world , with an estimated 1015% of us adults suffering from tinnitus . tinnitus is the number - one service related disability , with over 744,000 total veterans receiving disability compensation at a cost to american taxpayers over $ 2.26 billion in 2014 . the underlying etiology of tinnitus is not well - defined , yet it is typically associated with many forms of peripheral ear pathology , hearing loss , retrocochlear lesions , head and neck injury , dental and temporal - mandibular joint dysfunction , and drug toxicity . changes at the auditory periphery often lead to aberrant neural activity within central auditory pathways that may be localized within and influenced by multiple brain stations . as a result , a central etiology of tinnitus has been supported by persistence of phantom sound perception following auditory nerve transection . animal studies have been instrumental in elucidating potential central neuronal mechanisms of tinnitus . following noise damage , increased spontaneous and tone - evoked neural firing rates and enhanced neural synchrony have been found at multiple brain stations including primary auditory cortex . these putative neural correlates of tinnitus have largely been unexplored or translated within human central auditory pathways . one reason for this is due , in part , to the limitation of technology in capturing human central auditory circuits under real time conditions of aberrancy . optical imaging modalities used to study tinnitus include functional magnetic resonance imaging ( fmri ) , positron emission tomography ( pet ) with and without computer tomography ( pet - ct ) , electroencephalography ( eeg ) , and magnetoencephalography ( meg ) . meaningful data captured from these seemingly noninvasive imaging modalities have been limited by the potential confounding effect of extraneous noise ( mri ) , use of high production - cost radioisotopes for pet studies , and limited spatial resolution for eeg and meg studies [ 6 , 7 ] . although there are limitations to each imaging modality , reported results with current technology have identified changes in tinnitus brains that may reflect basic science neural correlates as described in animal models [ 8 , 9 ] . additional optical image technologies are needed to provide an alternative means to potentially further translate these putative correlates within the central human auditory pathway . functional near - infrared spectroscopy ( fnirs ) is an imaging modality that has emerged as a noninvasive metric of cortical hemodynamic activity in many human auditory and nonauditory studies [ 10 , 11 ] . this technology takes advantage of the changing optical properties of brain tissue using near - infrared light to extrapolate and quantify hemodynamic responses through neurovascular coupling , whereby activated brain regions trigger increased blood flow that can be captured and monitored for changes in oxygenated and deoxygenated hemoglobin . when a specific brain region is activated , fnirs measure changes in localized hemoglobin level as an index of neural activity within the specific region . by relying on the intrinsic optical properties of blood , fnirs provides a more direct metabolic marker relative to the widely used bold effect in fmri , which derives contrast only from the paramagnetic properties of deoxyhemoglobin . fnirs has reliably confirmed increased and decreased hemodynamic activity in auditory and surrounding nonauditory regions under conditions of acoustic stimulation [ 1316 ] and during rest / silence , respectively . recorded brain activation with fnirs while participants listened to pleasant , unpleasant , and neutral sounds and observed that unpleasant sounds increased auditory cortex activation when compared to neutral sounds . alternatively , the utility of fnirs to calculate temporal synchronization of spontaneous neuronal activity within various cortical regions , referred to as resting - state functional connectivity , has also been reported . to the best of our knowledge , that proof of concept study demonstrated the utility of fnirs to detect levels of oxygenation in auditory cortex in participants with tinnitus at rest and in response to transcranial magnetic stimulation ( tms ) . interestingly , participants with tinnitus demonstrated a decrease in hemodynamic activity following tms suggesting that increased auditory cortical activity as measured by fnirs may be related to tinnitus perception . . demonstrated higher oxygenation at baseline over the right auditory cortex in participants with tinnitus than in controls . the authors concluded that this increased activity is at least partially representative of the tinnitus percept and validated the use of fnirs as an investigative tool to study the pathophysiology and treatment response . the hypothesis of the present study is that participants with tinnitus and essentially normal hearing will show increased hemodynamic responses under conditions of silence indicative of physiologic neural correlates including increased spontaneous neural firing rates in animal models . our hypothesis using a randomized auditory stimulus and silence block paradigm will extend the current understanding of hemodynamic changes that occur in human tinnitus brains using fnirs technology as published . to test this hypothesis , we created two analytical approaches , one that involves a standard measurement of hemodynamic activation during auditory stimulation after subtracting the initial periods of stimulation ( thereby avoiding baseline activations in the analysis ) and secondly by measuring hemodynamic responses during intervening periods of silence . here we demonstrate that during periods of silence hemodynamic responses are maintained in auditory and adjacent nonauditory cortices in tinnitus participants , a response not seen in controls . interestingly , the elevated hemodynamic states during silence are subsequently decreased with broadband noise in tinnitus indicative of forward masking and residual inhibition . while these data derived from fnirs waveforms may not be directly representative of putative neural physiologic correlates of tinnitus from animal models , measurable changes using this innovative technology may provide a translational index of activation seen in auditory and surrounding associative auditory cortices . the university of michigan institutional review board approved the study and participants were reimbursed for participation . informed consent was obtained from each participant after an extensive explanation of the protocol using noninvasive fnirs technology . ten normal / near - normal hearing adults with subjective tinnitus ( average age : 48.7 16 years ) appreciated equally in both ears and seven normal / near - normal hearing nontinnitus controls ( average age : 25.7 7.8 years ) participated in the study . the selection of normal and near normal hearing tinnitus participants was based on the desire to rule hearing loss out as a factor influencing the fnirs results . all of the participants suffered from constant , nonpulsatile tinnitus percepts that were appreciated in the head or in both ears equally . exclusion criteria included prior otologic surgery , unilateral tinnitus , and any degree of conductive hearing loss , sensorineural hearing loss greater than 30 db at any frequency or other identified etiologies of tinnitus ( i.e. , glomus skull base tumors , retrocochlear lesions , and high dose aspirin ) . in order to maximize the ability to compare the two groups , participants were enrolled only if they demonstrated bilateral normal / near - normal hearing thresholds confirmed via an audiogram . both control and tinnitus participants were held to the same normal / near - normal hearing criteria defined as hearing thresholds less than 30 db for pure tone averages ( pta ) across all frequencies , including high - frequency regions such as 8000 hz . moreover the speech reception thresholds ( srts ) and word discrimination scores ( wds ) were also found to be within the normal range between both groups . the srt is defined as the lowest threshold ( in db ) at which 50% of spoken spondees the wds is based on the percentage of single - syllable words correctly understood when stimuli are presented at a normal conversational speech level ( 4060 db ) . prior to testing , individuals with tinnitus completed a subjective tinnitus assessment scale ( tinnitus handicap inventory ; thi ; ) to quantify and qualify the severity of tinnitus by evaluating both the emotional burden and degree of daily disruption caused by the condition . usa ) with two wavelengths ( 690 nm and 830 nm).a customized configuration of 30 optodes ( 15 per hemisphere ; source - detector pairs ; figure 1 ) inserted into a silicone band was wrapped circumferentially around the head and secured with velcro straps . emitters and detectors were arranged into 5 3 arrays over the frontal , temporal , and parietal cortices of the right and left hemispheres . the distance between each light emitter and detector was set at 3 cm . to identify if the probe / optode array had moved during testing , the positions of t3 and t4 were confirmed before and after experiment . additionally , postexperiment photographs were taken and reviewed for each participant to ensure consistent probe location . the data were collected at a sampling rate of 20 hz . since fnirs measures light intensity several conversation steps were made between the captured intensity values to derive the final data measured in units of micromolar ( m ) concentration . the region of interest ( roi ) included primary auditory cortex ( temporal lobe including superior temporal plane ) and surrounding auditory belt regions ( temporal and parietal cortices ) . acknowledging the spatial resolution limits of fnirs technology , both anatomical ( 1020 eeg system ) and functional ( normal brain response to auditory stimulation ) strategies were utilized to identify roi . first , the international 1020 system for eeg electrode placement with bilateral t3/t4 coordinates for temporal optode placements ( figure 1 ; [ 13 , 23 ] ) was utilized . second , anatomical localization of roi was achieved by isolating only those channels that in control participants showed an increased average group hemodynamic activity in response to auditory stimuli and subsequent declines in the presence of silence as previously reported [ 1317 , 23 ] . the roi ( and non - roi ; see below ) were separately derived from 2 respective channels from each hemisphere that were then averaged to obtain the final concentration for all test conditions . because there were no statistically derived differences between the hemispheres in all participants , the data was averaged and the roi corresponded to channels 13 and 15 ( right hemisphere ) and 23 and 29 ( left hemisphere ; figure 1 ) . to discern whether brain regions not directly associated with auditory processing were activated during periods of auditory stimulation and silence , the non - roi covered the area immediately outside / adjacent to roi minus an additional channel ( n 1 ) over the occipital region , which has known functional relationships with the auditory sensory systems [ 2427 ] . the channels corresponding to the non - roi were 3 and 5 ( right hemisphere ) and 34 and 39 ( left hemisphere ; figure 1 ) . based on that selection criteria channels were identified auditory ( roi ) and nonauditory ( non - roi ) cortical regions that equated to brodmann areas 19 and 37 . participants were engaged in a passive listening block paradigm that consisted of periods of silence ( interstimulus rest ; isr ) , broadband noise ( bbn ) , or tonal ( 750 or 8000 hz ) stimuli using audacity ( gnu general public license ) and normalized with praat 4.2 . auditory stimuli were presented via e - prime ( psychology software tools , inc . , pittsburgh , pa , usa ) and played at a standard fixed volume through two loudspeakers placed at a distance approximately 2 feet from the subject in a sound field orientation that was held constant for all participants at 70 db sound pressure level ( spl ; creative inspire t12 ) . this configuration achieved a consistent spl given participant selection criteria that included a srt range of 1020 db and comparable pure tone averages ( pta ) . participants were positioned at arm 's length from a desk mounted computer monitor with a projected plus sign image to maintain stable head position ( without formal head fixation / rest platform ) through visual fixation to prevent motion artifact during the recording session . participants were instructed to stay awake and to simply sit and listen with their gaze fixed on the screen . participants were initially presented with a sample of each of the testing condition stimuli as a demonstration prior to the start of the formal research paradigm . the auditory testing paradigm consisted of 9 rounds of randomly chosen 18-second blocks of 750 hz , 8000 hz , or bbn separated by intervening 18-second blocks of silence ( interstimulus rest ; isr ) between each auditory stimulus ( figure 2 ) . each experimental run per participant consisted of 54 blocks : 27 silent ( isr ) and 27 stimulation blocks equally distributed amongst the three experimental stimuli ( 750 hz , 8000 hz , or bbn ) . auditory stimuli were selected to evaluate hemodynamic responses during partial ( 750 hz and 8000 hz ) and complete ( bbn ) auditory cortical tonotopic activation as compared to isr in the two groups . all data were preprocessed using homer2 software based on matlab ( mathworks , ma , usa ) . the e - prune channel function was used to exclude channels with very low optical intensity from analyses . in addition , to remove motion artifact from the data , the wavelet - based motion artifact was used . the data was low - pass filtered at 0.5 hz in order to eliminate physiological fluctuations and high - pass filtered at 0.01 hz to remove instrumental noise . optical density data was then converted into concentration changes using a partial path length factor of 6.0 . since the hemodynamic response following auditory stimulation takes approximately 46 seconds to reach maximum level , the preprocessed signal was averaged across all blocks over a time range of 418 seconds for all conditions . normalization of the hemodynamic activity was achieved by subtracting the mean value of the first 3 seconds from the block hemodynamic activity average . this was done in order to prevent capture of any dynamic changes occurring in the immediate preceding block where at the end of the recording block the hemodynamic activity was still elevated . this procedure was conducted in each condition and group for oxy - hemoglobin ( hbo ) and deoxy - hemoglobin ( hbr ) , separately [ 30 , 34 ] . channels covering the roi and non - roi were subsequently used for the statistical analyses . standard deviations were calculated using the equation : =(a - a-2)/n ( with = standard deviation ; a = each value in the population ; a- = the mean of the values ; n = the number of values ) . standard errors were calculated using the equation : x = s/n ( s = sample standard deviation ; n = the number of values ) . statistical analyses were focused on the oxygenated hemoglobin ( hbo ) signal because hbo constitutes a greater portion of signal from the cortex ( 76% ) compared to deoxy - hemoglobin ( hbr ; 19% ; ) . moreover , hbo is sampled over a larger region of brain tissue , and the signal - to - noise contrast for hbo is better than hbr . the correlations between the canonical model of the hemodynamic response function and models of hbo ( versus hbr ) are consistently higher . the authors conducted testing of normality ( kolmogorov - smirnov test ) for the variables of interest including metabolic activity per group in the various experimental conditions . the results indicated normal data ; those results along with normal appearing histogram and boxplots facilitate the use of parametric tests . pearson correlation analyses were conducted between age , hearing threshold , and tinnitus handicap inventory ( thi ) scores with hemodynamic responses during the various experimental paradigms ( isr , bbn , 750 hz , and 8000 hz ) in both roi and non - roi . to assess for interhemispheric differences for each test condition and to rule out the possibility of optode asymmetry paired t - test comparisons between right and left rois and non - rois within specific experimental conditions were conducted . these analyses showed no interhemispheric differences and enabled hemodynamic responses to be pooled for each participant for final evaluation . in order to examine whether hbo differences existed between conditions , rois , and groups , a 2 4 2 repeated measures analysis of variance ( anova ) with bonferroni correction the anova allows for post hoc analyses to investigate the neurophysiological response to sound within group , roi , and condition . thus , we performed paired t - test analyses within each of the experimental groups between the hbo mean value following each auditory stimulation and isr in roi and non - roi . this allowed us to verify if tinnitus and control participants showed activation to the auditory stimuli , separately . paired t - tests were then conducted between the hbo mean values of roi and non - roi for each type of auditory stimulation ( 750 hz , 8000 hz , and bbn ) . this allowed us to verify if the response was limited to the auditory regions . following this , we performed independent t - test analyses to assess hemodynamic activity differences during the various experimental conditions , including isr . only those participants with normal / near - normal hearing were included in the study . within controls the average srt was 15 db with an average wds of 99% , while the tinnitus group had an average srt of 19 db and 100% wds . independent t - tests were conducted to assess differences in audiogram results between tinnitus and control participants . the analysis indicated no differences in hearing thresholds between the two groups ( p = 0.10 ) . to characterize the severity of tinnitus symptoms across the group two participants were grade 1 ( slight severity easily masked ) , two at grade 2 ( mild severity easily masked ; occasionally interferes with sleep ) , and one at grade 3 ( moderate severity difficult to mask ) with the final four participants scoring grade 4 ( severe severity always heard ; altered sleep ) . none of the participants in the study scored a grade 5 ( catastrophic severity always heard ; interfere with sleep and daily activities ) . pearson correlation analysis was conducted between age , hearing threshold , thi score , and audiogram findings with hemodynamic activity during the various experimental paradigms ( isr , bbn , 750 hz , and 8000 hz ) in both roi and non - roi . the only significant effect was a negative correlation between thi score and hemodynamic activity in non - roi during 750 hz tone stimulation ( pearson correlation of 0.7 ; p = 0.02 ) . none of the participants were excluded from the study due to inadequate fnirs signal or changes in headband position as determined by pre- and postexperimental photographs . paired t - tests on the right and left hemispheres within specific experimental conditions pooled across the length of the experiment did not reveal any significant differences . in the tinnitus group , a trend of increased hemodynamic activity in the left hemisphere was seen in response to bbn in both roi ( t = 1.80 ; p = 0.79 ) and non - roi ( t = 1.95 ; p = 0.67 ) but was not significant . paired t - tests analysis between pooled right and left hemispheric hemoglobin concentration across the experimental paradigm revealed a trend of left greater than right brain hemodynamic response in both groups in both roi and non - roi that was not significantly different ( roi control : right mean 0.08 0.49 ; left mean 0.31 0.39 ; non - roi control : right mean 0.96 0.70 ; left mean 0.70 0.48 ; roi tinnitus : right mean 0.28 0.34 ; left mean 0.09 0.29 ; non - roi tinnitus : right mean 0.72 0.41 ; left mean 0.58 0.27 ; p > 0.05 ) . the handedness of the participants was not determined and this variability may account for this observed trend . no other interhemispheric differences or trends were identified in either group . given the lack of interhemispheric asymmetry , all remaining analysis pooled total right and left hemispheric data for roi and non - roi analysis . during isr , controls demonstrated an as expected significant deactivation of hbo within roi during the entire interval , suggesting that hemodynamic activity decreased with the cessation of auditory stimulation ( figures 3 and 4(a ) ) . conversely , tinnitus participants maintained or slightly increased hbo concentration throughout the recording block during isr ( figure 4(a ) ) . t - test comparisons revealed that controls had a larger hbo decrease than tinnitus ( t = 2.9 ; p < 0.001 ) , suggesting that tinnitus participants have greater roi metabolic activity during periods of silence / absence of acoustic stimulation . interestingly , tinnitus participants also demonstrated maintenance of hemodynamic responses ( increased hbo ) within non - roi during isr ( figure 4(c ) ) indicating that central tinnitus origins may also exhibit physiological changes over other regions of brain not primarily dedicated to auditory function . t - test comparisons demonstrated that controls had more significant hbo decreases than tinnitus over the non - roi ( t = 2.5 ; p = 0.01 ) , indicating , as in the roi , that tinnitus participants have greater non - roi metabolic activity at rest . the results of the anova demonstrated a main effect for brain region ( f(1,68 ) = 10.80 ; p < 0.001 ) and revealed a significant interaction between the various experimental conditions ( isr , bbn , 750 hz , and 8000 hz ) and region of analysis ( roi versus non - roi ; f(3,66 ) = 4.78 ; p = 0.03 ) indicating that , as expected , the two regions had different hemodynamic responses under various experimental conditions ( table 1 ) . a significant interaction was found between the various experimental conditions and the two groups ( f(3,66 ) = 4.80 ; p = 0.01 ) , indicating that tinnitus and control participants responded to the experimental conditions differently . post hoc analyses revealed that control participants showed significant hbo increases during 750 hz , 8000 hz , and bbn when compared to the periods of silence in the roi , corresponding to the area of the auditory cortex ( 750 hz , t = 2.60 , p = 0.01 ; 8000 hz , t = 2.10 , p = 0.04 ; bbn , t = 2.30 , p = 0.03 ; table 1 ) . as expected , in the non - roi , control participants did not demonstrate any changes in hemodynamic activity during tone ( 750 hz , 8000 hz ) or noise ( bbn ) , indicating that this region was not involved in auditory processing ( 750 hz , t = 0.80 , p = 0.40 ; 8000 hz , t = 0.60 , p = 0.50 ; bbn t = 0.10 , p = 0.90 ) . these results indicate that our data are plausible with neurophysiological response to sounds . following stimulation with bbn , hbo concentrations that were maintained in the tinnitus group under conditions of silence showed a significant deactivation in roi relative to isr ( t = 2.2 , p = 0.04 ; figure 4(b ) ) . the suppressed activity in response to bbn in roi was maintained and actually continued to decline over time during the experimental condition suggesting long - term effects likely consistent with forward masking and residual inhibition of response to broad auditory stimulation ( figure 4(b ) ) . like the effects on roi , bbn also interestingly lead to a significant deactivation in measured hbo over the non - roi as compared to isr ( t = 3.30 , p < 0.001 ; figure 4(d ) ) . although a trend of hbo increase was found for 750 hz and 8000 hz in the tinnitus group within roi , the comparisons between these auditory stimulations and isr were not significant ( figures 5(a ) and 5(b ) ) . conversely , hbo responses to 750 hz and 8000 hz decreased in the non - roi ( 750 hz , t = 3.38 , p < 0.001 ; 8000 hz , t = 2.14 , p = 0.04 ; figures 5(c ) and 5(d ) ) . the comparison between roi and non - roi during acoustic stimulation ( 750 hz , 8000 hz , and bbn ) revealed a significant difference in response to 750 hz ( t = 3.7 ; p < 0.001 ) in controls and only a trend in response to 8000 hz ( t = 0.5 ; p = 0.6 ) and bbn ( t = 1.01 ; p = 0.32 ) , revealing the as expected result of increased auditory cortex activity following acoustic stimulations as compared to non - roi . similar results were found between roi and non - roi in tinnitus participants ( 750 hz , t = 2.7 , p = 0.01 ; 8000 hz , t = 0.52 , p = 0.61 ; bbn , t = 0.17 , p = 0.86 ; table 1 ) . figure 6 graphically summarizes the changes in hbo concentration in both groups under all tested conditions . our data demonstrated maintenance of elevated hemodynamic activity during periods of silence in tinnitus participants supporting our central hypothesis that auditory cortical regions are spontaneously active in the absence of stimulation as compared to nontinnitus controls . animal models of noise - induced tinnitus demonstrate increased spontaneous and tone - evoked neural firing rates and enhanced neural synchrony in auditory cortex and brainstem [ 37 , 38 ] . increased neuronal firing rates and enhanced synchrony have both been touted as putative physiologic correlates of tinnitus [ 5 , 38 ] . however , caution should be exercised in equating animal models of tinnitus with the human condition , in particular given the strong emotional and cognitive aspect in humans that can not be equated or demonstrated in an animal model . when combined with cortical tonotopic map reorganization [ 3941 ] and alterations between central auditory neural excitation and inhibition , the reported neural changes highlight plasticity within the central circuit that may underlie tinnitus etiology and ultimate phantom sound perception . our fnirs results showing alterations in waveforms representative of changes in neural hemodynamic properties in human tinnitus may directly reflect the basic science neural correlates . to our knowledge , only one other study utilizing fnirs technology has demonstrated the aberrant effects of tinnitus in the human brain while other neuroimaging studies have attempted to identify the equivalent purported basic science neural correlates from animal models . reviewed several pet and fmri studies that demonstrated enhanced steady state neural activity across multiple central auditory centers as a potential correlate of tinnitus . our data are consistent with the findings that primary auditory regions maintained hemodynamic activity under periods of silence while control subjects showed a deactivation in hemodynamic response . the maintenance of hemodynamic activity within primary auditory regions may reflect increases in spontaneous neural discharge rates found reliably in animal models suggesting that the human tinnitus brain may also exhibit this physiologic change in the absence of an actual acoustic stimulus . indeed , the perception of tinnitus is considered to be the result of increased neural ( cortical , subcortical ) activity . this can have two opposite types of effects on the magnitude of an evoked or event - related potential ( erp ) ; one may hypothesize that in the presence of elevated tonotopic activity at tinnitus frequency , the magnitude of an erp elicited by the same frequency may be increased as it is added to the existing synchronized activity . conversely , the result could also be a reduced response due to the availability of fewer neurons to generate the synchronized activity necessary to generate an ep or erp . one unexpected finding in the present study is the elevated hemodynamic activity in non - roi in tinnitus . while we show that human tinnitus may also influence nonauditory cortical regions , other imaging modalities have identified enhanced brain activity in regions outside auditory cortex , including areas dedicated to attention and emotion in tinnitus [ 8 , 21 ] . utilizing a single - photon emission computer tomography ( spect ) scan , which uses gamma rays and has the ability to provide 3d information , shulman et al . demonstrated significant abnormalities in cerebral perfusion in multiple brain regions including auditory , frontal , and parietal cortices in human tinnitus . others have also demonstrated changes in multiple brain regions in individuals with tinnitus using eeg , including primary and secondary auditory cortices , anterior cingulate cortex , dorsal lateral prefrontal cortex , insula , parahippocampus , and posterior cingulate cortex [ 7 , 43 ] . elevated waveforms during silence in non - roi in our data agree with data from other optical imaging modalities suggesting that potential maladaptive changes in tinnitus extend beyond primary auditory cortices to nonauditory areas . our non - roi channels corresponded to regions in occipital cortex , which has been identified by optical imaging studies in tinnitus brains to show altered processing [ 2527 , 44 , 45 ] . another maladaptive change identified in human tinnitus utilizing optical imaging is hemispheric asymmetry [ 8 , 4648 ] . using pet technology , langguth et al . found that 17/20 tinnitus participants displayed increased activity in left versus the right auditory cortex suggesting that interhemispheric differences may contribute to tinnitus etiology or perception . our analysis of waveforms from both roi and non - roi did not identify significant interhemispheric asymmetry in either group but did demonstrate an overall left greater thann right trend across both groups . while this may be , in part , a reflection in variability in handedness amongst the participants admitted limited power of this pilot study could explain the lack of statistical significance between hemispheres . together , these data suggest that fnirs as an imaging modality is capable of accurately measuring and discerning subtle and dynamic changes in cortical hemodynamic activity under conditions of stimulation and silence in both normal and aberrant neural circuits . the present results using fnirs technology demonstrate differences in auditory and adjacent nonauditory cortical hemodynamic responses in humans with subjective tinnitus . , we demonstrate the capability of fnirs to investigate brain changes that may underlie tinnitus etiology . our data show clear neurophysiological responses within the auditory cortex following acoustic stimulation in tinnitus participants as compared to regions not primarily associated with auditory processing . additionally , these results were also comparable with those of normal hearing controls and consistent with our predictions and previous reports utilizing alternative optical imaging modalities . indeed , the data showed a canonical hemodynamic response in roi with auditory stimulation and lack of response in non - roi under the same conditions [ 1317 , 23 ] . although increased tone - evoked firing rates to auditory stimuli are touted in animal models as potential neural correlates of tinnitus , our data did not demonstrate increased hemodynamic responses to auditory stimuli in tinnitus participants . this may be a relevant demonstration of the differences between tinnitus perceived by humans and animal models . while animal models are important in demonstrating neurophysiological phenomena in tinnitus , they have limitations in terms of describing the human condition . moreover , these results agree with findings from attias using other optical imaging techniques on humans , suggesting that this abnormal activity is unlikely to underpin tinnitus perception [ 4952 ] . interestingly , bbn significantly decreased hemodynamic responses in both roi and non - roi within the tinnitus group . this stands in contrast to controls , where an expected elevated response in roi and not in non - roi was seen . this suppression of cortical activation potentially represents forward masking and based on the permanence of temporal effect , also residual inhibition , where an external sound is used to suppress phantom perception . forward masking is thought to work through the interruption of abnormal synchronous activity among networks of neurons that generate tinnitus by the external sound stimulus . capitalizing on the well - recognized phenomenon of tinnitus modulation in certain participants , others have demonstrated with optical imaging techniques that tinnitus perception is associated with changes in activity within the auditory circuit [ 50 , 54 , 55 ] . melcher et al . utilized fmri with and without sound from the scanner pump to demonstrate that hemodynamic responses to auditory stimuli in tinnitus were blunted when the pumps were on relative to when the pumps were off , a finding absent in controls . utilized pet imaging during habitual and blunted tinnitus sensation , reporting that suppressing tinnitus perception decreased cortical activity . the decreased activity seen in our tinnitus group in response to bbn may reflect similar phenomena . future studies will be required to assess tinnitus perceptual changes during prolonged experimental paradigm to closer investigate residual inhibition . a longer block paradigm would also provide better insight into both initial and sustained hemodynamic responses to auditory stimuli as well as reducing confounding factors of the cortical measurements in various experimental conditions . a limitation of this study is the interpretability of optical imaging data from participants with heterogeneous brains . spatial normalization is an image processing step used with other optical imaging modalities , such as fmri or pet , to help standardize optical imaging data given the variability in human participant brain size and morphology . we did not utilize that approach as that would require the use of a second optical imaging modality . while many published fnirs reports have not implemented this technique as a standard practice in data collection , future studies will be designed to incorporate multiple optical imaging modalities . a potential limitation to this difference may influence the differential path length ( dpf ) that depends upon the proportions of different scatterers and absorbers in tissue , including the presence of bone , myelin , and muscle . although these proportions can be heterogeneous within any study group , dpf has been found to be approximately constant with the use of a source detector distance over 2.5 cm [ 5961 ] . with aging , there are changes in tissue properties that may alter the dpf . duncan et al . assessed differences in dpf at a fixed frequency and a source - detector separation for 283 subjects from 1 day to 50 years of age , finding that there is a slowly varying age dependence of dpf across all the study subjects . modeling studies have shown that an increase in scattering or lowering in absorption can lead to an increase in dpf ; the higher dpf values seen with increases in age are expected given the increases in myelin that occurs with aging . when analyzing dpf values of only adult subjects , duncan et al . found no correlation between dpf and age within this subgroup existing . given that all of our study subjects were adults , we feel that ultimately we had a consistent dpf in our study and essentially void of any bias form differences in age across the subjects . another limitation of the current study is the relatively small number of subjects in both groups . despite this limitation , the current responses are quite compelling and provide a solid foundation for future , potential multicentered trials to test more complex variables in this population . we also acknowledge that the reduced numbers also limit our ability to correlate tinnitus severity ( per the testing index ) and age differences with hemodynamic response . this will certainly be important going forward as fnirs could then have potential clinical diagnostic and potentially prognostic application . further limitation of this study includes the use of a fixed dpf value for both wavelengths . as demonstrated by strangman et al . and boas et al . , the use of the same dpf for 690 and 830 nm may lead to cross - talk in the estimates of hbo and hbr concentrations . future studies should consider cautiously the appropriate value for each wavelength in order to reduce systematic errors . the present study uses fnirs technology to investigate and report hemodynamic response changes that occur in auditory and select nonauditory cortices in the human brain in those suffering from subjective tinnitus . our data support the hypothesis that auditory cortices maintain and even increase hemodynamic activity during periods of silence , supporting , and even potentially representing putative neural correlates of tinnitus from animal models implicating increased spontaneous neural firing rates as a potential underlying etiology . while the measured changes in hemodynamic activity using fnirs may not be a direct reflection of physiologic correlates , our results support future application of noninvasive fnirs technology to further investigate translational correlates of central auditory aberrancy .

high - quality images are crucial to accurately diagnose a patient or determine treatment . in addition to requiring the best images possible many imaging systems use x - rays to provide a view of what is beneath a patient 's skin . x - ray radiation levels must be kept at a minimum to protect both patients and staff . as a result , raw image data can be extremely noisy . in order to provide clear images , algorithms designed to reduce noise are used to process the raw data and extract the image data while eliminating the noise . in video imaging applications , data often have to be processed at rates of 30 images per second or more . filtering noisy input data and delivering clear , high - resolution images at these rates require tremendous computing power . this gave rise to the need of developing high - end computing algorithms for image processing and analysis which are able to exploit the high performance of advanced computing machines . in this paper , we focus on the computational kernels which arise as basic building blocks of the numerical solution of medical imaging applications described in terms of partial differential equations ( pdes ) of parabolic / hyperbolic type . such pdes arise from the scale - space approach for description of most inverse problems in imaging . one of the main reasons for using pdes to describe image processing applications is that pde models preserve the intrinsic locality of many image processing operations . moreover , we can rely on standard and up - to - date literature and software about basic computational issues arising in such case ( such as the construction of suitable discretization schemes , the availability of a range of algorithmic options , and the reuse of software libraries that allow the effective exploitation of high - performance computing resources ) . finally , pdes appear to be effectively implemented on advanced computing environments . we consider two standard discretization schemes of nonlinear time - dependent pdes : semi - implicit scheme and fully implicit scheme . the former leads to the solution of a linear system at each time ( scale ) step , while the computational kernel of the fully implicit scheme is the solution of a nonlinear system , to be performed at each time ( scale ) step . taking into account that we aim to solve such problems on parallel computer in a scalable way , in the first case , we use , as linear solver , krylov iterative methods ( gmres ) with algebraic multigrid preconditioners ( amg ) [ 4 , 5 ] . regarding the fully implicit scheme , we use the jacobian - free newton - krylov ( jfnk ) method as nonlinear solver . in recent years we provide a multicore implementation of numerical algorithms arising from using the semi - implicit and the implicit discretization schemes of nonlinear diffusion models underlying most problems in image analysis . our implementation is based on parallel petsc ( portable extensible toolkit for scientific computation ) computing environment . parallel software uses a distributed memory model where the details of intercore communications and data managements are hidden within the petsc parallel objects . the paper is organized as follows . in section 2 , an overview of the pde model equation used in describing some of inverse problems in imaging applications section 4 is devoted to the discussion of numerical algorithms based on semi - implicit and implicit numerical schemes . in section 6 , we describe the experiments that we carried out to show both the accuracy and the performance of these algorithms , while section 7 concludes the work . the task in medical imaging is to provide in a noninvasive way information about the internal structure of the human body . the basic principle is that the patient is scanned by applying some sort of radiation and its interaction with the body is measured . most medical imaging problems lead to ill - posed ( inverse ) problems in the sense of hadamard [ 911 ] . a standard approach for dealing with such intrinsic instability is to use additional information to construct families of approximate solution . this principle characterizes regularization methods that , starting from the milestone tikhonov regularization , are now one of the most powerful tools for solution of inverse ill - posed problems . in 1992 , rudin et al . introduced the first nonquadratic regularization functional ( i.e. , the total variation regularization ) to denoise images . moreover , the authors derive the euler - lagrange equations as a time - dependent pde . in the same years scale - space theory has been developed by the computer vision community to handle the multiscale nature of image data . a main argument behind its construction is that if no prior information is available about what are the appropriate scales for a given data set , then the only reasonable approach for a vision system is to represent the input data at multiple scales . this means that the original image u(x ) , x should be embedded into a one - parameter family of derived images , in which fine - scale structures are successively suppressed : ( 1)ss:u(x, ) . a crucial requirement is that structures at coarse scales in the multiscale representation should constitute simplifications of corresponding structures at finer scales they should not be accidental phenomena created by the method for suppressing fine - scale structures . a main result is that if rather general conditions are imposed on the types of computations that are to be performed , then convolution by the gaussian kernel and its derivatives is singled out as a canonical class of smoothing transformations [ 15 , 16 ] . a strong relation between regularization approaches and the scale - space approach exists via the euler - lagrange equation of regularization functionals : it consists of a pde of parabolic / hyperbolic ( diffusion / advection ) type , defined as follows . nonlinear diffusion modelslet x = ( x , y ) and u(x , ) , defined in [ 0 , t ] , be the scale - space representation of the brightness function image u(x ) defined in describing the real ( and unknown ) object and u0(x ) the observed image ( the input data ) . let us consider the following pde problem : ( 2)u(x,)=|u|(g(u)u|u| ) [0,t ] , ( x , y)u(0,x)=u0(x ) =0 , ( x , y).[0 , t ] is the scale ( time ) interval ; g(v ) is a nonincreasing real valued function ( for v > 0 ) which tends to zero as v . initial and boundary conditions will be provided according to the problem to be solved ( denoising , segmentation , deblurring , registration , and so on ) . let x = ( x , y ) and u(x , ) , defined in [ 0 , t ] , be the scale - space representation of the brightness function image u(x ) defined in describing the real ( and unknown ) object and u0(x ) the observed image ( the input data ) . let us consider the following pde problem : ( 2)u(x,)=|u|(g(u)u|u| ) [0,t ] , ( x , y)u(0,x)=u0(x ) =0 , ( x , y).[0 , t ] is the scale ( time ) interval ; g(v ) is a nonincreasing real valued function ( for v > 0 ) which tends to zero as v . initial and boundary conditions will be provided according to the problem to be solved ( denoising , segmentation , deblurring , registration , and so on ) . equations in ( 2 ) describe the motion of a curve ( a moving front ) with a speed depending on a local curvature . such equations , known as level set equations , were first introduced in . n 1 ) , bounding an ( perhaps multiply connected ) open region , we wish to analyze and compute its subsequent motion under a velocity field v. this velocity can depend on position , time , the geometry of the interface ( e.g. , its normal or its mean curvature ) , and the external physics . the idea , as devised in 1988 by osher and sethian , is merely to define a smooth ( at least lipschitz continuous ) function (x , t ) , that represents the interface as the set where (x , t ) = 0 . thus , the interface is to be captured for all later time , by merely locating the set (t ) for which vanishes . the motion is analyzed by convecting the values ( levels ) with the velocity field v. this elementary equation is ( 3)t+v=0 . actually , only the normal component of v is needed : ( 4)vn = v|| , and the motion equation becomes ( 5)t+vn||=0 . taking into account that the mean curvature of (t ) is ( 6)cur=(|| ) , equation ( 5 ) describes the motion of (t ) under a speed vn proportional to its curvature cur ( mean curvature motion , mcm equation ) [ 1820 ] . this basic model has received a lot of attention because of its geometrical interpretation . indeed , the level sets of the image solution or level surfaces in 3d images move in the normal direction with a speed proportional to their mean curvature . in image processing , equations like ( 5 ) arise in nonlinear filtration , edge detection , image enhancement , and so forth , when we are dealing with geometrical features of the image - like silhouette of object corresponding to level line of image intensity function . finally , the level set approach instead of explicitly following the moving interface itself takes the original interface and embeds it in higher dimensional scalar function u , defined over the entire image domain . the interface is now represented implicitly as the zeroth level set ( or contour ) of this function , which varies with space and time ( scale ) using the partial differential equation in ( 2 ) , containing terms that are either hyperbolic or parabolic . the theoretical study of the pde was done by which proved existence and uniqueness of viscosity solutions . in this paper the task of image segmentation is to find a collection of nonoverlapping subregions of a given image . in medical imaging , for example , one might want to segment the tumor or the white matter of a brain from a given mri image . the idea behind level set ( also known implicit active contours , or implicit deformable models ) for image segmentation is quite simple . the user specifies an initial guess for the contour , which is then moved by image - driven forces to the boundaries of the desired objects . more precisely , the input to the model is a user - defined point - of - view u0 , centered in the object we are interested in segmenting . function u(x , ) in ( 2 ) is the segmentation function , u0 represents the initial contour ( initial state of the segmentation function ) , and the image to segment is i. moreover , as proposed in , instead of following evolution of a particular level set of u , the pde model follows the evolution of the entire surface of u under speed law dependent on the image gradient , without regard to any particular level set . suitably chosen , this flow sharpens the surface around the edges and connects segmented boundaries across the missing information . in [ 22 , 23 ] , the authors formalized such model as the riemannian mean curvature flow where the variability in the parameter also improves the segmentation process and provides a sort of regularization . thus , ( 2 ) becomes ( 7)u(x,)=2+|u|2(g(|i0|)u2+|u|2 ) [0,t ] , x=(x , y)u(x,0)=u0(x ) =0 , x=(x , y)u( , x)= 0 [0,t ] , x=(x , y) accompanied with initial condition u0 and zero dirichlet boundary conditions . regarding u0 , it is usually defined as a circle completely enclosed inside the region that one wish to segment . the term g(v ) , called edge detector , is a nonincreasing real function such that g(v ) 0 while v , and it is used for the enhancement of the edges . indeed , it controls the speed of the diffusion / regularization : if u has a small mean in a neighborhood of a point x , this point x is considered the interior point of a smooth region of the image and the diffusion is therefore strong . if u has a large mean value on the neighborhood of x , x is considered an edge point and the diffusion spread is lowered , since g(v ) is small for large v. a popular choice in nonlinear diffusion models is the perona and malik function : g(v ) = 1/(1 + v/ ) , > 0 . in many models , the function g(|u| ) is replaced by its smoothed version g(|gu| ) , where g is a smoothing kernel , for example , the gauss function , which is used in presmoothing of image gradients by the convolution . for shortening notations , we will use abbreviation ( 8)g = g(|gu|).in conclusion , we use the riemannian mean curvature flow , as model equation of the segmentation of medical structures : given i0 , the initial image and u0 equals to a circle contained inside an object of the image i0 , we are interested in segmenting , we compute u(x , ) by solving ( 7 ) . the level sets of u(x , ) , at steady state , provide approximations of the contour to detect . nonlinear pde in ( 7 ) can be expressed in a compact way as ( 9)u(x , y,)=f[u(x , y,,u(x , y,),i0 ) ] , where ( 10)f=2+|u|2(g(|i0|)u2+|u|2 ) . scale discretizationthat is discretization with respect to . if [ 0 , t ] is the scale interval and nscales is the number of scale steps , we denote by i the ith scale - step for all i = 1 , , nscales , so that i+1 = i + , where = t / nscales is the step - size . that is discretization with respect to . if [ 0 , t ] is the scale interval and nscales is the number of scale steps , we denote by i the ith scale - step for all i = 1 , , nscales , so that i+1 = i + , where = t / nscales is the step - size . using the euler forward finite difference scheme to discretize the scale derivative on the left hand side of ( 9 ) , we get ( 11)u(x , y,i)u(x , y,i1)=f[u(x , y, ) ] or , equivalently ( 12)u(x , y,i)=u(x , y,i1)+f[u(x , y, ) ] . let us denote as ui = u(x , y , i ) , i = 1 , , nscales , the function u evaluated at i . equation ( 12 ) is rewritten as ( 13)ui = ui1+f(u(x , y,))g[u(x , y, ) ] . depending on the collocation value , used to evaluate u(x , y , ) with respect to the parameter , inside the f function on the right hand side of ( 12 ) three iterative schemes derive : explicit scheme : ui = g[ui1 ] , that is , the function f is evaluated at ui1 = u(x , y , i1 ) ; semi - implicit scheme : ui = g[ui1 , ui ] , that is , we use ui to discretize the numerator |u| of the fraction u/2+|u|2 . other quantities are evaluated at ui1 ; implicit scheme : ui g[u(i ) ] = 0 , that is , the function f is evaluated at ui . in summary , the difference between the semi - implicit and the implicit scheme relies on the scale discretization of the term |u| at the numerator of u/2+|u|2 inside the function f. this term controls the diffusion process , and it plays the role of edge - enhancement . if we consider the three - dimensional ( 3d ) domain t = [ 0 , t ] , the semi - implicit scheme employs a sort of 2d + 1 discretization of t proceeding along nscales two dimensional ( 2d ) slices each one obtained at i , while the fully implicit scheme uses a fully 3d discretization of t . this difference suggests that the fully implicit scheme may provide a more accurate edge detection than the semi - implicit scheme . if is the space domain , we introduce a rectangular uniform grid on consisting of nx ny ( for simplicity we assume that is a rectangular of dimension 1 1 ; this means that hx = 1/nx and hy = 1/ny ) , nodes ( xi , yj ) = ( lx , my),l = 1 , , nx , m = 1 , , ny , and we use finite volumes to discretize the partial derivatives of u , as in [ 24 , 25 ] . that is discretization with respect to ( x , y ) . if is the space domain , we introduce a rectangular uniform grid on consisting of nx ny ( for simplicity we assume that is a rectangular of dimension 1 1 ; this means that hx = 1/nx and hy = 1/ny ) , nodes ( xi , yj ) = ( lx , my),l = 1 , , nx , m = 1 , , ny , and we use finite volumes to discretize the partial derivatives of u , as in [ 24 , 25 ] . scale - space discretizationlet ( 14)uil , m = u(xl , ym,i)nxnynscales be the vector obtained from the scale - space discretization of the function u , we have the following iteration formulas.explicit scheme : ( 15)uil , mui1l , m=2+|ui1l , m|2 (g(|i0|)ui1l , m2+|ui1l , m|2)uil , m=(i+[a]i1l , m)ui1l , m i=1,2, ,ne , where , for each i , the matrix [ a]i1 ny and i ny is the unit matrix , while ne is the scale steps number . let ( 14)uil , m = u(xl , ym,i)nxnynscales be the vector obtained from the scale - space discretization of the function u , we have the following iteration formulas.explicit scheme : ( 15)uil , mui1l , m=2+|ui1l , m|2 (g(|i0|)ui1l , m2+|ui1l , m|2)uil , m=(i+[a]i1l , m)ui1l , m i=1,2, ,ne , where , for each i , the matrix [ a]i1 ny and i ny is the unit matrix , while ne is the scale steps number . explicit scheme : ( 15)uil , mui1l , m=2+|ui1l , m|2 (g(|i0|)ui1l , m2+|ui1l , m|2)uil , m=(i+[a]i1l , m)ui1l , m i=1,2, ,ne , where , for each i , the matrix [ a]i1 ny semi - implicit scheme : ( 16)uil , mui1l , m=2+|ui1l , m|2 (g(|i0|)uil , m2+|ui1l , m|2)uil , m = ui1l , m+[a]i1l , muil , m(i+[a]i1l , m)uil , m = ui1l , m i , i=1, ,nsi where , for each i , the matrix [ a]i1 ny fully - implicit scheme : ( 17)uil , mui1l , m=2+|uil , m|2 (g(|i0|)uil , m2+|uil , m|2)uil , m = ui1l , m+[a]il , m(uil , m ) i=1, ,ni , where ni is the scale steps number and [ a]i , for each i , is a nonlinear vector operator on ny in particular , we apply the crank - nicholson scheme which uses the average of the forward euler method at step i 1 and the backward euler method at step i : ( 18)uil , mui1l , m=12[2+|uil , m|2 (g(|i0|)uil , m2+|uil , m|2)]+12 [2+|ui1l , m|2 (g(|i0|)ui1l , m2+|ui1l , m|2 ) ] , i=1, ,niuil , m = ui1l , m+[bi1l , mui1l , m+[a]il , m(uil , m ) ] , i=1, ,ni , where bi1 is a matrix on ny the effectiveness of these schemes depends on a suitable balance between accuracy ( scale - space discretization error ) , number of flop / s per iteration ( algorithm complexity ) , and the total execution time needed to reach a prescribed accuracy ( software performance ) . explicit scheme is accurate at the first order both with respect to scale and space , that is , p = q = 1 ; anyway , it is the one straightforwardly computable . this scheme requires very small time steps in order to be stable ( cfl ( courant - friedrich - levy ) condition that guarantees the stability of the evolution ) , and its use is limited rather by its stability than accuracy . this constraint is practically very restrictive , since it typically leads to the need for a huge amount of iterations . the accuracy , in terms of discretization error with respect to both scale and space , is of the first order , because p = 1 , q = 2 [ 24 , 25 ] . crank - nicholson provides a discretization error of second order , that is , p = q = 2 , but it requires extra computations , leading to a nonlinear system of equations , at every time step , while stability is ensured for all scale steps . in the following , we collect these results : ( 20)explicit scheme : p = q=1,semi - implicit scheme : p=1 , q=2,implicit scheme : p = q=2 . then , the fully implicit scheme provides an order of accuracy greater than that provided by the others . this difference may be important in those applications of image analysis where the edges are fundamental to recognize some pathologies . concerning the semi - implicit scheme , we employ krylov subspace methods , which are the most effective approaches for solving large linear systems . in particular , we use generalized minimal residual method ( gmres ) equipped with algebraic multigrid ( amg ) preconditioner . such techniques are convenient because they require as input only the system matrix corresponding to the finest grid . in addition , they are suitable to implement in a parallel computing environment . for the fully implicit scheme we use the jacobian free newton krylov method ( jfnk ) . jfnk methods are synergistic combinations of newton - type methods for superlinearly convergent solution of nonlinear equations and krylov subspace methods for solving the newton correction equations . the link between the two methods is the jacobian - vector product , which may be probed approximately without forming and storing the elements of the true jacobian . let us briefly describe the numerical algorithms that we are going to implement , which are based on the semi - implicit and the implicit discretization schemes , together with their complexity . algorithm si ( semi - implicit scheme)for all i = 1 , , nsi solution of ( 21)(i+[a]i1l , m)uil , m = ui1l , mhsi1l , muil , m = ui1l , m , with respect to ui . hsi1 , for each i , is a matrix ny . as space derivative we use the 2nd order finite covolume discretization scheme ( see [ 24 , 25 ] for convergence , consistence and stability ) . by this way , matrix [ a]i1 is a block pentadiagonal matrix with tridiagonal blocks along the main diagonal and diagonal blocks along the upper and lower diagonals . for all i = 1 , , nsi solution of ( 21)(i+[a]i1l , m)uil , m = ui1l , mhsi1l , muil , m = ui1l , m , with respect to ui . hsi1 , for each i , is a matrix ny algorithm i ( implicit scheme)for all i = 1 , , ni solution of ( 22)uil , m = ui1l , m+[bi1l , mui1l , m+[a]il , m(uil , m)]hiil , m(uil , m , ui1l , m)=0 , with respect to ui . hii , for each i , is a nonlinear vector operator on ny . for all i = 1 , , ni solution of ( 22)uil , m = ui1l , m+[bi1l , mui1l , m+[a]il , m(uil , m)]hiil , m(uil , m , ui1l , m)=0 , with respect to ui . hii , for each i , is a nonlinear vector operator on ny algorithm sifor each scale step , to solve the linear system ( 21 ) , we employ gmres iterative method ( see algorithm 1 ) . taking into account the structure of the coefficient matrix ( we assume that nx = ny = n , then h = 1/n = hx = hy ) , the computational cost of gmres is ( 23)tgmres(n2)=o(kgmressi5n2 ) , where kgmres is the maximum iterations of gmres ( over the scale steps ) . for each scale step , to solve the linear system ( 21 ) , we employ gmres iterative method ( see algorithm 1 ) . computational kernel of gmres is a matrix - vector product . taking into account the structure of the coefficient matrix ( we assume that nx = ny = n , then h = 1/n = hx = hy ) , the computational cost of gmres is ( 23)tgmres(n2)=o(kgmressi5n2 ) , where kgmres is the maximum iterations of gmres ( over the scale steps ) . algebraic multigrid ( amg ) method follows the main idea of ( geometric ) multigrid ( mg ) , where a sequence of grids is constructed from the underlying geometry with corresponding transfer operators between the grids . the main idea of mg is to remove the smooth error , that can not be eliminated by relaxation on the fine grid , by coarse - grid correction . the solution process then as usual consists of presmoothing , transfer of residuals from fine to coarse grids , interpolation of corrections from coarse to fine levels , and optional postsmoothing . in contrast to geometric multigrid , the idea of amg is to define an artificial sequence of systems of equations decreasing in size . the interpolation operator plv and the restriction operator rlv define the transfer from finer to coarser grids and vice versa . finally , the operator on the coarser grid at level lv + 1 is defined by ( 25)alv+1l , m = rlvl , malvl , mplvl , m . the amg method consists of two main parts , the setup phase and the solution phase . during the setup phase , the solution phase consists of a multilevel iteration . the number of recursive calls , which is the number of levels lv , depends on the size and structure of the matrix . for our case , we use the v - cycle pattern with the falgout - cljp coarse grid selection . looking at the algorithm si in table 1 , the preconditioner p is just alv+1 . computational cost of each iteration of gmres is that of the amg preconditioner plus the matrix - vector products : ( 26)tamg+gmres(n2)=o(kgmressi(tamg(lv)lvn2 + 5n2 ) ) , then , we get : ( 27)tsi+amg+gmres(n2)=o(nsikgmressilvn2 ) . following picture shows a schematic description of algorithm si that emphasizes its main steps and the most time consuming operation , that is , the matrix vector products needed at each step of gmres . algorithm ifor each scale step , to solve the nonlinear equations ( 22 ) , we employ the jacobian - free newton - krylov ( jfnk ) method . jfnk is a nested iteration method consisting of at least two and usually four levels . the primary levels , which give the method its name , are the loop over the newton method : ( 28)hiil , m(un+1l , m)=0hiil , m(unl , m)+ji(unl , m)(un+1l , munl , m)=0 , and the loop building up the krylov subspace out of which each newton step is drawn : ( 29)ji(unl , m)unl , m=hiil , m(unl , m ) , un+1l , m = unl , m+unl , m , outside of the newton loop , a globalization method is often required . for each scale step , to solve the nonlinear equations ( 22 ) , we employ the jacobian - free newton - krylov ( jfnk ) method . jfnk is a nested iteration method consisting of at least two and usually four levels . the primary levels , which give the method its name , are the loop over the newton method : ( 28)hiil , m(un+1l , m)=0hiil , m(unl , m)+ji(unl , m)(un+1l , munl , m)=0 , and the loop building up the krylov subspace out of which each newton step is drawn : ( 29)ji(unl , m)unl , m=hiil , m(unl , m ) , un+1l , m = unl , m+unl , m , outside of the newton loop , a globalization method is often required . forming each element of j which is a matrix of dimension n n requires taking derivatives of the system of equations with respect to u. this can be time consuming . using the first order taylor series expansion of hii(un + v ) , it follows that ( 30)ji(unl , m)unl , m=[hiil , m(unl , m+unl , m)hiil , m(unl , m)] + o(2 ) , where is a perturbation . jfnk does not require the formation of the jacobian matrix ; it instead forms a result vector that approximates this matrix multiplied by a vector . this jacobian - free approach has the advantage to provide the quadratic convergence of newton method without the costs of computing and storing the jacobian . algorithm 3 shows a schematic description of algoritm i that emphasizes its main steps and the most time consuming operation , that is , evaluations of the nonlinear operator hii at each innermost step . algorithm complexity of jfnk is ( 31)tjfnk(n2)=o(nnewkgmresi[f+o(n2)]),where nnew is the maximum number of newton 's steps , over the scale steps , kgmres is the maximum number of gmres iterations ( computed over newton 's steps and scale steps ) ) , f is the number of evaluations of hii . finally , we get ( 32)ti+jfnk(n2)=o(ninnewkgmresi[f+o(n2 ) ] ) . a straightforward comparison between the algorithm complexity of these algorithms shows that algorithm si asymptotically seems to be comparable with respect to algorithm si . of course , the performance analysis must also take into account the efficiency of these two schemes in a given computing environment . next section describes the petsc - based implementation of these algorithms that we have developed in a multicore computing environment . the software has been developed using the high - level software library petsc ( portable extensible toolkit for scientific computations ) ( release 3.1 , march 2010 ) . petsc provides a suite of data structures and routines as building blocks for the implementation of large - scale codes to be used in scientific applications modeled by partial differential equation . petsc is flexible : its modules , that can be used in application codes written in fortran , c , and c++ , are developed by means of object - oriented programming techniques . the library has a hierarchical structure : it relies on standard basic computational ( blas , lapack ) and communication ( mpi ) kernels and provides mechanism needed to write parallel application codes . petsc transparently handles the moving of data between processes without requiring the user to call any data transfer function . this includes handling parallel data layouts , communicating ghost points , gather , scatter and broadcast operations . our parallelization strategy is based on domain decomposition : in particular , we adopt the row - block data distribution , which is the standard petsc data distribution . n / p n of contiguous rows of matrices of dimension n n are distributed among contiguous processes . by the same way , vectors of size n are distributed among p processors as blocks of size n / p . such partitioning has been chosen because overheads , due to redistribution before the solution of the linear systems , are avoided . further , row - block data distribution introduces a coarse grain parallelism which is best oriented to exploit concurrency of multicore multiprocessors because it does not require a strong cooperation among computing elements : each computing element has to locally manage the blocks that are assigned to it . the computing platform that we consider is made of 16 blades ( 1 blade consisting of 2 quad core intel xeon e5410@2.33 ghz ) dell poweredge m600 , equipped with ieee double precision arithmetic . because high performance technologies can be employed in medical applications only to the extent that the overall cost of the infrastructure is affordable , and because we consider single images of medium size , we show results obtained by using 1 blade , that is , we run the parallel algorithms on up to p = 8 cores of a single blade . of course , in case of multiple images or sequences of images , the use of a greater number of cores may be interesting . in this section we present and discuss computational results obtained by implementing algorithm i and algorithm si in a multicore parallel computing machine . before illustrating experimental results , ( 1 ) test imageswe have carried out many experiments in order to analyze the performance of these algorithms . here we show results concerning the segmentation of a ( malignant ) melanoma ( see figure 7 ) . epiluminescence microscopy ( elm ) has proven to be an important tool in the early recognition of malignant melanoma [ 29 , 30 ] . in elm , halogen light is projected onto the object , thus rendering the surface translucent and making subsurface structures visible . as an initial step , then , a set of features containing shape and radiometric features as well as local and global parameters is calculated to describe the malignancy of the lesion . in order to better validate computed results and to analyze the software performance , we first consider a synthetic test image simulating the object we are interested in segmenting ( see figure 1 ) . we have carried out many experiments in order to analyze the performance of these algorithms . here we show results concerning the segmentation of a ( malignant ) melanoma ( see figure 7 ) . epiluminescence microscopy ( elm ) has proven to be an important tool in the early recognition of malignant melanoma [ 29 , 30 ] . in elm , halogen light is projected onto the object , thus rendering the surface translucent and making subsurface structures visible . as an initial step , then , a set of features containing shape and radiometric features as well as local and global parameters is calculated to describe the malignancy of the lesion . in order to better validate computed results and to analyze the software performance , we first consider a synthetic test image simulating the object we are interested in segmenting ( see figure 1 ) . ( 2 ) comparison criteria we compare the algorithms using the following criteria : distance from original solution . as measure of the difference between two curves , we use the hausdorff distance measured between the computed curve and the original one . it is well known that the hausdorff distance is a metric over the set of all closed bounded sets ( see ) , here we restrict ourselves to finite point sets because that is all that is necessary for segmentation algorithms . given two finite point sets c1 and c2 , the hausdorff distance dh between the sets c1 and c2 is defined as follows : ( 33)dh(c1,c2)=max{h(c1,c2),h(c2,c1)},h(c1,c2)=maxac1 minbc2||ab|| , where |||| is the euclidean norm . it identifies the point a c1 that is fastest from any point of c2 and vice versa , then it keeps the maximum , efficiency : execution time of ( serial ) algorithms , convergence history : behavior of residuals and iteration numbers of inner solvers , parallel performance : execution time , speedup , and efficiency versus cores number . we compare the algorithms using the following criteria : distance from original solution . as measure of the difference between two curves , we use the hausdorff distance measured between the computed curve and the original one . it is well known that the hausdorff distance is a metric over the set of all closed bounded sets ( see ) , here we restrict ourselves to finite point sets because that is all that is necessary for segmentation algorithms . given two finite point sets c1 and c2 , the hausdorff distance dh between the sets c1 and c2 is defined as follows : ( 33)dh(c1,c2)=max{h(c1,c2),h(c2,c1)},h(c1,c2)=maxac1 minbc2||ab|| , where |||| is the euclidean norm . it identifies the point a c1 that is fastest from any point of c2 and vice versa , then it keeps the maximum , efficiency : execution time of ( serial ) algorithms , convergence history : behavior of residuals and iteration numbers of inner solvers , parallel performance : execution time , speedup , and efficiency versus cores number . distance from original solution . as measure of the difference between two curves , we use the hausdorff distance measured between the computed curve and the original one it is well known that the hausdorff distance is a metric over the set of all closed bounded sets ( see ) , here we restrict ourselves to finite point sets because that is all that is necessary for segmentation algorithms . given two finite point sets c1 and c2 , the hausdorff distance dh between the sets c1 and c2 is defined as follows : ( 33)dh(c1,c2)=max{h(c1,c2),h(c2,c1)},h(c1,c2)=maxac1 minbc2||ab|| , where |||| is the euclidean norm . it identifies the point a c1 that is fastest from any point of c2 and vice versa , then it keeps the maximum , efficiency : execution time of ( serial ) algorithms , convergence history : behavior of residuals and iteration numbers of inner solvers , parallel performance : execution time , speedup , and efficiency versus cores number . let us explain how we select the values of the scale step size and the number of scale steps . regarding , its value is chosen according to that required to ed . taking into account that , in algorithm si , ed is accurate at the first order with respect to si , while it is accurate at the second order with respect to i , in algorithm i , by requiring that the discretization error is about the same , we get ( 34)ed = o(si)=o(i2)si=i . let us explain how we select the values of the scale step size and the number of scale steps . regarding , its value is chosen according to that required to ed . taking into account that , in algorithm si , ed is accurate at the first order with respect to si , while it is accurate at the second order with respect to i , in algorithm i , by requiring that the discretization error is about the same , we get ( 34)ed = o(si)=o(i2)si=i . finally , in algorithm si , the stopping criterion of the linear solver ( gmres ) uses the tolerance ( 35)tol=1010 , while the preconditioner amg uses the tolerance tol = 10 and 25 as maximum number of amg - levels . in the algorithm 2 , the stopping criterion of the nonlinear solver uses the tolerance ( 36)tol=1010 . regarding the number of scale steps ( nsi and ni ) , taking into account that ( 37)nsi , i = tsi , i , its choice depends on si , i and on the value of t , that is , the value of the scale parameter corresponding to steady state of the segmentation function u(x , ) , solution of the pde model . to check the steady state , we require that the residuals , corresponding to different scale steps , reach the tolerance we found that this corresponds to t = 0.4 ( see figure 2 ) for test 1 and to t = 2 for test 2 . test 1 : synthetic imagein tables 1 , 2 , and 3 , we show the hausdorff distance and execution time by requiring that discretization error is of the first , second , and third order , that is , ed = o(10 ) , ed = o(10 ) , and ed = o(10 ) , respectively . hence , we get the following values of the scale step size : ( 39)i=0.4si=0.16,i=0.04si=0.016,i=0.004si=0.0016 . in tables 1 , 2 , and 3 , we show the hausdorff distance and execution time by requiring that discretization error is of the first , second , and third order , that is , ed = o(10 ) , ed = o(10 ) , and ed = o(10 ) , respectively . hence , we get the following values of the scale step size : ( 39)i=0.4si=0.16,i=0.04si=0.016,i=0.004si=0.0016 . moreover , concerning the number of scale steps , it follows that ( 40)ed = o(101)ni=0.40.4=1 , nsi = int[0.40.16]=3,ed = o(102)nsi=0.40.04=10 , nsi=0.40.016=25,ed = o(103)nsi=0.40.04=100 , nsi=0.40.016=250 . note that while in the first case , that is , if we require ed = o(10 ) , these two algorithms are quite numerically equivalent , both in terms of execution time and of the computed result ; as discretization error decreases , algorithm i appears to be more robust than algorithm si , in the sense that algorithm i reaches the steady state with high accuracy ( the hausdorff distance is of 95% ) , while the computed results of algorithm si are less accurate , even though the execution time of algorithm i sometimes slightly increases . these results suggest that if it needs to get an accurate and reliable result , algorithm i should be preferable . finally , note that the execution time of these two algorithms asymptotically is the same , as stated by the analysis of computational cost carried on in section 4 . convergence historyconvergence history is illustrated by showing the behavior of relative residuals versus the scale steps ( see figures 4 , 5 , and 6 ) , and by reporting iteration number of the gmres and of newton 's method , respectively , ( see tables 4 , 5 , 6 , and 7 ) . convergence history is illustrated by showing the behavior of relative residuals versus the scale steps ( see figures 4 , 5 , and 6 ) , and by reporting iteration number of the gmres and of newton 's method , respectively , ( see tables 4 , 5 , 6 , and 7 ) . we consider si = 0.16 , 0.016 , and i = 0.04 . in the following , we show results corresponding to the segmentation of a melanoma ( see figure 7 ) . as expected , because this is a real image , the steady state is reached at a scale greater than that of the synthetic test image , that is , t = 2 , thus both algorithms require a greater number of scale steps to the reach the steady state . tables 8 , 9 , and 10 , and figure 8 compare results of algorithm si and algorithm i. parallel performancewe show the performance of the multicore - based parallel algorithms and their scalability as the number of cores increases . we run the parallel algorithms using up to p = 8 cores of the parallel machine . we show the performance of the multicore - based parallel algorithms and their scalability as the number of cores increases . we run the parallel algorithms using up to p = 8 cores of the parallel machine . following figures report execution time , speedup , and efficiency of algorithm si , at scale step size = 0.16 ( i.e. , ed = ( 10 ) ( i.e. , figures 9 , 10 and 11 ) , then same results are shown at scale step size = 0.016 , corresponding to ed = o(10 ) ( i.e. , figures 12 , 13 and 14 ) . finally , we report execution time , speedup , and efficiency of algorithm i , at scale step = 0.4 ( i.e. , figures 15 , 16 and 17 ) and = 0.04 ( i.e. , figures 18 , 19 , and 20 ) , respectively . note that parallel efficiency of both algorithms always is , at least , of 60% and , on average , of about 80% . execution time of both algorithms reduces to about 2 seconds on eight cores in the first case ( i.e. , ed = ( 10 ) , and to about 10 seconds in the second case ( ed = o(10 ) ) . this means that , in a multicore computing environment , both algorithms provide the requested solution within a response time that can be considered quite acceptable in medical imaging applications and , in particular , that algorithm i is competitive with algorithm si . figures 21 , 22 , 23 , 24 , 25 , and 26 show time , speedup , and parallel efficiency of two algorithms in case of test 2 . finally , we show results on scalability of parallel algorithms . let tp(n ) be the execution time of the parallel algorithm running on p cores for segmenting an image of size n n. we measure the scalability of these algorithms by measuring tp(n ) as n varies , once p is fixed , and by measuring tp(n ) as n and p grow . we note that , in case of algorithm si , the scaling factor is ( 41)tp(2n)tp(n)4.2 , while , for algorithm i , we get as scaling factor ( 42)tp(2n)tp(n)2.3 . this means that algorithm i scales better than algorithm si . in figures 27 and 28 ( for algorithm si ) and figure 29 and 30 ( for algorithm si ) , we report tp(n ) as n and p varies . in particular , each point of the graph refers to the execution time of the parallel algorithm at ( p = k p1 , n = k n1 ) , where p1 = 1 , n1 = 210 , and k = 1,2 , 3,4 . a straightforward comparison between the semi - implicit and the fully implicit discretization schemes of nonlinear pde of parabolic / hyperbolic type states that fully implicit discretization usually leads to too expensive algorithms . in this paper , we provide a multicore implementation of two numerical algorithms arising from using these two discretization schemes : semi - implicit ( algorithm si ) and fully implicit ( algorithm i ) . taking into account that we aim to solve such problems on parallel computer in a scalable way , in the first case , we use , as linear solver , krylov iterative methods ( gmres ) with algebraic multigrid preconditioners ( amg ) . regarding the fully implicit scheme , we use the jacobian - free - newton krylov ( jfnk ) method as nonlinear solver . we compare these two algorithms using different metrics measuring both the accuracy and the efficiency . we note that if we require that the discretization error ed is ed = o(10 ) , these two algorithms are quite numerically equivalent , both in terms of execution time and of the computed result ; while , as discretization error decreases , algorithm i appears to be more robust than algorithm si , in the sense that algorithm i reaches the steady state with high accuracy ( the hausdorff distance is of 95% ) , while the computed results of algorithm si are less accurate , even though the execution time of algorithm i sometimes slightly increases . these results suggest that if it needs to get accurate and reliable results , algorithm i should be preferable . the parallel efficiency of both algorithms always is , at least , of 60% and , on average , of about 80% . execution time of both algorithms reduces to about 2 seconds on eight cores if ed = ( 10 ) and to about 10 seconds if ed = o(10 ) . this means that , in a multicore computing environment , algorithm i is competitive with algorithm si . in conclusion , our results suggest that if it is required high accuracy of the computed solution in a suitable turnaround time , using a multicore computing environment fully implicit scheme provides an accurate and reliable solution within a response time of few seconds , quite acceptable in medical imaging applications , such as computer - aided - diagnosis .

thalassemia major is a congenital defect of globulin chain synthesis , ineffective erythropoiesis and intense anemia . the chronic anemia leads to growth retardation , bone marrow expansion , extramedullary hematopoiesis , splenomegaly , greater intestinal iron absorption , hypercoagulability and higher propensity to infection . although iron chelator therapy has resulted in incredible progress in the treatment of thalassemia major , cardiac complications such as heart failure and arrhythmias , caused by the so - called iron - induced cardiomyopathy , may be the primary feature of death in patients with thalassaemia major . the qt interval reveals the duration between the beginning of ventricular depolarization and the end of ventricular repolarization . prolongation of the qt interval may be correlated with arrhythmogenesis in amount of cardiac disorders . some reports demonstrated higher dispersal of the qtc interval in a population of young and asymptomatic patients with thalassemia major than in healthy control subjects . since cardiac function remains normal until late in the field of iron cardiomyopathy , other tools are essential to predict and prevent iron cardiomyopathy . although liver iron level has been used as a substitute for cardiac iron for many years , the relation between cardiac iron and liver iron is quite complicated . in this study , we sought to evaluate qt distribution and the relation between qt dispersion and body iron overload among patients with -thalassemia major . in this cross - sectional study , 60 patients with thalassemia major and thalassemia intermedia , who had been diagnosed since childhood , were enrolled . all these patients had received transfusions frequently and had referred to firouzgar center for cardiologic follow - up every 6 - 12 months . control group was assigned simply and non - randomly from medical students that were 19 - 28 years old and did not have any past medical history . demographic / clinical data and informed written consent were obtained from all of the patients . this study was conducted with the approval of our institution review board ( irb ) . bruce method consists of four stages with increasing velocity and in every stage , qt intervals are measured and recorded in electrocardiography papers during the test . in addition , blood pressure and heart rate are measured and recorded during every stage of the test . all data were presented as the mean sd . statistical analyses were performed by spss statistical software ( version 16.0 for windows ; spss inc , chicago , il , u.s.a ) . chi - square and independent sample t- tests were used to compare qualitative and quantitative variables respectively . p values less than 0.05 were considered statistically significant and 95% confidence intervals were reported . in this cross - sectional study , 60 patients with thalassemia major and thalassemia intermedia , who had been diagnosed since childhood , were enrolled . all these patients had received transfusions frequently and had referred to firouzgar center for cardiologic follow - up every 6 - 12 months . control group was assigned simply and non - randomly from medical students that were 19 - 28 years old and did not have any past medical history . demographic / clinical data and informed written consent were obtained from all of the patients . this study was conducted with the approval of our institution review board ( irb ) . bruce method consists of four stages with increasing velocity and in every stage , qt intervals are measured and recorded in electrocardiography papers during the test . in addition , blood pressure and heart rate are measured and recorded during every stage of the test . statistical analyses were performed by spss statistical software ( version 16.0 for windows ; spss inc , chicago , il , u.s.a ) . chi - square and independent sample t- tests were used to compare qualitative and quantitative variables respectively . p values less than 0.05 were considered statistically significant and 95% confidence intervals were reported . sixty thalassemia major and/or intermadia patients ( 40 thalassemia major and 20 intermadia ) including 32 males and 28 females and 62 healthy controls ( 30 males and 32 females ) were enrolled in this cross - sectional study . mean age of b thalassemia and control groups were 25.4 5.9 ( ranged 15 to 41 ) and 25.7 3.1 years old ( ranged 20 to 35 ) , respectively . demographic and hemodynamic characteristics of thalassaemia major / intermedia patients and control group additionally , there were no significant differences in serum creatinine and hemoglobin level between both groups . electrocardiographic ( ekg ) and standard echocardiographic findings were normal and all patients were in sinus rhythm . the systolic and diastolic blood pressure values were remarkably lower in thalassemia group ( p = 0.001 ) , while the heart rate was higher in thalassemia compared with control group ( 105.1 15.1 vs. 89.7 12.3 , p = 0.001 ) . as shown in [ table 2 ] , measurement of corrected qt ( qtc ) interval values before and during exercise test revealed that resting qtc interval was slightly longer in thalassemia group compared with the control with no statistical significance ( 422.3 30.2 vs. 413.6 23.1 , p = 0.08 ) . but qtc interval during exercise became prolonged in thalassemia patients that were considerably higher compared with control group [ table 2 ] . the electrocardiographic findings of the study and control groups we compared effect of serum ferritin levels > 1000 g / l and < 1000 g / l on qtc interval among thalassemia patients in which the results showed no significant difference between groups ( p > 0.05 ) . despite remarkable iron chelator therapy in the treatment of thalassemia major , cardiomyopathy and heart failure are the major causes of death in patients with thalassemia major.[13 ] in accordance with other studies , we showed that qtc interval values augmented during exercise test in the young and clinically asymptomatic patients with thalassemia suggesting early abnormal myocardial repolarization . several studies report that these qt indices are helpful for recognizing changes in cardiac repolarization as structural and ultra structural injury , especially in patients inclined to sudden cardiac death . similar to our findings , ulger et al . detected that although there were no clinical or echocardiographic signs of cardiac disease , qtc intervals were superior in thalassemia major patients compared to the control group . in our study , the resting qtc interval was slightly higher in thalassemia patients compared with the healthy controls with no statistical difference . in b thalassemia patients of our study , systolic and diastolic blood pressures were significantly lower than control group ; however , heart rate was more among b thalassemia patients . this could be explained by the fact that thalassemia major is a chronic high - output state , and therefore , maintaining a normal mean systemic blood pressure requires reduced vascular resistance . previous studies have reported that serum ferritin level less than 2500 ng / dl is associated with better cardiovascular prognosis . moreover , serum ferritin less than 1000 ng / dl is associated with normal left ventricular ejection fraction ( lvef ) . here in , the mean serum ferittin in thalassemia major and intermedia patients was 2806 and 2004 , respectively . thirty percent patients had serum ferritin upper than 2500 ng / dl , which indicates that compliance to chelation therapy was not uniform within the study group . this may also explain the absence of heart failure and significant valve diseases in the current study population . the prolongation of qt interval predicts increased risk of life threatening ventricular arrhythmias and sudden death . ulger and colleagues revealed that left ventricular myocardial infarction e(lvmi ) was higher in thalassaemia major patients compared to the control group and there was a positive association between lvmi and qtc dispersion . in this study , we detected that although there was no clinical or echocardiographic signs of cardiac disease , qtc interval and qtc dispersion were higher in thalassaemia major patients compared to the control group . . the cardiomyopathy may be reversible if iron chelating treatment is strengthened in time , but the early diagnosis of iron - induced cardiomyopathy with established clinical techniques such as echocardiography and stress radionuclide angiography has had imperfect success . in particular , explicit dysfunction on echocardiography presents late stage in the disease process and the progression from mildly abnormal echocardiographic parameters to fulminant cardiac failure is often rapid and insistent . prospective studies are considered necessary to elucidate the relationship between the prolongation of qtc dispersion and consequent cardiac events in thalassemia patients .

though well known , ruptured sinus of valsalva ( rsov ) aneurysms are rare comprising 0.3 - 3.56 % of all congenital heart diseases.1 they are thin walled outpouchings of the sinus of valsalva and may be tubular or saccular in shape . the right sinus of valsalva is most commonly involved and usually ruptures into right heart chambers . uncommonly they rupture into left heart chambers ( left atrium , left ventricle ) , pulmonary artery , interventricular septum or the pericardial cavity.23 rupture into the left ventricle ( lv ) is very rare.45 we report a young male patient who was diagnosed to have a large aneurysm of left sinus of valsalva with rupture into lv along with distortion of mitral valve requiring replacement of both aortic and mitral valve . to our knowledge , an aneurysm of the left valsalva sinus bulging into the left atrial ( la ) roof and damaging the mitral valve along with concomitant rupture into the left ventricular cavity leading to severe aortic regurgitation ( ar ) and thus requiring a double valve replacement ( dvr ) has not been reported . pk , a 25 year old male patient was referred to our hospital with chest pain and dyspnea on exertion for the previous two months . there was no history of prolonged fever or trauma . on examination , he was of an average built . there was pallor , tachycardia ( hr-110/minute ) , tachypnoea ( respiratory rate-24/minute ) and wide pulse pressure ( bp 124/40 mmhg in right upper limb ) . cardiac examination revealed cardiomegaly , a hyperdyanamic precordial impulse , an ejection systolic murmur grade 2/6 and a pandiastolic murmur at the left parasternal border . twelve lead electrocardiogram showed sinus tachycardia , qrs axis + 60 and lv hypertrophy , and st depression in the left sided leads ( figure 1 ) . transthoracic echocardiography done on phillip ie33 showed a large aneurysm of the left sinus of valsalva with rupture of the aneurysm into the lv ( figures 26 ) . 12 lead ecg showing sinus tachycardia(hr-90/minute ) , qrs axis + 60 and lvh , st depression in left sided leads apical 4 chamber view with anterior tilt ( 5 chamber view ) showing aneurysm from left sinus bulging into left atrium and impinging to mitral valve . parasternal short axis view at the level of great vessels showing large aneurysm arising from left sinus of valsalva standard parasternal long axis view showing aortic valve and small portion of aneurysm posterior to aortic valve and dilated left ventricle . anterior tilt from figure 4 opened up whole aneurysm extending anteriorly to right ventricle outflow and posteriorly bulging into left atrium . the aneurysm bulged into the mitral inflow distorting the mitral valve ( figures 2 and 3 ) . color flow mapping showed 3 jets of aortic regurgitation , one central jet and two other from the ruptured aneurysm ( figure 6 ) . it also revealed turbulent flow across mitral inflow and moderate mitral regurgitation ( mr ) . continuous wave doppler interrogation of the mitral inflow revealed a transmitral mean gradient of 7 mmhg . early surgical intervention was advised and was performed through midsternotomy under routine cardiopulmonary bypass . at surgery , a large aneurysm of the left sinus of valsalva measuring 5 by 6 cm was found originating just distal to the orifice of the left coronary artery and protruding into the lv cavity . posteriorly , the aneurysm had burrowed into the roof of the left atrium causing displacement of the anterior mitral leaflet , resulting in significant mr . the mouth of the aneurysm was closed with a gore - tex ( expanded ptfe ) patch and the aortic valve was replaced with a 19 mm st . jude aortic valve prosthesis .the left sinus portion of the valve was anchored to the gore - tex patch .the mitral valve was inspected through a left atriotomy to assess feasibility of repair . there was marked distortion of the mitral annulus by the aneurysm and the valve cusps were considerably thickened . feasibility of durable repair was ruled out and a valve replacement with 25 st jude mitral valve was performed . the earliest descriptions of sinus of valsalva were in the reports of hope and thurman.67 later , abbott described this malformation as congenital and not acquired in origin.8 most aneurysms are congenital in origin but they may be seen after bacterial endocarditis , atherosclerosis or chest trauma.29 aneurysms of sinus of valsalva are thought to result from the absence of normal elastic and muscular tissue , which leads to thinning of the wall of the aortic sinus.47 in a study of 332 patients collected from western and oriental literature by chu et al , pathological rupture of a sinus of valsalva most frequently involved the right sinus ( 76.8% ) , followed by the noncoronary ( 20.2% ) , and least commonly , the left sinus of valsalva ( 3%).1 on reviewing the english literature we could find only 26 cases of rsov ruptured into the left ventricle and out of them only 3 arose from a left sinus of valsalva aneurysm.5 in all of them , the aneurysm was repaired along with aortic valve replacement . presentation of sinus of valsalva aneurysm is varied .15 with rupture , the presentation is acute with chest pain and dyspnea . chest pain is due to acute ar from the ruptured sinus of valsalva , frank or dynamic coronary artery compression , or dissection into a coronary artery . unruptured sinus of valsalva aneurysms are usually asymtomatic but may sometimes present as right ventricular outflow tract obstruction when they bulge into right ventricular outflow tract , or subaortic stenosis when they bulge into the left ventricular outflow tract . rarely , complete heart block occurs when it erodes into the interventricular septum.12 associated heart defects that have been reported are ventricular septal defect ( in about 50% of cases ) , pulmonary stenosis , tetralogy of fallot , coarctation of aorta , bicuspid aortic valve , valvular and supravalvular aortic stenosis , left superior vena cava and one case report of anomalous origin of left coronary artery from pulmonary artery.2 initially , angiography was the gold standard for the diagnosis but now , noninvasive modes of diagnosis such as echocardiography ( transthoracic , transesophageal ) , spiral computed tomography and cardiac magnetic resonance imaging have taken over as preferred investigations . in our case we were able to delineate the detailed anatomy by transthoracic echocardiography and the patient underwent surgery requiring replacement of both aortic and mitral valve .

the survey used a random sample of 508 surgery residents from 7 ( dandong , dalian , yingkou , chaoyang , tieling , fushun , and shenyang ) of the 14 cities that constitute the liaoning province , covering the geographic diversity of the east , south , west , and north areas of liaoning province . the cross - sectional study was performed in 15 hospitals from march to october 2014 . seven patients as an assessment group completed the survey for each individual corresponding resident using the self - administered questionnaires . the patients were surveyed in the process of completing the discharge formalities at the admission office of hospital . in our study , if 1 or more patients in the group completed below 80% of the questionnaire , the survey on corresponding resident would be considered as invalid ; 2947 patient - assessment questionnaires on assessing 421 surgery resident competencies were analyzed , and were available for a mean of 7 per resident . the questionnaire included 2 parts , the demographic characteristics and the section assessing the resident competency . the questionnaire from the education outcomes service ( eos ) group of the arizona medical education consortium was developed for use by the patient . this questionnaire consists of 23 items , with a 5-point rating scale ( 1 = never to 5 = always ) . all questions provided the respondents with the option of indicating that they were unable - to - evaluate the resident for that item . if the percentage of unable - to - evaluate items exceeded 10% , the item should be examined for revision or deletion . for each item on the instrument , the percentage of people who scored < 4 over 5% was considered as poor competency . we used exploratory factor analysis to identify the factors for this instrument and to describe the relative variance accounted for by each factor . reliability was assessed using cronbach alpha ( ) coefficient , which enables an assessment of the overall instrument stability . statistical analysis used spss version 13.0 ( spss inc . , the investigator informed all participants about the purpose of study and assured them of anonymity before the research was initiated . the questionnaire was effectively completed by 2947 patients , with a response rate of 82.8% . the mean age was 28.12 years ( standard deviation : 2.56 ) . for 60.1% ( 253 ) , the highest level of education was equivalent to a bachelor 's degree , and the remaining ( 168 ) had a master 's degree or above . the 23-item instrument showed a standardized cronbach of 0.92 , indicating good internal consistency . olkin criteria for sample adequacy , with a result of 0.86 , as well as bartlett test of sphericity ( = 572.16 , df = 227 , p < 0.0001 ) . there were 4 factors on the instrument , including professionalism , patient care , system - based practice , and interpersonal and communication skills , which accounted for 66.98% of the total variance . the scores for majority of items were 4 , as presented in tables 3 - 6 . competency , the number of patients scored the items of promotes health maintenance ( talks about preventive care ) ( 206/7.0% ) , tells me about any side effects of the medicine ( 177/6.0% ) , and clearly explains my treatment choices ( 168/5.7% ) < 4 were relatively high . however , the suggests community resources for additional information and support ( 11.9% ) , demonstrates respect for my disability ( 11.6% ) , and demonstrates respect for my sexual orientation ( 14.3% ) had unable - to - evaluate rates of > 10% . in this study , we evaluated surgery residents for competency from the point of view of the patient . the exploratory factor analysis results showed that 4 factors emerged , including professionalism , patient care , system - based practice , and interpersonal and communication skills , accounting for a total of 66.98% of the variance . the internal consistency reliability analysis ( cronbach ) suggested that the instrument of evaluation was internally consistent . thus , our study confirmed that the instrument of evaluation was a valid and reliable tool to measure competency among chinese surgery residents from the patient perspective . with regard to competency on patient care , the number of patients scored the competency for preventive care ( 206/7.0% ) was higher . preventive healthcare reduces the disease incidence , which is considered as more cost - effective than a curative strategy . however , in china , the utilization of preventive healthcare is challenging for many people , particularly those living in rural areas and those without medical insurance , because preventive healthcare activities are associated with socioeconomic conditions . additionally , 1 policy , the new cooperative medical scheme initiated in 2003 , funds only a few preventive services , which may lead to a lower focus on preventive care . regarding another factor of patient care , many patients ( 177/6.0% ) regarded resident competency in relation to drug side effects as poor . a large number of people are injured or die in hospitals due to adverse drug events each year , and the delivery of unsafe medical care has resulted in a trend toward increasing conflict between physicians and patients in china . thus , patient safety should have particular importance , and clinical pharmacy services in particular can contribute to improving patient safety and reducing hospital costs associated with medication errors . these findings suggest a need for surgery residents to focus on preventive care and raise concerns regarding patient safety . as an important component of system - based practice , our study also found that the unable - to - evaluate percentages of competency related to suggesting community health resources were over 10% . although community health service ( chs ) organizations have become essential parts of the primary healthcare institutions , chs is relatively underdeveloped in urban areas . there are existing bottlenecks in the sustainable development of chs in china , such as the inequitable distribution of healthcare workers . so people 's awareness of chs is not strong . with the current inadequacy of health resources in china , it is necessary to further develop chs to provide additional information and support to patients under government guidance . our results indicate that surgery residents should pay more attention to spending enough time with patients and answering questions thoroughly . based on the findings of salib et al , the majority of participating residents regarded various communication skills , including the duration of communication with patients , to be highly important in patient care . thus , teaching communication skills at all levels of medical education should be promoted , and additional training in communication for healthcare workers is necessary . although all patients could answer the majority of items on the questionnaire , there were specific types of items that showed higher unable - to - evaluate percentages . these items tended to focus on aspects of professionalism , such as respecting the patient 's sexual orientation . besides the heterosexuality , discrimination against other sexual orientations exists in china . due to social stigma , respondents may have declined answering the related question about sexual orientation to protect their privacy . in china , along with advances in health sciences , increasingly complicated working conditions have led to additional ethical dilemmas . particularly in recent years these changes make the evolution of professional values more important and have drawn greater attention to these issues . effective strategies should be proposed to facilitate chinese physician professionalism , and professional development programs and medical ethics education should not only emphasize the general principles involved but also formulate guidelines on how these principles can be achieved in practice . first , our study used a cross - sectional design ; therefore , no causal conclusions can be drawn . in addition , the subjects were recruited from several cities in liaoning province , and they may not be representative of all surgery residents in the entire country . future studies should examine surgery residents in a larger sample size to enrich the data . one possible cause may be that the standardized training and evaluating system for chinese residents have been increasingly improved to enhance the quality of health service in recent years , so the scores on some items were high . the instrument of evaluation was acceptable for patients to evaluate the competency of chinese surgery residents . to improve surgery resident competency , it is necessary to place greater emphasis on enhancing competency on preventive care and raising concerns regarding patient safety . moreover ,

adenoid cystic carcinoma ( acc ) of the breast is a rare condition , and cases in male patients are even less common . we describe a case of acc of the breast with axillary lymph node metastasis , disseminated osteolytic bone metastasis and bone marrow involvement in a 41-year - old man . male breast acc is an extremely rare malignancy ; there can be difficulty in obtaining a final diagnosis . breast cancer in men is very rare , and the majority of male breast cancers show pathological findings similar to breast cancer in females . guidelines for diagnosis and treatment are based on those for female breast cancer due to the extremely low incidence of male breast cancer , i.e. < 1% of the rate in females . male breast cancer usually presents as a firm , painless mass , along with palpable axillary nodes , nipple retraction and ulceration of the skin at presentation . the mass is usually located in the subareolar region , but can also be seen in the upper outer quadrant . adenoid cystic carcinoma ( acc ) of the breast is a rare subtype of breast cancer , usually originating from the salivary gland . acc of the breast has very favorable biological characteristics for treatment and patients usually have an excellent prognosis . axillary lymph node dissection is not helpful for clinical management due to the rarity of metastasis . acc of the breast is a rare subtype of breast cancer , and cases in males are even less common . we report a rare case of advanced acc of the breast in a 41-year - old man , along with a review of the relevant literature . the patient was a 41-year - old male who presented at the department of rehabilitation with symptoms of cervical back pain that had been occurring for 2 months . he was healthy , with an unremarkable medical history and a 20-pack per year smoking history . a cervical spine ct scan showed osteolytic changes with bony fragmentations in the c3 , c5 and c6 transverse processes and vertebral bodies . these findings are suggestive of pathological fracture related to the presence of a tumor , such as metastatic carcinoma or multiple myeloma , rather than trauma ( fig . to differentiate between metastatic bone lesions and the presence of a solid tumor , we examined the bone scan labeled with tc , and the results showed only osteolytic bone lesions , with the exception of a small number of fractures . we did a serum and urine protein electrophoresis evaluation and immunofixation , but there was no evidence of monoclonal gammopathy . in the bone marrow examination for excluding hematologic malignancies such as multiple myeloma and lymphoma , the findings showed that the hematopoietic cells were almost replaced by neoplastic cells with a glandular pattern . neoplastic cells were stained with ck pan ( ae1/ae3 ) and were positive ( fig . metastatic carcinoma was suspected , but there were no abnormal findings in the esophagogastroduodenoscopy or colonoscopy . a pet - ct scan showed a disseminated hypermetabolic lesion ( maxsuv = 7.4 ) in the axial skeleton , multiple , small - sized lung nodules and a multifocal hypermetabolic lung lesion ( maxsuv = 2.6 ) as well as focal hypermetabolic lesions in the left axillary lymph nodes ( maxsuv = 2.3 ) ( fig . an ultrasound - guided core biopsy of the two 1.7-cm , low - echoic , pathological lymph nodes in the left level i axilla area was performed . otorhinolaryngology and head and neck screening were performed to attempt to ascertain the location of the primary tumor in the salivary gland , as most acc tumors in men are of salivary gland origin . however , this screening failed to identify specific lesions . to identify the primary origin of the cancer this revealed palpable and movable small nodular lesions in the periareolar area of the left breast , which had escaped interpretation on the pet - ct scan because the lesion was obscured ( fig . there were clustered small nodules at the 1 o'clock and 2 o'clock locations around the left nipple upon physical examination . in a breast ultrasound , an irregularly - shaped solid lesion , 1.7 cm in size , the margin of the lesion was irregular and the internal area was relatively homogeneous and hypoechoic and was suggestive of a bi - rads category 4c lesion . a core biopsy was performed and the pathology result was acc , the same as that of the auxiliary lymph node biopsy ( fig . 2i , j ) . immunohistochemical staining was performed and the sample was negative for estrogen receptor ( er ) , progesterone receptor ( pr ) and c - erbb2 , and positive for ck5/6 and p53 . we finally diagnosed acc of the breast with multiple lung and bone metastases and bone marrow involvement . the authors submitted a waiver from their institutional review board ( irb ) stating that this case report does not require irb approval or oversight . the overall incidence of acc of the breast is 0.050.1% [ 3 , 4 ] , and is exceedingly rare in men . acc commonly occurs at the following sites : the minor or major salivary glands , oral mucosa , larynx , lung and maxillary sinus . the incidence of all types of pathology of breast cancer in men is < 1% in western countries , but the exact incidence in korea is not known due to the rarity of this disease in men . according to a subsequent review of the relevant literature of acc of the breast , this cancer generally affects older female patients , with a median age range of 5864 years . the median age of male breast acc patients is not clear because few reports exist ; however , of the cases published [ 6 , 7 , 8 ] , 2 patients were diagnosed during adolescence and another at the age of 82 years . in our report , the case of a 41-year - old , relatively young man with a nontender , movable , left - breast lesion is described . acc of the male breast can develop at a relatively younger age than the common male breast cancer pathological type . the published tumor sizes of male breast accs range from 1.8 to 3.7 cm , and in this case , the maximum tumor diameter was 1.7 cm . acc of the breast is not associated with a bloody nipple discharge , even though it is present near the nipple areola region . our case also had no subjective symptoms , with the exception of the small palpable nodularity in the periareolar area . one hundred and fifty cases of female acc of the breast have been reported in the literature , only two of which had axillary lymph node metastasis [ 10 , 11 , 12 ] . however , our case showed pathologically confirmed left axillary lymph node metastasis and bone marrow involvement . this finding suggests the possibility of the long - term duration of the disease prior to the diagnosis , due to the small - sized primary tumor and lack of symptoms or signs except for the presence of aggressive multiple bone and lung metastasis . this case displays an extraordinary pattern of bone metastasis findings compared to other solid tumors : ct and pet - ct scans showed osteolytic bone lesions , but the findings of bone scans labeled with tc were negative . this reflects almost all lesions as having osteolytic activity without compensatory osteoblastic activity , like in multiple myeloma . the majority of breast accs are triple - negative ( i.e. er- , pr- and her2-negative ) , according to previous studies and reports . in this case , male breast acc is rare , and only a few cases have been reported [ 6 , 7 , 8 ] . it is difficult to trace the cancer 's origin to the male breast when initial pathological findings reveal acc in metastatic lymph nodes , as in this case . salivary - gland origin tumors are suspected first because acc with this site of origin is common in men . furthermore , it was difficult to identify the primary site as breast cancer in this case because there was no skin retraction or nipple discharge , unlike common types of breast cancer . in contrast to triple - negative breast carcinomas , acc of the breast has an excellent prognosis for female patients , as the incidence of lymph node metastasis is lower and distant metastasis is uncommon . acc of head or neck origin is also known to have a slow progression and thus a good prognosis for patients . however , male breast acc could be overlooked by the patient , and this , coupled with the difficulty of diagnosis , can lead to extended periods of time , with possible aggressive metastasis , until a diagnosis is made .

a 15-year - old iranian girl was admitted to tehran pediatrics medical center , tehran ( iran ) in june 2006 . on admission also , she had submandibular soft - tissue asymmetry , night sweats , and proptosis ( figure 1 ) and round shape painless patches on the arm and leg extensor surfaces ( figure 2 ) . a 15-year - old woman with proptosis a ) painless patches on extensor surface of the arm . b ) painless patches on extensor surface of the leg she had previous medical history of frequent pharyngitis , hearing loss since age 3 , a left - sided orbital dermoid cyst at age 4 , and herpes - zoster infection at age 11 . physical examination revealed two large bilateral submandibular lymph nodes with diameters of 32 cm and 11.5 cm that were persistent despite antibiotic therapy . laboratory examination revealed mild leukopenia[3,700/cu mm ( normal range = 5000 - 10000/cu mm ) ] , iron deficiency anemia [ hb = 10 g / dl ( normal , 12 - 14 g / dl ) , red blood cell = 3,800/cu mm ( normal , 3.9 - 6.1/cu mm ) ] , platelet count of 421,000/mm ( normal , 150,000 - 400,000/mm ) , erythrocyte sedimentation rate of 98 mm/1 hour ( normal , < 20 mm/1 hour ) , and a 4.3 g / dl gamma globulin rate ( normal , 0.7 - 1.2 g / dl ) . tuberculin test and serological tests for viral infections including hbs ag , hbs ab , hbc ab , hcv ab , hav ab ( igm ) , hev ab ( igm ) , and vca ( igm ) , and also the test for toxoplasmosis were all negative . we performed biopsy of the neck lymph nodes for the second time as the result of the first one performed two years before admission has been reported as reactive adenitis . histological examination demonstrated numerous histiocytes invading the lymph sinuses . on immunohistochemical examination , these were positive for s-100 protein and negative for cd1a , a marker of langerhans histiocytosis ( figure 3 ) . her skin biopsy showed a dense inflammatory infiltrate into dermis , mainly histiocytes , containing vesicular nuclei , lymphocytes , neutrophils , and plasma cells ; i.e. , panniculitis . a ) lymph node architecture is rather preserved but sheets of large histiocytes with pale cytoplasm fill the sinusoids . b ) closer view : large histiocytes with abundant pale cytoplasm are seen , some ingesting blood elements ( emperipolesis ) . with the classical features of massive lymphadenopathy and evidence of sinus histiocytosis and the patient was treated with prednisone ( 40 mg / d ) for a period of one month , and thereafter the dose decreased and azathioprine 1 mg / kg was added . after one year , prednisone discontinued and all of the symptoms and signs progressively resolved , although no change occurred in her proptosis . rdd predominantly affects children and young adults with relatively male sex predominance.2 although some other reports are available from iran including rdd cases with intracranial1415 and kidney involvement16 , the disease is less common in asian populations.2 its etiology still remains obscure , but two hypotheses have been proposed : a disturbance in inguinal , and mediastinal lymph nodes are also commonly affected . antecedent non - specific fevers and pharyngitis may herald the onset of rdd , occasionally accompanied by pain , tenderness , malaise , night sweats , or weight loss . extranodal disease is documented in 43% of cell mediated immunity17 , and a focus of infection due to viruses such as human herpes viruses ( hhv-6 and hhv-8).18 painless lymphadenopathy is the most frequent presenting symptom and involves the cervical region in up to 90% of the cases . axillary , para - aortic , the cases , in some of them , without associated lymphadenopathy which may or may not develop later in the disease course.2 hepatosplenomegaly which was present in our case is relatively an uncommon finding in rdd as very few cases with splenomegaly and or hepatomegaly have been reported.1920 widespread concurrent nodal and extranodal involvements have also been reported by other authors . reported a case of rdd presented with inguinal lymphadenopathy in which lung , renal and bone were also extensively involved.21 in such patients , numerous extranodal involvements pose a diagnostic challenge , as was the problem in our case . involvement of the neck area , skin , soft tissue , liver , spleen , orbit , and bone elicited a wide differential diagnosis . rdd in lymph nodes may clinically mimic hodgkin and non - hodgkin lymphoma , infectious processes , granulomatous lymphadenitis , reactive lymphadenopathy or other histiocytoses including langerhans cell histiocytosis ( lch ) and metastatic carcinoma . histiocytic disorders , particularly langerhans cell histiocytosis must be distinguished from rdd affecting skin , soft tissue , and bone . another external manifestation is orbital soft - tissue mass with proptosis , which can present without any sign suggestive of lymphadenopathy.22 skin lesions of rdd may be solitary or multiple , macular or papulonodular , xanthomatous or erythematous , dermal or subcutaneous , and found in virtually any location including the face , ears , trunk , extremities , or genitalia.5 histological findings of skin lesion biopsy specimens are similar to those found in lymph nodes . however , the lesions of rdd can be localized in the soft tissue mimicking a subcutaneous mass or panniculitis cutaneous . also , it has been clinically mistaken for other dermatologic disorders including vasculitis , acne vulgaris , lupus vulgaris , sarcoidosis , hidradenitis suppurativa , granuloma annulare , malignant breast neoplasm , and other histiocytosis.23 the histological differential diagnosis includes lch , storage disorders such as gaucher disease , classical hodgkin lymphoma , metastatic melanoma and carcinoma , histiocytic sarcoma , and infections caused by histoplasma and mycobacterial organisms involving the lymph node . in the nasal cavity , perhaps the most frequently faced histological challenge is that of distinguishing shml from reactive sinus histiocytic proliferations which occur as nonspecific responses to a variety of instigating agents . although erythrophagocytosis is seen in reactive and neoplastic histiocytic proliferations including lch , emperipolesis in a setting outside of shml is extremely rare . the appearance of lymph node sinuses expanded by a histiocytic proliferation is a feature common to lch and shml2 , but in our case lch was unlikely because langerhans histiocytes with their familiar folded nuclei and eosinophil infiltrates were absent , as was immunoreactivity for cd1a , a reliable marker of lch.23 since rdd recognition , the constellation of clinical findings has formed an ever - expanding almanac as more and more cases of rdd are diagnosed so it should be included in the differential diagnoses for patients , whoever admitted to our medical center with any of the mentioned extranodal involvements . given the wide range of clinical presentations and the broad pathologic differential diagnosis , the clinical hallmark of massive lymphadenopathy is often crucial for diagnosis of rdd , however , several pitfalls may complicate pathologic diagnosis . mns and hossein saneian participated in diagnosis and treatment of the patient and fm participated in pathological study of the patient . all authors participated in writing the draft and editing the final version of the report .

in 1987 , francine shapiro , a former english teacher , reportedly walked in a park , and noticed that unpleasant thoughts seemed to fade when she moved her eyes quickly back and forth ( luber , 2009 ; rosen , 1995 ) . two years later , she launched a new treatment for post - traumatic stress disorder ( ptsd ) , which is now known as eye movement desensitisation and reprocessing ( emdr ; shapiro , 1989a , 1989b ) . a key part of the procedure is that the patient retrieves traumatic memories while making horizontal eye movements by following the fingers of the therapist moving back and forth in front of the patient 's eyes . shapiro wrote about rapid eye movement ( rem ) sleep ( shapiro , 1989a ) , and of catalysing and rebalancing the nervous system , and shifting information that is dysfunctionally locked in the nervous system ( shapiro , 1995 ) . , she believed that her new treatment was very effective , and provided support for it with her own study of 22 participants who had experienced physical or sexual abuse or vietnam war combat incidents and had related symptoms ( e.g. , intrusive thoughts ; shapiro , 1989a ) . where previous therapies lasting an average of 6 years had failed , her single session ( of about 50 min ) apparently succeeded in desensitizing the anxiety related to traumatic memories . however , there were clear methodological problems : shapiro did not use standardised clinical measures , and did everything herself : the treatment and interviews . she received a doctoral degree for her research ( see mcnally , 1999 ) , from an unaccredited school . the academic community was skeptical about the introduction of emdr ( e.g. , herbert et al . , 2000 ; mcnally , 1999 ; muris & merckelbach , 1999 ) . the validity of shapiro 's wonder therapy and her cryptic explanation were critically questioned . shapiro went on to train therapists ; from 1990 in the usa , from 1991 in other countries ( maxfield , 2009 ) . nevertheless , the method quickly gained in popularity : many therapists were enthusiastic and convinced that the treatment worked . in 1995 , the first large randomised clinical trial ( rct ) was published : people with ptsd received emdr or were put on a waiting list ( wilson , becker , & tinker , 1995 ) . still , such a comparison does not take into account non - specific factors ( placebo ) . a next important step was to compare the effects of emdr with the most effective treatment for ptsd : cognitive behavioural therapy ( cbt ) . in 1998 , the first meta - analysis appeared . it showed that emdr and cbt were , in general , equally effective ( van etten & taylor , 1998 ) . this was good news , because there were now more than 25,000 emdr - trained therapists ( greenwald , 1999 ) . why can we not just rely on the experiences of therapists : see if the treatment works in one patient , see if it works in other patients who are treated the same way , and draw a conclusion : the treatment works ( or it does not ) . i will try to clarify this with an example.friendly dolphinnow and then we hear a story about a shipwrecked sailor or a surfer who is saved by a dolphin . according to the social psychologists tavris and aronson ( 2007 ) , pp . 108109 ) , it is tempting to conclude that dolphins must really like human beings , enough to save us from drowning . but wait are dolphins aware that humans do n't swim as well as they do ? are they actually intending to be helpful ? to answer this question , we would need to know how many shipwrecked sailors have been gently nudged further out to sea by dolphins , there to drown and never be heard from again . we do not know about those cases , because they do not live to tell us about their evil - dolphin experiences . if we had that information , we might conclude that dolphins are neither benevolent nor evil ; they are just being playful . now and then we hear a story about a shipwrecked sailor or a surfer who is saved by a dolphin . 108109 ) , it is tempting to conclude that dolphins must really like human beings , enough to save us from drowning . but wait are dolphins aware that humans do n't swim as well as they do ? are they actually intending to be helpful ? to answer this question , we would need to know how many shipwrecked sailors have been gently nudged further out to sea by dolphins , there to drown and never be heard from again . we do not know about those cases , because they do not live to tell us about their evil - dolphin experiences . if we had that information , we might conclude that dolphins are neither benevolent nor evil ; they are just being playful . this fallacy is known as confirmation bias : information that fits with our own hypothesis is weighted more heavily than conflicting information , and evidence to the contrary is not sought . to determine whether a treatment works , a clinician must not rely only on his or her own experience and intuition , because you never know how a different treatment would have fared in the same patient : a clinician only makes uncontrolled observations . consider a cold : treated , it will go away after one week ; untreated , after seven days . the great philosopher of science , karl popper , reported a nice anecdote about this.popper and adleradler believed that all sorts of psychological distress are rooted in the inferiority complex . in the interbellum , it took adler little time to analyse the case in terms of the inferiority complex . popper then writes : i could not help saying : and with this new case , i suppose , your experience has become thousand - and - one - fold. each previous observation was coloured by the observation that preceded it , but was advanced as new evidence . and so it started to dawn on popper that this apparent strength ( that the theory could explain everything ) was in fact a weakness ( popper , 1963/2002 , pp . if every result confirms your hypothesis , then your ideas are a matter of belief rather than science . a theory must be falsifiable ( that is , refutable ) , and in testing a hypothesis we must look for evidence to the contrary . adler believed that all sorts of psychological distress are rooted in the inferiority complex . in the interbellum , it took adler little time to analyse the case in terms of the inferiority complex . popper then writes : i could not help saying : and with this new case , i suppose , your experience has become thousand - and - one - fold. each previous observation was coloured by the observation that preceded it , but was advanced as new evidence . and so it started to dawn on popper that this apparent strength ( that the theory could explain everything ) was in fact a weakness ( popper , 1963/2002 , pp . if every result confirms your hypothesis , then your ideas are a matter of belief rather than science . a theory must be falsifiable ( that is , refutable ) , and in testing a hypothesis we must look for evidence to the contrary . his criticism was aimed at marxism , naive empiricism , freud , plato 's ideas on polity and much more . but crucial was his notion that confirmation through ( clinical ) observation does not count in favour of the theory . at least , it only counts if the observations are set up such that they can serve as falsification . so what counts , are critical tests . and those who make extraordinary claims , for example , about a miracle cure , also need to have extraordinary evidence . returning to emdr : the treatment now consists of several sessions , and its efficacy has been demonstrated in meta - analyses ( e.g. , bisson et al . , 2007 ; bradley , greene , russ , dutra , & westen , 2005 ; see also wampold et al . , 2010 ) . the research findings have been so convincing that emdr is now registered alongside cbt as the preferred treatment for ptsd in , for instance , the netherlands ( trimbos , 2011 ) and united kingdom ( nice , 2005 ) . 1 these guidelines are evidence based , meaning that the best evidence is used to guide decisions related to care . of course , this should not detract from the fact that more than one third of patients have little benefit from these treatments ( bradley et al . , 2005 ) , and that further improvement is still needed . interim conclusions : emdr came directly from practice and has been studied in a number of rcts ( which is consistent with the first aim of academisation ) . for that , knowledge transfer is needed ( e.g. , training ) and guidelines need to be promoted within the organisation ( this is consistent with the second aim ) . it was initially thought that emdr is cbt plus eye movements , and you could just as well leave out the latter , but this does not seem to be the case : eye movements appear to be doing something ( lee & cuijpers , 2012 ) . if we can figure out what they do , we would know why and when to use emdr ( e.g. , only for ptsd or also for other disorders ? ) . shapiro thought that other alternating stimulation would work just as well , such as sounds ( beeps ) coming alternately from left and right headphones . many therapists started using these beeps , which seemed convenient , but they had not yet been researched . to determine how eye movements actually work in emdr the renowned dutch psychologist linschoten ( 1964 ) wrote that the starting point of science is that the occurrence of a phenomenon is determined systematically and can thus be explained by way of reduction . it is also very useful to work with a model in the lab : you can change one variable , hold others constant , and assess the effect . there are lab models that are well suited to examining clinical phenomena . in research on eye movements , after that , they recall the image for a longer period in two conditions : without or with eye movements ( by looking at a dot on a computer screen that moves from left to right and back again ) . after a break of several minutes or days , the memory is recalled and assessed again . in recent years many experiments have been done using this model ( see van den hout & engelhard , 2011 ) . they do : in more than 15 experiments , memory recall with eye movements worked best . this was also found with an objective memory task ( van den hout , bartelski , & engelhard , in press ) and with physiological measurements2 ( engelhard , van uijen & van den hout , 2010 ) . then it was examined whether eye movements work by promoting communication between the left and right brain hemispheres ( interhemispheric communication ) . this theory is popular among emdr therapists and prompted many of them to switch to using the beeps mentioned earlier . canadian researchers gunter and bodner ( 2008 ) tested this hypothesis by adding one condition : recalling a memory with vertical eye movements . just recalling the memory had no effect . in the case of horizontal eye movements vividness and emotionality decreased . but this also happened with vertical eye movements ; they were equally effective . thus , the popular explanation of interhemispheric communication turned out to be a misconception . here , research serves to correct practice . the idea arose that it might have something to do with working memory . this is used to perform cognitive tasks , and it has limited capacity ; when we do two tasks at the same time that both require attention , these tasks compete for this limited capacity . accordingly , if you think of a memory and move your eyes back and forth at the same time , there is less capacity available for the memory . as a result , the memory should become more vague ( see andrade , kavanagh , & baddeley , 1997 ) . it is important to note that whenever we retrieve a memory , this memory is temporarily susceptible to change and is then reconsolidated in long - term memory . imagination inflation.imagination inflationthe memory researcher elizabeth loftus ( loftus & palmer , 1974 ) showed participants a film of a car accident , and then asked some of them : how fast were the cars going when they hit each other ? others were asked the same question , except that hit each other was replaced by smashed into each other . this word influenced the estimated speed : with hit , participants guessed approximately 55 km per hour ; with smashed they thought 66 km . a week later , the participants were asked if there was any broken glass at the scene . those who had been asked the question with smashed most often thought ( incorrectly ) that broken glass had been shown in the film . later it was shown that you can also get people to recall unpleasant things that never happened ( false memories ) . this gives rise to a vivid , detailed ( pseudo-)memory , which soon becomes confused with a real memory ( see crombag & merckelbach , 1996 ; mcnally , 2003 ) . in the 1990s , a great deal of attention was paid to recovered memories of people who were searching for repressed traumas . these often proved to be pseudo - memories , which were recalled out of nothing during recovered memory there were far fewer cases involving disputed recovered memories , perhaps because therapists who used such techniques were being more cautious ( schacter , 2001 , p. 130 ) . the memory researcher elizabeth loftus ( loftus & palmer , 1974 ) showed participants a film of a car accident , and then asked some of them : how fast were the cars going when they hit each other ? others were asked the same question , except that hit each other was replaced by smashed into each other . this word influenced the estimated speed : with hit , participants guessed approximately 55 km per hour ; with smashed they thought 66 km . a week later , the participants were asked if there was any broken glass at the scene . those who had been asked the question with smashed most often thought ( incorrectly ) that broken glass had been shown in the film . in other words , later it was shown that you can also get people to recall unpleasant things that never happened ( false memories ) . this gives rise to a vivid , detailed ( pseudo-)memory , which soon becomes confused with a real memory ( see crombag & merckelbach , 1996 ; mcnally , 2003 ) . these often proved to be pseudo - memories , which were recalled out of nothing during there were far fewer cases involving disputed recovered memories , perhaps because therapists who used such techniques were being more cautious ( schacter , 2001 , p. 130 ) . returning again to emdr . according to the theory , the combination of retrieving memories and eye movements should lead to the opposite that is , to imagination deflation . from this theory , first , the theory predicts that memories fade not only through horizontal eye movements , but also through other ( demanding ) dual tasks . this is correct , and applies , among other things , to vertical eye movements ( as we have already seen ) , drawing a complex figure ( gunter & bodner , 2008 ) , mental arithmetic ( engelhard , van den hout , & smeets , 2011 ; van den hout et al . , 2010 ) , playing the computer game tetris ( engelhard , van uijen , et al . , 2010 ) , and ( van den hout , engelhard , beetsma , et al . , 2011 ) . the latter is an important component of mindfulness therapy , which like emdr consists of a package of interventions . in attentional breathing , if people get unpleasant thoughts or images , they should allow them , but focus their attention on their breathing . this seems to place demands on our working memory , just as eye movements do ( van den hout , engelhard , beetsma , et al . , 2011 ) . so there seems nothing special about eye movements , as other demanding tasks work just equally well . second , many people are not ( only ) plagued by bad memories ( flashbacks ) , but also by future - oriented images ( flashforwards ) . for example , a person with panic disorder may imagine having a heart attack in a busy store , and a person with hypochondrias may imagine receiving a life threatening diagnosis . the theory predicts that flashforwards , too , should fade when working memory is taxed . this was indeed found to be the case ( engelhard , van den hout , janssen , & van der beek , 2010 ; see also engelhard , ven den hout , dek , et al . , 2011 ; 2012 ) . this potentially important finding could lead to an extension of the indication range of working memory taxing therapy : from trauma in the past to pathological the theory also predicts a dose - response relationship : the dual task should not be too easy ( this leaves too much capacity available for the memory ) , but also not too hard ( this makes it too difficult to recall the memory ) . shortly after the queen 's day tragedy on april 30 , 2009 in the netherlands , participants recollected distressing images they had seen in the media . four groups performed tasks that were increasingly demanding : group 1 only recalled the memory , groups 2 and 3 recalled the memory while doing simple or slightly harder arithmetic , and group 4 recalled the memory while doing difficult arithmetic . as predicted , the largest effect ( i.e. , the biggest decrease in the emotional intensity of the memories ) was observed in the middle groups ( engelhard , ven den hout , & smeets , 2011 ) . , people with lower working memory capacity should benefit more from dual tasks than those with higher working memory capacity . this was indeed found ( e.g. , gunter & bodner , 2008 ; van den hout et al . , 2010 ) . based on current research , individual tailoring seems to be important : gearing the degree of working memory load to the individual patient . if the mental image remains too salient , the load can be increased ( e.g. , faster eye movements , more difficult arithmetic ) , and if the image can not be retrieved vividly ( e.g. , in the case of people with lower working memory capacity or children ) the load can be reduced . therefore , you would expect that it barely taxes working memory and is thus less effective than , for instance , eye movements . this is exactly what we found in recent research in the lab ( van den hout , engelhard , rijkeboer , et al . , 2011 ) and in research among patients with ptsd ( van den hout et al . , 2012 ) . thus , there is clear evidence for a new theory on the effect of eye movements , and this may have relevant clinical implications . these suggestions still need to be examined in clinical practice ( so it is important that universities have access to patients for research purposes ) , but it is better to improvise on the grounds of a strong theory , supported by experimental data , than on the basis of untested assumptions . these research findings have been translated by dutch emdr trainers into guidelines for practice and training ( beer et al . , 2011 ) . to summarise : due to scientific research , over the course of 20 years emdr has transformed from an unconventional intervention on the basis of an unusual theory into a proven treatment for ptsd , which we are only just now beginning to understand . it is rare that a treatment works its way from the periphery to the centre of regular healthcare . due to francine shapiro faced a lot of criticism , but to her credit , she always has been willing to subject her treatment to scientific examination ( mcnally , 2001 ) . the example of emdr illustrates four important things about academisation.practice often forges ahead of science , and new treatments can spread rapidly . this is often how things work in mental healthcare : new ideas are accepted faster than new insights from research.it rarely happens that a single study changes healthcare . typically , many studies are needed to develop knowledge that can actually be implemented.although it is important to know whether a technique or a therapy works , it is also important to know why it works . a better understanding of the working mechanism not only improves the treatment ( consider the beeps and arithmetic ) , but also creates other areas of application such as the extension from flashbacks to flashforwards . and if patients understand how it works , the therapy is more credible ( which in itself has a therapeutic effect ; kazdin , 2005 ) , and they can apply the insights themselves.sometimes clinically relevant research is done outside mental healthcare ( e.g. , fundamental or translational research in the lab or epidemiological research ) . if it has passed this phase , it needs to return to mental healthcare so that important results can be put into practice quickly . sometimes a lot is known through research , but this knowledge does not find its way directly into therapeutic practice . the path of implementation is bendy : it is unusual that researchers find something and then transfer that knowledge to therapists . for instance , it took years for knowledge about false memories to find its way into practice . this is often how things work in mental healthcare : new ideas are accepted faster than new insights from research . typically , many studies are needed to develop knowledge that can actually be implemented . although it is important to know whether a technique or a therapy works , it is also important to know why it works . a better understanding of the working mechanism not only improves the treatment ( consider the beeps and arithmetic ) , but also creates other areas of application such as the extension from flashbacks to flashforwards . and if patients understand how it works , the therapy is more credible ( which in itself has a therapeutic effect ; kazdin , 2005 ) , and they can apply the insights themselves . sometimes clinically relevant research is done outside mental healthcare ( e.g. , fundamental or translational research in the lab or epidemiological research ) . if it has passed this phase , it needs to return to mental healthcare so that important results can be put into practice quickly . sometimes a lot is known through research , but this knowledge does not find its way directly into therapeutic practice . the path of implementation is bendy : it is unusual that researchers find something and then transfer that knowledge to therapists . for instance , it took years for knowledge about false memories to find its way into practice . after this illustration i would like to focus more on reasons for promoting the academisation of mental healthcare . the importance of control conditions and the need to be wary of incidental observations belong to the basic do 's and don'ts of methodology . in daily therapeutic practice , this importance quickly fades from view . consider once more confirmation bias ( we often see what we want to see ) . methodology is a set of rules that we can follow to counter confirmation bias ( see also lilienfeld , 2010 ) . and academisation means regularly asking yourself : what works , how does it work , and what method will give us the greatest benefits ? sometimes you have to put aside your intuition and personal experiences and say : i have the feeling that this is true , but i know through research that it is n't . after all , we all see that the world is as flat as a pancake . second , various care guidelines are being developed in the netherlands and in other countries . in line with research , we expect the largest effects from these treatments . but clinicians often offer these treatments suboptimally or not at all ( shafran et al . , 2009 ) . it has frequently been said that the guidelines need to be used more often , but we do not know how often this should be . bad reasons might be that a clinician wrongly believes that the effects of treatment studies will not apply to his or her patients ( effectiveness ) . there are all sorts of misunderstandings about guidelines : that they are protocols , that everything in them is contrived , that they are all evidence based . this is not the case : guidelines are a snapshot , a great deal is based on consensus and effectiveness is not known for all recommendations . moreover , many patients do not benefit much from application of the guidelines . we need to do more research on this . but this does not detract from the fact that controlled data should be taken seriously . patients are disadvantaged if they are treated with techniques or procedures that have not been proven effective or have been proven not effective , while there are better alternatives . an important third reason is that university research agendas are determined more by an assessment of new opportunities and continuity of traditions than by a general responsibility towards healthcare . as a result fourth , academisation corrects the undesired tradition in which researchers have little incentive to share their findings with practitioners . however , it should also be reiterated that much of the research in mental healthcare has no practical implications and many studies are needed to effect changes in care . finally , the disease burden of psychological problems is enormous ( world health organization , 2003 ) , and this is a further argument why academisation deserves to be prioritised . efficiency studies can help to make mental healthcare less expensive , although , of course , there is a limit to this . academisation is a common topic at conferences , as can be seen in recent themes such as translating science into practice ( wcbct in boston , june 2010 ) and translating research into care ( epa in vienna , march 2011 ) . it is also a common theme in professional associations aiming to strengthen the role of science in training and practice via scientific committees , for example , the board of scientific affairs of the european federation of psychologists associations ( efpa ) , and it is commonly discussed in journals . a great deal has been done , but there is still more to do . according to the law of the handicap of a head start , a head start often means there is little stimulus for further improvement . from this is derived the law of the stimulative arrears ; precisely because you can be fresh , you can gain a lot of momentum . the challenge is to keep the momentum going , in spite of the government 's austerity measures . it is important that managers facilitate , safeguard priorities and promote an academic culture ; that clinicians are open to a scientific approach ; and that researchers have an eye for the questions and problems that emerge in practice . bridge - builders are neededamphibious psychologists , psychiatrists and nurses who are familiar both with practice and with research . the foundation , of course , is training , in which students and trainees learn not only clinical skills but also , for example , how to read research papers well . the number of publications out there is not equal to the amount of valuable knowledge ( see lilienfeld , lynn , ruscio , & beyerstein , 2010 ) . however , many students choose clinical psychology , not so much out of an interest in science , but because they want to do something for suffering people . i want to fly a helicopter , not look at a bunch of crazy dials . for years he has dreamt of flying a helicopter , and he starts taking flying lessons . but during these lessons he has to pay attention to the buttons and the lights on the dashboard . he wants to use his intuition and judgement ; if he sees a tree he will fly over it ; an electricity pole he will go around . his passion is for flying . just like pilots , therapists , too , need knowledge about the methods and techniques in order to be able to treat their patients . students need to be taught this in their training , so they can be better prepared to help those in need . the author 's research is funded by the netherlands organisation of scientific research with a vidi innovational research grant .

malaria is among the leading causes of childhood mortality in guinea - bissau , comprising 18% of mortality of children less than five years of age as of 2010 ( who , 2010 ) . however , the human malaria incidence rate in guinea bissau varies considerably from year to year with a general decrease in recent years to about 3 children ( < 5 yrs of age ) per thousand in some locations ( ursing et al . , plasmodium falciparum predominates , causing 98% cases , followed by a few cases of plasmodium malaria and plasmodium ovale . mixed infections of p. malariae , and to a lesser extent p. ovale , have been recorded but appear to be rare and highly variable in both guinea - bissau ( snounou et al . , 1993 ) and neighboring senegal ( fontenille et al . limited research has been conducted on the vectors and malaria parasite infection rates in guinea - bissau populations of anopheles species in general and there is no data on comparative infection rates between a. gambiae and a. coluzzii and members of the a. gambiae complex . implicated as vectors in this region of west africa in terms of both their temporal population dynamics as well as species composition among study sites ( carnevale et al . , 2010 ; fontenille et al . , 1997a ; jaenson et al . , 1994 ; snounou et al . , 1993 ) here we present much needed data on p. falciparum infection of anopheles spp . specimens collected from inside and around associated human habitations at eight sites in guinea - bissau ( table 1 ) . numbers ( # ) indicate site locations on the map of guinea - bissau in figure 1 . all mosquitoes were collected indoors with a single exception ; samples in ponta anabaca were opportunistically collected outside . mosquitoes were collected by mouth aspiration from both the island and inland areas of guinea - bissau ( figure 1 ) in 2009 between october and november , which corresponds with the time of year previously observed to have the highest infection rate in anopheles species ( jaenson et al . , 1994 ) . the mosquito was dissected and the head and thorax were preserved in 100% ethanol for subsequent elisa . species determination of mosquitoes from the a. gambiae complex were made with the combination of species diagnostic assays ( fanello et al . , 2002 ; favia et al . , 2001 ; santolamazza et al . , 2008 ; scott et al . , 1993 ) and a divergence island snp ( dis ) genotyping assay ( lee et al . , 2014 ) while other species were identified by morphology . 1 : canjufa ( 12.43n , 14.13w ) , 2 : bambadinca ( 12.02n , 14.86w ) , 3 : antula ( 11.91n , 15.58w ) , 4 : prabis ( 11.80n , 15.74w ) , 5 : abu ( 11.46n , 15.91w ) , 6 : brus ( 11.23n , 15.88w ) , 7 : ponta anabaca ( 11.18n , 16.14w ) and 8 : eticoga ( 11.16n , 16.14w ) . the fanello rflp ( fanello et al . , 2002 ) , we used four primers ( un [ 5'-gtg tgg ccc ttc ctc gat gt-3 ' ] , ga [ 5'-ctg gtt tgg tcg gca cgt tt-3 ' ] , me [ 5'-tga cca acc cac tcc ctt ga-3 ' ] and ar [ 5'-aag tgt cct tct cca tcc ta-3 ' ] ) . we excluded qd primer ( scott et al . , 1993 ) because our study site is well outside of the geographic range of this species ( east africa ) . a 25 l pcr reaction containing 1x geneamp pcr buffer ( applied biosystems ) , 1 mm mgcl 2 , 0.2 mm of each dntp , 0.12 m of each primer and 0.05u amplitaq dna polymerase ( applied biosystems ) was carried out for each individual . scott pcr products were digested using hha1 enzyme ( new england biosystems ) following the protocol stated in ( fanello et al . , 2002 ) . thermocycler conditions were 95c for 5 min followed by thirty - five cycles of 95c for 45 s , 50c for 30 s and 72c for 45 s , with a final elongation at 72c for 7 min , and a 4c hold . for the favia pcr ( favia et al . , 2001 ) , we used four primers ( r5 [ 5'-gcc aat ccg agc tga tag cgc-3 ' ] , r3 [ 5'-cga att cta ggg agc tcc ag-3 ' ] , mopint [ 5'-gcc cct tcc tcg atg gca t-3 ' ] and b / s int [ 5'-acc aag atg gtt cgt tgc-3 ' ] . a 25 l pcr reaction containing 1x pcr buffer ( applied biosystems ) , 1.5 mm mgcl 2 , 0.2 mm of each dntp , 0.2 m of primer r5 , 0.2 m of primer r3 , 0.16 m of primer mopint , 0.1 m of primer b / s int and 0.02u dna polymerase amplitaq ( applied biosystems ) was carried out for each individual . thermocycler conditions were 95c for 5 min followed by thirty - five cycles of 95c for 30 s , 64c for 30 s and 72c for 30 s , with a final elongation at 72c for 7 min , and a 4c hold . for the sinex pcr ( santolamazza et al . , 2008 ) , we used s200 x6.1 forward [ 5'-tcg cct tag acc ttg cgt ta-3 ' ] and reverse [ 5'-cgc ttc aag a 25 l pcr reaction containing 1x pcr buffer ( applied biosystems ) , 2 mm mgcl 2 , 0.4 mm of each dntp , 0.2 m of each primer and 0.1u dna polymerase amplitaq ( applied biosystems ) was carried out for each individual . thermocycler conditions were 95c for 5 min followed by thirty - five cycles of 95c for 30 s , 60c for 30 s and 72c for 30 s , with a final elongation at 72c for 10 min , and a 4c hold . the resulting pcr products were analyzed on a qiaxcel capillary electrophoresis instrument ( qiagen ) using a dna screening cartridge ( qiagen ) . for dis genotyping , we used sequenom iplex gold genotyping reagent set ( catalog number : sequenom 10158 ) and ran on massarray ( sequenom ) mass spectrometer at uc davis veterinary genetics laboratory . a mosquito was considered a hybrid if at least 5 out of 7 dis on the x chromosome were in a heterozygous state . p. falciparum infection was determined by enzyme linked immunosorbent assay ( elisa ) of circumsporozoite protein ( csp ) ( burkot et al . , 1984 ; wirtz et al . , 1987 ) from the head and thorax of mosquito specimens in an attempt to capture the parts of the mosquito that would indicate they were infective mosquitoes . all chemicals except for substrate solutions ( item 5 on page 5 of the supplemental elsa protocol document ) were ordered from sigma - aldrich . monoclonal antibodies ( capture and conjugate ) were obtained from kirkegaard & perry laboratories . p. falciparum sporozoite protein for positive control was ordered from the centers for disease control and prevention ( cdc ) . we followed the sporozoite elisa directions provided by the cdc ( sep , 2009 version ) with a few modifications ( see supplemental document for the modified elisa protocol ) . samples were considered positive if absorbance values were three or more standard deviations from the negative control samples ( 99% ci ) on each elisa plate ( beier et al . the results of the elisa were analyzed for both csp concentration , adjusted for plate - to - plate variation , with an analysis of variance and for a binary outcome using a test implemented in spss 16.0 ( spss , 2007 ) . the data were analyzed for differences between species and among collection sites , using g - test implemented in deducer library under r software ( http://www.r-project.org/ ) . species and p. falciparum infection state and csp concentration for each individual is provided in dataset 1 . elisa results identifying plasmodium falciparum infection status in anopheles spp . collected in guinea - bissaumosquitoes were collected at eight different sites in guinea - bissau between october and november 2009 . mosquitoes were collected at eight different sites in guinea - bissau between october and november 2009 . four species were collected during sampling ; a. coluzzii , a. gambiae , a. melas , a. pharoensis and a. coluzzi x a. gambiae hybrids were observed . all mosquitoes were collected indoors with a single exception ; samples in ponta anabaca were opportunistically collected outside of a human habitation while apparently host - seeking immediately after sunset at about 18:00 hr , which is earlier than reported observations for members of the a. gambiae complex in the gambia ( west africa ) ( lindsay et al . , 1989 ; snow et al . , 1988 ) . all species were collected at multiple sites except a. pharoensis , which was only collected at the more inland site of bambadinca . a. pharoensis is not generally considered a significant vector in west africa but the distribution observed in this study matches the previously observed pattern in senegal ( carrara et al . , 1990 ) . no significant differences were observed for csp concentration or in the analysis of positive samples with . this is probably due to the variation in the distribution of vector species and p. falciparum in the environment at the time of sampling . table 1 presents csp rate data and the total number of each individual species collected at each site . the percentage of p. falciparum positive samples from members of the a. gambiae species complex observed in this study ( overall 10.2% ) were similar to earlier studies in other regions in guinea - bissau ( 12.0% ( snounou et al . , 1993 ) and 9.612.4% ( jaenson et al . , 1994 ) ) . the overall csp positive rate for a. gambiae was 12.6% and 11.1% for a. melas . (= a. gambiae and a. coluzzii ) range between 2.24% in guinea ( carnevale et al . , 2010 ) to 9.6% in guinea - bissau ( jaenson et al . , 1994 ) . earlier studies when individual species within the a. gambiae complex were not identified , infection rate of a. gambiae s.l . ranged from as high of 17.73% in the eastern regions of the gambia ( thomson et al . , 1994 ) the csp positive rate was significantly higher in a. gambiae ( 12.6% ) than a. coluzzii ( 4.3% ) ( wilcoxon rank sum test p - value=0.0384 ) . this is consistent with the earlier study in senegal ( ndiath et al . , 2011 ) but differs from a recent survey conducted in mali ( fryxell et al . , the study site in senegal located in the village of dielmo ( 1343'n , 1624'w ) ( ndiath et al . , 2012 ) ) was geographically closer ( 200 km ) than mali sites ( > 800 km ) to our collection sites in guinea - bissau . the senegal study site at dielmo and nine of our study sites were proximal ( < 50 km ) to the atlantic ocean , while mali is a land - locked country at least 500 km away from the atlantic ocean . therefore , the discrepancy among studies may be due to climatic and environmental pressure on the different genetic backgrounds of a. gambiae observed in this area of west africa ( lee et al . , 2013 ) . more robust sampling over a larger number of collection sites would help in confirming this trend . in this study , a few a. pharoensis ( n=6 ) were collected , half of which were csp positive . other studies in this region of west africa have found that a. funestus and a. arabiensis may also be important vector species at different times in nearby senegal ( fontenille et al . a. arabiensis was not collected in our study while a small number ( n<10 ) of a. funestus were observed but not collected . recent studies on the prevalence of malaria parasites in humans have suggested that infection rates in guinea - bissau may be in decline due to widespread use of effective treatment and insecticide treated bed nets ( itns and long lasting insecticide treated bed nets , llins ) by the most high - risk groups ( rodrigues et al . , 2008 ; ursing et al . , 2014 ) . the malaria parasite life cycle is complicated and may not directly relate to the prevalence of human cases but it is possible that the lack of data during periods of political unrest has concealed a more stochastic pattern than was previously observed in guinea - bissau ( ursing et al . , 2014 ) . outdoor mosquito collection was not the focus of this survey and was only made at ponta anabaca hotel grounds when we fortuitously noted mosquitoes biting . consequently no general comments about the degree of exophily of a. gambiae in guinea - bissau can be made . however , evidence of exophily by the major malaria vector a. gambiae in this study and by others in west africa ( reddy et al . , 2011 ; tchouassi et al . , 2012 ) raises the concern of the long term effectiveness of indoor residual spraying ( irs ) and long lasting insecticide - treated nets ( llins ) in reducing outdoor transmission of malaria especially before bedtime and by people sleeping outdoors . the relatively high infection rate of 11.1% of a. melas in guinea - bissau together with its tendencies to be both endophilic and exophilic and have a high human blood index ( sharp et al . , 2007 ; tuno et al . , 2010 ) make the species a significant vector , which may also be hard to control by reliance on itns and llins . the high csp rate of 33.3 % in the 4 indoor collected a. pharoensis might implicate a significant role in malaria transmission in drier inland guinea bissau , however this should be viewed with caution due the small sample size . very low infection rates and absence of malarial parasites , traditionally found in west and central african populations of a. pharoensis has always led to the conclusion that this mosquito plays little role in malaria transmission despite its anthropophilic habits and that it can be easily experimentally infected ( demeillon , 1947 ; ndiath et al . , 2012 ; tchouassi et al . , 2012 ) . in drier sahel regions of africa where the major vectors of malaria are absent or very rare and irrigated rice and other crop lands are increasing , a. pharoensis is considered more important at maintaining low levels of malaria ( kerah - hinzoumbe et al . , 2009 ; kibret et al . , 2010 ) . figshare : elisa results identifying plasmodium falciparum infection status in anopheles spp . collected in guinea - bissau .

ampk , or 5'-adenosine monophosphate - activated protein kinase , is an evolutionarily conserved serine / threonine kinase that plays a crucial role in regulating energy homeostasis . in higher order eukaryotes , it is expressed in multiple systems and serves as an integrator of cellular and organismal energy balance and energy - dependent responses . as its name implies , ampk is activated by physiological or pathophysiological stimuli that cause elevations in cellular amp concentration , as well as hormones , drugs , and xenobiotics . upon activation , ampk phosphorylates its targets with the goal of acutely stimulating catabolic processes while inhibiting anabolic processes to restore cellular energy homeostasis , and chronically altering gene transcription and controlling cellular fate . in this review , as the ampk field is rapidly growing , for further discussion of the regulation of ampk and its role in coordinating various aspects of cell and whole organism function , readers are directed towards several recent reviews [ 1 - 4 ] . ampk is a heterotrimeric enzyme complex , comprised of a catalytic subunit and regulatory and subunits . there are two alpha subunit genes ( 1 and 2 ) , two beta subunit genes ( 1 and 2 ) , and three gamma subunit genes ( 1 , 2 , and 3 ) which are capable of assembling in different combinations in a tissue - specific manner . in the rodent brain , all the isoforms have been detected except the 3 , which is primarily thought to be a muscle - specific subunit . activation of the ampk complex requires phosphorylation of the subunit at threonine-172 ( t172 ) in the kinase loop by one of two identified upstream kinases , lkb1 ( liver kinase b1/serine threonine kinase 11 ) or camkk ( ca / calmodulin - dependent kinase kinase ) ( fig . the subunits contain a carbohydrate - binding domain that acts as a cellular glycogen sensor . in addition , a myristoylation site in the subunit has been identified that is required for subunit phosphorylation and allosteric activation by amp [ 7 , 8 ] . the subunits contain four cystathionine -synthase domains arranged as tandem pairs ( also known as cbs or bateman domains ) , which serve as amp , adp , and atp binding sites . more recently , through biochemical and structural studies , evidence has been provided that ampk also responds to elevations in both cellular amp and adp . initially , phosphorylation of t172 by the upstream kinase increases ampk activity by several hundred - fold . additionally , binding of amp or adp to site 1 and site 3 of the cbs domains induce further allosteric activation ( induced by amp alone ) and protect the kinase from dephosphorylation ( induced by both amp and adp ) . in this manner , several thousand - fold increases in ampk activity can be achieved in the presence of elevated levels of amp after subunit phosphorylation . in contrast , high concentrations of atp bind to the subunit and promote subunit dephosphorylation and kinase inactivation . therefore due to the fact that ampk can directly monitor amp and adp levels and indirectly sense atp levels it has been proposed that ampk acts at the cellular level through changes in adenine nucleotides via lkb1 phosphorylation , and at the whole organism level through elevations in intracellular ca and camkk activation . in addition to lkb1 and camkk , it has also been suggested that tak1 ( transforming growth factor -activated kinase 1 ) may serve as an upstream ampk kinase [ 12 , 13 ] . however , the exact mechanism of this regulation and its in vivo significance in the brain is less well understood . these targets can be broadly categorized into enzymes involved in glucose and lipid metabolism , protein synthesis , cell growth , autophagy , cell polarity , and gene transcription . based on numerous studies , it appears that the overall consequence of ampk activation is context and stimulus - specific . under normal physiological situations in the brain , ampk serves as a master energy sensor and integrator of signals in response to long - term energy deficits . in pathophysiological situations such as ischemia , over activation of ampk early histological studies revealed that ampk subunits are highly expressed in the developing and adult rodent brain [ 5 , 14 ] . although neurons represent a minority of cells in the brain , it appears that ampk subunits are enriched in these cells irrespective of brain area . in particular , hypothalamic ampk is proposed to be an " energy integrator " and is poised to sense not only cellular energy status , but also to monitor whole - organism energy balance . the role of ampk in the regulation of food intake and energy balance was elucidated by several groups . it was shown that signals such as fasting and the orexigenic hormone ghrelin , increased hypothalamic ampk activity , whereas refeeding and the anorexigenic hormone leptin , decreased ampk activity [ 15 , 16 ] . as further evidence that it is indeed alterations in hypothalamic ampk that mediate changes in food intake , it was demonstrated that direct pharmacological activation of ampk by i.c.v . injection of aicar ( 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside ) or expression of a constitutively active ampk adenovirus increase food intake and body weight . similarly , injection of a dominant negative ampk adenovirus produced a reduction in food intake and body weight . previously , we developed the compound c75 ( 3-carboxy-4-octyl-2-methylenebutyrolactone ) as a fatty - acid synthase inhibitor / carnitne palmitoyltransferase-1 stimulator and demonstrated its ability to reduce food intake and body weight in mice [ 17 - 19 ] . it was observed that c75 caused rapid profound anorexia , reversible weight loss , and increased peripheral energy expenditure . subsequently , when examining the mechanism of action of c75 , we determined that c75 , either given i.p or i.c.v , mediates its effects through ampk inhibition in the hypothalamus [ 20 , 21 ] . we demonstrated that direct stimulation of ampk with aicar reverses the effects of c75 on ampk inhibition and feeding reduction . injection of the ampk inhibitor compound c ( 6-[4-(2-piperidin-1-yl - ethoxy)-phenyl)]-3-pyridin-4-yl - pyrrazolo[1,5-a]-pyrimidine ) , reduced hypothalamic ampk activation and food intake . furthermore , it was demonstrated the altering hypothalamic neuronal energy balance and inhibiting ampk activity with c75 treatment produced long - term effects on neuropeptide y ( npy ) gene expression and feeding behavior , suggesting a mechanism for c75 action in the cns . in the subsequent years , it has been demonstrated by many laboratories that hypothalamic ampk can be regulated by numerous orexigenic signals which indicate " energy deficit " and anorexigenic signals which indicate " energy surplus . " fasting , hypoglycemia , ghrelin , adiponectin , agouti - related peptide ( agrp ) , endocannabinoids , thyroid hormone , glucocorticoids , are among the orexigenic cues that increase hypothalamic ampk and feeding [ 22 - 30 ] . in contrast , refeeding , hyperglycemia , leptin , insulin , resistin , -melanocyte stimulating hormone ( -msh ) , ciliary neurotrophic factor ( cntf ) , glucagon - like peptide 1 ( glp-1 ) , and angiopoeitin - like protein 4 ( angpt14/fiaf ) are among the anorexigenic signals that inhibit hypothalamic ampk and reduce food intake [ 15 , 16 , 26 , 31 - 34 ] . several transgenic and knockout mouse model systems provided further evidence supporting the importance of hypothalamic ampk in energy balance . initially , global deletion of either 1 or 2 subunits did not reveal any differences in food intake , body weight , or energy expenditure [ 35 - 37 ] . this lack of central effect could be attributed to compensatory up - regulation or redundancy in function of the subunits . to overcome this limitation , regard , mice with a targeted deletion of 2 ( on an global 1-null background ) in pro - opiomelanocortin ( pomc ) neurons of the arcuate ( arc ) nucleus developed obesity and exhibited a reduced metabolic rate . by contrast in the same study , mice with a deletion of 2 in agouti - relate peptide ( agrp ) neurons were leaner than wild type mice . unexpectedly , neurons from both of these knockout mouse models retained acute sensitivity to leptin and insulin signaling , but were blunted in their response to glucose alterations . these findings suggest that ampk is important for glucose - sensing in pomc and agrp neurons but is not required for hormonal responses . as an alternative strategy , ampk subunit knockout mouse models have also provided interesting insight into enzyme regulation in feeding centers . it was recently observed that mice with a global 1 deletion have reduced food intake , adiposity , and total body mass when fed either a standard or high fat diet . in contrast , mice with a global deletion of 2 subunits that were fed a high fat diet gained more weight and developed hyperinsulinemia and glucose intolerance . the finding that each of the subunit deletion mice exhibits defined phenotypes is interesting and may be explained by differential subunit expression in the various tissues involved in metabolic regulation ( e.g , hypothalamus , muscle , liver , adipose tissue ) . furthermore , while these knockout models have yielded significant and interesting findings that are sometimes surprising , they underscore the complexity of regulation of ampk in different hypothalamic nuclei and neuronal subpopulations . to further address the role of ampk in the hypothalamus , it would be useful to have conditional and inducible control of gene deletion in specific neuronal populations with appropriate cre - recombinase mouse lines . it is likely that such tools are already in development and studies will yield valuable information soon . as ischemic brain injury is essentially an acute disorder of energy deficit , it is not surprising that ampk would be implicated in this process . stroke injury involves multiple cellular and tissue stresses including hypoxia , hypoglycemia , lactic acidosis , calcium overload , and oxidative and nitrosative stresses , apoptosis , and necrosis [ 41 - 43 ] . accumulating evidence suggests that ampk is activated by various metabolic stresses in the brain and elicits downstream compensatory responses . however , the overall consequence of ampk activation appears to be dependent on the degree of activation , the cell - type in which it is activated and the specific metabolic status of the cell . in vitro , it was demonstrated that ampk activation with a low dose of aicar protects primary hippocampal neurons from hypoglycemic stress and glutamate excitotoxicity and protects primary astrocytes from ceramide - mediated apoptosis [ 14 , 45 ] . in contrast , studies using cell lines determined that aicar - stimulated ampk activation was detrimental to overall cell survival in sh - sy5y neuroblastoma cells and mouse neuroblastoma cells [ 46 , 47 ] . similarly , it was found that ampk activation mediated by tributlyltin induced primary cortical neuronal cell death . furthermore , it was shown that the ampk inhibitor compound c was able protect neurons from tributyltin toxicity . studies from another group showed that aicar could protect rat primary cortical neurons from hypoxic death in a concentration - dependent manner . most recently , another study provided further mechanistic insight into the role of ampk in excitotoxic injury . it was found that nmda or glutamate excitotoxic stress produced activation of ampk in primary cortical or cerebellar granule cells . moreover , overactivation of ampk was found to be involved in increased gene transcription and activation of the pro - apoptotic protein bim , a bcl-2 family member . in this regard , constitutively active ampk or aicar were found to activate an atp - dependent cell death process known as " excitotoxic apoptosis . " based on in vitro studies , it is evident that discrepancies exist regarding the role of ampk in the neuronal response to stress . one important consideration is that many studies typically use neuronal culture medium containing 25 mm glucose whereas the physiological concentrations of glucose in vivo have been estimated to be 0.82~2.4 mm [ 51 - 53 ] . to facilitate the study of ampk , neuronal metabolism , and their responses to physiological and pathophysiological stress conditions we established a new neuronal culture system that would more closely mimic the in vivo situation . we observed that when neurons are cultured in 3 mm glucose as opposed to 25 mm , they respond to metabolic alterations more similar to that seen in vivo . more recently , we have also optimized this culture paradigm by culturing cells at 5% o2 tension ( as opposed to 21% or ambient o2 tension ) , which is more reflective of that seen in the brain . interestingly , when neurons were grown in both 3 mm glucose and 5% o2 they exhibit increased survival , reduced reactive oxygen species , improved mitochondrial function . moreover , cellular atp levels were increased through increased glucose uptake and flux through the glycolytic pathway . while activated ampk levels were elevated , oxidation of both glucose and fatty acids were decreased . these data suggest that under physiological glucose and oxygen culture conditions , activated ampk is reset at a new setpoint where it is better poised to respond to and integrate energy flux signals as it may in vivo . in addition to accumulating data from in vitro experiments , several studies have examined the role of ampk in ischemia reperfusion injury in vivo . work from our group demonstrated that ampk is activated by oxygen - glucose deprivation ( ogd ) stress in rat hippocampal slices . similar findings were observed in the mouse cortex using a middle cerebral artery occlusion ( mcao ) model of transient focal ischemia . furthermore , it was found that administration of c75 , which inhibits ampk indirectly , or compound c , which inhibits ampk directly , produced significant reductions in infarct size and behavioral defects , suggesting that ampk over - activation is detrimental to overall stroke outcome . additional data to support the involvement of ampk in ischemic damage was provided in studies using 1- or 2-deletion mice . it was found that mice with global 2 deletion had significantly smaller cortical , striatal , and total infarct volumes compared to wild - type mice . in contrast 1-deletion mice were not significantly neuroprotected compared to wild - type controls . furthermore , compound c , which was shown to be neuroprotective in wt mice , lost its effects in 2 knockout mice . it was also found that the neuroprotective effect of ampk 2 deletion was equally evident in male and female mice . in another series of experiments , it was demonstrated that acute treatment with the commonly used anti - diabetic drug and ampk activator , metformin , exacerbated stroke outcome after mcao . this detrimental effect was lost in both nnos and 2 knockout mice , demonstrating that over - activation of this pathway is harmful in ischemic injury . most intriguingly , it was also shown that chronic metformin actually improved stroke outcome by reducing ampk activation . it was suggested that the duration and degree of ampk activation are the critical factors that determine cellular responses in ischemia . based on these data , it was proposed that over - activation of ampk especially in astrocytes enhances glycolysis and produces extreme lactic acidosis which exacerbates ischemic injury . however , it can not be ruled out that over - activation of ampk in neurons induces a cell - autonomous death pathway such as autophagy . indeed it has recently been demonstrated that ampk activation is a key signal in the initiation of this cascade [ 61 , 62 ] . although the precise mechanism remains unclear , accumulating in vivo data using a combination of genetic and pharmacological tools has shown that over activation of ampk is deleterious in stroke and that ampk inhibition is neuroprotective . while the role of ampk in hypothalamic energy - sensing and ischemic / metabolic injury is well studied , recent work implicates ampk in other aspects of brain physiology . as the complete deletion of both ampk subunits results in embryonic lethality , several authors have investigated the role of ampk in neuronal development using transgenic and alternate methods . these mice exhibited a striking neurodegeneration phenotype and early post - natal lethality suggesting that 1 subunit is required for normal brain development . however using an alternative 1 deletion strategy , another group was unable to confirm these severe neurological deficits . given the lack of a striking neurological phenotype in the 1 , 2 , or the 2 deletion animals , it is likely that the phenotype observed by dasgupta and milbrandt reflects the expression of a 1-fusion protein rather than a 1 deletion [ 37 , 40 ] . clearly additional studies are required to test the role of the 1 subunit in neuronal development . more recently , using a conditional neuronal deletion of 2 on a whole body 1-deletion background , it was shown that ampk activity is not required for cortical neuron development but regulates axon formation during metabolic stress . it was also shown that overactivation of ampk during a critical phase of neuronal development inhibits polarization through phosphorylation of kif5 and disruption of pi3-kinase transport in growing axons . these data also lend support to the notion that over - activation of ampk in neurons is detrimental and suggest a mechanism of damage to developing neurons in conditions such as perinatal hypoxic - ischemic injury . ampk has also been implicated in neurodegenerative diseases such as alzheimer 's ( ad ) and huntington 's disease ( hd ) [ 66 , 67 ] . in this regard , it has been shown that there is overactivation of ampk in striatal neurons in a transgenic mouse model of hd . more recently , a mechanism was identified by which the 1 subunit abnormally translocates to the nucleus to mediate neurotoxicity via suppression of expression of the anti - apoptotic protein bcl2 . it was also found that a pharmacological agent that ameliorates hd symptoms acts in part by preventing 1 translocation to the nucleus . initially , it was demonstrated that ampk activation , either directly or indirectly , inhibits tau phosphorylation in rat cortical neurons . in contrast , others demonstrated that ampk could phosphorylate tau on multiple sites and disrupt the binding of tau to microtubules . interestingly , it was demonstrated that exposure of mouse cortical neurons to amyloid peptide ( a.a . 1 - 42 ) induced ca - camkk-dependent activation of ampk and hyperphosphorylation of tau . in human patients it was observed that ampk is abnormally activated and translocalized from the nucleus to the cytoplasm in ad tangles as well as other tauopathies . thus it is evident that although ampk is activated in the alzheimer 's tangle formation process , it is not yet established if this is a primary cause or a cellular response in an attempt to repair disordered metabolic balance and signaling . other studies have examined the role of ampk on amyloid plaques , the other major component of ad pathology . several studies in this field have shown that ampk activation represses amyloidogenesis in neurons [ 68 , 71 , 72 ] . furthermore , it was observed that amyloid- production is increased in cortical neurons from ampk 2 knockout mice , whereas activators of ampk such as aicar and resveratrol are able to reduce a production and facilitate its clearance . as alzheimer 's disease is a complex neurodegenerative process encompassing numerous molecular players , a complete understanding of its details is lacking . however , based on accumulating data over the past few years , it is likely that ampk plays an important role in several aspects of ad pathogenesis . therefore , selective modulation of ampk signaling may be a potential consideration for ad therapy . ampk is a multi - functional energy sensor in many organ systems . in the hypothalamus , ampk serves as an energy integrator which responds to signaling by hormonal and nutrient cues . in pathological conditions such as stroke and neurodegenerative diseases , information from various transgenic and knockout ampk mouse model systems will help to elucidate precise cellular mechanisms of ampk activation in both normal and pathophysiology . due to the fact the ampk is implicated in a variety of processes ,

one of the best examples of how autocrine pdgf signaling contributes to malignancy is from studies of malignant gliomas . co - expression of pdgf -receptor ( pdgfr ) and pdgf ligands are common in glioblastoma multiforme ( gbm ) , and frequently associated with p53 loss . enforced expression of pdgfr and pdgf - a confers growth advantages to mouse and human glioma cells in the brain of immunodeficient mice . activating mutations of pdgfr the role of pdgf signaling in normal central nervous system ( cns ) development and human gliomas will be discussed in the first review article of this issue . in addition to its role in gliomas , autocrine pdgf signaling is also involved in the development of soft tissue sarcomas . because pdgf is a potent mitogen for various types of mesenchymal cells including fibroblasts and myofibroblasts , several studies using mouse models have been carried out to determine if pdgf overexpression can induce a malignant phenotype in these cells . as expected , a coding region in the pdgf - b gene corresponding to the v - sis oncogene was capable of inducing fibrosarcomas at the site of inoculation in newborn mice . subsequent studies showed that pdgfrs and pdgfs are expressed in various types of soft tissue tumors , , , suggesting a critical role for autocrine pdgf signaling in the development of these tumors . emt is a process of switching polarized epithelial cells to a migratory mesenchymal type of cells , which are largely responsible for the progression and metastasis of human cancers that originate from epithelial cells . studies involving the emt process in breast cancers are discussed in the second review article in this issue . components of the pdgf signaling pathway are up - regulated during transforming growth factor- ( tgf-)-induced emt in breast cancer . this observation is corroborated by studies demonstrating that autocrine pdgf signaling maintains emt and promotes metastasis in mouse mammary carcinomas . interestingly , in gliomas , high tgf- signaling confers poor prognosis in patients and promotes glioma cell proliferation by activating pdgfb / pdgfr signaling , suggesting an evolutionarily conserved mechanism of regulating pdgf signaling in different types of human cancers . constitutive autocrine pdgf signaling can also be achieved by mutational activation of the genes encoding pdgf receptors or ligands . for example , a gene translocation event occurs in dermatofibrosarcoma protuberans ( dfsp ) , a rare form of dermal sarcoma resulting in a fusion gene between collagen 1a1 ( col1a1 ) and pdgf - b . the col1a1/pdgf - b fusion protein over - produces pdgf - b and creates an autocrine loop that transforms nih3t3 cells in vitro and in vivo , . a subgroup of chronic myeloid leukemia without bcr - abl fusion gene harbors a translocation - mediated fusion between bcr gene and pdgfra gene , leading to over - activation of pdgfr. gene fusion between flip - like 1 ( fip1l1 ) and pdgfra is also found in patients with hypereosinophilic ( he s ) symdrome , resulting in a fusion protein with constitutive tyrosine kinase activity and transforming capacity . consequently , these he s patients often develop acute myeloid leukemia ( aml ) . on the other hand , gene fusion events between pdgfrb and the transcription factor tel / etv6 as well as several other fusion partners take place in patients with chronic myeloid leukemia , , ] . additionally , a subset of gastrointestinal stromal tumors ( gist ) with wild - type c - kit often harbor intragenic mutations in pdgfra , leading to a ligand - independent activation of the receptor and its downstream signaling pathways , . lastly , gene mutations that constitutively activate pdgfr are found in malignant human gliomas , which are discussed in the following review article of this issue . pdgf signaling not only plays an essential role in promoting tumor malignancy but also contributes to the processes of tumor stromal recruitment , angiogenesis , and drug resistance . prevalent pdgfr expression has been found in the stromal compartments of various types of solid tumors . pdgf ligands expressed by the neoplastic components of the tumor mass stimulate the recruitment of surrounding non - neoplastic stromal cells expressing pdgfr , including endothelial cells , pericytes , and fibroblasts , to the tumor . in mouse fibrosarcomas , gliomas , and melanomas , paracrine pdgf / pdgfr signaling enhances pericyte recruitment to the tumor vasculature , thereby promoting tumor cell growth , survival , and vessel stabilization . in gliomas , paracrine pdgf signaling induces neo - angiogenesis by directly stimulating proliferation of pdgfr-positive tumor endothelial cells , . these studies suggest that inhibition of pdgf signaling by pharmacologic intervention can potentially suppress growth and survival of both tumor and tumor - associated stromal and endothelial cells , . pdgf signaling also supports tumor metastasis to the bone . using an experimental prostate cancer mouse model , researchers demonstrated that blocking pdgfr thwarted the angiogenesis of tumors that had metastasized to the bone . additionally , as discussed in the third review article in this issue , pdgfr signaling induces prostate cancer cell metastasis to the bone , further demonstrating that paracrine pdgf signaling enhances tumor growth , angiogenesis , and metastasis . previous data suggests that recruited tumor fibroblasts are an integral part of various types of solid tumors and influence malignant transformation , angiogenesis , and tumor cell proliferation . therefore , blocking pdgf signaling in tumor stroma is effective in suppressing tumor growth in experimental tumor models . furthermore , the benefit of pdgf inhibition in tumor stroma could extend beyond suppression of tumor growth , angiogenesis , and metastasis . some solid tumors frequently show high ifp , which restricts fluid convection rate across the capillary walls and results in low drug uptake and tumor resistance to cytotoxic drug treatments . one of the factors that contributes to high ifp within the tumor mass is pdgfr. indeed , inhibition of pdgfr signaling by imatinib reduces tumor ifp and enhances the drug uptake into tumor cells , . autocrine and paracrine pdgf signaling contribute to several hallmarks of human cancers , including self - sufficient tumor growth , angiogenesis , stromal recruitment , and tumor invasion and metastasis to distant organs . although inhibition of pdgf signaling holds promises for treatments of human cancers with activated pdgf signaling , carefully designed pre - clinical / clinical trials are necessary for the success of targeted therapies against components of this signaling pathway .

anorectal malignancies are commonly adenocarcinoma or squamous cell carcinoma , with melanoma having a relative incidence of 0.54% of all malignancies in this region [ 1 , 2 ] . this site is the third most common primary location for melanoma after skin and retina and yet only 0.41.6% of all primary melanomas arise here [ 2 , 3 ] about 1% of all anorectal carcinomas are melanomas , typically presenting in the fifth or sixth decade of life and predominantly in women . prognosis is very poor with a median survival of 24 months and a 5-year survival of 10% . due to rarity of this entity there is no consensus on which surgical approach is favourable . the surgical procedure of choice ranges from an abdominoperineal resection ( apr ) to wide local excision ( wle ) with or without adjuvant radiotherapy . we report a case of parm in a 50-year - old caucasian man presenting intermittent prolapse of an anorectal mass during 6 months with occasional bleeding . the patient was in good condition with no weight loss and past medical and family history unremarkable . proctoscopy confirmed the physical examination and showed a 3 cm bleeding villous polyp that originated above the dentate line . endorectal ultrasound showed a fusion of all layers , infiltration of the sphincters and no suspect lymph nodes . magnetic resonance imaging of the pelvis highlighted a high suspicion of internal sphincter infiltration and no inguinal or pelvic lymph nodes . computed tomography ( pet ct ) showed an intensely hypermetabolic lesion centred on the lower rectum ( standardized uptake value ( suv ) = 35.1 ) and no evidence of disease extension loco - regionally or at distance ( figs 1 and 2 ) . the histopathology of the biopsy concluded to a primitive malignant melanoma due to the tumour profile in immunohistochemistry : human melanoma black ( hmb-45 ) + + + , s100 protein + + + , melan a + , wide spectrum cytokeratins negative . the oncologic multidisciplinary council validate a surgical treatment without neoadjuvant radio or chemotherapy . due to high suspicion of internal sphincter involvement an apr the histopathology reported a pedicle , mostly exophytic tumour of the anorectal junction , measuring 2.5 2.5 1.5 cm . this tumour is associated to a tubulo - villous polyp in low grade dysplasia coexisting on the tumour pedicle . follow up , the patient presented a single hypermetabolic lymphadenopathy localized in the left obturator region , measuring 13 mm long axis ( suv = 9.5 ) and several spots of increased glycolytic activity in the hepatic parenchyma , which only one was corresponding to a small hypodense lesion ( suv = 4.5 ) . the patient was included in a study protocol comparing a new treatment anti - pd1 ( nivolumab ) with ipilimumab ( monoclonal antibody anti - ctla-4 ) or both . about 1% of all anorectal carcinomas are melanomas , typically presenting in the fifth or sixth decade of life and predominantly in women . it may be difficult to identify , and may be misdiagnosed as a haemorrhoid , rectal polyp , or as an ulcerative lesion following prolapse through the anal orifice . macroscopically , the tumours are polypoidal and pigmented while microscopically , the cells are arranged in nests with characteristic immunostaining specific for melanosome protein . however the histologic markers of s-100 , hmb-45 and vimentin are frequently identified and help clarify the diagnosis . if biopsy shows a specimen suspicious for sarcoma ( e.g. leiomyosarcoma ) , s-100 staining should be performed and if positive the tumour is most likely to be a melanoma . tumour histology includes epitheliod or spindle cells but as in our case can be a mixed tumour . tumours with pure epithelioid histology were less likely to recur [ 1 , 2 , 4 , 5 ] . figure 1:(a ) t2-weighted axial , coronal and transverse mri images , demonstrating the polypoid 3 cm anorectal mass . ( b ) pet ct showed an intensely hypermetabolic lesion centered on the lower rectum.figure 2:histologic illustration of the collision tumour . ( a ) t2-weighted axial , coronal and transverse mri images , demonstrating the polypoid 3 cm anorectal mass . ( b ) pet ct showed an intensely hypermetabolic lesion centered on the lower rectum . the rarity of this case is based on the observation of a tubulo - vilous polype on the tumour pedicle . actually there was a small in situ melanoma on the anal side that turn into a polypoid and invasive melanoma upwards to the rectal mucosa . collision tumour. we do not know to this date if there is any association of those two tumours on the anorectal melanoma pathogenesis . to our knowledge , there is no other similar observation reported in the exception of case reporting synchronous anorectal melanoma and rectal adenocarcinoma in a distance of 3 - 4 cm between them . in the absence of metastasis surgical therapy however , several recent studies suggest that , if possible , sphincter - sparing local excision and adjuvant radiation is well tolerated and can effectively control loco - regional disease while avoiding the functional morbidity of the apr [ 35 , 8 , 9 ] . on the other hand , mesorectal lymph nodes are involved in preference to inguinal lymph nodes in contrast to squamous cell carcinoma of the anus . if an apr is performed , the mesorectal lymph node resection may contribute to a better staging of the disease . there is no value of prophylactic inguinal lymph node resection [ 2 , 3 , 5 ] . patients without lymph node metastasis have a survival advantage with a 5-year survival rate of 20 versus 0% in patients with metastasis . a worse prognosis was also associated with tumour thickness , tumour necrosis ( important histologic feature , representing a biologically more aggressive tumour ) and perineural invasion . this reported survival is not up to date due to the small number of cases and does not take into account new medical treatments ( nivolumab and ipilimumab ) .

the participant of this study is a male subject ( 37 years old , 1.70 m , 67.40 kg and manual preference for right hand ) with complete sensory - motor thoracic traumatic spinal cord injury ( t9 ) , ais a ( american spinal injury association impairment scale ) and with 3 years post - sci . he is a manual wheelchair user who is capable to perform a spt without physical or technical assistance , routinely using this type of transfer in daily life for both sides ( right / left ) . additionally , he does not have any condition that affects the ability to transfer or the use of electrical stimulation . the spt were performed by the participant using two seats with the same characteristics ( 42 cm x 42 cm ) and with an angle of 20 between them . the height of both seats ( 48 cm ) resembles that of the wheelchair used by the participant . beneath each seat , a force plat ( bertec corporation ; columbus , ohio ) additionally , another force plat was placed beneath the participant s feet , so the force caused by the stimulation could be also recorded . the seats were firmly attached to a wooden case placed between the seats and the force plates . the additional weight imposed by the seats and the wooden structure was compensated by software before running the experiments . kinetic data were recorded with a sampling frequency of 200 hz . the bilateral femoral quadriceps ( rectus femoral ) of the participant were electrically stimulated ( hasomed , rehastim ) by surface electrodes with the following parameters : frequency of 50hz and pulse width of 450s , while the intensity varied according to the desired muscle contraction grades from the manual muscle test ( mmt ) . the intensity levels were identified while the individual was sitting on his wheelchair : 38ma to reach grade 3 in mmt ; 48ma to reach grade 4- in mmt ; 52ma to reach grade 4 in mmt . the parameters were tested in the individual before running the spt experiments . the start and stop of fes the participant was encouraged to use his usual transfer strategy during the experiments . after a familiarization period , figure 2 shows the force data of the three force plates during the spt for both scenarios : with and without fes . force plates 1 and 3 correspond to the ones placed beneath each seat , while force plate 2 recorded the feet reaction forces . the force data was normalized to the participant body weight and the time was expressed as a percentage of the total movement time , corresponds to the total trial duration . the participant performed a total of eight trials with fes and four trials without fes . the trials chosen for analysis correspond to the ones in which the individual had the best performance in the spt , based on some conditions verified through video documentation : transfer performed on the same side , hand maintenance in the same position along the transfer and a single lift movement with respect to the pelvis motion . according to the analysis of the video recorded for each trial , the best performance of the spt was when the participant transferred from the left to the right side , therefore three trials with fes and two without fes were selected for further analysis . in this study the spt division is the same as the one used in previous works . hence , the pivot transfer is defined by the occurrence of three phases : pre - lift , lift - pivot and post - lift . for this preliminary study , only the force information was used to delimit the beginning and the end of each phase . the beginning of the pre - lift and the end of the post - lift , which delimit the overall transfer motion were determined by the start and the end of motion dynamics looking at the forces provided by the force plates 1 and 3 , while the lift pivot resultant takes place when the resultant force of plate 1 starts to decrease ( after attaining a certain threshold ) and ends when the resultant from the force plate 3 reaches its maximum value . figure 3 shows the transfer motion selected by each trial according to the lift - pivot phase detection explained above . in order to assess the effect of the fes during the spt based on the force data from the three platforms , a calculation of the force impulse ( the integral of force over time ) for the resultant forces during the lift - pivot phase the idea of this calculation is to use this parameter to quantify the effect of fes on the ll . if the fes is capable to provoke a force on the ll , then the total force during the transfer will increase on the data recorded from force plate 2 when compared to the trials without fes . the force impulse of the rest of the plates should be the same or , in the best scenario , will decrease as a positive effect of the fes in aiding the subject to perform the spt . the participant of this study is a male subject ( 37 years old , 1.70 m , 67.40 kg and manual preference for right hand ) with complete sensory - motor thoracic traumatic spinal cord injury ( t9 ) , ais a ( american spinal injury association impairment scale ) and with 3 years post - sci . he is a manual wheelchair user who is capable to perform a spt without physical or technical assistance , routinely using this type of transfer in daily life for both sides ( right / left ) . additionally , he does not have any condition that affects the ability to transfer or the use of electrical stimulation . the spt were performed by the participant using two seats with the same characteristics ( 42 cm x 42 cm ) and with an angle of 20 between them . the height of both seats ( 48 cm ) resembles that of the wheelchair used by the participant . beneath each seat , a force plat ( bertec corporation ; columbus , ohio ) additionally , another force plat was placed beneath the participant s feet , so the force caused by the stimulation could be also recorded . the seats were firmly attached to a wooden case placed between the seats and the force plates . the additional weight imposed by the seats and the wooden structure was compensated by software before running the experiments . kinetic data were recorded with a sampling frequency of 200 hz . the bilateral femoral quadriceps ( rectus femoral ) of the participant were electrically stimulated ( hasomed , rehastim ) by surface electrodes with the following parameters : frequency of 50hz and pulse width of 450s , while the intensity varied according to the desired muscle contraction grades from the manual muscle test ( mmt ) . the intensity levels were identified while the individual was sitting on his wheelchair : 38ma to reach grade 3 in mmt ; 48ma to reach grade 4- in mmt ; 52ma to reach grade 4 in mmt . the parameters were tested in the individual before running the spt experiments . the start and stop of fes the participant was encouraged to use his usual transfer strategy during the experiments . after a familiarization period , figure 2 shows the force data of the three force plates during the spt for both scenarios : with and without fes . force plates 1 and 3 correspond to the ones placed beneath each seat , while force plate 2 recorded the feet reaction forces . the force data was normalized to the participant body weight and the time was expressed as a percentage of the total movement time , corresponds to the total trial duration . the participant performed a total of eight trials with fes and four trials without fes . the trials chosen for analysis correspond to the ones in which the individual had the best performance in the spt , based on some conditions verified through video documentation : transfer performed on the same side , hand maintenance in the same position along the transfer and a single lift movement with respect to the pelvis motion . according to the analysis of the video recorded for each trial , the best performance of the spt was when the participant transferred from the left to the right side , therefore three trials with fes and two without fes were selected for further analysis . in this hence , the pivot transfer is defined by the occurrence of three phases : pre - lift , lift - pivot and post - lift . for this preliminary study , only the force information was used to delimit the beginning and the end of each phase . the beginning of the pre - lift and the end of the post - lift , which delimit the overall transfer motion were determined by the start and the end of motion dynamics looking at the forces provided by the force plates 1 and 3 , while the lift pivot resultant takes place when the resultant force of plate 1 starts to decrease ( after attaining a certain threshold ) and ends when the resultant from the force plate 3 reaches its maximum value . figure 3 shows the transfer motion selected by each trial according to the lift - pivot phase detection explained above . in order to assess the effect of the fes during the spt based on the force data from the three platforms , a calculation of the force impulse ( the integral of force over time ) for the resultant forces during the lift - pivot phase the idea of this calculation is to use this parameter to quantify the effect of fes on the ll . if the fes is capable to provoke a force on the ll , then the total force during the transfer will increase on the data recorded from force plate 2 when compared to the trials without fes . the force impulse of the rest of the plates should be the same or , in the best scenario , will decrease as a positive effect of the fes in aiding the subject to perform the spt . the total duration of spt varies between 1.43 s to 2.45 s , while the lift pivot phase in trials 1 , 2 , 3 , 4 , and 5 corresponds to 57% , 64% , 69% , 68% and 45% of the total spt , respectively ( see table 1 ) . the overall movement pattern was similar in all trials ( figure 2 ) with the resultant force changing from plate 1 to 3 . a closer analysis of only the lift phase of the spt ( figure 3 ) reveals some differences between the spt with and without fes . specifically , the force values in platform 2 are higher in trials 3 , 4 and 5 , compared to trials 1 and 2 . no differences in terms of force amplitude were observed on platforms 1 and 3 for all trials . figure 4.a and 4.c show the results for the impulse in all platforms , as a stacked bar chart , in body weight units and normalized by the total amount of impulse in all the platforms . the calculation of impulse just for force plate 2 ( ll forces ) is also displayed on figures 4.b and 4.d . , there is no study in the literature that uses the fes to assist the spt , so it is not possible to compare our data with any other previous study . the total time of spt are similar to that shown by gagnon et al . ranging on average from 1.65 to 2.6 s , so fes did not modify the total transfer time , demonstrating that can be a good resource that does not affect the agility during the paraplegic transfer . this is important to be considered , because if the assistive technology device in potential increases the total transfer time , it will not be practical on daily activities and the individual may have some resistance to use it . found that the lift pivot phase has an average duration of 40% of the total spt cycle . in our experiments , the closest value was found in the fes trial with the highest intensity ( trial 5 ) . it seems that a higher stimulation intensity reduces the lift phase duration , which in turn may reduce ul overload . in more a detailed study of the lift pivot phase it is observed that the higher the current intensity of fes , the higher the muscular strength and greater the impulse on plate 2 . this may indicate that the degree 4- in mmt of quadriceps muscle with fes is a good intensity to assist spt . this threshold will be better studied when the research sample is enlarged . additionally , the use of fes did not hinder the spt movement and there were no clear negative effect , according to our preliminary analysis . the kinetics curves in figure 3 indicate an increase in forces applied by the ll in the spt during the trials with fes activation , which means that the muscular activity induced by the fes was converted into forces that may have a positive effect during the spt favoring the movement . during the lift - pivot phase therefore , the increase of the participation of the ll in the total impulse during the lift - pivot phase , as displayed in figure 4 is an indication of alleviated effort in the ul . we need to investigate more if the increase of fes will produce a better participation of ll and a lower muscular demand of shoulder joint muscles , as well as a decrease impingement of the subacromial structures , which may minimize the risk of the joints during transfers . physiotherapists and occupational therapists have little knowledge and clinical resources to assess spt in clinical practice . there are few studies of this ability and little progress has been made in this area of expertise over the past years . the sliding board is the unique low cost assistive technology resource used worldwide , but few progresses have been made on new technological possibilities in this area . fes can be a potential alternative since it can be portable , lightweight , personalized and easy to use with surface electrodes . this study shows some evidence that fes of quadriceps may favor participation of ll during spt , but the hypothesis that fes in ll decreases overload on ul should be further investigated in future studies .

periodontitis is a multifactorial disease with microbial dental plaque as the initiator of periodontal disease.1 however , the manifestation and progression of periodontitis is influenced by a wide variety of determinants and factors including subject characteristics , social and behavioral factors , systemic factors , genetic factors , tooth - level factors , microbial composition of dental plaque and other emerging factors.2 smoking is one of the major environmental risk factors for periodontal diseases.311 in studies where plaque accumulation was similar in smokers and non - smokers or was adjusted , current smokers had deeper probing depths,5,6,1214 greater attachment loss,10,12,13,15 more bone loss,4,11,16 and fewer teeth.13,15 smokers also exhibit more supragingival calculus deposits.17 it is widely accepted that smoking impairs various aspects of innate and immune host responses.18,19 numerous functions of oral or peripheral neutrophil are negatively affected by smoking or nicotine exposure , including phagocytosis,20 superoxide and hydrogen peroxide generation,21,22 integrin expression,23 and protease inhibitor production.24 alterations in gingival crevicular fluid2527 and peripheral blood mononuclear cell28 levels of various cytokines in smokers , tipping the balance in favor of tissue breakdown , have been noted . until relatively recently , less attention has been devoted to exploring the role that chronic oral diseases may have on systemic health . the hypothesis that oral conditions , such as periodontal infections , may be risk factors or indicators for important medical outcomes represents a paradigm shift in thinking about causality and the directionality of oral and systemic associations . the subgingival microbiota in patients with periodontitis provides a significant and persistent gram - negative bacterial challenge to the host . these organisms and their products , such as lipopolysaccharide ( lps ) , have ready access to the periodontal tissues and to the circulation via the sulcular epithelium , which is frequently ulcerated and discontinuous.29 it has therefore been speculated that periodontitis results in a low grade systemic inflammation . anemia of chronic disease ( acd ) is a cytokine - mediated anemia commonly encountered in clinical practice and characterized by hypoferramia with adequate reticuloendotelial iron stores , normal to elevated ferritin concentrations and it is a frequent complication of chronic inflammatory conditions.30,31 hutter et al32 suggested that periodontitis has chronic and systemic effects and that periodontitis may tend towards anemia . the aim of present study was to evaluate the influence of cigarette smoking on clinical parameters and the signs of acd in patients with chronic periodontitis . the study population included 88 patients , 45 smokers and 43 non - smokers in the age range of 3069 ( 45.58.5 ) and 3261 years ( 45.87.9 ) , respectively . the patients had chronic periodontitis as evidenced a probing depth of 6 mm or more at 80% of the proximal sites and bone loss > 50% by radiographs.33 all participants were in principal periodontally untreated and had not previously received surgical therapy and were drawn from the patients with chronic periodontitis at the department of periodontology . all subjects were systemically healthy , with no medical condition that would effect their participation in the study . exclusion criteria applied were a course of anti - inflammatory or antimicrobial therapy within the previous 3 months , a history of use of vitamin or iron supplementation within the previous 3 months , and any special dietary requirements ( e.g. coeliac disease ) . pregnant women and individuals who suffered , apart from periodontitis , from any given acute or chronic medical condition , including diabetes , viral , fungal or bacterial infections , or had recent trauma or tooth extractions were also excluded . the purpose and nature of the study , including the types of clinical measurements and sample collection , were explained to all potential subjects . after reading and signing the consent form , for all participants smoking habits were recorded and patients were classified as either current smokers [ s ( + ) ] , i.e. , regular daily smoke 20 cigarettes ( 45 patients ) , or non - smokers [ s ( ) ] , i.e. , who had never smoked tobacco ( 43 patients ) . smoking condition of the patients was calculated as : number of cigarette per day / number of years smoked . the mean age of current smokers and non - smokers was 45.58.5 and 45.87.9 , respectively . body mass index ( bmi ) measures of the patients were also recorded and there was not statistically significant difference between groups ( p>.05 ) . supragingival plaque was scored using plaque index ( pi)34 gingival inflammation was scored using gingival index ( gi)35 bleeding on probing ( bop ) was measured dichotomously.36 probing depth ( pd ) and clinical attachment level ( cal ) measures were obtained from the six points of the teeth using a conventional periodontal probe ( hu - friedy , chicago , il , usa ) . cal measurements were made from the cemento - enamel junction to the bottom of the sulcus . venous blood samples were obtained between 8.30 and 11.00 am by venepuncture in the antecubital fossa without excessive venous stasis . the blood was taken into edta containing vacuum tubes ( hema , germany ) in the faculty of medicine , department of biochemistry . the laboratory analysis of differential blood count was performed immediately with the sysmex xt 2000i ( roche , switzerland ) . in standardized and automated procedures , numbers of erythrocytes , hemoglobin , hematocrit , iron , ferritin levels , mean corpuscular volume of erythrocytes ( mcv ) , mean corpuscular hemoglobin ( mch ) and the mean corpuscular hemoglobin concentration ( mchc ) were calculated . the study population included 88 patients , 45 smokers and 43 non - smokers in the age range of 3069 ( 45.58.5 ) and 3261 years ( 45.87.9 ) , respectively . the patients had chronic periodontitis as evidenced a probing depth of 6 mm or more at 80% of the proximal sites and bone loss > 50% by radiographs.33 all participants were in principal periodontally untreated and had not previously received surgical therapy and were drawn from the patients with chronic periodontitis at the department of periodontology . all subjects were systemically healthy , with no medical condition that would effect their participation in the study . exclusion criteria applied were a course of anti - inflammatory or antimicrobial therapy within the previous 3 months , a history of use of vitamin or iron supplementation within the previous 3 months , and any special dietary requirements ( e.g. coeliac disease ) . pregnant women and individuals who suffered , apart from periodontitis , from any given acute or chronic medical condition , including diabetes , viral , fungal or bacterial infections , or had recent trauma or tooth extractions were also excluded . the purpose and nature of the study , including the types of clinical measurements and sample collection , were explained to all potential subjects . after reading and signing the consent form , for all participants smoking habits were recorded and patients were classified as either current smokers [ s ( + ) ] , i.e. , regular daily smoke 20 cigarettes ( 45 patients ) , or non - smokers [ s ( ) ] , i.e. , who had never smoked tobacco ( 43 patients ) . smoking condition of the patients was calculated as : number of cigarette per day / number of years smoked . the mean age of current smokers and non - smokers was 45.58.5 and 45.87.9 , respectively . body mass index ( bmi ) measures of the patients were also recorded and there was not statistically significant difference between groups ( p>.05 ) . supragingival plaque was scored using plaque index ( pi)34 gingival inflammation was scored using gingival index ( gi)35 bleeding on probing ( bop ) was measured dichotomously.36 probing depth ( pd ) and clinical attachment level ( cal ) measures were obtained from the six points of the teeth using a conventional periodontal probe ( hu - friedy , chicago , il , usa ) . cal measurements were made from the cemento - enamel junction to the bottom of the sulcus . venous blood samples were obtained between 8.30 and 11.00 am by venepuncture in the antecubital fossa without excessive venous stasis . the blood was taken into edta containing vacuum tubes ( hema , germany ) in the faculty of medicine , department of biochemistry . the laboratory analysis of differential blood count was performed immediately with the sysmex xt 2000i ( roche , switzerland ) . in standardized and automated procedures , numbers of erythrocytes , hemoglobin , hematocrit , iron , ferritin levels , mean corpuscular volume of erythrocytes ( mcv ) , mean corpuscular hemoglobin ( mch ) and the mean corpuscular hemoglobin concentration ( mchc ) were calculated . the clinical characteristics of this study are shown in table 1 . when the clinical parameters were compared between groups , in s ( + ) group , pi , pd and cal were significantly higher compared to s ( ) ( p<.05 ) . there were not any differences between smokers and non - smokers in the mean values of gi and bop ( p>.05 ) . both study groups had a comparable number of teeth present ( mean 24.6 smokers and 24.5 for non - smokers ) . the number of erythrocytes and the levels of hemoglobin , hematocrit and iron were lower in smokers compared to non - smokers ( p<.05 ) . correlations between mean levels of serum and clinical parameters are shown in tables 3 and 4 for s ( + ) and s ( ) , respectively . there were no significant correlations between the mean levels of serum parameters and clinical parameters in s ( ) group ( p>.05 ) . in s ( + ) group , there were negative correlation between cal and the level of rbc in serum values ( p<.05 ) , and positive correlations with fe , mch and mchc ( p<.01 ) . in this study group , there were also positive correlations between mean pd values and the levels of mcv ( p<.05 ) , mch and mchc in serum ( p<.01 ) . the clinical characteristics of this study are shown in table 1 . when the clinical parameters were compared between groups , in s ( + ) group , pi , pd and cal were significantly higher compared to s ( ) ( p<.05 ) . there were not any differences between smokers and non - smokers in the mean values of gi and bop ( p>.05 ) . both study groups had a comparable number of teeth present ( mean 24.6 smokers and 24.5 for non - smokers ) . the number of erythrocytes and the levels of hemoglobin , hematocrit and iron were lower in smokers compared to non - smokers ( p<.05 ) . correlations between mean levels of serum and clinical parameters are shown in tables 3 and 4 for s ( + ) and s ( ) , respectively . there were no significant correlations between the mean levels of serum parameters and clinical parameters in s ( ) group ( p>.05 ) . in s ( + ) group , there were negative correlation between cal and the level of rbc in serum values ( p<.05 ) , and positive correlations with fe , mch and mchc ( p<.01 ) . in this study group , there were also positive correlations between mean pd values and the levels of mcv ( p<.05 ) , mch and mchc in serum ( p<.01 ) . it is now well established from a large body of epidemiologic evidence that cigarette smoking is the major preventable risk factor in the incidence progression of periodontal disease.9,10 it is suggested that periodontitis is associated with an increased risk for systemic diseases like cardiovascular diseases , cerebrovascular ischemia and atherosclerosis.37 it has been also shown that periodontitis patients have elevated levels of white blood cells , and elevated plasma levels of c - reactive protein.3842 substantial scientific data indicate that the localized infections characteristic of periodontitis can have a significant effect on the systemic health of both humans and animals.43 just as the periodontal tissues mount an immunoinflammatory response to bacteria and their products , systemic challenge with these agents also induces a major vascular response . this host response may offer explanatory mechanisms for the interactions between periodontal infection and a variety of systemic disorders.44 in the present study , smokers with chronic periodontitis have lower levels of hemoglobin , hematocrit , iron and number of erythrocytes than non - smokers . a depressed level of mcv ( microcytosis ) relates anemia to iron deficiency and elevated level of mcv ( macrocytosis ) relates anemia to vitamin deficiency.45,46 in our study , mcv levels were between the reference values , as mostly seen in acd and called as normocytosis . it has been proposed that hepcidin is a primary factor in the pathogenesis of the acd , a cytokine - mediated anemia commonly encountered in clinical practice and characterized by hypoferraemia with adequate reticuloendothelial iron stores.31 previous studies indicated that il-6 mediates hepcidin increase and consequent hypoferremia during inflammation.47 it was found that hepcidin mrna was increased in the livers of lipopolysaccharide ( lps)-treated mice and lps - treated hepatocytes.48 this also suggested that hepcidin could be the pathogenic mediator of acd . kemna et al49 showed the importance of il-6-hepcidin axis in development of hypoferremia in inflammation and highlight the rapid responsiveness of this iron regulatory system . nemeth et al50 found that patients with acd due to inflammatory disorders or infections had markedly increased excretion of urinary hepcidin . in vitro stimulation of fresh human hepatocytes with a panel of cytokines showed strong induction of hepcidin mrna by il-6 , but not il- or tnf- , indicating that il-6 may be the mediator of hepcidin induction by inflammation . although there are nt any studies about the relationship between hepcidin and periodontal diseases or the effect of smoking on hepcidin , it is well known that pro - inflammatory cytokines and mediators are significantly elevated , with gingival inflammation during the destructive phase of periodontitis.5156 there is also strong evidence for cytokines eliciting the systemic acute - phase response in various chronic inflammatory diseases.57,58 hutter et al32 suggested that periodontitis also needs to be considered as a chronic disease which may cause lower numbers of erythrocytes and consequently lower hemoglobin levels . they mentioned that the pathogenesis for their findings is similar as reported for rheumatoid arthritis , i.e. depressed erythropoiesis by systemically circulating pro - inflammatory cytokines resulting from a local chronic inflammatory process . elevated concentrations of il-6 were observed in the plasma of smokers,59 as well as in the alveolar cells of healthy donors stimulated by tobacco glycoprotein.60 nicotine , one of the most deleterious products of cigarette , has been shown to increase release of il-6 by cultured murine osteoblasts.61 giannopoulou et al26 indicated that smoking interferes with cytokine production . it has also been reported that release of cytokines from peripheral neutrophils and various parameters of inflammation in plasma seem to be affected more by cigarette smoking than periodontal disease.62 such alterations in host response may affect the reparative and regenerative potential of the periodontium in tobacco smokers . in the literature it has been identified that smoking is an important factor to affect erythrocytes and related parameters.63,64 in the present study , our first aim was to detect the effect of smoking on acd in the existence of chronic periodontitis . but further studies are needed that support the findings of our study with these measurements . the current study indicates periodontitis also needs to be considered as a chronic disease and together with the effect of cigarette smoking it may cause lower numbers of erythrocytes and the levels of hemoglobin , hematocrit and iron . the bmi measures were also collected due to well recognized effect of adiposity on systemic host response.65,66 nishida et al67 suggested that the immunological disorders or inflammation might be the reason that obese smokers tend to exhibit escalating poor periodontal status relative to non - obese and non - smoking individuals . because of that obese patients were excluded from the study and also the difference between the groups was not significant . some of the studies interpreted the effect of cigarette smoking on the periodontium to be indirect and due to inadequate levels of oral hygiene and increased plaque accumulation among smokers relative to non - smokers.12,68,69 in this study , pi levels of s ( + ) were higher than s ( ) . the studies searching the effect of smoking on clinical parameters suggest that non - smokers have higher gi and bop values than smokers.3,6,15 but , there are conflicting results those show no significant difference between smokers and non - smokers70 and smokers have higher values than non - smokers.71 pucher et al72 reported that gi and bop values were similar in smokers and non - smokers 9 months after periodontal therapy . in this study , however , the effects of nicotine has been disputed , some claim that the blood flow is reduced73 and others claim it is significantly increased74 or unchanged.75 the studies which show that pd values were higher in smokers than non - smokers are also present in literature.3,5,12,16,69,70,76,77 a significant positive correlation has been shown between smoking and cal.11,15,69,70,77,78 in the present study , pd and cal values were also higher in s ( + ) than s ( ) . the reason of increased pd and cal levels in smokers may be depend on accumulation of dental plaque and poor oral hygiene.68,69,79 in s ( + ) group , it was found negative correlation between cal and the level of rbc in serum values and positive correlations with mch and mchc . in this study group , it was found positive correlations between mean pd values and the levels of mcv , mch and mchc in serum . according to the results of this study , it seems that smoking is an important factor to affect erythrocytes and related parameters . from the results of the present study , it can be stated that cigarette smoking could have an effect on the numbers of erythrocytes and the levels of hemoglobin hematocrit and iron in serum . additionally , although cigarette smoking does not affect the degree of gingival inflammation , the amount of dental plaque , probing depth and clinical attachment loss were higher in smokers .

even though laryngotracheal strictures that are due to inhalation injuries frequently occur , the current treatment options for this medical condition are limited . the management of laryngotracheal strictures that are caused by damage to the tracheal tissue from heat and intubation remains challenging . the acute treatment goal is preservation of airway access , while the later treatment focuses on restoring a proper laryngotracheal lumen ( 1 ) . slide tracheoplasty has several advantages for reconstructing a stenotic segment of the trachea , and especially in children with congenital long - segment tracheal stenosis . the primary advantage of this technique is reduction of the tension on the anastomosis ; this alleviation of pressure is obtained by reducing the approximation distance . furthermore , the vascularity of the tracheal cartilage is preserved , and this allows for better healing . a few articles have recently suggested that slide tracheoplasty could be a useful method for treating upper tracheal stenosis in adults ( 2 - 4 ) . we report here on a case of a patient who had recurrent upper tracheal stenosis after an inhalation injury . repetitive symptoms of stenosis were present even after balloon dilatation , several bouts of laryngomicrosurgery ( lms ) and resection with end - to - end anastomosis surgery . she had a history of endotracheal intubation for a week to maintain her airway after an inhalation burn , and she then developed dyspnea after the treatment . prior to the inhalation injury , she had no family history of disease or any notable medical conditions . on physical examination , the mobility of both vocal folds was found to be intact ; however , we observed upper tracheal stenosis . the patient 's airway was severely compromised with less than 30% of the normal function due to the stenosis . a neck computed tomography ( ct ) scan showed a tracheal stenosis lesion around the 6th cervical spinal level . the narrowest diameter of the lesional trachea was about 0.7 cm and the lesion length was about 3 cm . slight enhancement was also visible on the stenotic lesion , which most likely due to granulation tissue . the 10 mm balloon dilatation was initially successful , but further procedures could not be performed because of the patient 's unstable vital signs . we decided to perform a resection with end - to - end anastomosis in november 2002 . we performed a hyoid release and resection of a 2 cm segment of the trachea ( from the 2nd to 5th tracheal rings ) 2 months after the burn injury . tracheal ballooning and stent insertion were then performed , but the stent was removed after 1 month due to displacement . lms was performed next to remove the granulation tissue and mitomycin c was applied . in 2006 , upper tracheal stenosis due to a fibrous stricture of the tracheal mucosa was found . a neck ct scan revealed a narrowed airway with hour - glass - shaped tracheal rings on the 6th cervical spinal level ( fig . the narrowest diameter of the trachea was about 0.6 - 0.7 cm and lms then was performed two more times . five years after the inhalation injury , we performed slide tracheoplasty to treat the stenotic tracheal lesion . the procedure was performed under general anesthesia and the airway was secured using an endotracheal tube . additional dissection was performed 4 to 5 cm distal to the site of the stenosis . three circumferential narrowed tracheal rings were observed , and transection of the stenotic trachea was performed at the narrowest midpoint ; thick fibrous mucosal changes were observed . the proximal half of the trachea was split along its anterior wall , while the distal segment was split along its posterior wall . the right angle corners , where the vertical incisions met the transverse incision , were trimmed . on postoperative day 7 , we confirmed that the anastomosis site was healing and that the upper tracheal lumen was maintained with about 70% patency by a telescopy - guided examination . three months after the operation , the tracheal anastomosis area was visually healthy and the upper trachea was enlarged . objective improvement of the airway was confirmed on the ct image with using a 3d volume rendering technique at the levels 15 mm above and below the stenosis area . the preoperative selected airway volume of 1,082.6 mm had increased to 1,767.8 mm postoperatively ( fig . the patient has been followed up for 18 months and there has been no tracheal narrowing or symptoms of dyspnea . various factors can cause tracheal stenosis , including prolonged endotracheal intubation , severe inflammation , autoimmune disorders , iatrogenic disorders and inhalation burns . many factors can adversely affect a tracheal stenosis and increase the growth of granulation tissue in patients suffering with inhalation burns . the initial insults caused by an inhalation injury can resolve easily if no airway stricture is present ; however , if a transmural injury initially occurs or it occurs as a consequence of additional trauma caused by intubation , then granulation and scar tissue formation may occur , leading to stenosis ( 1 , 5 , 6 ) . the duration of the intubation and the motion of the intubation tube caused by the ventilator are also possible reasons why stenosis might occur . tracheal infection can also inhibit wound healing , causing granulation tissue to form as a result of an abnormal healing process ; this tissue can eventually develop into a stenosis ( 7 ) . in most patients , the tracheal inflammation subsides over a period of 3 to 5 yr , and the granulation tissue on the tracheal wall becomes firm . even though the optimal time for resection of a tracheal stenosis is unclear , we performed resection with end - to - end anastomosis surgery 2 months after the burn . it is possible that at this time point , the granulation tissue was still friable and that it still had great proliferative potential . these may be the main reasons for the failure of the resection with performing end - to - end anastomosis surgery . another possible reason for the failure may have been the presence of the narrow , hourglass - shaped airway and tracheal rings . schweizer et al . ( 8) mentioned that tension may tear an anastomosis , which is the weakest part of the trachea , resulting in an hour - glass shape after end - to - end anastomosis surgery ( 8 , 9 ) . selection of the type of treatment depends on the patient 's clinical status and the anatomical pattern of the stenosis . the technique of slide tracheoplasty was developed to repair supracarinal long - segment tracheal stenosis . this technique includes transecting the trachea in the mid - stenosis and performing an oblique side - to - side anastomosis ( 10 ) . this reduces the tracheal length , but it doubles the circumference , quadruples the cross - sectional area and decreases the resistance to airflow . this technique can be used for the management of acquired subglottic and upper tracheal stenosis with a relatively long segment ( 2 - 4 ) . in our case , the patient had already undergone resection with end - to - end anastomosis surgery and we could not perform that surgery again because her trachea was too short at this stage . the surgery was completed successfully and the patient 's postoperative checkup - findings and telescopy - guided examination indicated that the patient 's airway volume had increased as a result of the slide tracheoplasty . in conclusion , even though the optimal time for performing resection of a tracheal stenosis in inhalation burn patients is unclear , delaying the surgery for an appropriate period of time is important for fewer recurrences . slide tracheoplasty is a safe , effective procedure that can be used to successfully treat problematic recurrent stenotic upper tracheal lesions , and even in patients with a trachea that has been shortened by resection with end - to - end anastomosis .

age estimation is of great importance for the identification of victims of accidents and crimes . in case of living individual such as refugees and adopted children who have no acceptable identification documents , confirmation of chronological age postmortem age estimation involves analyzing the remains of bones and teeth directly and also by using radiographs . teeth show great resistance to postmortem alterations caused by humidity , high temperature , microbial activities , and mechanical forces . also , developmental and physiological changes of the tooth can be related to chronological age . hence , for these reasons teeth can be better predictors of age compared to bone . with advancing age , secondary dentin is deposited along the walls of the dental pulp chamber , leading to a reduction in the size of the pulp cavity . this age - related change can be evaluated from ground sections and from dental radiographs of the teeth . these methods are destructive in nature and time consuming . also , they may not be acceptable for ethical , religious , cultural , and scientific reasons . dental radiography is a simple technique , used regularly in dental practice which can be employed in forensic age estimation as a nondestructive method . intraoral periapical radiographs have been employed for age estimation for various reasons . though age estimation using radiographs by kvaal and coauthors has shown to be reliable method , possible variations in ethnicity this has also been substantiated by the study conducted by babshet and coauthors in local population . essentially while age estimation using radiographs is undoubtedly a practical , convenient , and fairly accurate method ; questions remain on the uniformity of the results obtained while using a single formula for varied ethnicity . our study comprised of two parts . in the first part , we evaluated the accuracy of kvaal 's formula in sample indian population while comparing the results with those obtained by them in the norwegian population . in the second part of the study , we developed a new formula ( modified kvaal 's formula ) for a sample indian population by using the ratios from first part of the study and tested it again on another group of subjects and compared the results obtained by using kvaal 's formula and modified kvaal 's formula to establish the need for local indian - based formulae . our study comprised of two parts . in the first part , we evaluated the accuracy of kvaal 's formula in sample indian population while comparing the results with those obtained by them in the norwegian population . in the second part of the study , we developed a new formula ( modified kvaal 's formula ) for a sample indian population by using the ratios from first part of the study and tested it again on another group of subjects and compared the results obtained by using kvaal 's formula and modified kvaal 's formula to establish the need for local indian - based formulae . the 200 study subjects included were males / females between the age group 20 and 50 years visiting the department of oral medicine and radiology , for routine check - up . exact chronological age of the patient was recorded with date of birth , as per patient 's information . in the first part of the study , intraoral periapical ( iopa ) radiograph of maxillary central incisors was taken from ( 1 - 100 ) 100 subjects using paralleling cone technique to avoid angulations changes . these iopa radiographs were digitized using hp scanner ( x - ray scanner a3 ) , and images were transferred to computer . length and width of the teeth were measured using image - pro plusii software ( media , cybernetics , usa ) [ figures 15 ] . pulp width at three different levels root width at three different levels length of the toothlength of the rootlength of the pulppulp and root width at three different levels : at the enamel cementum junction ( ecj)at the midpoint between the ecj and mid root levelat the mid root level . pulp and root width at three different levels : at the enamel cementum junction ( ecj)at the midpoint between the ecj and mid root levelat the mid root level . at the enamel cementum junction ( ecj ) at the midpoint between the ecj and mid root level at the mid root level . ratios were calculated based on measurements taken in order to avoid errors due to differences in magnification of the image on the radiograph . the ratios calculated were : p = pulp length / root lengthr = pulp length / tooth length . . ratios of the pulp / root width at three different levels : at the ecj ( a)at the midpoint between ecj and mid root level ( b)at the mid root level ( c ) . at the midpoint between ecj and mid root level ( b ) at the mid root level ( c ) . the obtained values were applied to the formula developed to estimate the age from maxillary central incisor given by kvaal and coauthors . the estimated age was compared with the chronological age recorded and the efficacy of the formula in estimating the age was evaluated . it was found that the kvaal 's formula produced less accurate results in our population when compared to the results obtained by them in norwegian population . in the second part of the study , modified kvaal 's formula the obtained formula is as below : age = 33.5 - 18.6 ( m ) -3.49 ( w l ) m = mean value of all the ratios excluding t ( t = tooth length ) w = mean value of width ratios from level b and c l = mean value of length ratios p and r w l = differences between w and l the study was repeated by applying modified kvaal 's formula on another 100 subjects ( 101 - 200 ) to compare its efficacy against the kvaal 's formula in indian population . length of the toothlength of the rootlength of the pulppulp and root width at three different levels : at the enamel cementum junction ( ecj)at the midpoint between the ecj and mid root levelat the mid root level . pulp and root width at three different levels : at the enamel cementum junction ( ecj)at the midpoint between the ecj and mid root levelat the mid root level . at the enamel cementum junction ( ecj ) at the midpoint between the ecj and mid root level at the mid root level . ratios were calculated based on measurements taken in order to avoid errors due to differences in magnification of the image on the radiograph . the ratios calculated were : p = pulp length / root lengthr = pulp length / tooth length . . ratios of the pulp / root width at three different levels : at the ecj ( a)at the midpoint between ecj and mid root level ( b)at the mid root level ( c ) . at the midpoint between ecj and mid root level ( b ) at the mid root level ( c ) . the obtained values were applied to the formula developed to estimate the age from maxillary central incisor given by kvaal and coauthors . the estimated age was compared with the chronological age recorded and the efficacy of the formula in estimating the age was evaluated . it was found that the kvaal 's formula produced less accurate results in our population when compared to the results obtained by them in norwegian population . in the second part of the study , modified kvaal 's formula the obtained formula is as below : age = 33.5 - 18.6 ( m ) -3.49 ( w l ) m = mean value of all the ratios excluding t ( t = tooth length ) w = mean value of width ratios from level b and c l = mean value of length ratios p and r w l = differences between w and l the study was repeated by applying modified kvaal 's formula on another 100 subjects ( 101 - 200 ) to compare its efficacy against the kvaal 's formula in indian population . the correlation between the age and ratios b ( pulp and tooth width ratio at the midpoint between ecj and mid root level ) , c ( pulp and root width ratio at the mid root level ) , and w ( mean value of width ratios from level b and c ) were highly significant . the correlation between the age and ratios a ( pulp and root width at the enamel cement junction ) , p ( pulp length / root length ) , r ( pulp length / tooth length ) , l ( mean value of length ratios p and r ) , and m ( mean value of all the ratios excluding t ) were significant [ table 1 ] . correlation coefficients between chronological age and ratios of measurements from dental radiographs and mean of the ratios from either of maxillary central incisor , n=100 ( 1 - 100 ) in the first part of the study these ratios were applied to age estimation formula using maxillary central incisor given by kvaal and co - authors . standard error of estimated age ( 12.3 years ) from sample indian population [ table 2 ] was more compared to standard error of estimated age ( 9.5 year ) in norwegian population as the study conducted by kvaal and coauthors . age estimation of the sample indian population using kvaal 's formula ( subjects 1 - 100 ) in the second part of the study , modified kvaal 's formula for sample indian population was derived . that is , age = 33.5 -18.6 ( m ) -3.49 ( w l ) using another 100 subjects same ratios were calculated and applied to the modified kvaal 's formula and age estimation was done . standard error of estimated age of these subjects was 6.5 years [ table 3 ] . standard error of estimated age by modified kvaal 's formula ( 6.5 years ) was less when compared to standard error of estimated age of kvaal and coauthors formula in norwegian population ( 9.5 years ) . standard error of estimated age by modified kvaal 's formula ( 6.5 years ) was less when compared to standard error of estimated age of kvaal and coauthors formula ( 12.3 years ) in sample indian population [ table 3 ] . estimation of the human age is a procedure adopted by anthropologists , archeologists , and forensic scientists . for age estimation , different methods are available ; however , invasive methods using extracted teeth , ribs , or femur can not be used in living individuals . radiographic age estimation methods using teeth are advantageous when compared to other methods , by being noninvasive and providing collectability of data and simplicity in estimation . kvaal and coauthors developed an age estimation method by using measurement of six teeth ( maxillary central incisor , maxillary lateral incisor , maxillary second premolar , mandibular lateral incisor , mandibular canine , and first premolar ) observed on orthopantomogram ( opg ) or periapical radiographs . the measurement included length and width ratios such as pulp - root length ( p ) , pulp - tooth length ( r ) , tooth - root length ( t ) , pulp - root width at ecj ( a ) , pulp - root width at mid - root level ( c ) , pulp - root width at midpoint between level c and a ( b ) , mean value of all the ratios excluding t ( m ) , mean value of width ratios b and c ( w ) , mean value of length ratios p and r ( l ) . these ratios are used in order to compensate for magnification and angulations errors of teeth and the radiograph . age estimation formula was formulated by using six teeth , three teeth , and also individual teeth . the present study was conducted to analyze applicability of age estimation formula of kvaal and coauthors on sample indian population using maxillary central incisor with age groups 20 - 50 years , using iopa radiographs . since the error of age estimation was higher with kvaal 's formula , a new age estimation formula was derived from obtained length and width ratios . maxillary central incisor was selected because it is a single - rooted tooth with the largest pulp area , which is often present in old age . maxillary anterior teeth show considerably less crowding and attrition as compared to their mandibular counterpart , and contain more secondary dentin tissue than canines . intraoral periapical radiograph were used because they are fast , inexpensive , and routinely used in dentistry . ratios between the tooth and pulp measurements as suggested by kvaal 's and coauthors were calculated and used in the sample indian population . estimated age was found to have a standard error of 12.3 years , which was more when compared to standard error of 9.5 years in study conducted by kvaal and coauthors in norwegian population . ethnic differences and variation in the pattern of secondary dentine deposition in sample indian population have been considered to have large differences between estimated and actual ages . a recent study by babshet and coauthors highlighted the need for population specific equations due to differences in ethnicity . this lack of correlation can be attributed to the fact that with advancing age , secondary dentin is deposited along the wall of the dental pulp chamber leading to a reduction in the size of the pulp cavity . the quantity of secondary dentin deposition is influenced by factors like racial , ethnic , diet , and lifestyle . in the present study , correlation coefficient between the chronological age of the subjects 1 - 100 and width ratios b , c , and w were highly significant when compared to the length ratios . similar results were shown by and bosmansa et al . using the regression analysis of the data recorded a modified kvaal 's formula was devised , that is , age = 33.5 -18.6 ( m ) -3.49 ( w - l ) . then the modified kvaal 's formula was evaluated by applying the tooth , root , pulp length , and width ratios from the sample indian population and age estimation was done . when compared with chronological age , standard error of estimation was 6.5 years . standard error of estimated age by using modified kvaal 's formula was also less ( 6.5 years ) when compared to standard error of estimated age of kvaal 's formula in sample indian population ( 12.3 years ) . standard error of estimated age by modified kvaal 's formula was also less ( 6.5 years ) when compared to standard error of estimated age of kvaal 's formula used in norwegian population ( 9.5 years ) . from this study we conclude that kvaal 's age estimation formula developed on norwegian population , when applied to a different population such as sample of indian population , shows higher error of age estimation . hence , formula which was derived from norwegian population ( caucasian ) is not applicable to other population .

a model for the sequential generation of distinct cell types in the vertebrate cns . neural stem cells ( nscs ) from the rat embryonic brain give rise to progenitors that are restricted to neuronal or glial fates . in vitro treatment of glial - derived precursors ( grps ) with members of the bone morphogenetic protein ( bmp ) family of secreted signaling molecules drives their differentiation into a distinct subtype of astrocyte ( type 1 astrocyte , asti ) that promotes repair when transplanted to the injured adult spinal cord . in contrast , treatment of grps with the secreted protein sonic hedgehog ( shh ) , a member of a different family of signaling molecules , causes their differentiation into type ii astrocytes ( astii ) and oligodendrocytes .

considerable clinical evidence suggests that renin - angiotensin system ( ras ) inhibitors ( e.g. , ramipril and benazepril ) are beneficial as first - line agents for the treatment of hypertension in patients with chronic kidney disease ( ckd ) 1 - 4 . however , by themselves , ras inhibitors are unable to maintain an adequate blood pressure ( bp ) in these individuals . to maintain the bp , second - line depressor agents are therefore required . accordingly , dihydropyridine - type calcium channel blockers ( ccbs ) ( e.g. , felodipine and amlodipine ) are frequently used in combination with ras inhibitors in hypertensive patients with ckd because of their strong bp - lowering properties and minimal adverse effects 5 . for example , in the renoprotection in patients with nondiabetic chronic renal disease ( rein)-2 trial 6 , felodipine did not decrease the incidence of end - stage renal disease in ramipril - treated patients with ckd , despite further lowering the bp . furthermore , the gauging albuminuria reduction with lotrel in diabetic patients with hypertension ( guard ) trial 7 showed that the antialbuminuric effect of amlodipine was weaker than that of hydrochlorothiazide , a thiazide diuretic , in benazepril - treated hypertensive patients with type 2 diabetic nephropathy . in addition , urinary protein was not significantly decreased in a meta - analysis of the antiproteinuric effects of dihydropyridine ccbs 8 . on the other hand , certain ccbs , including benidipine ( a multifunctional l - type ( long - lasting)/t - type ( transient)/n - type ( neural ) ccb ) , cilnidipine ( a dual l - type / n - type ccb ) , and azelnidipine ( an l - type and sympatholytic ccb ) , have stronger antialbuminuric effects than others . for example , benidipine decreased urinary protein levels to a greater extent than amlodipine ( an l - type ccb ) in ras inhibitor - treated hypertensive patients with stage 3 - 5 ckd 9 . benidipine was also more effective than amlodipine in terms of reducing urinary albumin in patients with mild to moderate stage ckd and albuminuria 10 . a more pronounced antiproteinuric effect of benidipine compared to amlodipine has also been reported in hypertensive patients with early - stage ckd 11 . the above studies provide comparisons among ccbs , but not between ccbs and other types of antihypertensives , such as the thiazide diuretics . as noted above , the guard trial 7 indicated the inferiority of amlodipine versus hydrochlorothiazide in benazepril - treated hypertensive patients with type 2 diabetic nephropathy . however , there is a paucity of data concerning the non - inferiority of other so - called renoprotective ccbs such as benidipine relative to the antihypertensive diuretics . for this reason , we set out to clarify whether benidipine , with demonstrated renoprotective actions in patients with ckd 9 - 11 , could decrease urinary albumin with similar efficacy as hydrochlorothiazide . hydrochlorothiazide is often used together with an angiotensin receptor blocker ( abr ) and/or a thiazide diuretic in ckd patients . hence , the goal of this study was to examine the non - inferiority of benidipine compared with hydrochlorothiazide when administered to ras inhibitor - treated hypertensive patients with ckd and macroalbuminuria . a prospective , multicenter , open - labeled , randomized trial , the combination strategy on renal function of benidipine or diuretics treatment with ras inhibitors in chronic kidney disease hypertensive population ( cosmo - ckd ) trial , was performed in clinics and hospitals in japan from july 2009 to march 2013 . the trial was registered with the university hospital medical information network - clinical trials registry ( umin - ctr ) under trial identification number umin000002143 . the trial was approved by the institutional review board of the university of tokyo clinical research center ( reference number p2008042 - 11x ) and by the review boards of all other participating medical facilities . written informed consent was obtained from all participating patients after they received oral and written explanations about the study protocol . hypertensive patients with albuminuria under treatment with an ras inhibitor ( arb or angiotensin - converting enzyme ( ace ) inhibitor ) were recruited for this study . the inclusion criteria were : 1 ) outpatient systolic and diastolic bp readings of 130/80 mmhg ; 2 ) a pretreatment urinary albumin to creatinine ratio ( uacr ) ( the average of two measured values ) of 300 mg / g ; 3 ) an estimated glomerular filtration rate ( egfr ) 12 of 30 ml / min/1.73 m ; 4 ) between 20 and 80 years of age ; 5 ) a duration of antihypertensive treatment with ras inhibitors of 3 months prior to enrollment ; and 6 ) no treatment with ccbs or diuretics of any kind for at least 3 months prior to enrollment . the exclusion criteria were : 1 ) outpatient systolic and diastolic bp readings of 180/110 mmhg ; 2 ) a hypertensive emergency requiring intravenous administration of any antihypertensive agent ; 3 ) administration of an adrenocorticosteroid or an immunosuppressant , or long - term ( 2 weeks ) administration of nonsteroidal anti - inflammatory drugs ( nsaids ) ; 4 ) a past history of severe adverse reaction to ccbs , thiazide diuretics , arbs , or ace inhibitors ; 5 ) type 1 diabetes or type 2 diabetes requiring hospitalization due to high hemoglobin a1c content ( 9.0% ) , extremely high blood glucose , or diabetic ketoacidosis ; 6 ) cerebrovascular disease occurring within 6 months of enrollment ; 7 ) severe heart failure ( new york heart association ( nyha ) class iii ) , severe arrhythmia ( frequent ventricular or atrial extrasystole , prolonged ventricular tachycardia , atrial tachyarrhythmia with severe tachycardia , atrial fibrillation or flutter with severe tachycardia , sick sinus syndrome with severe bradycardia , or atrio - ventricular block with severe bradycardia ) , angina , or myocardial infarction within 6 months of enrollment ; 8) aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) levels of > 5 times the upper limit ; and 9 ) pregnancy , the possibility of pregnancy , or a desire to become pregnant . after confirming patient eligibility during the observation period ( figure 1 ) , each individual was randomly allocated to one of two groups , the benidipine group or the hydrochlorothiazide group . benidipine was initiated at 4 mg / day , followed by adjustment to 4 - 8 mg / day , while hydrochlorothiazide was initiated at 6.25 mg / day , followed by adjustment to 6.25 - 12.5 mg / day . each drug was given in combination with one or more ras inhibitors ( an arb and/or an ace inhibitor ) during the treatment period ( figure 1 ) , and the dose of the arb or the ace inhibitor was not changed during the treatment period . the following factors were used for stratified randomization : 1 ) systolic bp ( < 140 mmhg , 140 mmhg ) and 2 ) uacr ( < 1000 mg / g , 1000 mg / g ) . the target bp was < 130/80 mmhg . if benidipine or hydrochlorothiazide plus the ras inhibitor failed to reduce bp to the target level , additional antihypertensive drugs of a different type ( i.e. , not a ccb , diuretic , or ras inhibitor ) were administered . the primary endpoint was the change in the uacr ( mg / g ) from the baseline to the endpoint ( endpoint / baseline ratio ) . the uacr at endpoint was determined in spot urine samples after 12 months of benidipine or hydrochlorothiazide treatment , and the uacr at baseline ( the average of two consecutive measurements ) was determined during a 4-week pretreatment period . the resulting endpoint / baseline ratios were then compared between the benidipine and hydrochlorothiazide arms . the urinary albumin level was measured by using the bromcresol green photometric method ( iatrofine alb ii ) , and the urinary creatinine level was measured by using an enzymatic colorimetric assay ( iatrolq cre(a ) ii ) . secondary outcomes included the absolute values of the uacr at each time point ; the egfr , calculated according to the modified modification of diet in renal disease ( mdrd ) formula set forth by the japanese society of nephrology ( male : egfr ( ml / min/1.73 m ) = 194 age serum creatinine ; female : same as for male , with further multiplication by a factor of 0.739 ) 12 ; the ckd stage 13 ; urinary liver - type free fatty acid - binding protein ( l - fabp ) levels ; serum creatinine and blood urea nitrogen ( bun ) content ; bp readings ; renal events ( initiation of dialysis or renal transplantation ) ; cerebro - cardiovascular events ( cerebro - cardiovascular death ( fatal myocardial infarction , fatal heart failure , sudden death , fatal stroke , or death due to other cardiovascular causes ) or hospitalization due to a cerebro - cardiovascular disease ( nonfatal myocardial infarction , angina , heart failure , cerebral bleeding , cerebral infarction , or transient cerebral ischemic attack ) ) ; and other adverse events . all analyses were performed using sas version 9.1 ( sas institute , cary , nc , usa ) . subjects who did not meet the eligibility criteria , who were not administered the assigned drugs ( benidipine or hydrochlorothiazide ) , or who had no relevant data after randomization were excluded from the full analysis . all data are given as the mean standard deviation ( sd ) or the mean together with the 95% confidence interval . for the analysis of the non - inferiority of the antialbuminuric effect of benidipine versus hydrochlorothiazide when prescribed in combination with ras inhibitors , the value of the endpoint / baseline ratio of the uacr in the benidipine group was divided by that of the uacr in the hydrochlorothiazide group . one - hundred - and - seventy patients were required in each treatment group for a power of 80% with a significance level of 2.5% ( one - sided test ) to detect the non - inferiority of benidipine when the sd of the natural logarithm of the uacr was assumed to be 0.73 . with the added expectation of 15% of the patients withdrawing from the study , hypertensive patients with albuminuria under treatment with an ras inhibitor ( arb or angiotensin - converting enzyme ( ace ) inhibitor ) were recruited for this study . the inclusion criteria were : 1 ) outpatient systolic and diastolic bp readings of 130/80 mmhg ; 2 ) a pretreatment urinary albumin to creatinine ratio ( uacr ) ( the average of two measured values ) of 300 mg / g ; 3 ) an estimated glomerular filtration rate ( egfr ) 12 of 30 ml / min/1.73 m ; 4 ) between 20 and 80 years of age ; 5 ) a duration of antihypertensive treatment with ras inhibitors of 3 months prior to enrollment ; and 6 ) no treatment with ccbs or diuretics of any kind for at least 3 months prior to enrollment . the exclusion criteria were : 1 ) outpatient systolic and diastolic bp readings of 180/110 mmhg ; 2 ) a hypertensive emergency requiring intravenous administration of any antihypertensive agent ; 3 ) administration of an adrenocorticosteroid or an immunosuppressant , or long - term ( 2 weeks ) administration of nonsteroidal anti - inflammatory drugs ( nsaids ) ; 4 ) a past history of severe adverse reaction to ccbs , thiazide diuretics , arbs , or ace inhibitors ; 5 ) type 1 diabetes or type 2 diabetes requiring hospitalization due to high hemoglobin a1c content ( 9.0% ) , extremely high blood glucose , or diabetic ketoacidosis ; 6 ) cerebrovascular disease occurring within 6 months of enrollment ; 7 ) severe heart failure ( new york heart association ( nyha ) class iii ) , severe arrhythmia ( frequent ventricular or atrial extrasystole , prolonged ventricular tachycardia , atrial tachyarrhythmia with severe tachycardia , atrial fibrillation or flutter with severe tachycardia , sick sinus syndrome with severe bradycardia , or atrio - ventricular block with severe bradycardia ) , angina , or myocardial infarction within 6 months of enrollment ; 8) aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) levels of > 5 times the upper limit ; and 9 ) pregnancy , the possibility of pregnancy , or a desire to become pregnant . after confirming patient eligibility during the observation period ( figure 1 ) , each individual was randomly allocated to one of two groups , the benidipine group or the hydrochlorothiazide group . benidipine was initiated at 4 mg / day , followed by adjustment to 4 - 8 mg / day , while hydrochlorothiazide was initiated at 6.25 mg / day , followed by adjustment to 6.25 - 12.5 mg / day . each drug was given in combination with one or more ras inhibitors ( an arb and/or an ace inhibitor ) during the treatment period ( figure 1 ) , and the dose of the arb or the ace inhibitor was not changed during the treatment period . the following factors were used for stratified randomization : 1 ) systolic bp ( < 140 mmhg , 140 mmhg ) and 2 ) uacr ( < 1000 mg / g , 1000 mg / g ) . the target bp was < 130/80 mmhg . if benidipine or hydrochlorothiazide plus the ras inhibitor failed to reduce bp to the target level , additional antihypertensive drugs of a different type ( i.e. , not a ccb , diuretic , or ras inhibitor ) were administered . the primary endpoint was the change in the uacr ( mg / g ) from the baseline to the endpoint ( endpoint / baseline ratio ) . the uacr at endpoint was determined in spot urine samples after 12 months of benidipine or hydrochlorothiazide treatment , and the uacr at baseline ( the average of two consecutive measurements ) was determined during a 4-week pretreatment period . the resulting endpoint / baseline ratios were then compared between the benidipine and hydrochlorothiazide arms . laboratory tests were performed at a central laboratory ( mitsubishi chemical medicine inc . , tokyo , japan ) . the urinary albumin level was measured by using the bromcresol green photometric method ( iatrofine alb ii ) , and the urinary creatinine level was measured by using an enzymatic colorimetric assay ( iatrolq cre(a ) ii ) . secondary outcomes included the absolute values of the uacr at each time point ; the egfr , calculated according to the modified modification of diet in renal disease ( mdrd ) formula set forth by the japanese society of nephrology ( male : egfr ( ml / min/1.73 m ) = 194 age serum creatinine ; female : same as for male , with further multiplication by a factor of 0.739 ) 12 ; the ckd stage 13 ; urinary liver - type free fatty acid - binding protein ( l - fabp ) levels ; serum creatinine and blood urea nitrogen ( bun ) content ; bp readings ; renal events ( initiation of dialysis or renal transplantation ) ; cerebro - cardiovascular events ( cerebro - cardiovascular death ( fatal myocardial infarction , fatal heart failure , sudden death , fatal stroke , or death due to other cardiovascular causes ) or hospitalization due to a cerebro - cardiovascular disease ( nonfatal myocardial infarction , angina , heart failure , cerebral bleeding , cerebral infarction , or transient cerebral ischemic attack ) ) ; and other adverse events . all analyses were performed using sas version 9.1 ( sas institute , cary , nc , usa ) . subjects who did not meet the eligibility criteria , who were not administered the assigned drugs ( benidipine or hydrochlorothiazide ) , or who had no relevant data after randomization were excluded from the full analysis . all data are given as the mean standard deviation ( sd ) or the mean together with the 95% confidence interval . for the analysis of the non - inferiority of the antialbuminuric effect of benidipine versus hydrochlorothiazide when prescribed in combination with ras inhibitors , the value of the endpoint / baseline ratio of the uacr in the benidipine group was divided by that of the uacr in the hydrochlorothiazide group . one - hundred - and - seventy patients were required in each treatment group for a power of 80% with a significance level of 2.5% ( one - sided test ) to detect the non - inferiority of benidipine when the sd of the natural logarithm of the uacr was assumed to be 0.73 . with the added expectation of 15% of the patients withdrawing from the study , as noted above , 585 patients were recruited into the cosmo - ckd trial . of these 585 individuals , nineteen individuals were not administered an allocated drug and were therefore eliminated from the trial . data corresponding to the remaining 346 patients ( benidipine , n = 176 ; hydrochlorothiazide , n = 170 ) were used for the analysis of drug safety . however , only 344 of these individuals ( benidipine , n = 175 ; hydrochlorothiazide , n = 169 ) were included in the analysis of efficacy ( table 1 ) , because two patients ( benidipine group , n = 1 ; hydrochlorothiazide group , n = 1 ) did not meet the eligibility criteria . furthermore , only 277 of 346 subjects ( benidipine , n = 143 ; hydrochlorothiazide , n = 134 ) completed the study regimen . the patient characteristics at baseline ( n = 344 ) are shown in table 1 . baseline uacr was 930.8 ( 95% confidence interval , 826.1 , 1048.7 ) mg / g in the benidipine group and 883.1 ( 781.7 , 997.7 ) mg / g in the hydrochlorothiazide group ( figure 2a ) . after 12 months of drug treatment , the uacr values were reduced to 783.1 for benidipine and 383.6 mg / g for hydrochlorothiazide . the last observation carried forward ( locf ) value of the uacr was 790.0 ( 668.1 , 934.2 ) mg / g for benidipine and 448.5 ( 372.9 , 539.4 ) mg / g for hydrochlorothiazide , resulting in endpoint / baseline uacr ratios of 0.85 ( 0.76 , 0.95 ) ( benidipine group ) and 0.51 ( 0.44 , 0.58 ) ( hydrochlorothiazide group ) . the ratio ( benidipine / hydrochlorothiazide ) of the endpoint / baseline data sets was 1.67 ( 1.40 , 1.99 ) . thus , the non - inferiority of benidipine versus hydrochlorothiazide was not demonstrated ( figure 2b ) . serum creatinine levels were similar at baseline ( 0.961 ( 0.919 , 1.005 ) mg / dl , benidipine ; 0.967 ( 0.922 , 1.014 ) mg / dl , hydrochlorothiazide ) and at the locf ( 1.039 ( 0.986 , 1.096 ) mg / dl , benidipine ; 1.054 ( 0.998 , 1.114 ) mg / dl , hydrochlorothiazide ) . however , serum creatinine levels were slightly and similarly increased by both treatments ( figure 3a ) . by contrast , egfr values were also similar at baseline ( 57.91 ( 55.31 , 60.64 ) ml / min/1.73 m , benidipine ; 57.01 ( 54.05 , 54.05 ) ml / min/1.73 m , hydrochlorothiazide ) and at the locf ( 52.81 ( 49.94 , 55.84 ) ml / min/1.73 m , benidipine ; 51.55 ( 43.36 , 54.95 ) ml / min/1.73 m , hydrochlorothiazide ) ( figure 3b ) , but the egfr values were slightly and similarly decreased . table 2 shows that the distribution of ckd stages , as assessed by the egfr , was largely comparable between baseline and the locf for both drug groups . however , a marked increase ( from 1.2 ( 0.1 , 4.2)% at baseline to 10.1 ( 6.0 , 15.7)% at the locf ) was observed in the number of stage 4 patients after hydrochlorothiazide treatment . at the same time point , the number of stage 4 patients in the benidipine group increased from 1.2 ( 0.1 , 4.2)% at the baseline to 5.3 ( 2.4 , 9.8)% at the locf . bun values were similar between the two drugs at baseline ( 17.57 ( 16.85 , 18.32 ) mg / dl , benidipine ; 17.28 ( 16.49 , 18.11 ) mg / dl , hydrochlorothiazide ) and at the locf ( 18.14 ( 17.31 , 19.01 ) mg / dl , benidipine ; 18.84 ( 17.84 , 19.89 ) mg / dl , hydrochlorothiazide ) . however , although bun levels did not appreciably change over the course of the study in the benidipine group ( endpoint / baseline ratio , 1.02 ( 0.98 , 1.07 ) ) , they were slightly albeit significantly increased in the hydrochlorothiazide group ( 1.09 ( 1.05 , 1.13 ) ) . hence , the non - inferiority of benidipine was demonstrated in terms of bun content ( benidipine arm / hydrochlorothiazide arm , 0.98 ( 0.89 , 1.00 ) ) . urinary l - fabp values were also comparable between the two drug groups at baseline ( 15.20 ( 12.92 , 17.88 ) g / g , benidipine ; 13.94 ( 11.70 , 16.60 ) g / g , hydrochlorothiazide ) and at the locf ( 18.67 ( 15.47 , 22.53 ) g / g , benidipine ; 13.44 ( 11.22 , 16.11 ) g / g , hydrochlorothiazide ) . l - fabp levels showed a slight but significant increase in the benidipine group ( endpoint / baseline ration , 1.27 ( 1.10 , 1.46 ) ) , but not in the hydrochlorothiazide group ( endpoint / baseline ratio , 0.98 ( 0.84 , 1.13 ) ) . the non - inferiority of benidipine was not suggested ( benidipine arm / hydrochlorothiazide arm , 1.30 ( 1.06 , 1.59 ) ) . the systolic bp was 144.4 ( 142.5 , 146.2 ) mmhg for benidipine and 143.7 ( 141.9 , 145.6 ) mmhg for hydrochlorothiazide , and the diastolic bp was 83.6 ( 82.2 , 84.9 ) mmhg for benidipine and 84.5 ( 83.1 , 85.9 ) mmhg for hydrochlorothiazide , respectively . systolic and diastolic bp both decreased with drug treatment , and their decrements were similar between the two groups ( figure 4 ) . adverse events occurred in 65 of 176 patients ( 36.9% ) in the benidipine group and 74 of 170 patients ( 43.5% ) in the hydrochlorothiazide group . severe adverse events included one case of vitreous hemorrhage with aggravation of diabetic retinopathy and one case of cerebral bleeding in the benidipine group , and one case each of arteriosclerotic obliteration , gangrene of right third toe , and cerebral infarction with death in the hydrochlorothiazide group . the present study failed to demonstrate the non - inferiority of the antialbuminuric effect of benidipine versus hydrochlorothiazide in ras inhibitor - treated hypertensive patients with macroalbuminuria . the effect of benidipine appeared to be rather inferior to that of hydrochlorothiazide in terms of uacr ( figure 2b ) , even though the previous studies report that benidipine decreases urinary albumin / protein more efficaciously in patients with a wide range of ckd stages than other ccbs , such as amlodipine 9 - 11 . therefore , although we did not directly compare antialbuminuric actions between benidipine and amlodipine , the current investigation together with the previous data 9 - 11 suggest that the potency of benidipine to decrease urinary albumin might be intermediate between the less effective ccbs and the more effective thiazide diuretics . benidipine and hydrochlorothiazide decreased the egfr to a similar extent in the present cosmo - ckd trial , although its actions of both drugs on the kidney were fairly weak . on the other hand , amlodipine had a slightly but significantly lower propensity than hydrochlorothiazide to decrease the egfr in the guard study 7 . these results indirectly suggest that amlodipine , but not benidipine , exclusively dilates the afferent artery via l - type calcium channel blockade to increase glomerular pressure , as shown previously 14 . hence the blockade of t- and n - type calcium channels by benidipine , aside from its l - type calcium channel - blocking effects , may dilate both afferent and efferent arteries , leading to decreasing glomerular pressure , which might protect kidney . however , weaker antialbuminuric effect of benidipine than hydrochlorothiazide suggest that benidipine is less beneficial on the kidney , although both drugs did similarly reduce office systolic and diastolic bp . recently , the combination of losartan and hydrochlorothiazide decreased morning bp to a greater extent than high - dose losartan . furthermore , the effects of the thiazide diuretic to ameliorate circadian bp ( from a non - dipper pattern to a dipper pattern ) and to suppress proteinuria have been demonstrated in ras inhibitor - treated patients with immunoglobulin a nephropathy 16 . daytime salt retention is proposed to cancel the normal nighttime reduction in the bp ; nighttime high bp accelerates pressure natriuresis to excrete sodium retained during the day . however , diuretics are thought to attenuate daytime sodium retention , resulting in reduced nighttime bp . nonetheless , daytime and nighttime bp were not examined in the present study . at any rate , thiazide diuretics , which have a different mechanism of action from ras inhibitors to decrease urinary albumin , may confer greater renoprotective in combination with ras inhibitors than l-/t-/n - type ccbs such as benidipine , which has a similar renoprotective mechanism ( efferent arteriole vasodilation ) as ras inhibitors . another recent study 17 demonstrated the non - inferiority of the antialbuminuric effect of azelnidipine , another so - called renoprotective ccb 18 , compared with the thiazide diuretic , trichloromethiazide . because several different mechanisms have been attributed to the renoprotective ccbs ( e.g. , t - type calcium channel blockade ( benidipine ) , n - type calcium channel blockade ( benidipine and cilnidipine ) , and sympatholytic effects ( azelnidipine ) ) , the extent of antialbuminuric action might be different among these agents . in addition , the subjects of the present study and the above - mentioned study 17 showed different degrees of albuminuria ; 300 mg / g and 30 - 600 mg / g , respectively . recently , ogawa et al . demonstrated that the antialbuminuric effects of arb were weakened in patients with 1,000 mg / g of uacr 19 . thus , amelioration of glomerular microcirculation may not effectively decrease urinary albumin in ckd patients with the advanced renal dysfunction . therefore , the non - inferiority of benidipine versus hydrochlorothiazide might be observed in ras inhibitor - treated ckd patients with lower uacr values . another consideration is that the majority of the patients included in the present study had diabetes ( table 1 ) . macroalbuminuric patients with diabetes may have advanced diabetic vascular damage , which might also suppress the effectiveness of other renoprotective ccb 20 . in addition , there are few reports that compare the antialbuminuric effects between so - called renoprotective ccb and thiazide diuretics . moreover , future investigations are required to examine whether the antiproteinuric effect of benidipine and other renoprotective ccbs diuretic is non - inferior compared with thiazides in patients with microalbuminuria . first , uacr values in spot urine samples can vary with each measurement , even in the same patient with the same drug treatment . in this regard , measurement of urinary albumin excretion in samples collected over a 24-h period or repeated measurements of the first morning void sample may be more accurate than a spot sample . second , sample size estimation was done in the absence of previous reports comparing the antialbuminuric effects of renoprotective ccbs and thiazide diuretics . for this reason , we can not guarantee that the sample size was sufficient . in conclusion , the cosmo - ckd trial failed to demonstrate the non - inferiority of the antialbuminuric effect of benidipine relative to hydrochlorothiazide . several previous studies showed the renoprotective actions of benidipine in ckd patients with various stages of disease , as well as its potentially beneficial actions on glomerular microcirculation . thus , the renoprotective effect of benidipine might be limited compared with more potent thiazide diuretics , but more pronounced than that of other ccbs . further studies are required to clarify in the subset of ckd patients in which benidipine most effectively decreases urinary albumin content .

a 53-year - old man presented with a 10-year history of a nodule that had recently started growing and producing an itching sensation on the right occiput . a neck computed tomography scan suggested a benign soft tissue mass in the subcutaneous layer , so a local excision was performed . the tumor measured 2.61.71.5 cm , and there was no evidence of erosion or ulcer on the skin surface . the cut surface showed a relatively well demarcated white solid mass , which was located in the dermis and subcutaneous fat tissue . a histopathological examination revealed a nodular lesion composed of duct - like structures lined by cuboidal epithelium ( fig . 1a ) in the upper portion of the dermis and numerous nests of squamoid cells with amphophilic cytoplasm and keratinization in the deeper portion ( fig . the periphery of the lesion revealed more prominent squamoid differentiation and showed plump eosinophilic cytoplasm and keratinization with dense lymphocytic infiltration . most of the tumor cells were slightly pleomorphic ( variation of cellular and nuclear size , high n / c ratio , and hyperchromatic nucleoli ) , but focally more pleomorphism was seen in the infiltrating lesion . immunohistochemistry showed positive staining for cytokeratin ( ck ) , ck5/6 , ck7 , carcinoembryonic antigen ( cea ) ( fig . 2b ) but was negative for s-100 protein , gross cystic disease fluid protein-15 , and cd15 . the lateral resection margin was free , but the deep resection margin was very close to the tumor . after 5 months , the patient returned with a palpable mass at the previous excision site and on the right side of the neck . sono - guided fine needle aspiration of the right neck mass revealed a metastatic adenocarcinoma probably from an eccrine ductal adenocarcinoma of the skin . the dissected neck lymph nodes were involved with tumor cells and showed ductal structure and squamous differentiation ( fig . eccrine carcinomas uncommonly originate from eccrine glands . a review at the mayo clinic , covering more than 75 years of skin tumors , reported 14 cases of eccrine carcinoma.9 these carcinomas have a slow growth rate and a high potential for recurrence . diverse variants of eccrine carcinoma exist , including a variant with abundant fibromyxoid stroma , a spindle cell variant with myoepithelial differentiation , a basal cell carcinoma - like variant , and a sedc variant characterized by squamous metaplasia.1,7 classification of eccrine neoplasms is problematic because no universally accepted classification scheme exists , and the variable clinical appearance and histological features are often not predictive of clinical behavior.10 therefore , the diagnostic identification of sweat gland carcinomas is hampered by their rarity and their histological resemblance to various visceral tumors , leading to confusion with metastatic lesions . sedc shows a predilection for the head , neck , and extremities and presents most often in the middle - aged and elderly as a hard , usually nonulcerated cutaneous nodule . the histological features of sedc are duct - like structures resembling the dermal part of the eccrine duct and atypical squamous cell clusters . follicular or sebaceous differentiation can be seen.11 the histological differential diagnosis for sedc includes microcystic adnexal carcinoma ( mac ) with squamous features . the tumor cells express cea and ema.10,12,13 in our case , the absence of a keratinous cyst and a dense fibrous stroma surrounding tumor cells made it less likely that it was a mac . but if ductal differentiation is not obvious in shave or punch biopsy specimens , sedc may be mistaken for a scc . recent studies have reported that positive p63 and ck5/6 staining is relatively useful for distinguishing primary adnexal neoplasm with sweat gland differentiation from metastatic adenocarcinoma at various sites.14 p63 is a member of the p53 gene family and may be essential for regulating the differentiation of cells in normal human skin . p63 is normally expressed in basal and spinous cell nuclei of the epidermis , peripheral cells of the eccrine dermal ducts , germinative cells of sebaceous glands , and myoepithelial cells of the terminal portion of the eccrine and apocrine glands.15 ck5/6 is a basic high molecular weight ck . ck5/6 are normally expressed in stratified squamous epithelium , the basal - myoepithelial cell layer of the prostate , the salivary gland , and the breasts , as well as in myoepithelial cells of secretory glands and both layers of the epithelial cells of excretory ducts of eccrine and apocrine glands.16 in our case , the tumor cells were p63 negative but ck5/6 positive . however , a wide surgical excision with a wide negative margin ( with or without mohs 's technique ) is the treatment of choice . a high rate of local recurrence and subsequent metastasis after conventional surgical excision of eccrine carcinoma is observed . in our case , the significance of squamous differentiation in eccrine carcinoma is unclear.17 some believe that it shows more aggressive biological behavior , whereas others believe that it is an incidental finding and has no impact on disease biology . a study by kohda et al.17 reported that both benign and malignant eccrine neoplasms had foci of squamous differentiation .

the first land plant rooting structures comprised systems of tip - growing filamentous cells ( rhizoids ) . comparing the genetic mechanism that controls the development of filamentous rooting cells ( rhizoids and root hairs ) among different groups of land plants allows us to reconstruct the mechanism that controlled the development of these first land plant rooting systems . to identify genes required for the growth of rhizoids in one of the earliest diverging taxa of land plants , we generated a mutant population of 336,000 lines by transforming germinating marchantia polymorpha spores with the pcambia1300 t - dna vector and screened for plants with defective rhizoid morphology ( drm ) . 301 drm mutants were isolated ( table s1 ) ; 165 mutants were crossed to wild - type and the mutant phenotype was inherited in the f1 generation , whereas 136 were not successfully crossed ( table s1 ) . the approximate 1:1 segregation of wild - type to drm mutant rhizoid phenotypes in the f1 generation for each of the 165 inherited mutants indicated that the drm phenotypes were caused by single nuclear mutations ( table s2 ) . the drm mutant rhizoid phenotype co - segregated with the hygromycin resistance encoded by the hygromycin phosphotransferase gene on the t - dna in 62 of the 165 inherited mutant lines ( table s2 ) . this is consistent with the hypothesis that the insertion of a t - dna carrying a functional hygromycin resistance gene caused the mutation that resulted in defective rhizoid growth in 37% of the inherited mutants ( table s2 ) . to identify t - dna insertion sites , we first generated a draft assembly of the m. polymorpha genome . because the plants used in the mutant screen grew from spores generated in a cross between wild - type male ( takaragaike-1 [ tak-1 ] ) and female ( tak-2 ) accessions , dna was isolated from tak-1 and tak-2 plants , pooled , and sequenced . illumina hiseq technology was used to generate 84,554,420 short - insert paired - end reads and 32,963,957 long - insert paired - end reads . the draft genome comprised 4,137 scaffolds with a total scaffold length of 206 mb ( data s1 ) , scaffold n50 length of 376 kb , and estimated coverage of 64 ( data s1 ) . to identify protein - coding genes in this draft genome , we sequenced , assembled , and mapped an m. polymorpha gametophyte transcriptome onto the genome assembly . the transcriptome was generated using pooled rna isolated from mature dorsal thallus epidermis ( excluding midrib region and gemma cups ) , the meristematic zone , rhizoids , and 0- and 1-day - old gemmae . rna was sequenced using illumina hiseq in 183,475,609 short - insert paired - end reads and assembled into contigs ( data s1 ) ; 29,453 gametophyte - expressed contigs were mapped to the genome assembly . the whole - genome shotgun assembly ( ddbj : lvlj00000000 ) and transcriptome shotgun assembly ( ena : gefo00000000 and genbank : gefo01000000 ) have been deposited at the dna data bank of japan , european nucleotide archive , and genbank . the genomic locations of 57 of the 62 t - dnas linked to drm mutations were identified by thermal asymmetric interlaced ( tail ) pcr ( data s2 ) . the t - dna insertion sites of the 57 drm mutants were distributed among 31 different genes ( figure s1 ) . tail pcr was also carried out on drm mutants that were sterile and could not be crossed , and this resulted in identification of the three alleles of mpcsld1 and two alleles of mpscd . mpalba-3 , mpemb2756 - 2 , mpexl-1 , mppi4ka1 - 5 , mppti-2 , mpsri1 - 1 , mpsri3 - 2 , and mpxut-3were identified by sequencing dna flanking t - dna insertions in sterile drm mutants . the mpire mutation was complemented with a transgene expressing the wild - type mpire - coding sequence ( figure s3 ) . phylogenetic analysis was conducted to assign putative functions and identify related genes in arabidopsis thaliana ( table 1 ) . trees were constructed with maximum - likelihood statistics using protein sequences predicted from the m. polymorpha transcriptome assembly and published a. thaliana genome ( figure s2 ; data s3 and s4 ) . in total , we identified between one and five alleles in 33 genes ; multiple independent mutant alleles were identified for 17 genes , and single alleles were identified for 16 genes . mpcellulose synthase - like class d 1 ( mpcsld1 ) , mpcsld2 , mpxyloglucan - specific galacturonosyltransferase 1 ( mpxut1 ) , mpgdp - mannose pyrophosphorylase ( mpgmp ) , and mprhamnose biosynthesis 1 ( mprhm1)encode proteins that are predicted to function in the synthesis of cell wall polysaccharides . mpcsld1 , mpcsld2 , mpxut1 , mpgmp , and mprhm1develops shorter rhizoids than wild - type , and mpcsld1 and mpxut1 mutant rhizoids also burst at their tips ( figure 1 ; table 1 ) . atcsld3 , atxut1 , and atrhm1are expressed in root hairs and required for root hair growth because atcsld3 , atxut1 , and atrhm1 mutants develop short root hairs [ 3 , 4 , 5 , 6 , 7 ] . a role for atgmp1 ( at2g39770 ) in root hair development has not yet been defined . this is most likely because loss of atgmp1 function is lethal and mutants do not survive to the stage where root hairs develop . taken together , these data demonstrate that the same molecular mechanism for wall synthesis operates in m. polymorpha rhizoids and a. thaliana root hairs . the sensing of cell wall integrity requires a signal transduction cascade that has been defined in a. thaliana . receptor kinases in the catharanthus roseus receptor kinase 1-like ( crrlk1l ) subclass are required for cell wall integrity sensing in tip - growing cells [ 9 , 10 , 11 ] . maximum - likelihood phylogenetic trees were constructed using crrlk1l protein sequences from a. thaliana and m. polymorpha ( figure s2 ) . there are 17 crrlk1l family members in a. thaliana , which include attheseus ( atthe ) and atferonia ( atfer ) . there is a single member of this family in m. polymorpha that we designated mpthe because it is more similar to atthe than to any other a. thaliana protein in this family ( figure s2 ) . mpthe mutants develop short and irregularly shaped rhizoids , indicating that the mpthe protein is required for rhizoid elongation ( figure 1b ) . to test the hypothesis that the mpthe protein controls cell wall integrity in m. polymorpha , we examined rhizoid tips in mpthe mutants for evidence of bursting . the tips of mpthe rhizoids are brown as a result of rhizoid rupture during elongation and do not reach the same length as wild - type ( figure 1b ) . the defective elongation and cell integrity phenotypes of mpthe and atfer mutant rhizoids and root hairs , respectively , suggest that crrlk1l proteins carry out similar functions in cell wall integrity signaling in both species and in their last common ancestor . the atmaris pti kinase is a receptor - like cytoplasmic kinase ( rclk ) required for cell wall integrity signaling , and acts downstream of crrlk1l proteins in a. thaliana . m. polymorpha has a single pti protein that is sister to a group of six a. thaliana pti proteins that includes maris . mutants with defective mppti function develop short rhizoids ( figure 1a ) , just as mutants that lack atmri activity develop short root hairs in a. thaliana . taken together , these data suggest that at least some of the components associated with wall integrity sensing rlck and crrlk1l proteins have been conserved since m. polymorpha and a. thaliana last shared a common ancestor . ten of the 33 genes identified in this screen encode proteins involved in vesicle transport or cytoskeleton function ( table 1 ; figure 2 ) [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] . close homologs of nine are highly expressed in root hairs , and three of these are required for root hair growth in a. thaliana . mutations in the gene encoding the plant - specific class xi myosin , mpxi , result in the development of short rhizoids ( table 1 ; figure 2 ) , just as triple and quadruple myosinxi mutants develop short root hairs in a. thaliana [ 16 , 17 ] . mpren is a predicted rop - gap protein ( table 1 ; figure 2 ) , and because rops control microfilament dynamics in a. thaliana [ 24 , 25 , 26 , 27 ] it is likely that this protein modulates microfilament dynamics during rhizoid growth . consistent with this role is the observation that mp wave dampened - like ( mpwdl ) mutants developed wavy - shaped rhizoids similar to oryzalin- or taxol - treated root hairs ( table 1 ; figure 2 ) . mpzwi rhizoids are shorter than wild - type , indicating that the zwichel - like ( zwi ) kinesin motor is required for rhizoid growth ( table 1 ; figure 2 ) . whereas homologs of mpwdl and mpzwi have not been shown to be required for root hair growth in a. thaliana , the expression of atzwi and two atwdl genes ( at2g35880 and at4g32330 ) is enriched in root hairs ( arabidopsis efp browser 2.0 [ 29 , 30 ] ) compared to other root cells , suggesting that they are active during root hair growth . three genes were identified that encode proteins predicted to be involved in endocytosis but that have not been functionally characterized in green plants to date . their predicted function is based on the roles of similar proteins in yeast and mammals ( table 1 ; figure 2 ) . mpsri2 ( mp short rhizoids2 ) encodes an ef - hand - containing protein that is similar to s. cerevisiae pan1 . pan1 is required for association of the arp - actin polymerization complex with clathrin - coated vesicles during endocytosis in yeast . mp short rhizoids1 ( mpsri1 ) encodes a protein similar to s. cerevisiae ric1 , which is a guanine exchange factor involved in activating ypt6p gtpase and required for trafficking from early endosomes to the golgi late in the endocytosis pathway . mpap5 m is predicted to encode the subunit mu of the adaptor protein 5 ( ap5 ) complex . ap5 is a tetrameric protein complex that coats vesicles acting as a cargo adaptor complex and is likely to be involved in endocytosis , but its precise function remains to be defined in any organism . mpsri3 encodes a protein found in green plants but whose function has not been defined ; it is similar to atemb2756 , which mutates to an embryo lethal phenotype ( table 1 ; figure s4 ) [ 31 , 32 ] . mpsri4 is similar to the yeast efr3 gene that codes for a protein required for the localization of phosphatidylinositol-4-phosphate ( pi4 ) kinase alpha at the plasma membrane , but no similar genes have been functionally characterized in green plants ( table 1 ; figure s4 ) . consistent with this hypothetical role is the observation that rhizoid development is also defective in mutants in which the pi4 kinase alpha is defective ( table 1 ) . alba proteins are nucleic - acid - binding proteins that form chromatin in archaea and bind rna in a number of animal parasites [ 34 , 35 , 36 ] . not only is mpalba required for rhizoid development because mpalba mutants develop short rhizoids but we discovered that loss - of - function alba mutants in a. thaliana develop shorter root hairs than wild - type ( table 1 ; figure 3 ) . this indicates that alba proteins are required for tip growth in both m. polymorpha and a. thaliana , and therefore are likely to be required for tip growth in rhizoids or root hairs throughout the land plants . these data demonstrate that genes in the same orthogroups control the synthesis of new cell surface in liverwort rhizoids and angiosperm root hairs . this conservation suggests that this mechanism acted during the growth of the first land plant rooting structures at or soon after the colonization of the land by streptophytes . these data also indicate that some of these genes such as the and pti were co - opted during the evolution of pollen tubes , one of a suite of traits that evolved during the evolution of the seed plant life cycle . some genes previously shown to be involved in root hair growth have not been identified in this screen . this may be because the screen was not carried out to saturation and other rhizoid development genes remain to be discovered . furthermore , many arabidopsis gene families or subfamilies that contain genes implicated in root hair growth were present as a single - copy gene in m. polymorpha . therefore , the m. polymorpha homologs of some genes involved in root hair growth are likely to have more general developmental roles than their arabidopsis counterparts , and consequently result in severe growth defects or lethality when mutated . moreover , it is likely that the function of some genes involved in root hair growth diversified in the lineage leading to the tracheophytes after the divergence of the last common ancestor of liverworts and angiosperms . such divergence of function is supported by the observation that the phenotypes of some loss - of - function mutants in genes from the same orthogroup are different in m. polymorpha and a. thaliana . these data are consistent with the hypothesis that the evolution of the land plant body and life cycle involved a core set of genes with conserved functions that were active in the earliest land plants and underwent duplication followed by neofunctionalization . these novel functions programmed the development of novel structures and contributed to increased life cycle diversity during the subsequent radiation of land plants . s.h . screened 150,000 t - dna lines and identified 38 tagged mutants ; v.a.s.j . screened 105,000 lines and identified 8 tagged mutants ; g.m . screened 81,000 lines and identified 15 tagged mutants ; g.m . and h. proust isolated dna for genome sequencing and rna for rna sequencing ; c.c . and a.j.h . to determine co - segregation of 4 mutants and carried out tail pcr ; h. prescott established all m. polymorpha growth and transformation protocols ; s.h . comments from other authors ; genes were grouped according to the classification established by s.h . ; and l.d . conceived and designed the project .

these are usually patients who are seriously ill with sepsis or children and adults with cardiovascular infections and paucibacillary disease . appropriate antibiotic treatment is based on isolation , identification , and antimicrobial susceptibility pattern of an organism isolated from blood culture . the conventional blood culture methods currently being used are time consuming , labor intensive , and suffer from poor sensitivity due to the lack of use of nutritionally enriching substances , inability to neutralize the inherent antimicrobial components in blood and/or patients receiving antimicrobial treatment . in patients who are receiving antimicrobials , various approaches have been employed that remove antimicrobials from blood or otherwise render the drugs inactive . bactec plus media are reported to effectively remove all penicillins , most cephalosporins , and vancomycin . they utilize ion exchange and nonionic adsorbent resins to remove antimicrobials thereby enhancing the recovery of organisms . bactec 9050 is a fully automated system , which monitors increase in co2 concentration produced by growing microorganisms by means of a fluorescent sensor present at the bottom of the bottle . being a fully automated system for detecting growth , it also has the advantage of earlier detection time . various studies have demonstrated the higher isolation rate of bactec automated system over conventional culture methods . ours is a large tertiary care multi - specialty teaching hospital where patients are more likely to be on antibiotics at the time of blood collection because patients are either referrals from other public or private health care settings with very few direct walk - ins . hence , this study was undertaken to determine the utility of bactec 9050 in our setting by comparing its recovery rate and time to detection with the existing conventional blood culture . a prospective study was conducted in the department of microbiology from march 2011 to june 2013 after obtaining permission from institutional review board . all patients in whom blood culture was clinically indicated and who provided written informed consent were included . indications for blood culture were suspected cases of sepsis , meningitis , osteomyelitis , arthritis , endocarditis , peritonitis , pneumonia , and fever of unknown origin . age - specific volume of blood was collected aseptically from patients , such that each system was inoculated with 810 ml in adults , 35 ml in children aged 212 years old , 23 ml in infants , and toddlers aged 1 month to 2 years and 1 ml in neonates and corresponded to manufacturer 's instructions . we have also included specimens other than blood such as cerebrospinal fluid , pericardial fluid , ascitic fluid , synovial fluid , pleural fluid , pus , bronchoalveolar lavage , tissue , bone marrow aspirate , and bile . for specimens other than blood , where volume was at least 2 ml , each was divided equally into two and inoculated into peds plus bactec vials and conventional pediatric blood culture bottle . clinical details of the patients were recorded with respect to age , gender , diagnosis at investigation , ward , unit , and present or recent past antibiotic treatment . for each isolate recovered , a dialogue with the clinician was established for its pathogenic potential . conventional culture bottles contained either brain heart infusion or trypticase soya broth with volumes of 50 ml in adults , 30 ml in children , and 10 ml in neonates and sodium polyanethol sulfonate at a concentration of 0.025 g/100 ml of broth . subcultures were made at the end of 24 h , 72 h , and 7 day of incubation on 5% sheep blood agar . if no growth was detected at the end of 7 days of incubation , it was reported negative . bd bactec 9050 ( becton and dickinson microbiology system , sparks , md , usa ) blood culture system is a continuous monitoring automated culture system . aerobic f and peds plus were incubated until signal - positive or till the end of day 5 , whichever was earlier . when positive signal was observed , bottles were unloaded from instrument and gram 's stain and cultures were performed as per standard microbiological protocol . an isolate was considered significant if the same organism was recovered from both the systems and was not a contaminant or a known pathogen was recovered from either of the systems and for other isolates recovered from only one of the two systems , after clinical consultation . growth was considered concordant if the same isolate was obtained in both the culture systems and discordant if both systems were culture positive but different isolates were recovered in each system . methicillin resistance in staphylococcus aureus ( mrsa ) spp . and vancomycin resistance in enterococcus spp . these strains were procured from microbiologics supplied by hi - media . the time to culture positivity , i.e. , time taken from incubation to growth detection for conventional and signal positive for bactec 9050 , was noted . mean time to detection ( mttd ) is the average of the time to culture positivity for all the isolates in a particular category . growth was considered false positive when a bactec 9050 vial was signal positive by instrument but negative on gram 's stain as well as culture after 72 h of incubation . data were analyzed for time to detection and isolation rate , by comparing both systems , bactec 9050 and conventional . the significance of the difference between the two systems was determined by the chi - square test , and p < 0.05 was considered significant . data were analyzed for time to detection and isolation rate , by comparing both systems , bactec 9050 and conventional . the significance of the difference between the two systems was determined by the chi - square test , and p < 0.05 was considered significant . from 835 patients , 835 paired specimens were processed of which 797 were blood [ table 1 ] and 38 were nonblood specimens [ table 2 ] . a total of 497 aerobic / f and 338 peds plus / f vials were used for the study . totally , 509 males and 326 females were included of which 62% ( 517 ) were adults , 22% ( 181 ) were children , and 16% ( 137 ) were neonates . percent positivity of bactec in adults , children , and neonates was 29.6% ( 153/517 ) , 32% ( 58/181 ) , and 38.7% ( 53/137 ) respectively whereas in conventional it was 21.7% ( 112/517 ) , 17.1% ( 31/181 ) , and 16.8% ( 23/137 ) , respectively . distribution of specimens and comparison of culture positivity ( n=835 ) the details of isolates recovered from specimens other than blood ( n=38 ) * nfgnb : non - fermenting gram - negative bacilli , cons : coagulase negative staphylococcus , e. coli : escherichia coli , s. aureus : staphylococcus aureus overall , culture positivity for bactec 9050 and the conventional system was 32% and 19.88% , respectively . monomicrobial growth was observed in 91.6% ( 242/264 ) and 96% ( 160/166 ) of positive bactec 9050 and conventional systems , respectively . culture positivity was higher both for blood ( 31% ) as well as for other specimens [ 42% , table 2 ] as compared to conventional ( 19.94% and 18.42% ) . the difference was found to be statistically significant ( p < 0.0001 and p = 0.02463 ) . of the cultured paired specimens , 533 specimens were culture negative by both systems , 85 demonstrated concordant growth , 43 demonstrated discordant growth , 136 specimens were culture positive by bactec only and 38 specimens were culture positive by conventional only . the same contaminant was recovered once from both systems . using bactec 9050 , significantly higher isolation [ table 3 ] , coagulase - negative staphylococcus ( cons ) , streptococcus spp . and candida spp . comparison of isolates recovered nfgnb : non - fermenting gram - negative bacilli , cons : coagulase negative staphylococcus , vre : vancomycin resistant enterococcus , s. aureus : staphylococcus aureus , b. cepacia : burkholderia cepacia , e. coli : escherichia coli a total of 410 patients were on antimicrobial treatment . culture positivity was 41.95% ( 172/410 ) by both methods or either of the methods [ table 4 ] . 18.29% ( 75 ) showed positive growth by bactec only and 3.90% ( 16 ) by conventional culture only . although a higher isolation was observed for most pathogens using bactec 9050 , none of them were statistically significant except in candida spp . however , a significantly higher rate of isolation by bactec 9050 was observed when all gram - positive cocci and gram - negative bacilli were grouped ( gpc , 58 vs. 33 , p = 0.00544 ; gnb , 80 vs. 60 , p = 0.06343 ) . comparison of isolates in patients on antimicrobial treatment n=410 nfgnb : non - fermenting gram - negative bacilli , cons : coagulase negative staphylococcus , vre : vancomycin resistant enterococcus , s. aureus : staphylococcus aureus of the 43 discordant paired specimens , in 12 , growth was polymicrobial by bactec and monomicrobial by conventional with recovery of one of the organisms being common to both systems . a contaminant in one of the two systems with a corresponding potential pathogen in other was observed in 4 ( 3 conventional and one bactec ) . in the remaining 27 , different pathogens were recovered by each system . using bactec , majority ( 173 ) of the culture positives [ table 5 ] were detected on / before day 2 whereas , during the same time , only three were culture positive by conventional method . comparison of time to detection nfgnb : non - fermenting gram - negative bacilli , cons : coagulase negative staphylococcus , vre : vancomycin resistant enterococcus , s. aureus : staphylococcus aureus , b. cepacia : burkholderia cepacia , e. coli : escherichia coli a comparison of mttd for those organisms that grew concordantly in both systems is depicted in figure 1 . it ranged from 15 h to 50 h by bactec 9050 and 40 h to 106 h by the conventional system . in both systems mean time to detections of concordant and concordant at least one organism similar in both system n = 85 there has been a continuous evolution of methodologies and principles to improve and increase the sensitivity for the detection of organisms in bloodstream infections . there is a need for every institute to evaluate the method , which is best suited for their setup . considering that ours is a tertiary care and referral center where a greater proportion of patients are likely to be on antimicrobials , we compared the fully automated bactec 9050 culture system having the capability of neutralizing or removing commonly used antibiotics , with the conventional culture system in terms of recovery rate , species wise significant recovery if any , contamination rate , mean time to detection including patients receiving antibiotics . as expected , bactec 9050 demonstrated a significantly higher rate of isolation overall ( 32% ) , in blood ( 31% ) , in nonblood specimens ( 42% ) , and in patients receiving antibiotic treatment ( 38.04% ) when compared with the conventional culture . however , in the group of patients with discordant growth , the isolation of two different potential pathogens , one in each of the two systems , made clinical correlation difficult and both organisms were reported to the clinician . maximum concordant growth between the two systems was observed when the isolate was a gnb . in this study , the reported reasons for culture negativity could be inappropriate timing of blood collection ( recommended is 3090 min before the fever spikes or in practice , as soon after the fever spike ) , inadequate volume ( each milliliter increase in volume of blood cultured increases recovery of organisms by 3% ) , concurrent administration of antimicrobials , presence of fastidious organisms , delay in transportation and processing , blood broth dilution ratio , number of culture sets , media used , use of anticoagulants ( sodium polyanethol sulfonate [ sps ] ) , duration of incubation , temperature of incubation , and use of agents to remove effect of antimicrobials . in this study , adequate volume , immediate transportation and processing , blood broth dilution ratio , use of sps , media used , and temperature of incubation were as per recommendation . forty - nine percent of patients had received antibiotics due to suspected diagnosis of sepsis or case mix before blood collection . bactec 9050 was used due to its capability to remove antimicrobials most commonly administered . in this study , which included adults , children , and neonates , this may be because of the magnitude of bacteremias in infants and children which is generally greater than in adults , a high index of suspicion in high - risk neonates and the practice of collecting specimen before starting antimicrobial treatment as a policy . the culture positivity reported in indian literature in neonatal sepsis ranges from 5.6% to 47% . various studies have highlighted the effectiveness of bactec 9050 system in terms of increase in organism yield , decrease in admission time , reduction in inappropriate antibiotic therapy , facilitating an early diagnosis and saving life by reducing morbidity . in the present study , overall culture positivity for bactec 9050 was 32% as compared to 19.88% by conventional system , which is similar to the findings reported in literature . bactec 9050 recovered any organism in an additional 136 specimens as compared to the conventional system , which was expected . however in 38 patients , only conventional culture was positive of which significant isolates were recovered in 25 , a finding not reported in most of the studies that have compared automated culture system with conventional . even though conventional system does not have resins to neutralize the antimicrobial effect in specimens , it was still able to detect organisms . contamination rate was 1.4% ( 12 ) by bactec and 1.07% ( 9 ) by conventional . the lower contamination rate in the present study may be attributed to the supply of alcohol - impregnated swabs that were provided along with the blood culture bottles . in this study , isolation of gram - negative bacteria was more as compared to gpc , which is similar to other studies . individual pathogen comparison between the two systems showed significantly higher recovery of acinetobacter spp , streptococcus spp , and cons [ table 3 ] by bactec 9050 . there was a significant difference in the mttd with bactec 9050 recovering 86.8% of isolates within 48 h. using bactec , the mean detection time was 31.12 h for all isolates as compared to 67.80 h by conventional method . other studies have reported a mean detection time of 1924 h hours by bactec and 57 days by conventional method . since bactec is a continuous monitoring system , detection occurs earlier and can be reported in hours . however , conventional blood culture , by its very nature of processing , can not be reported positive before 24 h and that too when a gram 's stained primary smear reveals the presence of microorganisms . based on culture , the earliest result would be at the end of 48 h. when mttd of concordant isolates were analyzed , it was observed to be 25.08 h using bactec and 67.56 h using conventional . in this study , only one streptococcus pneumoniae and one haemophilus spp . time to detect these fastidious organisms was 19 h and 50.83 h respectively by bactec and 48 h and 96 h respectively by conventional system . other studies have reported a recovery time from 10 h to 24 h for s. pneumoniae . one of the observations of the present study was that in 32 patients with native or prosthetic valve endocarditis , conventional blood culture recovered three isolates ( one each of s. pneumoniae , haemophilus influenzae , and enterococcus faecalis ) whereas bactec , in addition , recovered two candida spp . and two streptococcus spp . of 137 clinically suspected cases of neonatal sepsis , a significantly higher recovery ( p < 0.0001 ) was obtained using bactec 9050 ( 51 ) as compared to conventional culture ( 17 ) . in both systems , cons , klebsiella pneumoniae and candida spp . bactec 9050 recovered a significantly higher number of gnb ( 22 vs. 8 , p = 0.0068 ) and candida spp . ( 18 vs. 6 , p = 0.0103 ) as compared to the conventional system . cons recovered in blood are not considered clinically significant in some studies . of the 38 acinetobacter spp . isolated by bactec 9050 , 30 were recovered from icu patients at a risk of acinetobacter spp . a similar observation was seen with cons with only ten that could be considered as clinically nonsignificant . the type of setting , the presence of devices or lines and other risk factors should be considered while defining a contaminant rather than the type of organism . the cost per test ( not including the cost of equipment and manpower ) using bactec 9050 was rs . 400 ( age unrelated ) and using conventional culture ( cost of reusable bottles and media ) was approximately rs . . the limitations of this study are as follows . for cons to be considered significant in adults and children , the same species should be repeatedly isolated either from specimens collected from two different sites or at two different times which was not possible in the present study . however , only those that had the potential of association after clinical consultation have been classified as significant . cons were speciated ( staphylococcus warneri , staphylococcus lugdenensis , staphylococcus hemolyticus , staphylococcus epidermidis ) and staphylococcus saprophyticus has been classified as a contaminant . furthermore , actual cost benefit of earlier detection of organisms by bactec 9050 with respect to treatment , its outcome and length of stay has not been considered in the present study . by demonstrating a significantly higher recovery including in the group of patients receiving any antimicrobial , in less than half the time as compared to the conventional culture , bactec 9050 will be an investment worth the cost . at the same time , recovery by conventional culture even in patients receiving antimicrobials suggests that an appropriately performed blood culture with adequate volumes can provide the much - needed information in resource - constrained settings on the possible etiology and also provide the isolate which can be further tested to facilitate choice of therapy . the provision of skin disinfectant wipes along with blood culture bottles also reduces contamination rate .

osteosarcoma constitutes a small percentage of rare malignant non - epithelial neoplasms of the thyroid , and has been reported in only several dozen cases . one of the conclusive tests is immunohistochemical staining , which excludes epithelial differentiation of the tumour . we present a case of a female patient who underwent surgery due to a fast - growing tumour of the right thyroid lobe , with an unexpected final diagnosis . a female patient , aged 76 , was qualified for surgical removal of a tumour of the right thyroid lobe , within the substernal goitre , due to its rapid growth over a period of two months . the estimated size of the goitre , measured in cross - section through both lobes , was 9 6 cm . no qualifying biopsy was carried out before surgery , opting for radical strumectomy . with an incision at the base of the neck , having cut through the short muscles , the thyroid lobes were identified . the left lobe was the size of a plum , with nodular reconstruction , whilst the right lobe was the size of a large fist , expanding down the lower pole , past the sternum and pushing the trachea to the left ( approx . having freed the lateral parts of the lobes of the thyroid from the trachea , the small pyramidal lobe was removed . the early post - operative period was complicated by acute respiratory failure requiring intensive care treatment . the impairment of vocal cord mobility resulted in the necessity to insert a tracheotomy tube on the laryngology ward . the evaluation of the single - piece surgical material revealed a nodular tissue structure , with an irregular external surface and dimensions of 9 6 7.5 cm , without separated thyroid lobes . a correct thyroid texture was observed only in the left lobe , with a diameter of approx . the remaining part of the material , corresponding to the right thyroid lobe of a diameter of approx . 7 cm , was a greyish - white tumour of increased density , with extensive calcified foci . a high proliferative activity of the tumour was observed ( mib-1 reaction ) in the form of a positive reaction in approx . staining h&e , magnification 100 a month after the operation , in quite good general condition , the patient reported for oncological treatment . the postoperative scar at the base of the neck was fully healed , with no infiltration . the peripheral lymph nodes were slightly enlarged , with small oedema of the subcutaneous tissue . computed tomography with local staging dab , magnification 100 the reconsultation revealed negative staining with cytokeratin , and positive with vimentin , thereby confirming the mesenchymal origin of the tumour , with the final diagnosis being primary thyroid osteosarcoma . taking into consideration the histopathological diagnosis , the extremely low radiation sensitivity of the tumour , the patient 's age , the radical surgical treatment and persisting respiratory failure , radiotherapy was rejected in favour of further follow - up . osteosarcoma , the most frequent malignant bone tumour , may also occur in various organs outside the osteoarticular system . however , these are rare incidents , described in single cases and involving the heart , lung , prostate , urinary bladder and breasts [ 14 ] . 5% ) , especially in the thyroid , where , so far , it has been diagnosed in 28 cases , and in only two cases with fine - needle biopsy [ 6 , 7 ] . this tumour is characterised by fast growth and requires quick surgical intervention due to pressure - related symptoms of dyspnoea . poorly differentiated thyroid carcinoma and anaplastic carcinoma with osseous metaplasia , the differentiating diagnosis should additionally involve medullary thyroid carcinoma , spindle epithelial tumour with thymus - like differentiation ( settle ) , synovial sarcoma and metastasis of osteosarcoma . the tumour is characterised by a variety of histological images , from extensive monomorphic infiltrations of primary mesenchymal cells with high mitotic activity , in which it is generally difficult to observe osteoplasia , through spindle - cell and epithelioid areas with accompanying giant tumour cells with multiple nuclei . the presence of bone matrix and mature bone trabeculae is a frequent component of the tumour . cytological material is usually rich in cells and appears in compact tissue fragments with slight discohesion , frequently showing a high degree of cell polymorphism . there are spindle cells and epithelioid cells with varying amounts of cytoplasm , from trace to abundant , frequently with the presence of intracytoplasmic vacuoles . the nuclei of these cells are usually elongated or oval - shaped , with coarse - grained chromatin and quite distinct chromocentres , often eccentric as in plasmocytes . in the background , there are usually binuclear and polynuclear cells with considerable polymorphism , some of which resemble osteoclasts [ 6 , 7 ] . beside cells , there may be masses partially composed of fibrillar structures , adjacent to tumour cells . thus , the cytological image may be ambiguous and requires histopathological confirmation , supported by immunohistochemical examinations . immunohistochemical examinations with vimentin ( positive reaction ) , cytokeratin , ttf1 , calcitonin , chromogranin , synaptophysin and s-100 ( negative staining ) are of decisive significance . the clinical course is characterised by rapid progression , which , within weeks , leads to death in the mechanism of lung metastasis or the superior vena cava syndrome as a result of extensive infiltration of the perithyroid tissue and large carotid vessels .

we enrolled 181 type 2 diabetic subjects using the following inclusion criteria : 1 ) ages > 35 and < 75 years , 2 ) serum creatinine levels less than 1.4 mg / dl and albumin excretion rate less than 300 mg / day , 3 ) hepatic enzymes levels less than three times upper normal , and 4 ) alcohol consumption less than 20 g / day . patients with known hepatic disease , cardiovascular disease , acute or chronic inflammation , and malignancy the mean age of the subjects was 54.3 10.4 years , and 55.2% of the total subjects were male . the protocol was approved by the ethics committee of yonsei university wonju college of medicine . all of the subjects gave written informed consent , and all of the reported investigations were carried out according to the principles of the declaration of helsinki ( the year 2000 revision ) . alcohol intake , smoking habits , medication history , and medical history were assessed using a standardized questionnaire . anthropometric data including weight , height , waist and hip circumference , and blood pressure were assessed . bmi was calculated as weight in kilograms divided by the square of height in meters ( kg / m ) . fasting plasma glucose , insulin , a1c , urine albumin excretion rate , hepatic enzyme levels , high- sensitivity c - reactive protein ( crp ) , and lipid profiles were measured . the severity of nafld on ultrasound was graded as follows : mild ( grade 1 ) , defined as a slight diffuse increase in liver echogenicity in the hepatic parenchyma with normal visualization of the diaphragm and the portal veins ; moderate ( grade 2 ) , defined as a moderately diffuse increase in liver echogenicity with a slightly impaired visualization of the diaphragm and the portal veins ; and severe ( grade 3 ) , defined as a marked increase in liver echogenicity with poor or no visualization of the diaphragm and the portal veins . we defined overt nafld in this study as more than a moderate degree of fatty liver . adiponectin and retinol - binding protein-4 ( rbp-4 ) levels were determined by elisa ( adipogen , seoul , korea ) . a - fabp levels were also assessed by elisa ( bio vendor laboratory medicine , modrice , czech republic ) . insulin resistance was measured by the homeostasis model of assessment for insulin resistance ( homa - ir ) . the homa - ir index was calculated using the following formula : fasting plasma glucose ( mg / dl ) fasting insulin /405 ( u / ml ) . statistical analysis was performed using spss ( version 13.0 ; spss , chicago , il ) . data are presented as means sd and as a number ( in percentages ) for categorical measures . for continuous variables , the differences between groups were compared using either an unpaired student 's t test or one - way anova . correlations of a - fabp with various metabolic parameters were analyzed using pearson correlation and multiple regression analysis after adjustments for age and sex . logistic regression analysis was performed to assess the odds ratio ( or ) of the metabolic parameters for the presence of overt nafld after adjustments for age and sex . multiple logistic regression analysis was used to assess the or for the presence of overt nafld in subjects with the higher a - fabp tertiles compared with those with the lowest tertile . statistical analysis was performed using spss ( version 13.0 ; spss , chicago , il ) . data are presented as means sd and as a number ( in percentages ) for categorical measures . data that were not normally distributed were logarithmically transformed before analysis . for continuous variables , the differences between groups were compared using either an unpaired student 's t test or one - way anova . correlations of a - fabp with various metabolic parameters were analyzed using pearson correlation and multiple regression analysis after adjustments for age and sex . logistic regression analysis was performed to assess the odds ratio ( or ) of the metabolic parameters for the presence of overt nafld after adjustments for age and sex . multiple logistic regression analysis was used to assess the or for the presence of overt nafld in subjects with the higher a - fabp tertiles compared with those with the lowest tertile . duration of diabetes and mean a1c levels for all of the subjects were 5.1 years and 8.6% , respectively . of the 181 type 2 diabetic patients , the users of peroxisome proliferator activated receptor- ( ppar- ) agonists , insulin , statins , and angiotensin - converting enzyme inhibitors and/or angiotensin ii receptor blockers were 7.2 , 4.4 , 17.1 , and 26.5% , respectively . the percentage of patients with metabolic syndrome and nafld were 66.5 and 76.8% , respectively . as shown in table 1 , all subjects were divided into three subgroups according to the severity of their fatty liver disease : normal , mild degree , and more - than - moderate degree . patients with overt nafld had higher bmi , waist circumference , waist - to - hip ratio ( whr ) , hepatic enzymes , crp , a - fabp , and homa - ir ( p < 0.05 ) and had lower adiponectin levels ( p < 0.05 ) compared with those without nafld . also , patients with overt nafld were more likely to have metabolic syndrome than those without . serum a - fabp levels were significantly higher in women than men ( 24.0 16.7 vs. 13.9 9.0 g / l ; p < 0.001 ) . also , a - fabp levels in patients with overt nafld and metabolic syndrome were significantly higher than in those without nafld ( 24.7 17.9 vs. 15.3 10.2 g / l ; p < 0.001 ) and those without metabolic syndrome ( 20.6 14.4 vs. 14.2 12.2 g / l ; p = 0.004 ) . a - fabp levels in users of ppar- agonists were slightly higher compared with nonusers , but this difference was not significant . as shown in table 2 , age- and sex - adjusted a - fabp showed significant positive correlations with bmi , whr , waist circumference , triglycerides , -glutamyltransferase , fasting insulin , homa - ir , a1c , and crp . however , a - fabp was negatively correlated with hdl cholesterol ( p < 0.05 ) . however , there were no significant correlations between age- and sex - adjusted a - fabp and adiponectin , rbp-4 , and the use of ppar- agonists , statins , or antihypertensive drugs ( data not shown ) . in multivariate linear regression analysis after adjustment for age and sex , a - fabp was significantly associated with overt nafld independent of bmi , waist circumference , homa - ir , and a1c ( p < 0.01 ) ( data not shown ) . in multiple logistic regression analysis after adjustment for age and sex , high a - fabp was associated with overt nafld ( or 2.87 [ 95% ci 1.475.61 ] ; p = 0.002 ) . also , waist circumference , bmi , whr , homa - ir , crp , triglycerides , aspartate aminotransferase , alanine aminotransferase , and -glutamyltransferase were significantly associated with the presence of overt nafld ( table 3 ) . as shown in table 4 , the patients in the highest tertile of sex - specific a - fabp had significantly higher bmi , waist circumference , whr , triglycerides , a1c , and serum creatinine but had lower hdl cholesterol and estimated glomerular filtration rate ( gfr ) compared with those in the lowest tertile ( p < 0.05 ) . patients in the higher tertiles of sex - specific a - fabp had higher or for the presence of overt nafld compared with those in the lowest tertile ( 2.90 [ 1.157.29 ] vs. 7.87 [ 3.2019.38 ] ) . the or in the highest tertile of sex - specific a - fabp remained significant after adjustment for bmi , waist circumference , a1c , hdl cholesterol , homa - ir , crp , triglycerides , and hepatic enzymes ( 8.53 [ 2.6327.65 ] ) ( table 5 ) . duration of diabetes and mean a1c levels for all of the subjects were 5.1 years and 8.6% , respectively . of the 181 type 2 diabetic patients , the users of peroxisome proliferator activated receptor- ( ppar- ) agonists , insulin , statins , and angiotensin - converting enzyme inhibitors and/or angiotensin ii receptor blockers were 7.2 , 4.4 , 17.1 , and 26.5% , respectively . the percentage of patients with metabolic syndrome and nafld were 66.5 and 76.8% , respectively . as shown in table 1 , all subjects were divided into three subgroups according to the severity of their fatty liver disease : normal , mild degree , and more - than - moderate degree . patients with overt nafld had higher bmi , waist circumference , waist - to - hip ratio ( whr ) , hepatic enzymes , crp , a - fabp , and homa - ir ( p < 0.05 ) and had lower adiponectin levels ( p < 0.05 ) compared with those without nafld . also , patients with overt nafld were more likely to have metabolic syndrome than those without . serum a - fabp levels were significantly higher in women than men ( 24.0 16.7 vs. 13.9 9.0 g / l ; p < 0.001 ) . also , a - fabp levels in patients with overt nafld and metabolic syndrome were significantly higher than in those without nafld ( 24.7 17.9 vs. 15.3 10.2 g / l ; p < 0.001 ) and those without metabolic syndrome ( 20.6 14.4 vs. 14.2 12.2 g / l ; p = 0.004 ) . a - fabp levels in users of ppar- agonists were slightly higher compared with nonusers , but this difference was not significant . as shown in table 2 , age- and sex - adjusted a - fabp showed significant positive correlations with bmi , whr , waist circumference , triglycerides , -glutamyltransferase , fasting insulin , homa - ir , a1c , and crp . however , a - fabp was negatively correlated with hdl cholesterol ( p < 0.05 ) . however , there were no significant correlations between age- and sex - adjusted a - fabp and adiponectin , rbp-4 , and the use of ppar- agonists , statins , or antihypertensive drugs ( data not shown ) . in multivariate linear regression analysis after adjustment for age and sex , a - fabp was significantly associated with overt nafld independent of bmi , waist circumference , homa - ir , and a1c ( p < 0.01 ) ( data not shown ) . in multiple logistic regression analysis after adjustment for age and sex , high a - fabp was associated with overt nafld ( or 2.87 [ 95% ci 1.475.61 ] ; p = 0.002 ) . also , waist circumference , bmi , whr , homa - ir , crp , triglycerides , aspartate aminotransferase , alanine aminotransferase , and -glutamyltransferase were significantly associated with the presence of overt nafld ( table 3 ) . as shown in table 4 , the patients in the highest tertile of sex - specific a - fabp had significantly higher bmi , waist circumference , whr , triglycerides , a1c , and serum creatinine but had lower hdl cholesterol and estimated glomerular filtration rate ( gfr ) compared with those in the lowest tertile ( p < 0.05 ) . patients in the higher tertiles of sex - specific a - fabp had higher or for the presence of overt nafld compared with those in the lowest tertile ( 2.90 [ 1.157.29 ] vs. 7.87 [ 3.2019.38 ] ) . the or in the highest tertile of sex - specific a - fabp remained significant after adjustment for bmi , waist circumference , a1c , hdl cholesterol , homa - ir , crp , triglycerides , and hepatic enzymes ( 8.53 [ 2.6327.65 ] ) ( table 5 ) . in the present study , we demonstrate that serum a - fabp levels in type 2 diabetic patients are closely associated with nafld independent of bmi , waist circumference , homa - ir , a1c , triglycerides , hdl cholesterol , and crp levels . patients in the highest tertile of a - fabp were eight times more likely to have overt nafld compared with those in the lowest tertile . in animal studies , there was no protection against fatty liver disease in ap2-deficient mice because of compensation through increased expression of mal1 ( 20 ) . however , a profound protection against fatty liver disease was shown in ap2-mal1 combined deficient mice on high - fat diet ( 19 ) . also , cao et al . ( 21 ) demonstrated that there was striking protection from liver fatty infiltration in ob / ob - ap2-mal1deficient mice with strong suppression of liver stearoyl - coa desaturase-1 . furthermore , furuhashi et al . ( 22 ) reported that fatty infiltration of the liver was attenuated and total liver triglyceride content was reduced in ap2-inhibitor treated ob / ob mice . however , the relationship between a - fabp and fatty liver disease has not yet been established in a human study . this study is the first to demonstrate an association between a - fabp and nafld in type 2 diabetic patients . taken together , these findings suggest that chemical inhibition of a - fabp might show beneficial effects against fatty liver disease . like previous studies , serum a - fabp levels in our type 2 diabetic patients were associated with markers of obesity , dyslipidemia , hyperglycemia , insulin resistance , and inflammation . however , there are discrepancies in the correlation between a - fabp and adiponectin . xu et al . ( 13 ) reported that a - fabp in nondiabetic subjects was positively correlated with homa - ir but was negatively correlated with adiponectin . on the contrary , cabre et al . ( 23 ) reported that a - fabp in type 2 diabetes was positively correlated with adiponectin but was not correlated with homa - ir . in our type 2 diabetic patients , a - fabp was not correlated with adiponectin but was positively correlated with homa - ir . differences in the adiposity of the populations and sex difference of a - fabp and adiponectin levels might partly explain this discrepancy . recently , cabre et al . ( 24 ) reported that high a - fabp plasma concentrations were associated with high plasma creatinine and low gfr in type 2 diabetic patients . in our study , patients with estimated gfr < 60 ml / min per 1.73 m were only 2.8% of the total subjects . in multiple regression analysis , a - fabp was associated with serum creatinine after adjustments for age , sex , and bmi but was not associated with estimated gfr ( data not shown ) . similar to previous studies ( 15,23 ) , sex difference in a - fabp was observed in our study . the sex difference is explained partly by the higher fat and subcutaneous fat percentages in women compared with men because adipose tissue is a major source of circulating a - fabp and a - fabp expression is higher in subcutaneous fat than in visceral fat . in our data , patients with nafld had higher a - fabp levels than those without nafld . in women , a - fabp levels in patients with overt nafld these findings suggest that a - fabp is a more specific marker of nafld in women than in men . we could not prove a causal link between serum a - fabp levels and the development of nafld . second , we could not analyze our data stratified by sex because of the small sample size . nevertheless , we assessed the or for the presence of nafld according to the sex - specific tertiles of a - fabp . third , the severity of nafld was assessed by ultrasound in this study but was not confirmed pathologically . although liver biopsy is the gold standard to assess pathologic grading of nafld , it is difficult to perform liver biopsies for the assessment of nafld in clinical practice . it has been reported that the sensitivity and specificity of ultrasound in the diagnosis of fatty liver , as assessed on liver biopsy , were 6094 and 8495% , respectively ( 25 ) . in conclusion , we demonstrated that serum a - fabp was closely associated with nafld in type 2 diabetic patients . our data suggest that a - fabp may be an independent marker of nafld in type 2 diabetes , independent of bmi , waist circumference , homa - ir , a1c , triglycerides , hdl cholesterol , and crp levels . large population - based prospective studies are warranted to confirm whether a - fabp is an independent predictor of nafld and whether it plays a causative role in the pathogenesis of nafld .

gait refers to moving the body to targeted points using the legs as a complex and integrated activity with low energy consumption involving alternative and harmonious movements with normal tension of the antigravity muscles1 . during gait , the smaller the step length difference ( sld ) , stance phase difference ( stpd ) , swing phase difference ( swpd ) , single support difference ( ssd ) , and step time difference ( std ) between the left and right feet are , the higher the stability of the gait will be . securing the stability of gait is an important factor when it comes to preventing falls . a fall may not only cause fractures and brain damage but mat also reduce mobility and motility , thereby bringing about many restrictions on living activities and leading to a declined quality of life2 . improvements in muscle endurance and gait velocity have been reported after performance of lower extremity exercises , and recent studies have emphasized that strengthening of not only the lower extremity muscles but also the lumbar muscles would improve the functional stability of the body and consequently enhance balance and gait ability3 . the pelvis is a structure located between the hip joint and the lumbosacral region to which many muscles are attached to adjust the movements of the hip joint and the lumbosacral region . therefore , the position of the pelvis is the most important element determining the body s sagittal alignment and posture4 . the lumbosacral joint affects the lumbar vertebrae , and the position of the lumbar vertebrae above the lumbosacral joint is determined by the position of the lumbosacral joint . the angle of the lumbosacral joint may vary depending on the position of the pelvis . dynamic problems of the musculoskeletal system can be said to arise from the lumbar vertebrae , depending on the position of the pelvis . anteroposterior gradient movements of the pelvis may affect the stability of the vertebrae and the alignment of the body . many studies have been conducted on the stability and postures of the pelvis , which are important for gait . there have been some cases in which lumbo - pelvic stabilization and posture were improved through bridge exercises6 , and there have been others in which lumbo - pelvic stabilization and posture were improved through diverse mat exercises7 . however , these methods have drawbacks in that they require time , space , and active participation from the subjects . on the other hand , although many previous studies have examined the effects of pelvic adjustment on functional leg length inequality8 , balance9 , posture10 , and so on , no previous research has examined the extent of changes in gait brought about by one session of adjustment . therefore , the aim of this study was to examine the immediate effects of one session of pelvic adjustment on the gait variables of female university students . thirty female university students were selected and randomly assigned to a pelvic adjustment group of 15 subjects as an experimental group and a stretching group of 15 subjects as a control group . those who had problems with their muscular , skeletal , or nervous system ; those who felt pain in their lumbar region or pelvis during daily living ; those who had improper postures due to burns or postoperative scars ; and those who could not walk normally were excluded from the study . all included patients understood the purpose of this study and provided written informed consent prior to their participation in accordance with the ethical standards of the declaration of helsinki . the adjustment group that participated in this study had a mean age of 21.54.9 years , a mean height of 163.07.3 cm , and a mean weight of 54.17.1 kg ; the stretching group had a mean age of 20.24.3 years , a mean height of 163.46.6 cm , and a mean weight of 57.19.5 kg . chi - squared tests were used to analyze the groups with regard to sex , and independent t - tests were used to analyze the groups with regard to age , height , and weight . the analyses showed no statistically significant difference ( p>0.05 ) , indicating that there was no problem in the homogeneity between the two groups . in this study , the pelvic adjustment applied to the pelvic adjustment group was the high - velocity , low - amplitude technique for prone positions among gonstead s theories . the direction of adjustment was set up before correction through gait analysis and gillet tests11 . each subject was instructed to take a prone position and the height of the pelvis of the subject was checked to provide anterior rotation impacts on the innominate bone if it was posterior rotation displaced and to provide posterior rotation impacts on the innominate bone if it was anterior rotation displaced . when giving the impacts , the therapist overlapped her two hands and placed them on the posterior - superior iliac spine or ischial spine of the subject to give the impacts using her body weight , gravity , and acceleration8 . this adjustment was conducted 35 times by one physical therapist with at least 10 years of clinical experience . in the case of the stretching group , to relieve any imbalance in muscles that may result in the imbalance of the pelvis , the muscles around the pelvis specifically the erector spinae muscle , the rectus abdominis , the iliac muscle , the psoas major muscle , the quadriceps femoris muscle , the leg adductor muscle , and the quadratus lumborum muscle were stretched . during the stretching , the therapist helped the subjects so that each muscle could be extended a little further than the maximum extended posture , and each subject was instructed to maintain each maximum extended posture for 1015 seconds . after each stretching posture , the subject slowly restored the initial posture , took a rest for five seconds , and stretched once again . each stretching posture was carried out three times in total , and the time spent for each session of stretching was 15 minutes . the optogait ( microgate italy , bolzano - bozen , italy ) , used to measure gait variables , is gait analysis equipment made up of an optical detection system having 96 leds that transmit information using infrared frequencies via a transmission bar and the same number of leds on the receiving bar on the opposite side . the transmission and receiving bars of the optogait are installed on the two sides of a treadmill , and the sld , stpd , swpd , ssd , and std during gait are calculated based on communication between the two bars being blocked by the patient s movements . the lengths of time a subjects runs , jumps , performs a series of jump tests , makes contact with the floor , or remains in the air can be calculated accurately from data captured with 1,000 instances of transmission / receiving per second . the dedicated software analyzes these basic data measured in real time and converts them into a series of movements . the measured data were analyzed using spss 12.0 ko ( spss , chicago , il , usa ) , and the collected data are presented as means and standard deviations . the significance of differences between before and after the experiment in each group was tested using paired t - tests and the significance of differences between the two groups was tested using independent t - tests . the pre- and post - intervention measurement results in the adjustment group and the stretching group were compared . the results revealed that the adjustment group showed statistically significant differences in all items ( sld , stpd , swpd , ssd , and std ; p<0.05 ) , while the stretching group showed no statistically significant difference in any of the items ( p>0.05 ) ( table 1table 1 . comparison of sld , stpd , swpd , ssd and std between before and after intervention in each group ( meansd ) ( unit : sld - cm , stpd , swpd , ssd , std-%)categorygroupbefore interventionafter interventionsldadjustment group*3.31.60.70.5stretching group3.74.03.12.3stpdadjustment group*2.42.90.70.6stretching group2.11.62.41.6swpdadjustment group*2.91.91.51.4stretching group2.61.83.13.0ssdadjustment group*3.12.31.20.9stretching group2.71.92.82.2stdadjustment group*3.12.11.61.1stretching group3.21.82.82.0 * p<0.05 . sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference ) . sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference the pre- and post - intervention measurement results and changes between before and after intervention in the adjustment group and the stretching group were compared with each other . the results revealed that the pre - intervention measurements showed no statistically significant differences in any of the items ( p>0.05 ) , while the post - intervention measurements showed statistically significant differences in all of the items , and the changes between the pre- and post - intervention measurements showed statistically significant differences in all of the items except for std ( p<0.05 ) ( table 2table 2 . comparison of sld , stpd , swpd , ssd , and std between the adjustment group and stretching group ( meansd ) ( unit : sld - cm , stpd , swpd , ssd , std-%)categoryadjustment groupstretching groupbefore interventionsld3.31.63.74.0stpd2.42.92.11.6swpd2.91.92.61.8ssd3.12.32.71.9std3.12.13.21.8after interventionsld*0.70.53.12.3stpd*0.70.62.41.6swpd*1.51.43.13.0ssd*1.20.92.82.2std*1.61.12.82.0change between before and after interventionsld*2.51.50.64.2stpd*1.63.00.21.3swpd*1.32.40.52.5ssd*1.92.70.21.7std1.42.50.42.7 * p<0.05 . sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference ) . * sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference humans are exposed to gravity because they walk upright ; this exposure easily leads to misalignment in the pelvis and the lower extremities , which affects posture , gait patterns , and balance . the pelvis supports the abdomen , connects the vertebrae to the lower extremities , transmits body weight from the vertebrae to the lower extremities while standing , and enables proper posture to be maintained in order to generate smooth movements of the upper extremities12 . studies have reported that proper posture can be maintained and the upper body and the lower body can be controlled during movement to enhance gait ability only when the pelvis is in the neutral position13 . differences in lower extremity lengths can be found in all clinical cases , and these differences cause imbalance in the joints , vertebrae , and pelvis and disturb normal biodynamic functions14 . a normal position and the stability of the pelvis are thus important for a balanced gait . although many previous studies have analyzed the gait of hemiplegia15 or cerebral palsy patients16 after diverse interventions using three - dimensional gait analysis , no study has analyzed the gait of such patients after pelvic adjustment ; moreover , no studies that analyzed the gait of healthy adults after pelvic adjustment could be found . therefore , the aim of this study was to apply pelvic adjustment to healthy adults and see if immediate changes in gait would occur . accordingly , changes in gaits were analyzed after pelvic adjustment was applied using gonstead techniques . as a result , the sld , stpd , swpd , ssd , and std between the left and right sides decreased , indicating that pelvic adjustment had immediate effects on balanced gait in the female university students . in particular , the pre - intervention difference in sld , which was 3.31.6 in the adjustment group and 3.74.0 cm in the stretching group , resulted from the anterior and posterior rotation rates of the left and right innominate bones being different . the post - intervention sld of the adjustment group decreased to 0.70.5 cm because the posteriorly rotated innominate bone was imposed with impact for anterior rotation and the anterior rotation pelvis was imposed with impact for posterior rotation through pelvic adjustment . in previous papers related to pelvic adjustment , gong et al . ( 2011 ) advised that functional leg length inequality was reduced through pelvic adjustment among gonstead s techniques , and these changes in leg length inequality reduced differences in foot pressure8 . 2012 ) reported that the balance of 20 elderly males was improved through pelvic adjustment using gonstead techniques9 , while cho ( 2013 ) stated that pelvic adjustment improved posture in female university students10 . the results of previous studies indicating that pelvic adjustment using gonstead techniques improved postures , reduced differences in leg lengths , enhanced balance , and reduced differences in foot pressure are similar to the results of this study , which indicated that pelvic adjustment applied with gonstead techniques had immediate effects on balanced gait in the female university students . the changes balanced gait in the female university students brought about by pelvic adjustment , as mentioned above , are considered to have occurred because the pelvis is an important structure that supports the vertebrae and transmits body weight to the lower extremities . functional leg length inequality is considered to have been reduced through pelvic adjustment , and these changes in leg length inequality are considered to have induced proper postures , as well as affects on gait variables , so that balanced gaits were shown . therefore , the author recommends utilizing pelvic adjustment in patients who require properly balanced gaits . however , how long pelvic adjustment will enable balanced gait to be maintained should be examined through follow - up studies .

lupus nephritis ( ln ) is characterized by immune complex deposition and inflammation in glomeruli and the tubulointerstitium , but its precise pathogenesis is not very much clear until now . many studies have indicated that systemic loss of b - cell tolerance results in the local deposition of immune complexes [ 1 , 2 ] . with the successful application of targeted b - cell therapy in the treatment of refractory ln , it is known that b lymphocytes are involved in the pathogenesis of systemic lupus erythematosus ( sle ) in autoantibody - dependent mechanisms . while some recent studies suggested that autoantibody - independent mechanisms might be more important , the specific mechanism is still unclear [ 35 ] . investigations in the mrl / lpr mouse model of lupus nephritis have indicated that b cells exert a pathogenic role in the absence of soluble autoantibody production . local b - cell infiltration and related abnormal expression of ectopic lymphoid tissue ( elt ) in the renal tissues of ln mice models was related to the severity of renal impairment . these findings suggested that excessive intrarenal b - cell infiltration and related elt formation might play an important role in ln occurrence and development . cxcl13 is a small - molecule cytokine and belongs to the chemokine family , which can regulate transport of various types of white blood cells by binding to its receptor , cxcr5 . because of the ability to attract b cells , research performed in nzb / w - f1 mice and ln patients suggested that cxcl13 played an important role in the initiation and development of ln as a b - lymphocyte chemokine . whether cxcl13 is related to the renal pathological damage observed in ln and the formation of abnormal b - lymphocyte infiltration - related elt thus , we speculated that an abnormally increased serum cxcl13 level could induce excessive chemotaxis of b cells , t cells , and dendritic cells into renal tissues of ln and subsequent excessive elt formation , which promotes a vicious cycle of the expansion and perpetuation of inflammatory cell aggregates resulting in persistence and chronicity of the renal inflammation in ln patients . this study demonstrated intrarenal b - cell infiltration and related elt formation in the ln patients with different who pathological classifications and showed serum cxcl13 levels pre- and posttreatment and their role in the process of intrarenal elt formation . moreover , we explored the role of intrarenal b - cell infiltration and related elt formation , as well as an increase in serum cxcl13 levels in the pathogenesis , diagnosis , and treatment of ln ; cxcl13 may function as a reliable and practical marker of serology and excessive elt formation as a marker of histology for the clinical diagnosis of ln . a prospective study of 114 patients who attended the department of rheumatology and immunology of guangdong general hospital was carried out . all patients fulfilled the american college of rheumatology classification criteria for the diagnosis of sle . clinical evidence of ln was obtained in all cases and ln diagnosis was confirmed by examination of renal biopsy specimens . all sle patients included in this study are with age of no more than 70 years without hepatitis b - associated nephritis or the following complications including infection , a tumor , and severe cardiopulmonary dysfunction . the following demographic , clinical , and serologic data were collected at the time of renal biopsy : sex , age , duration of sle and ln , systemic lupus erythematosus disease activity index ( sledai ) , 24 h proteinuria , levels of blood urea nitrogen , serum creatinine , serum c3 , c4 , antinuclear antibodies ( ana ) , anti - sm , anti - ribonucleoprotein ( anti - rnp ) , anti - double - stranded dna ( anti - dsdna ) , and antinucleosome antibodies were determined . this study was performed according to the principles of the declaration of helsinki and each participant completed written informed consent before measurements . this study was also approved by the local ethics committee ( institutional review board of guangdong general hospital ) . all ln patients underwent combination therapy with glucocorticoids ( gc ) and immunosuppressants ( is ) . is mainly referred to cyclophosphamide ( ctx ) , mycophenolate mofetil ( mmf ) , and cyclosporine a ( csa ) . after 6 months of treatment , responses in ln patients were evaluated that included complete remission ( cr ) and no cr . other reagents included clone ps1 , novacastra as a cd3 antibody ( t - cell surface marker ) ; clone l26 , dako company as a cd20 antibody ( b - cell surface marker ) ; and clone 2g9 , gene company as a cd21 antibody [ follicular dendritic cell ( fdc ) surface marker ] . all enrolled clinically diagnosed ln patients undertaken renal biopsy with b - mode ultrasound - guidance . native kidney biopsies will need to be divided into three parts for conventional light microscopy , electron microscopy , and immunofluorescence studies . two - thirds of renal biopsy samples were stereotyped in 10% neutral buffered formalin fixative , dewatered gradually , and soaked in paraffin . serial sample sections were marked with alternating hematoxylin and eosin ( he ) , periodic acid - schiff ( pas ) , masson 's trichrome , and periodic acid - silver methenamine . one - third of fresh renal tissue was quickly frozen , and 4 m unfixed and frozen sections were stained with fluorescein isothiocyanate- ( fitc- ) conjugated rabbit antisera against human igg , iga , igm , c1q , and c3 ( dako , denmark ) , and the direct immunofluorescence of these sections was observed . the pathological diagnosis for biopsy specimens was established using the international standards for pathological classification of ln . a routine pathological examination was carried out , and cd3 , cd20 , and cd21 coexpression were detected using immunohistochemical staining kits ( dako envision plus ) . serial paraffin - fixed tissue sample sections were cut to a thickness of 3 m and mounted on positively charged glass slides and kept in a stove at 55c overnight . slides were dewaxed and hydrated through a series of washes with xylene and graded alcohols . antigen retrieval was carried out by dipping the slides into antigen repair solution ( ph 9.0 tris - edta ) and heating at 100c in a microwave at 650 w for 25 min . first mouse monoclonal anti - human cd20 , cd3 , and cd21 antibodies were added to the slides at dilution of 1 : 800 , 1 : 100 , and 1 : 200 , respectively , in 1% bovine serum albumin in phosphate buffered saline ( bsa / pbs ) and subsequently stored them 70 min at room temperature . slides were rinsed with pbs ( ph 7.4 ) between each step ( 2 times for 5 min ) . slides were then mounted with a secondary goat anti - mouse igg antibody ( envision / hrp ; dako ) for 40 min at room temperature . slides were rinsed in pbs ( ph 7.4 ) between each step ( 2 times for 5 min ) , then incubated in dab solution for 13 minutes , and rinsed in distilled water . finally , sections were counterstained with mayer 's hematoxylin , air - dried , cleared in xylene , and coverslipped with mounting medium . tonsil tissue was stained as a positive control , and primary antibodies were replaced with pbs as a negative control . positive results manifest with brown particles localized at cytomembrane or cytoplasm of cd3 expression , or cytoplasm of cd20 and cd21 expression . based on the cd20 , cd3 , and cd21 coexpression patterns in the kidney , the ln patients could be divided into 4 categories of elts : class 0 , class 1 , class 2 , and class 3 . class 0 had scattered cd3 + cells infiltration only , but without cd20 + cells and cd21 + cells expression ; class 1 had scattered cd20 + cells and cd3 + cells infiltration , but without cd21 + cells expression ; and class 2 had a nodular aggregation of cd20 + cells and cd3 + cells without cd21 + cells expression . class 3 demonstrated a nodular aggregation of cd20 + cells and cd3 + cells with cd21 + cells expression . 2 ml venous blood samples were drawn from an enrolled individual in the morning before breakfast and then centrifuged at 3000 r / min for 5 minutes to obtain serum ( stored at 80c until analysis ) . cxcl13 was tested using an elisa kit , and quantification was accomplished according to the manufacturer 's protocol . all measurement data with normal distribution are expressed as mean standard deviation ( xs ) and analyzed by t - test between two groups or by anvoa and snk test between multiple groups . the measurement data with abnormal distribution such as serum cxcl13 , 24-hour urine protein , and anti - dsdna quantitation are presented as medians ( quartile ) and were compared using the mann whitney - wilcoxon test between 2 groups and using nonparametric kruskal wallis test among multiple groups . categorical data are presented as percentages and were compared using crosstabs and chi - square test among multiple groups . a p value of < 0.05 was considered significant . firstly , in this prospective study , 114 sle patients ( 89 ln patients and 25 sle patients without ln ) and 21 healthy individuals were enrolled . 89 ln patients ( 78 women and 11 men ; mean age sd : 35 12.8 years ) were separated into 2 groups according to the renal cd20 expression : ln with intrarenal b cells ( ln - b group ) and ln without intrarenal b cells ( ln non - b group ) . the ln - b group comprised 69 ( 77.5% ) patients ( 61 women and 8 men , 30.6 12.2 years ) and the ln non - b group comprised 20 ( 22.5% ) patients ( 17 women and 3 men , 41.5 12.9 years ) . no significant difference was detected between the two groups in terms of age or gender ( p > 0.05 ) . when compared with the ln - non - b - cell group , the ln - b - cell group had a longer disease course ( 21.8 9.9 months versus 9.8 6.2 months , resp . ; t = 2.185 , p = 0.045 ) and a poor complete remission ( cr ) rate after 6 months of treatment ( 63.8% versus 90% , resp . ; = 2.384 , p = 0.047 ) . other clinical characteristics ( age , sex , sledai , urine protein quantitation ( mg/24 h ) , serum creatinine , c3 , c4 , and anti - dsdna level ) between both groups demonstrated no differences . 89 ln patients were diagnosed clinically and confirmed by light microscopy , immunofluorescence , and electron microscopy . according to the isn / rps classification , 21 patients were diagnosed with type iii ( 23.6% ) , 53 patients with type iv ( 59.6% ) , and 15 patients with type v ( 16.8% ) . lymphocytes infiltration was mainly distributed in the renal interstitium of 77 patients ( 86.5% ) , around the renal tubules of 9 patients , and around the glomeruli of 3 patients . lymphocytes aggregation was regionally distributed in 51 patients ( 57.3% ) , while no typical lymph follicles as in tonsil tissue were found . the activity index ( ai ) score for the renal pathology was 10.7 4.8 ( 319 ) , and the chronicity index ( ci ) score was 3 1 ( 14 ) . there was no significant difference at the distribution of ln pathological classifications , ai score , and ci score between the ln - b - cell group and the ln - non - b - cell group . see table 1 . based on the cd20 , cd3 , and cd21 coexpression patterns in the kidney , the 89 ln patients could be divided into 3 categories : class 0 , class 1 , and class 2 . no formation of class 3 elt was found in the ln patients enrolled in our study . the elt class 2 group showed significantly longer disease duration than the elt class 0 and the elt class 1 ( p = 0.017 and 0.039 , resp . ) , while there was no significant difference ( p = 0.280 ) between the elt class 0 and the elt class 1 . the difference was significant in the ai score between the elt class 2 and the elt class 0 ( p = 0.047 ) , while it was not significant between the elt class 1 and the elt class 2 ( p = 0.056 ) . other clinical parameters ( sledai score , ci score , c3 , c4 , 24-h urine protein , and anti - dsdna antibody ) in the 3 elt classes groups demonstrated no differences . see figure 1 and table 2 . we noted that the ln kidney tissues for different who ln pathological types had different proportions of the three classifications of elt . the proportions of elt classes composition in type iii , iv , and v ln group were compared using a test , and the differences were significant ( = 28.04 , p < 0.0001 ) . the proportions of elt classes involving types iii and iv were significant ( = 10.78 , p = 0.0046 < 0.0167 [ 0.05/3 ] ) , as were those with types iv and v ( = 28.29 , p < 0.0001 < 0.0167 [ 0.05/3 ] ) . the proportions of elt classes involving types iii and v demonstrated no differences ( = 5.07 , p = 0.0793 > 0.0167 [ 0.05/3 ] ) . according to the presence of renal involvement , sle patients ( n = 114 ) were divided into the ln group ( n = 89 ) and the sle group without renal involvement ( sle - no - ln ) ( n = 25 ) . serum cxcl13 levels in sle patients ( 230.65 [ 145.92365.40 ] pg / ml ) were significantly higher than those of healthy individuals ( 85.13 [ 62.23131.58 ] pg / ml ) ( z = 4.42 , p < 0.001 ) . moreover , serum cxcl13 levels in the ln patients ( 258.86 [ 185.50392.18 ] pg / ml ) were higher than those of the sle - no - ln group ( 136.39 [ 104.23257.50 ] pg / ml ) ( z = 2.53 , p = 0.01 ) ( figure 2(a ) ) . after 6 months of treatment , serum cxcl13 levels and sledai were significantly decreased in the ln group ( cxcl13 : 258.86 [ 185.50392.18 ] pg / ml versus 195.54 [ 165.98233.01 ] pg / ml , resp . ; z = 2.59 , p = 0.009 ; sledai : 10 [ 8.7512 ] versus 6 [ 46.5 ] , z = 2.56 ; p = 0.01 ) . after 6 months of immunosuppressive therapy , the ln patients were divided into a complete remission group ( n = 56 ) and noncomplete remission group ( n = 33 ) . serum cxcl13 levels for the ln group in complete remission ( 189.46 [ 114.23216.50 ] pg / ml ) were significantly lower than those of the ln group with noncomplete remission ( 286.75 [ 181.40386.56 ] pg / ml ) ( z = 2.48 , p = 0.048 ) . the correlation of serum cxcl13 with sledai in sle patients was analyzed . serum cxcl13 levels in sle patients were positively correlated with sledai ( r = 0.55 , p < 0.001 ) ( figure 2(b ) ) and negatively correlated with c3 ( r = 0.39 , p < 0.001 ) ( figure 2(c ) ) , and there was no significant correlation with the anti - dsdna antibody titer ( r = 0.037 , p = 0.72 ) . serum cxcl13 levels for type iii and iv ln group were higher than those of type v ln group ( type iii : 287.58 [ 198.25355.44 ] pg / ml ; type iv : 271.96 [ 189.71434.99 ] pg / ml ; type v : 169.71 [ 101.09252.69 ] pg / ml ; type iii versus type v , p = 0.013 ; type iv versus type v , p = 0.002 ) , while serum cxcl13 levels in type iii and iv ln patients demonstrated no difference ( p = 0.39 ) ( figure 2(d ) ) . serum cxcl13 levels in the ln patients with class 2 elt were significantly higher than those of the ln patients with class 0 and 1 elt ( class 0 : 180.96 [ 101.93218.88 ] pg / ml ; class 1 : 216.91 [ 158.47327.15 ] pg / ml ; class 2 : 351.80 [ 251.13470.36 ] pg / ml ; class 2 versus class 0 : z = 4.79 , p < 0.001 ; class 2 versus class 1 : z = 2.32 , p = 0.02 ) ( figure 2(f ) ) . further , serum cxcl13 levels in the patients with class 1 elt were higher than those of the patients with class 0 elt ( z = 2.13 , p = 0.033 ) . moreover , serum cxcl13 levels were positively correlated with the number of b cells / hp in the renal tissue of ln patients ( r = 0.41 , p = 0.048 ) . the role of long - lived memory plasma cells and b - cell hyperactivity has been recently elucidated in the pathogenesis of sle [ 14 , 15 ] . a high prevalence of intrarenal b cells has been noted in immune - mediated diseases , such as renal transplant rejection and glomerulonephritis [ 1618 ] thus indicating that local b - cell infiltrates play a role in tissue injury such as tissue fibrosis , neolymphangiogenesis , and ectopic lymphogenesis . it is hypothesized that intrarenal b cells associated elts form part of a local autoimmune system with pivotal involvement in the pathogenesis of lupus nephritis . our study suggested that the abnormal aggregation of b lymphocytes and related elt formation in the renal tissue of ln patients might play a unique role in promoting ln persistent progression . elevated serum cxcl13 level might take part in this process . in this study , these patients , especially those with focal accumulation of b lymphocytes and t lymphocytes ( type 2 elt ) , had a significantly longer disease course than those ln patients without b - lymphocyte infiltration . moreover , ln patients with regional b - cell accumulation had a poor response to combination therapy with gc and is , suggesting that the abnormal intrarenal b - cell accumulation was related to the longer disease course and poor treatment response in ln . steinmetz and colleagues observed the renal biopsy specimens of 32 patients with ln and 16 patients with anca - associated vasculitis and found that traditional treatment failed to eliminate b - lymphocyte accumulations in the renal tissue of ln patients . in patients with active or refractory ln , more b lymphocytes accumulated in renal tissues and interacted with t lymphocytes and fdc to promote elt formation , thus leading to chronic renal injury and ln recurrence . blocking or removing abnormally accumulated b lymphocytes in renal tissues as early as possible might be important to the effective treatment of refractory ln and might be the basis for the therapeutic effect of cd20 monoclonal antibodies in refractory ln [ 2022 ] . this study showed that type 2 elt was mostly seen in ln patients ( n = 51 , 57.3% ) , but no typical lymphoid follicles were found . lymphocyte infiltration was mostly found in the renal interstitium ( n = 77 , 86.5% ) that was consistent with a study by shen et al . . the study enrolled 192 patients with ln and found no cd21 + cell expression in renal tissues . chang and colleagues conducted a study that enrolled 64 ln patients and found cd20 + b - lymphocyte and cd3 + cd4 + t - lymphocyte infiltration in the renal interstitium and lymphatic germinal center - like structures composed of cd138 + plasma cells and cd21 + dendrite cells . steinmetz and colleagues observed the renal biopsy specimens of 32 patients with ln and 16 patients with anca - associated vasculitis and also found the expression of cd21 + follicular dendrite cells . the diverse elt patterns demonstrated in various studies may be related to different regions , ethnicities , and inclusion criteria . all the above - mentioned points indicate that preventing elt formation in the renal tissue of ln patients is an important therapeutic target . since the renal elts in ln patients are obviously different from the secondary lymph node structure such as tonsil , the types of their core immune cell components are different , and their interaction modes may be different , and the roles and abilities of these intrarenal elts during ln onset and development may differ from typical secondary lymph tissue , thus necessitating more studies to elucidate . as compared with type v ln , type iii and iv ln showed more class 2 elt distribution . today , the role of cxcl13 in the pathogenesis of sle has attracted wide attention . we observed changes in serum cxcl13 levels in different who classifications of ln and different elt classes to determine the role of cxcl13 in the pathogenesis of ln and the formation of b - cell - infiltration - related elt . the results showed that ln patients with who types iii and iv ( especially who - iv ) and class 1 and class 2 elt ( especially class 2 ) had significantly higher serum cxcl13 levels . moreover , serum cxcl13 levels were positively correlated with sledai , the degree of b - cell infiltration in renal tissue , and ai but were negatively correlated with the c3 level , suggesting a possible correlation between abnormally increased serum cxcl13 levels and elt formation and renal impairment in ln patients . how blood cxcl13 induces locating of b cells mainly in the renal tubular interstitial region and thus forming elt deserves further study . in conclusion , this study suggested that abnormal b - cell infiltration and related elt formation in renal tissues might play an important role in ln occurrence and persistent development , and increased serum cxcl13 levels might take part in elt formation and the pathological renal impairment process in ln patients . blocking the biological effects of cxcl13 and the formation of elt in renal tissues might be important targets in the medical treatment of ln in the future .

the prevalence of obesity in the united states has reached epidemic proportions and continues to rise . numerous harmful sequelae of obesity including increased cardiometabolic risk and overall mortality have been demonstrated . variations in body fat distribution , independent of generalized adiposity , may be associated with differential metabolic risk . visceral adipose tissue ( vat ) is a fat depot that has been associated with an increased risk of metabolic syndrome , insulin resistance , and type 2 diabetes . however , studies have demonstrated only modest correlations between cardiometabolic risk factors and visceral adiposity , leading investigators to hypothesize that other fat depots may confer additional risk for the development of cardiovascular disease . upper body subcutaneous neck fat ( ubsf ) is a unique fat depot located in a separate anatomic compartment and has been associated with insulin resistance independent of vat . consequently , this fat depot may be associated with differential cardiometabolic risk above and beyond generalized adiposity and visceral fat . traditional measures of obesity have included anthropometric measurements including body mass index ( bmi ) and waist circumference , both of which have been correlated with increased risk of obesityrelated morbidity . further research , including data from our group , has shown that neck circumference has been correlated with cardiometabolic risk factors even after adjusting for levels of visceral adiposity and bmi , suggesting the significance of ubsf in the pathogenesis of obesity . in addition , we have also shown that neck circumference is associated with carotid atherosclerotic disease above and beyond other measures of adiposity ; however , neck circumference is only a proxy for ubsf . we aimed to develop a protocol to quantify ubsf using multidetector computed tomography ( mdct ) in a communitydwelling sample from the framingham heart study . the original framingham heart study cohort was first established in 1948 with the original cohort recruited from framingham , massachusetts . the offspring and third generation cohorts were recruited subsequently and have been well described previously . the mdct substudy consists of 1333 participants from the offspring cohort and 1431 participants from the third generation cohort who underwent computed tomography imaging as part of a comprehensive assessment of vascular calcification . participants for our current analysis are drawn from the mdct 2 substudy who underwent scanning from september 2008 to december 2011 . inclusion criteria included an age of > 35 years for men and > 40 years for nonpregnant women . a sample of 100 participants was selected from the study population for the purposes of the present analysis . this sample was randomly selected to include 50 women and 50 men evenly spread across the ages and divided between the offspring and third generation cohorts . the institutional review boards of boston university medical center and massachusetts general hospital approved the study protocol , and all participants provided written consent . subjects underwent mdct imaging of the thorax in a supine position using a general electric discovery vct 64slice pet / ct scanner ( ge healthcare ) . the entire chest from the lung base to the apices was imaged using a prospectively ecgtriggered computed tomography scanning protocol during a single inspirational breath hold to enable lung and coronary assessment of the apices . the gantry rotation time was 0.35 seconds , the tube voltage was 120 kvp , and the tube current was 300 ma ( subject weight 220 lb ) or 350 ma ( subject weight > 220 lb ) . a detector width of 0.625 mm was used . the 210 scan reconstruction algorithm was used , providing 210 of raw scan data ( tube rotation ) with an optimized reconstruction technique that provided images of 175ms temporal resolution depending on the speed of gantry rotation ( 0.35 seconds ) . mdct slices were loaded onto a dedicated offline workstation ( aquarius 3d workstation ; terarecon inc ) . a predefined image display setting ( 195 to 45 hounsfield units ; center : 120 hounsfield units ) was used to identify adipose tissue within these slices . the anatomic region of interest was defined by 40 contiguous 0.625mm slices superior to the body of the sternum and covering an area of 25 mm . the sternal landmark was determined by the first single slice to show the bowtie shape of the sternum filled completely with trabecular bone ( figure 1a ) . forty contiguous slices above the sternum were selected , and every 10th slice starting with the first slice was manually reviewed . the reader manually excluded the adipose tissue within the mediastinum ( figure 1b ) for the total neck fat . the reader then outlined the adipose tissue exterior to the chest wall ( figure 1c ) to exclude breast tissue . the final region of interest was defined as ubsf and was volumetrically measured ( cubic centimeters ) . chest computed tomography scans demonstrating upper body subcutaneous neck fat ( ubsf ) measurement technique . a , example of the initial slice displaying the sternal landmark . b , example of manual tracing to exclude mediastinal fat ( total neck fat ) . bmi , waist circumference , neck circumference , systolic blood pressure , diastolic blood pressure , fasting plasma glucose , and fasting lipids were determined based on study visit evaluations . bmi was calculated by dividing the weight ( kilograms ) by the square of height ( meters ) . waist circumference was measured to the nearest 0.25 inch at the level of the umbilicus . neck circumference was measured to the nearest 0.25 inch below the laryngeal prominence and perpendicular to the long axis of the neck . diabetes was defined as a fasting plasma glucose 126 mg / dl or treatment with insulin or a hypoglycemic agent . participants were considered to be current smokers if they smoked 1 cigarettes per day for the last year . among the representative preselected sample of 100 participants used for this analysis , 2 scans did not have adequate slices through the anatomic region of interest , 3 scans did not have the required 40 scans superior to the sternal landmark , and 3 had unusual anatomy disrupting the landmarks , resulting in a readable sample size of 92 . one participant did not attend the clinical examination , and no baseline characteristics were available ; therefore , the sample characteristics are based on a sample size of 91 . abdominal subcutaneous adipose tissue ( sat ) and vat volumes were previously determined using a protocol for abdominal adipose tissue quantification that was described previously . , k.e.t . ) for interobserver variation and then repeated by the first reader ( k.j.r . ) for intraobserver variation . inter and pearson correlation coefficients were calculated to determine the association between ubsf and cardiometabolic risk factors . the original framingham heart study cohort was first established in 1948 with the original cohort recruited from framingham , massachusetts . the offspring and third generation cohorts were recruited subsequently and have been well described previously . the mdct substudy consists of 1333 participants from the offspring cohort and 1431 participants from the third generation cohort who underwent computed tomography imaging as part of a comprehensive assessment of vascular calcification . participants for our current analysis are drawn from the mdct 2 substudy who underwent scanning from september 2008 to december 2011 . inclusion criteria included an age of > 35 years for men and > 40 years for nonpregnant women . a sample of 100 participants was selected from the study population for the purposes of the present analysis . this sample was randomly selected to include 50 women and 50 men evenly spread across the ages and divided between the offspring and third generation cohorts . the institutional review boards of boston university medical center and massachusetts general hospital approved the study protocol , and all participants provided written consent . subjects underwent mdct imaging of the thorax in a supine position using a general electric discovery vct 64slice pet / ct scanner ( ge healthcare ) . the entire chest from the lung base to the apices was imaged using a prospectively ecgtriggered computed tomography scanning protocol during a single inspirational breath hold to enable lung and coronary assessment of the apices . the gantry rotation time was 0.35 seconds , the tube voltage was 120 kvp , and the tube current was 300 ma ( subject weight 220 lb ) or 350 ma ( subject weight > 220 lb ) . a detector width of 0.625 mm was used . the 210 scan reconstruction algorithm was used , providing 210 of raw scan data ( tube rotation ) with an optimized reconstruction technique that provided images of 175ms temporal resolution depending on the speed of gantry rotation ( 0.35 seconds ) . mdct slices were loaded onto a dedicated offline workstation ( aquarius 3d workstation ; terarecon inc ) . a predefined image display setting ( 195 to 45 hounsfield units ; center : 120 hounsfield units ) was used to identify adipose tissue within these slices . the anatomic region of interest was defined by 40 contiguous 0.625mm slices superior to the body of the sternum and covering an area of 25 mm . the sternal landmark was determined by the first single slice to show the bowtie shape of the sternum filled completely with trabecular bone ( figure 1a ) . forty contiguous slices above the sternum were selected , and every 10th slice starting with the first slice was manually reviewed . the reader manually excluded the adipose tissue within the mediastinum ( figure 1b ) for the total neck fat . the reader then outlined the adipose tissue exterior to the chest wall ( figure 1c ) to exclude breast tissue . the final region of interest was defined as ubsf and was volumetrically measured ( cubic centimeters ) . chest computed tomography scans demonstrating upper body subcutaneous neck fat ( ubsf ) measurement technique . a , example of the initial slice displaying the sternal landmark . b , example of manual tracing to exclude mediastinal fat ( total neck fat ) . bmi , waist circumference , neck circumference , systolic blood pressure , diastolic blood pressure , fasting plasma glucose , and fasting lipids were determined based on study visit evaluations . bmi was calculated by dividing the weight ( kilograms ) by the square of height ( meters ) . waist circumference was measured to the nearest 0.25 inch at the level of the umbilicus . neck circumference was measured to the nearest 0.25 inch below the laryngeal prominence and perpendicular to the long axis of the neck . diabetes was defined as a fasting plasma glucose 126 mg / dl or treatment with insulin or a hypoglycemic agent . participants were considered to be current smokers if they smoked 1 cigarettes per day for the last year . among the representative preselected sample of 100 participants used for this analysis , 2 scans did not have adequate slices through the anatomic region of interest , 3 scans did not have the required 40 scans superior to the sternal landmark , and 3 had unusual anatomy disrupting the landmarks , resulting in a readable sample size of 92 . one participant did not attend the clinical examination , and no baseline characteristics were available ; therefore , the sample characteristics are based on a sample size of 91 . abdominal subcutaneous adipose tissue ( sat ) and vat volumes were previously determined using a protocol for abdominal adipose tissue quantification that was described previously . the protocol was completed independently by 2 readers ( k.j.r . , k.e.t . ) for interobserver variation and then repeated by the first reader ( k.j.r . ) for intraobserver variation . inter and pearson correlation coefficients were calculated to determine the association between ubsf and cardiometabolic risk factors . the mean bmi was 27.1 kg / m for women and 27.8 kg / m for men . study sample characteristics data presented as mean ( sd ) for continuous characteristics and mean ( sd ) or median ( quartile 1 , quartile 3 ) for categorical characteristics . bmi , indicates body mass index ; hdl , highdensity lipoprotein ; sat , subcutaneous adipose tissue ; sbp , systolic blood pressure ; vat , visceral adipose tissue . based on a sample size of 91 because 1 participant did not attend the clinical examination and thus no baseline clinical characteristics were available . data on neck circumference was available for 42 patients because it was not measured in offspring exam 8 . the mean volume of total neck fat was 438 cm for women and 429 cm for men . the mean volume of breast tissue fat was 128 cm for women and 83 cm for men . the anatomic area of interest , ubsf , was defined by the total neck fat minus the breast fat ( figure 1 ) . the mean volume of ubsf was 310 cm for women and 345 cm for men . intra and the intrareader intraclass correlation coefficient was 0.99 , and the interreader intraclass correlation coefficient was 0.99 . intrareader ( a ) and interreader ( b ) upper body subcutaneous neck fat measurement . age and sexadjusted pearson correlation coefficients for ubsf and cardiometabolic risk factors are presented in table 2 . ubsf was strongly correlated with waist circumference ( r=0.90 ) , bmi ( r=0.89 ) , sat ( r=0.87 ) , and vat ( r=0.86 ) . sexadjusted pearson correlation coefficients for upper body subcutaneous neck fat and cardiometabolic risk factors bmi , indicates body mass index ; hdl , highdensity lipoprotein ; na , not available ; sat , subcutaneous adipose tissue ; sbp , systolic blood pressure ; vat , visceral adipose tissue . data on neck circumference was available for 42 patients because it was not measured in offspring exam 8 . the mean bmi was 27.1 kg / m for women and 27.8 kg / m for men . study sample characteristics data presented as mean ( sd ) for continuous characteristics and mean ( sd ) or median ( quartile 1 , quartile 3 ) for categorical characteristics . bmi , indicates body mass index ; hdl , highdensity lipoprotein ; sat , subcutaneous adipose tissue ; sbp , systolic blood pressure ; vat , visceral adipose tissue . based on a sample size of 91 because 1 participant did not attend the clinical examination and thus no baseline clinical characteristics were available . data on neck circumference was available for 42 patients because it was not measured in offspring exam 8 . the mean volume of total neck fat was 438 cm for women and 429 cm for men . the mean volume of breast tissue fat was 128 cm for women and 83 cm for men . the anatomic area of interest , ubsf , was defined by the total neck fat minus the breast fat ( figure 1 ) . the mean volume of ubsf was 310 cm for women and 345 cm for men . the intrareader intraclass correlation coefficient was 0.99 , and the interreader intraclass correlation coefficient was 0.99 . intrareader ( a ) and interreader ( b ) upper body subcutaneous neck fat measurement . age and sexadjusted pearson correlation coefficients for ubsf and cardiometabolic risk factors are presented in table 2 . ubsf was strongly correlated with waist circumference ( r=0.90 ) , bmi ( r=0.89 ) , sat ( r=0.87 ) , and vat ( r=0.86 ) . sexadjusted pearson correlation coefficients for upper body subcutaneous neck fat and cardiometabolic risk factors bmi , indicates body mass index ; hdl , highdensity lipoprotein ; na , not available ; sat , subcutaneous adipose tissue ; sbp , systolic blood pressure ; vat , visceral adipose tissue . data on neck circumference was available for 42 patients because it was not measured in offspring exam 8 . first , the quantification of ubsf using computed tomography is feasible and reproducible in a communitydwelling sample from the framingham heart study . second , ubsf is correlated with cardiometabolic risk including strong correlations with other measures of adiposity in our feasibility study . traditional measures of obesity include anthropometric measurements such as bmi and waist circumference , both of which have been correlated with increased cardiometabolic risk and increased risk of obesityrelated morbidity . although bmi is an excellent indicator of obesity , it does not account for regional fat distribution and can not differentiate among fat compartments . fat distribution studies have used imaging modalities such as computed tomography to quantify and differentiate abdominal adipose tissue depots . both vat and sat have been associated with cardiometabolic risk , but vat has been shown to be a particularly pathogenic fat depot . data from the framingham heart study showed that waist circumference was highly correlated with vat ( r=0.78 for women and r=0.73 for men ) but also with sat ( r=0.87 for women and r=0.88 for men ) , suggesting that waist circumference can not discriminate between vat and sat . similar to previous studies of waist circumference , studies of neck circumference have shown an association between ubsf and insulin resistance , elevated blood pressure , and dyslipidemia ; however , neck circumference is only a proxy for ubsf . direct volumetric quantification of this novel depot is needed to understand the full clinical implications . we have developed a protocol for the quantification of ubsf that has high inter and intrareader reproducibility . the present study adds a reproducible technique to the literature that allows for further evaluation of upper body fat . through the use of this protocol , characterization of ubsf can be quantified , leading to the ability to assess the correlations of this fat depot with clinical characteristics and obesity outcomes . ultimately , implementation of this protocol more broadly may help provide further insight into the role of regional fat distribution and better mechanistic understanding of obesityrelated complications related to ubsf . major strengths of our study include the use of a large , communitydwelling population with comprehensively defined clinical characteristics . the use of 2 independent readers helped to establish the reproducibility of our protocol . to our knowledge , there are no other gold standard measurements of ubsf , and that limits our ability to assess measurement of validity . the study sample is predominantly of european ancestry , and generalizability to other ethnicities or populations enriched with ubsf is uncertain . ubsf can be reproducibly quantified using computed tomography in a communitydwelling sample from the framingham heart study . quantification of this unique fat depot may add to the mechanistic understanding of regional fat distribution .

transmembrane transport proteins often function in conjunction with auxiliary proteins that facilitate their vectorial activities [ 1 , 2 ] . these proteins are the membrane fusion proteins ( mfps ; transporter classification number ( tc # ) 8.a.1 in the transporter classification database ( tcdb ; http://www.tcdb.org/ ) , which function in conjunction with a variety of transport system types in gram - negative and gram - positive bacteria and the outer membrane auxiliary ( oma ; 1.b.18 ) proteins which function in conjunction with a different set of transporter types , exclusively in gram - negative bacteria [ 4 , 5 ] . adaptors , connecting a primary porter in the cytoplasmic membrane , belonging to one of four families of exporters ( mfs ( tc # 2.a.1 ) , rnd ( 2.a.6 ) , abc ( 3.a.1 ) , or aae ( 2.a.85 ) ) with an outer membrane factor ( omf ; 1.b.17 ) that provides a porin or channel function in the outer membrane . thus , in conjunction with an mfp and an omf , the primary porter in the cytoplasmic membrane pumps molecules out of the cytoplasm , across both membranes of the cell envelope , and into the external milieu without equilibration of solutes in the periplasm , all in a single energy coupled step . crosslinking studies of the mfp , acra of e. coli ( 8.a.1.6.1 ) , with its cognate transporter , acrb ( 2.a.6.2.2 ) , and its omf , tolc ( 1.b.17.1.1 ) , revealed that acra could be crosslinked to both acrb ( via the c - terminal portion of acra ) and tolc ( via the central coiled - coil region of acra ) . most mfps are about 350500 residues in length and either span the cytoplasmic membrane once at their n - termini or are anchored to the cytoplasmic membrane via a lipoyl moiety . these proteins cluster phylogenetically into subfamilies in accordance with the type of cytoplasmic membrane transporter with which they interact , although the omfs do not follow this pattern . these transport complexes export a variety of substrates such as toxins , drugs , aromatic acids , peptides , and proteins . the high - resolution 3d structures of two mfps , mexa of p. aeruginosa and acra of e. coli , have been solved by x - ray crystallography [ 8 , 9 ] . these proteins consist of three linearly arranged subdomains as suggested by earlier secondary structural predictions . each of these molecules consists of an n - terminal lipoyl domain , a central 47 long alpha - helical coiled - coil hairpin domain , and a c - terminal six - stranded beta - barrel . in the crystal structures , hairpins of neighboring mexa or acra mfps may assemble and control conformational channel opening in the complex [ 6 , 8 ] . they stimulate some but are absolutely required for the functions of other abc exporters [ 10 , 11 ] . other transmembrane transporters function together with another type of auxiliary protein that facilitates the export of exo- and capsular polysaccharides . these outer membrane auxiliary proteins ( omas ) comprise a distinct protein family ( oma ; 1.b.18 ) . omas are about the same size as mfps but function with secondary active exporters of the polysaccharide transporter ( pst ) family and primary active exporters of the abc superfamily ( tc # s 2.a.66.2 and 3.a.1 , resp . ) . they are found exclusively in gram - negative bacteria , and in addition to spanning the periplasm , they form pores in the outer membranes . omas are , in general , not homologous to mfps , and their 3-dimensional x - ray structures show no obvious similarities [ 8 , 9 , 13 , 14 ] . the best studied member of the oma family , wzak30 , has been shown to be required for export of the group 1 k30 capsular polysaccharide in e. coli strain e69 . mutations in the encoding gene do not interfere with the synthesis or polymerization of the polysaccharide repeat unit but prevent appearance of the polysaccharide on the cell surface . wzak30 is a surface - exposed outer membrane lipoprotein , which forms sds - stable octomeric ring - like structures superficially resembling secretins ( 1.b.22 ) . mature wzak30 is a 359-residue lipoprotein that is synthesized as a precursor with a cleavable 20-residue amino - terminal signal sequence . the thiol group in the processed n - terminal cys 21 is modified by a thioether - linked diacylglyceryl moiety , and the amino group of cys21 is acylated . omas are believed to form channels through which polysaccharides pass to reach the cell surface . the ring - like homo - octamers ( tetramers of dimers ) have an outer diameter of ~9 nm and a central cavity of about 2 nm [ 13 , 16 ] . the 2.26 resolution structure of the 340 kda octamer of wza has been reported by dong et al . . the bulk of the wza structure is located in the periplasm and comprises three novel domains forming a large central cavity . the revealed wza structure is open to the extracellular environment but closed to the periplasm . the route and mechanism of capsular polysaccharide translocation have been proposed . except for the central -helical domains , omas and mfps lack significant structural similarity . another member of the oma family , kpsd of e. coli , has been reported to distribute between the periplasm and the two membranes . together with the primary transporter , kpse , it was suggested to facilitate transport across the periplasm as well as the outer membrane . mcnulty et al . reported that kdpd is an outer membrane protein involved in the export of group 2 capsular polysaccharides across this membrane . interestingly , kdpd , kdpe , and the biosynthetic complex comprise a metabolon that is located at the cell poles . the large rhsa protein , previously of unknown function , is a component of this complex and is required for normal polysaccharide export . recently , cuthbertson et al . have reviewed the roles of omas in polysaccharide export . they have suggested changing the name of the family to opx ( outer membrane polysaccharide exporters ) . they have performed useful phylogenetic analyses , contributing insight into the distribution and structural relationships of these proteins . figure 1 shows an alignment of a large portion of an oma homologue , an oma - like protein from sinorhizobium meliloti , with the corresponding region of an mfp protein from nitrococcus mobilis . this alignment displays 30% identity and 41% similarity with no gaps and a comparison score based on the gap program of 23 standard deviations ( s.d . ) . this value is far in excess of what is required to establish homology . using similar criteria , both of these proteins proved to be homologous to established members of their respective families in the transporter classification database ( tcdb ) [ 3 , 5 , 22 ] as well as in the nonredundant ncbi protein database . thus , the oma homologue of s. meliloti , used to establish homology with the mfp of n. mobilis , is homologous to the established oma , exof of s. meliloti ( 1.b.18.1.1 ; 34% identity , 55% similarity , and a comparison score of 87 s.d . ) , while the mfp of n. mobilis , used in the same comparison , is homologous to the mfp hlyd of e. coli ( tc # 8.a.1.3.1 ; 26% identity , 45% similarity with a comparison score of 53 s.d . ) . to confirm this conclusion of homology , we aligned dozens of -helical regions of -proteobacterial omas with the corresponding regions of dozens of mfps . figure 2 shows the results of a second example obtained between a different -proteobacterial oma with an -proteobacterial mfp . although the sequences are very different from those shown in figure 1 , this alignment also gave a comparison score of 23 s.d . the results summarized above establish that central regions of mfps are homologous to the corresponding domains in a restricted group of omas , those present in -proteobacteria . surprisingly , the sequence similarity with mfps was not observed in the omas from other gram - negative proteobacteria . the hydropathy plots for the oma ( a ) and mfp ( d ) proteins are shown , as are secondary structure predictions ( b ) and ( c ) generated with the what program and the sopma program , respectively . specifically , we see n - terminal regions in both proteins , which show predicted mixtures of - and -structure followed by central regions , which are entirely -helical . following these helical regions , we again observe regions predicted by the sopma program to contain mixtures of - and -structure with a predominance of -structure . the three - dimensional structures of representative mfps and omas are surprisingly different [ 8 , 9 , 14 ] . in fact , these structures are so different that no parallels were noted by the investigators conducting the x - ray crystallographic studies . our results lead to the suggestion that the portions of these proteins that include the central -helical regions , evolved from a common ancestral sequence . indeed , the portions aligned in figures 1 and 2 are the only parts to show significant sequence similarities between the -proteobacterial omas and the gram - negative bacterial mfps . this , of course , does not prove , but it suggests that other portions of these proteins are nonhomologous . in gram - negative bacterial mfps , of the three regions noted above , the -helical region shows the least sequence similarity when compared with other mfps . moreover , in some but not all gram - positive mfps , this region is deleted , and these mfps are consequently much smaller . the most logical explanations for these findings are ( 1 ) that the long coiled - coil region of an mfp replaced the corresponding helical region in an oma , specifically in -proteobacteria , ( 2 ) the specific amino acyl sequence of this region is minimally important for function in all gram - negative bacteria , and ( 3 ) in some gram - positive bacteria , where there is no periplasm and no outer membrane , it is not required at all . the substitution event that occurred in -proteobacteria probably occurred late , after the separation of -proteobacteria from other proteobacteria . this would account for the high comparison scores obtained , scores , for example , for the alignments shown in figures 1 and 2 . this suggestion is also in agreement with the observation that of the mfps , those from -proteobacteria are most similar to the -helical regions of -proteobacterial omas , clearly implying that the intragenic transfer event occurred in -proteobacteria . it will be interesting to evaluate the detailed three - dimensional structural differences as well as the functional differences of these -proteobacterial oma homologues in light of these observations . of particular interest will be the specific roles of the central -helical regions of the proteins within these two families in executing their various functions , which may be multifaceted . our observations illustrate how poorly understood the structures of diverse members of a protein family can be , even after eucidation of high resolution protein structures for representative members of this family . they also provide unusual and unexpected examples concerning the progression of protein structural divergence during the evolutionary process . thus , the molecular basis underlying the differences and similarities between the various mfps and oma proteins should provide intriguing material for future studies .

periodontitis is an immune - associated inflammatory disease of the periodontium characterized by progressive destruction of the supporting tissues of teeth . it can destroy the surrounding connective tissue and adjacent alveolar bone and eventually causes tooth loss [ 2 , 3 ] . the immune and inflammatory responses that are critical to the pathogenesis of periodontitis are modified by a number of host - related factors . therefore , while microbial plaque is the primary etiology and initiates the host immune response , which induces the signs of periodontitis , the major component of the outer membrane of gram - negative bacteria , porphyromonas gingivalis , lipopolysaccharide ( lps ) , initiates the production of various cytokines such as interleukin-8 ( il-8 ) and tnf- which infiltrate gingival connective tissue and elicit a local inflammatory response . it further causes an increase in number and activity of polymorphonucleocytes ( pmn ) along with these cytokines . pmns also produce reactive oxygen species ( ros ) superoxide via the respiratory burst mechanism as part of the defense response to infection . the neutrophil - produced high levels of ros are associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis . the ros has the capacity to activate macrophages to synthesize and secrete inflammatory cytokines and hydrolytic enzymes , which manifest potent proinflammatory and catabolic activities and play key roles in periodontal tissue breakdown . periodontitis has been shown to link other chronic diseases such as diabetes , cardiovascular diseases , and rheumatoid arthritis . studies indicate that treatment of rheumatoid arthritis may improve the associated periodontal disease and vice versa . lately natural compounds capable of modulating the host inflammatory response have received considerable attention . naturally occurring orally related antibacterial and anti - inflammatory actions of polyphenols are capable of modulating the host inflammatory response . of these , certain flavonoids appear to stand out because of their beneficial profile and clinical evidence . resveratrol ( trans-3,4,-5-trihydroxystilbene ) is a widely existing stilbenoid in grape skin and in other plants such as berries and peanuts [ 12 , 13 ] . its supplementation directly suppresses the release of proinflammatory cytokines such as tnf- , il-1 , il-6 , il-10 , mcp-1 , ifn , and ifn in a wide range of tissues [ 1519 ] , including the brain in rodents . it has been shown to significantly inhibit nf-b - dependent cell adhesion molecules in monocyte adhesion to the endothelium induced by p. gingivalis lps suggesting that resveratrol has therapeutic effect in periodontal pathogen - induced vascular inflammation . the stilbene glucoside 2,3,5,4-tetrahydroxystilbene-2-o--d - glucoside ( thsg ) is one of the major bioactive components of polygonum multiflorum thunb . it also has protective effects on experimental colitis by reducing the level of oxygen and nitrogen free radicals . mechanism involved in p. multiflorum - induced antiatherosclerosis may be caused by thsg - induced antagonistic effects on oxidation of lipoprotein , proliferation , and decrease of no content of coronary arterial smooth muscle cells . it also has been shown to exert protective effect on cardiotoxicity induced by doxorubicin in vitro and in vivo . in addition , thsg can diminish peroxidation levels in the brain in a mouse model of alzheimer 's disease or cerebral ischemia - reperfusion . the protective effects of thsg are mediated by modulation of jnk , sirt1 , and nf-b pathways . in animals , experimental periodontitis can be induced by dietary manipulation , bacterial toxin injection , laying of peridental silk ligatures or orthodontic elastics for bacterial colonization [ 29 , 30 ] , and surgical removal of alveolar bone [ 31 , 32 ] . in the present study , we investigate the role of ethanol extract of p. multifloriand pure 2,3,5,4-tetrahydroxystilbene-2-o - beta - glucoside on the degree of alveolar bone loss associated with periodontitis in rats and its signal transduction pathways involved in the preventative and inhibitory effects in the development of periodontitis . 2,3,5,4-tetrahydroxystilbene-2-o - beta - glucoside ( thsg , purity : 95% , dissolved in 50% ethanol for animal feeding ; dissolved in dmso for cell treatment ) was kindly provided by dr . louis , mo , usa ) was dissolved in 50% ethanol for animal feeding and dissolved in dmso for cell treatment . p. multiflora thunb was purchased from a traditional chinese medicine market in china and identified by industrial technology research institute , taiwan . moreover , voucher specimens ( he shou wu 01 ) of dried rhizoma were deposited in the graduate institute of pharmacognosy , college of pharmacy , taipei medical university , taipei , taiwan . the rhizome of p. multiflora was cut into small pieces , immersed in a 10-fold amount of 50% ethanol , and refluxed for 2 hour at 65c , twice . and then the twice filtrate was combined and concentrated under a vacuum using a rotary evaporator to remove the ethanol . the ethanolic extract was freeze - dried to yield the powder ( pm - e ) . the yield of pm - e was about 27.82% ( w / w ) and 2,3,5,4-tetrahydroxystilbene-2-o - beta - glucoside ( thsg ) amount is 8.0% in pm - e by hplc analysis . the substance marker , thsg ( purity : 95% ) , was kindly provided by dr . human gingival fibroblasts ( hgfs ) were obtained from five patients ( males aged 22 to 32 years ; mean age : 24 years ) , and all experiments were repeated three times as described in our previous study . briefly , gingival specimens were taken from gingivectomy , crown lengthening , or distal wedge procedure of noninflamed periodontal tissues . specimens were directly submerged in leibovitz l-15 medium ( invitrogen , grand island , ny , usa ) containing 2 mg / ml dispase ii ( roche diagnostics , indianapolis , in , usa ) and 10% fetal bovine serum ( fbs ) ( invitrogen ) at 4c for 2 days . after separating and removing the outer epithelial layer , the connective tissue was minced and digested in leibovitz l-15 medium containing 10% fbs and 2 mg / ml collagenase ( sigma - aldrich , inc . ) for 24 hours . tissue was then placed in flasks containing 10% fbs in dulbecco modified eagle medium / f12 ( dmem / f12 ) ( invitrogen ) to allow the cells to migrate from the explants . when hgfs reached 80% confluence , cells were starved in serum - free dmem / f12 for 24 hours before experiments . similar patterns were examined in different cell origins , and one of them was selected to report . approval of all procedures was granted by the ethics committee of the faculty of medicine , tri - service general hospital , taipei , taiwan ( tsghirb number 2 - 103 - 05 - 067 ) . all participants included in the study signed a written consent form before the procedure . to evaluate the toxic effects of resveratrol and thsg on hgfs , hgfs were placed at a density of 1 10 cell / well in the wells of 96-well plates containing dmem / f12 and 10% fbs and cultured until 80% confluence . the cells were washed once with phosphate - buffered saline , and the medium was replaced with serum - free medium for 24 hours ' starvation . the cell viability of hgfs was tested after treatment of thsg ( 0 , 10 , 25 , 100 , or 200 m ) or resveratrol ( 0 , 10 , 25 , 100 , or 200 m ) in 0.25% stripped fbs medium for 72 h with reflashed medium with agents daily . the cell viability was performed with celltiter 96 aqueous one solution ( promega , madison , wi , usa ) according to the manufacturer 's protocol . to examine the effects of resveratrol and thsg on the mrna expression of proinflammatory cytokines , we treated hgfs with vehicle , 1 g / ml p. gingivalis lps ( porphyromonas gingivalis lipopolysaccharide ) ( invivogen , san diego , ca , usa ) , 1 and 10 m resveratrol , and 1 and 10 m thsg or combined treatments for 6 h. furthermore , in order to investigate the impacts of thsg on proinflammatory cytokines and inflammatory markers in ligature - induced experimental periodontitis of rats , silk - surrounded gingival tissues were removed and homogenized by a high - throughput tissue ( precellys 24 , bertin corp . , rockville , md , usa ) . total rna was extracted and genomic dna was also eliminated with illustra rnaspin mini rna isolation kit ( ge healthcare life sciences , buckinghamshire , united kingdom ) . 1 g of dnase i - treated total rna was reverse - transcribed with revertaid h minus first strand cdna synthesis kit ( life technologies corporation , carlsbad , california , usa ) into cdna and used as the template for real - time pcr reactions and analysis . the real - time pcr reactions were performed using quantinova sybr green pcr kit ( qiagen ) on cfx connect real - time pcr detection system ( bio - rad laboratories , inc . , hercules , ca , usa ) . this involved an initial denaturation at 95c for 5 min , followed by 45 cycles of denaturing at 95c for 5 sec and combined annealing / extension at 60c for 10 sec , as described in the manufacturer 's instructions . the primer sequences were shown as follows : homo sapiens tumor necrosis factor - alpha ( tnf- ) , forward 5-gcccatgttgtagcaaaccc-3 and reverse 5-tatctctcagctccacgcca-3 ( accession number nm_000594.3 ) ; homo sapiens interleukin-1 beta ( il-1 ) , forward 5-cttcgaggcacaaggcaca-3 and reverse 5-gcttcagacacttgagcaatga-3 ( accession number nm_000576.2 ) ; homo sapiens interleukin-6 ( il-6 ) , forward 5-aatcatcactggtcttttggag-3 and reverse 5-gcatttgtggttgggtca-3 ( accession number nm_000600.3 ) ; homo sapiens sirtuin 1 ( sirt1 ) , forward 5-ttgggtaccgagataaccttct-3 and reverse 5-ttgcatgtgaggctctatcc-3 ( accession number nm_012238.4 ) ; homo sapiens 18s ribosomal rna ( 18s ) , forward 5-gtaacccgttgaaccccatt-3 and reverse 5-ccatccaatcggtagtagcg-3 ( accession number nr_003286 ) ; rattus norvegicus tumor necrosis factor - alpha ( tnf- ) , forward 5-ttctcattcctgctcgtggc-3 and reverse 5-gcccatttgggaacttctcct-3 ( accession number nm_012675.3 ) ; rattus norvegicus interleukin-1 beta ( il-1 ) , forward 5-agctttcgacagtgaggagaa-3 and reverse 5-tcatctggacagcccaagtc-3 ( accession number nm_031512.2 ) ; rattus norvegicus inducible nitric oxide synthase ( inos ) , forward 5-aaaaccccaggtgctattccc-3 and reverse 5-tccagggattctggaacattctgt-3 ( accession number nm_012611.3 ) ; rattus norvegicus cyclooxygenase-2 ( cox-2 ) , forward 5-ttccaaaccagcaggctcat-3 and reverse 5-aaaagcagctctgggtcgaa-3 ( accession number l20085.1 ) ; rattus norvegicus sirtuin 1 ( sirt1 ) , forward 5-aggccacggataggtccata-3 and reverse 5-gaattgttcgaggatcggtgc-3 ( accession number xm_006223877.2 ) ; rattus norvegicus 18s ribosomal rna ( rn18s ) , forward 5-cctggttgatcctgccagtag-3 and reverse 5-ggccgtgcgtacttagacat-3 ( accession number nr_046237.1 ) . calculations of relative gene expression ( normalized to 18s reference gene ) were performed according to the ct method . fidelity of the pcr reaction was determined by melting temperature analysis . to examine the effects of thsg in p. gingivalis lps - induced nf-b activation , nuclear and cytoplasm proteins were extracted and isolated from hgfs by using of the thermo scientific ne - per nuclear and cytoplasmic extraction kit protocol ( thermo scientific , rockford , il , usa ) . briefly , hgfs were lysed in cytoplasm extraction reagent and were centrifuged at 16,000 g for extraction of the nuclear material . proteins from the nuclear material were further extracted by the addition of nuclear extraction reagent to the nuclei and centrifuged at 16,000 g . the protein concentration of the nuclear and cytoplasmic extracts was determined with the bca protein assay kit ( thermo scientific ) . to examine the effects of thsg in p. gingivalis lps - induced nf-b activation in nucleus , we performed western blot analysis to quantify the protein expression levels of nf-b in the nuclear extracts of hgfs which had been pretreated with 25 m thsg and followed by the treatment with 1 g / ml p. gingivalis lps for 3 h. nuclear protein samples were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel ( sds - page ) . a 20-g quantity of protein was loaded in each well with 5x sample buffer , and the protein samples were resolved by electrophoresis at 100 v for 2 hrs . the resolved proteins were transferred from the polyacrylamide gel to millipore immobilon - psq transfer pvdf membranes ( millipore , billerica , ma , usa ) with the mini trans - blot cell ( bio - rad laboratories , inc . , hercules , ca , usa ) . the membranes were blocked with a solution of 2% fetal bovine serum in tris - buffered saline . the membranes were incubated with primary antibodies to nf-b , phospho - p44/42 mapk ( perk1/2 ) , erk2 , glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) ( genetex international corporation , hsinchu city , taiwan ) , phospho - ampk , ampk ( cell signaling technology , inc . , beverly , ma , usa ) , and lamin b1 ( abcam , cambridge , ma , usa ) , at 4c overnight and washed , and the proteins were detected with hrp - conjugated secondary antibodies and immobilon western hrp substrate luminol reagent ( millipore ) . images of the western blots were visualized and recorded by biospectrum imaging system ( uvp , llc , upland , ca , usa ) . all rats were housed in a reserved , pathogen - free facility and were handled in accordance with protocols approved by the institutional animal care and use committee , national defense medical center , taipei , taiwan ( iacuc-15 - 361 ) . a group of 8-week - old male sprague - dawley rats weighing between 200 and 250 g ( n = 50 ) were randomly divided into eleven treatment groups defined as follows : nonligature ( control , n = 10 ) ; ligature ( n = 10 ) ; ligature + 0.1 mg / kg / d of thsg ( n = 5 ) ; ligature + 10 mg / kg / d of thsg ( n = 5 ) ; ligature + 25 mg / kg / d of resveratrol ( n = 5 ) ; ligature + 12.5 mg / kg / d of ethanol extracts ( n = 5 ) ; ligature + 25 mg / kg / d of ethanol extracts ( n = 5 ) ; ligature + 50 mg / kg / d of ethanol extracts ( n = 5 ) . for the ligation , thsg , and ethanol extracts groups , experimental periodontitis was induced by placing 3 - 0 silk sutures around mandibular first molars at the gingival level ( two molars in each rat ) , as described in our previous study . the rats in the thsg and ethanol extracts groups also received these extracts by gavage feeding , starting 1 d before ligation . the rats in the control and ligature groups received sterile water daily . on day 8 , all rats were sacrificed by co2 inhalation , and the mandibles were excised and silk - surrounded gingiva was also collected for further mrna examination , and then the mandibles were fixed in 4% paraformaldehyde . the mandibular specimens were prepared for dental radiography . to verify the level of bone loss for these treatment groups , the mandibular molars were examined by digital radiography using a computerized imaging system ( asahi xspot ; asahi roentgen ind . the x - ray tube was operated at 70 kv , with a current of 6 ma , for 0.64 s , and the source - to - sensor distance was 50 cm . the images were processed using an image management system ( infinitt dental pacs image system ; infinitt north america inc . , phillipsburg , nj , usa ) . we determined the dental alveolar bone level , defined as the distance from the cementoenamel junction ( cej ) to the most coronal level of the alveolar bone crest ( cej - bone distance ) , using image j processing software ( image j software , national institutes of health , bethesda , md , usa ) . the two parameters of the first mandibular molar were recorded along the mesial or distal root surfaces , respectively . in this study , all of the collected data of immunoblot and nucleotide densities were analyzed by ibm spss statistics software version 19.0 ( spss inc . , chicago , il , usa ) . two - tail student 's t - test was conducted and considered significant at p values < 0.05 ( or # ) , 0.005 ( or # # ) , and 0.001 ( or # # # ) . hgfs ( 1 10 cells / well ) were cultured in 96-well plate for 24 h and then starved with serum - free dmem / f12 medium for 24 h. after starvation , hgfs were refed with medium containing 0.25% stripped fbs with different concentrations of resveratrol ( 0 , 10 , 25 , 100 , or 200 m ) or thsg ( 0 , 10 , 25 , 100 , or 200 m ) reflashed daily for 72 h. resveratrol significantly enhanced cell proliferation of hgfs at low concentration ( 10 m ) but inhibited cell proliferation at high concentrations ( 100 and 200 m ) significantly ( figure 1(a ) ) . on the other hand , thsg significantly enhanced hgfs cell proliferation when the concentration was over 25 m and did not show any cytotoxic effect in hgfs ( figure 1(b ) ) . hgfs were cultured in 6-well plate . after starvation , cells were treated with 1 g / ml of lps extracted from p. gingivalis for 0 , 3 , 6 , and 24 h prior to harvest . lps induced the expression of tnf- at 3 h and maximum expression at 6 h ( figure 2(a ) , upper panel ) . lps stimulated the expression of il-1 at 6 h and expressed more at 24 h. lps stimulated the expression of il-6 at 3 h , 6 h , and 24 h ( figure 2(a ) , middle and lower panel ) . hgfs were cultured in 100 mm petri dish and starved with serum - free dmem / f12 medium for 24 h before studies . hgfs were pretreated with resveratrol or thsg ( 1 and 25 m ) for 30 min and then treated with 1 g / ml p. gingivalis lps for another 6 h. resveratrol and thsg significantly attenuated lps - induced expression of proinflammatory cytokines such as tnf- , il-1 , and il-6 ( figure 2(b ) ) . the inhibitory effect on the expression of proinflammatory cytokines by low concentration of thsg ( 1 m ) was more than that of resveratrol . activation of sirt1 is involved in anti - inflammatory process [ 16 , 35 ] . it is of interest to investigate if activation of sirt1 also plays a role in thsg - inhibited proinflammatory cytokines expression . hgfs were cultured in 6-well plate and were starved with serum - free dmem / f12 medium for 24 h when the cell confluence was reached to 80% . hgfs were treated with 1 g / ml of lps extracted from p. gingivalis , 100 m resveratrol , or thsg , and then rna was extracted . results shown in figure 3(a ) indicate that lps significantly induced sirt1 expression at 24 h. however , resveratrol significantly reduced the mrna expression of sirt1 at 1 h but enhanced it at 5 h to 24 h dramatically ( figure 3(b ) ) . on the other hand , thsg constantly enhanced the mrna expression of sirt1 significantly from 1 h to 24 h ( figure 3(c ) ) . hgfs were pretreated with resveratrol or thsg ( 1 and 25 m ) for 30 min and then treated with 1 g / ml p. gingivalis lps for another 6 h. the mrna expression of sirt1 was significantly enhanced by 25 m thsg but significantly reduced by 1 m resveratrol when treated with p. gingivalis lps ( figure 3(d ) ) . the enhanced effect on the expression of sirt1 by thsg was more than that of resveratrol when cells were treated with p. gingivalis lps . signal transductions involved in the inhibitory effect of thsg on lps - induced inflammation were also studied . hgfs were pretreated with resveratrol or thsg ( 25 m ) for 30 min and then treated with 1 g / ml p. gingivalis lps for another 3 h. the cytosolic and nuclear proteins of hgfs were extracted . in nuclear protein , however , thsg did not only significantly reduce lps - augmented nuclear translocation of nf-b but also decrease that when thsg was presented alone in hgfs ( figures 4(a ) and 4(b ) , upper panel ) . in cytosolic protein of hgfs , the activation of ampk and erk1/2 was significantly enhanced by thsg . on the other hand , treatment of lps pretreatment of thsg significantly enhanced lps - reduced ampk activation and increased erk1/2 activation even in the presence of lps ( figures 4(a ) and 4(b ) , middle and lower panel ) . ligature - induced experimental periodontitis was performed in rats ( 8-week - old ) at their first molar . rats were fed with thsg ( 0.1 or 10 mg / kg / day dissolved in sterile water ) or sterile water by gavage for 7 days . radiographic films were taken and the lost alveolar bone level was evaluated ( figure 5(a ) ) . however , administration of thsg significantly attenuated ligature - induced alveolar bone level lost ( figure 5(b ) ) . the mrna expression of proinflammatory cytokines and mediators , including tnf- , il-1 , inos , cox-2 , and sirt1 , in rat gingival tissues was evaluated by qpcr . thsg significantly attenuated ligature - induced tnf- , il-1 , inos , and cox-2 mrna expressions , even in low concentration ( 0.1 mg / kg ) ( figures 6(a)6(d ) ) . in addition , administration of thsg significantly enhanced the mrna expression of sirt1 in rat gingival tissues ( figure 6(e ) ) . to evaluate whether the crude extracts of p. multiflori would also have ameliorated effect on periodontitis , ligature - induced experimental periodontitis was performed in rats at their first molar and rats were fed with resveratrol ( 25 mg / kg / day ) , ethanol extracts ( 12.5 , 25 , or 50 mg / kg / day ) , or 50% ethanol by gavage for 7 days . according to our previous reports [ 29 , 34 ] the radiographic image demonstrated the mandible from a ligatured rat and the measurements used to measure the periodontal bone - supporting ratio ( figure 7(a ) ) . the periodontal bone - supporting ratio was significantly reduced in ligature group at first molar . ethanol extracts of p. multiflori significantly ameliorated ligature - induced periodontal bone - supporting ratio in all concentrations ( figure 7(b ) ) . damage of periodontal bone in rats receiving 25 mg / kg resveratrol was also reduced but there was no significant difference between resveratrol - treated group and control . among all herb medicines , resveratrol contributes the most in terms of health benefits due to its antioxidant properties . it has been described as a scavenger of superoxides , hydroxyl radicals , and peroxynitrites . furthermore , resveratrol has been shown to induce activation of antioxidant enzymes [ 36 , 37 ] and activates the nuclear factor e2-related factor ( nrf2 ) antioxidant defense pathway . activated nrf2 translocates to the nucleus and mediates the transcription of target genes such as heme oxygenase 1 ( ho-1 ) and nad(p)h : quinine oxidoreductase 1 ( nqo-1 ) which are involved in cellular resistance to oxidative stress which confers protection against inflammation . when human gingival fibroblasts were treated with different concentrations of resveratrol or thsg , resveratrol only stimulated cell proliferation at low concentration ( figure 1 ) but thsg stimulated cell proliferation at all concentrations ( 1 to 250 m ) examined ( figure 1 ) . resveratrol suppressed p. gingivalis lps - stimulated ib phosphorylation and nuclear translocation of the p65 subunit of nfb in hmecs . the concentration of il-17 in the resveratrol group is lower than that in control group gingival tissue ( p < 0.05 ) ; however , there is no difference in the il-1 and il-4 levels of the groups ( p > 0.05 ) . in addition , both resveratrol and thsg inhibited nfb activation which is essential for the expression of inflammatory cytokines . resveratrol is also able to downregulate the cell adhesion molecules , icam-1 and vcam-1 , and significantly inhibits the p. gingivalis lps - induced adhesion of leukocytes to endothelial cells and to the aortic endothelium . in addition , resveratrol is able to reduce the inducible no synthase ( inos ) expression and further prevents no production . this characteristic contributes to oxidative stress reduction to decrease the systemic levels of some proinflammatory cytokines and result in resveratrol 's immunomodulatory effect [ 40 , 41 ] . in addition , resveratrol can stimulate mitochondrial biogenesis by increasing the expression of nad - dependent nuclear class iii histone deacetylase , sirtuin 1 ( sirt1 ) via the activated amp - activated protein kinase ( ampk ) [ 42 , 43 ] . our studies also indicate that thsg and resveratrol were able to activate sirt1 and showed a role in the inhibitory effect on lps - induced expression of inflammatory cytokines ( figure 3(b ) ) . both ethanol extract thsg and pure compound did inhibit ligature - induced periodontitis ( figures 5 and 7 ) . on the other hand , the treatment of resveratrol did not attenuate ligature - induced periodontitis of rats ( p = 0.054 ) in our studies ( figure 7 ) . mg / kg resveratrol per day for 19 days pretreatment indicated that there were higher bone loss values in ligated molars and unligated teeth in the control group than in the resveratrol - fed group ( p < 0.05 ) . resveratrol added in drinking water has been shown to lead to relieve ligature placement associated alveolar bone resorption . those studies and our results confirm the positive effects of resveratrol on ligature - induced periodontitis animal model . studies have also found a powerful effect of resveratrol in preventing lipid peroxidation , specifically low density lipoprotein ( ldl ) . improved endothelial function and enhanced cardioprotective effects have been observed with resveratrol through its anti - inflammatory and antioxidant properties in various animal models of myocardial injury , hypertension , and type 2 diabetes [ 39 , 45 , 46 ] . in summary , although both resveratrol and thsg activated erk1/2 and ampk and inhibited nfb activation in human gingival fibroblasts , regenerating capacity of thsg is better than that of resveratrol . thsg and resveratrol both inhibited the expression of proinflammatory cytokines such as il-1 and tnf- , which are involved in pathogenesis of periodontitis . yet again , the efficiency of thsg on the prevention of periodontitis is better than that of resveratrol . from all those evidences , it suggests that thsg either in crude extract or as pure compound may be used in the prevention and treatment of periodontitis in the future .

hyponatremia and an increased production of urine are frequently observed after calvarial remodeling in patients with craniosynostosis . hypoosmotic conditions result due to sodium loss in the body when these symptoms are not corrected appropriately , leading to dysfunctional cerebral blood flow , circulation problems , cerebral edema , or increased intracranial pressure . ( 1 ) described hyponatremia associated with cerebral disease for the first time in 1950 . initially , the loss of central nervous system control on renal sodium regulation was thought to be the cause so that hyponatremia was termed cerebral salt wasting ( csw ) . in 1957 , however , schwarz et al . ( 2 ) reported the syndrome of inappropriate antidiuretic hormone ( siadh ) secretion as hyponatremia after brain disease or brain surgery , and the clinical entity of csw was viewed as either an extremely rare disorder or a misnomer for what was truly siadh . only in recent years has csw been thought of as a distinct entity , and this recognition has been particularly striking in the field of neurosurgery , where it is viewed as a more common disorder than siadh ( 3 - 5 ) . the clinical findings are more consistent with csw than siadh as the cause of postoperative and postinjury fluid and electrolyte changes . berger et al . showed that the release of one or more natriuretic factors could also play a role in renal salt wasting seen in csw , in addition to decreased neural input to the kidney ( 6 ) . atrial natriuretic peptide ( anp ) and brain natriuretic peptide ( bnp ) are important factors in clinically diagnosing csw . csw and siadh are two conditions that show hyponatremia after central nervous system ( cns ) injury . the causes and treatment principles of the two conditions are totally different , and thus the differential diagnosis is critical . many authors reported the disorder of salt and water homeostasis in patients with brain lesions such as traumatic brain injury , subarachnoid hemorrhage , and brain tumor . however , not much data are available on transient hyponatremia and an increased urinary output after calvarial remodeling ( 7 ) . in this study , we evaluated pediatric patients with hyponatremia developing after calvarial remodeling due to premature fusion of cranial sutures to investigate the method of early differential diagnosis between csws and siadh and appropriate treatment methods . calvarial remodeling was performed in 9 patients with craniosynostosis from february 2002 to october 2004 in the department of plastic surgery at st . the average age of the patients was 34 months , with a range from 14 months to 6 yr . anterior 2/3 calvarial remodeling was performed in 5 cases of metopic synostosis , 1 case of bicoronal synostosis , and 1 case of frontonasal dysplasia . although postoperative fluid therapy , intensive care unit stay , and total hospital stay were slightly different the basic method of surgery and treatment protocol after surgery were the same in all patients . central vein catheter was inserted in all patients . immediately after surgery in intensive care unit , vital signs , urinary output per hour , oral intake , and fluid intake urinary sodium concentration , urine osmolarity , urine specific gravity , rbc , wbc , prothrombin time ( pt ) , activated partial thrombin time ( aptt ) , bun , levels of creatine , albumin , serum sodium and potassium , weight change , and chest radiography were checked every day . the levels of antidiuretic hormone , anp , and bnp were monitored by postoperative days 1 , 3 , and 5 . after surgery 0.9% normal saline was administered in 9 patients under the strict monitoring of the volume of saline input and amount of urine output . although transient hyponatremia was seen during the postoperative period , the serum sodium concentrations ( 139 - 141 meq / l ) were within the normal range ( 135 - 148 meq / l ) after postoperative day 1 due to normal saline resurscitation that had started immediately after surgery . urine output was increased in all patients between postoperative 1 and 2 days ( > 1 ml / kg / hr ) . urinary sodium concentration was increased significantly by postoperative 1 and 3 days at 164.8967.34 meq / l and 127.8936.65 meq / l , respectively , showing a two - fold increase compared with 6052.59 meq / l measured before the procedure ( p<0.05 ) . it returned to the preoperative level by postoperative day 5 at 53.2233.98 meq / l . no significant changes were observed in serum and urine osmolarity during the 5 days after surgery ( fig . the anp level was increased significantly by postoperative days 1 and 3 at 108.1146.01 pg / ml and 8761.31 pg / ml , respectively ( p<0.05 ) . but it fell into the normal range ( 25 - 77 pg / ml ) by postoperative day 5 at 29.5618.24 pg / ml , recovering to the preoperative level at 29.5721.32 pg / ml . the bnp level was also increased dramatically by postoperative day 1 at 149.6680.49 pg / ml , but decreased to 36.7621.63 pg / ml by postoperative day 3 and dropped further to 5.022.98 pg / ml by postoperative day 5 . these figures all showed statistically significant differences ( p<0.05 ) . the level of antidiuretic hormone was normal in all patients ( 0.0 - 6.7 pg / ml ) and showed no statistical significance ( fig . calvarial remodeling was successful without apparent problems in all 9 patients , who recovered and discharged without serious complications . hyponatremia is frequently encountered in pediatric patients after calvarial remodeling surgery due to premature fusion of sutures . when this condition is not corrected in a timely manner , it leads to cerebral edema and an increased intracranial pressure as blood flow to cns is decreased due to low osmolarity . in 1950 ( 1 ) coined the term cerebral salt wasting syndrome ( csws ) to describe a decreased volume of extracellular fluid resulting from a decreased serum sodium concentration in not only patients who underwent calvarial remodeling but also those with traumatic cns injury and those with cns lesions such as cerebral hemorrhage and brain lesion . it was misunderstood as hyponatremia due to the siadh reported by schwarz et al . in 1957 recently , however , many authors reported that these two metabolic changes are completely different entities having different mechanisms and treatment approaches ( 3 - 5 ) . siadh is a condition where an increased ecf volume results from an excess production of adh , which promotes renal water reabsorption . thus , urine output decreases and urine osmolarity increases since water reabsorption is increased . as a result , weight gain and edema or engorged neck veins are observed , and hyponatremia is also seen due to the effect of dilution ( 2 ) . however , most cases of siadh do not typically show edema or engorged neck veins because only 1/3 of total body fluid is extracellular fluid . siadh results in an increased intravascular volume so that it brings about not only increased renal flow and glomerular filtration rate but also decreased proximal sodium reabsorption and urinary sodium excretion ( 8) . the gold standard protocol for treating siadh , which show increased body fluid , decreased bun / creatine ratio , and lower osmolarity , is to restrict water intake . on the other hand , csws is defined as a condition presented with hyponatremia and decreased extracellular fluid volume due to renal loss of sodium in the presence of a disorder of cns ( 5 ) although the mechanism of csws development is poorly understood , the possible mechanisms involve the disruption of neural input into the kidney and/or central elaboration of circulating natriuretic factors . decreased sympathetic input by disruption of neural input to the kidney could be an explanation for impaired proximal reabsorption because the sympathetic nervous system ( sns ) has been shown to alter salt and water handling in the kidney ( 4 ) . the release of one or more natriuretic factors such as anp and bnp increases urinary sodium excretion without causing hypokalemia , and anp and bnp can also directly decrease the autonomic outflow through effects at the brain stem level ( 6 , 8) . either or both of these mechanisms increase urinary sodium excretion , eventually decreasing the effective arterial blood volume and inducing csws ( 8) . berger et al . thought that adh , anp , and bnp are the common controlling factors of siadh and csws , which maintain the equilibrium in most cases . however , hyponatremia is induced when these controlling factors lose the equilibrium in pathologic states ( 6 ) . in siadh , the adh secretion is increased , whereas the plasma concentrations of anp and bnp are normal . however , a normal plasma adh concentration , markedly increased plasma level of natriuretic peptides , and particularly bnp are observed in csws . the patients in this study showed significantly increased levels of anp and bnp after surgery , with bnp showing a larger increase . on the other hand the major features of csws include a decreased plasma sodium concentration , increased urinary sodium concentration , increased urine output , decreased extracellular fluid , increased levels of anp and bnp , and increased osmolarity ; thus , volume - for - volume fluid and sodium replacement is essential for the treatment of csws ( 4 ) . the appropriate treatment for csws is maintaining the body fluid volume and balancing the electrolyte concentrations . when csws is misdiagnosed as siadh and treated with fluid restriction , which is the initial method of treatment for siadh , decreased cerebral perfusion is induced with the development of hemoconcentration , vasospasm and hypotension . furthermore , if postoperative hyponatremia is not corrected early , it may induce decreased cns blood flow , cerebral edema , and increased intracranial pressure due to low osmolarity ( 3 ) . oral intake of sodium drink is ideal but fluid therapy is needed when oral intake is not possible . when the water and sodium are supplied through intravenous injection , 0.9% normal saline solution or 3% salt solution is the choice . here , the correction of hyponatremia needs to be done to the levels to prevent the occurrence of central pontine myelinolysis . in addition , the rate of injection of saline solution should be at 1 - 2 mm / l per hour and should be monitored not to exceed 25 occasionally , an injection of a normal saline solution with blood diuretics such as furosemide is considered ( 9 ) . corticosteroids directly act on the distal tubule of the kidney , thereby directly increasing the sodium absorption and improving hyponatremia . however , the use of these drugs should be considered carefully since fatal side effects such as hypertension , hypocalcemia , and pulmonary edema could develop ( 10 ) . many authors reported that csws developed in patients with brain lesions such as craniocerebral injuries , subarachnoid hemorrhage , and brain tumor and patients who under went neurosurgery ( 3 - 5 ) . reported that it is also related with pediatric patients who underwent calvarial remodeling ( 7 ) . these pediatric patients showed hyponatremia despite appropriate normal saline injection after surgery , natriuresis and increased level of anp . they stated that the injection of usual pediatric fluid , i.e. , 0.25% normal saline , worsened hyponatremia . the pediatric patients in this study showed similar findings after calvarial remodeling as those in the study by levin et al . 11 ) , but we could maintain the optimal sodium levels through strict monitoring and resuscitation . thus , csws develops easily in not only patients with brain lesions such as craniocerebral injuries , subarachnoid hemorrhage , and brain tumor and patients who underwent neurosurgery but also pediatric patients who have underwent calvarial remodeling so that complications should be minimized through appropriate early intervention . in conclusion , the possibility of csws should be considered initially in the patients who develop hyponatremia after calvarial remodeling due to craniosynostosis . although the exact mechanism of the development of csws has not been elucidated , the disruption of neural input into the kidney and/or central elaboration of circulating natriuretic factors are considered to be the possible mechanisms . unlike siadh requiring fluid restriction , csws requires fluid and sodium replacement ; thus , differential diagnosis between these two conditions is critical , and the serum adh , anp , and bnp levels and urinary sodium level are very important for the differential diagnosis .

genetic nature of allergy and complex mechanisms governing the development of atopic phenotype already in fetal life have since long been recognized . the process of hypersensitization begins synchronously with the initiation of fetal production of immunoglobulin e in the 11th gestational week . the ige level in umbilical cord blood may have a significant influence on the future development of atopic diseases . therefore , factors influencing ige level in umbilical blood plasma may modify the course of allergy and the development of atopic symptoms . the aim of the present study was to evaluate the influence of genetically determined susceptibility and of selected environmental factors on the total ige level and on the presence of selected antigen - specific ige in umbilical cord blood plasma . this is a retrospective study in which the enrollment of patients depended on obtaining informed parental consent . the exclusion criteria were : low birth weight ( less than 2500 g ) , severe co - morbidity , e.g. , congenital defects , perinatal trauma , intracranial hemorrhage , other life - threatening conditions in the perinatal period . a retrospective survey of pregnancy and labor and family history was performed using a self - developed questionnaire . we collected data on pregnancy complications ( especially infections ) , type of delivery , gender , birth weight , gestational age , apgar score , mother and father lifestyle and habits , and environmental factors . after collecting the family history , notably about atopic diseases , we stratified the estimated risk of developing atopy into 4 groups : no risk - no atopy diseases in the child 's family ; mild risk -atopy disease in the extended family ; moderate risk - father and/or siblings with atopy disease ; severe risk - mother and/or father and siblings with atopy diseases . the presence of atopy was considered a basis for the physicians ' diagnosis of the following atopic diseases : bronchial asthma , atopic dermatitis , hay fever , urticaria , atopic conjunctivitis , food allergy . total serum ige levels were determined by the electrochemiluminescense immunoassay eclia - sandwich principle in an elecsys 2010 analyzer ( roche diagnostics , mannheim , germany ) with a detection level of 0.1 iu / ml . specific umbilical cord blood ige was assessed for the following allergen kits : children 's food : egg , milk , wheat meal , and peanuts ; soya ; grass and grain pollen ( the most common in poland ) ; house dust mite ( hdm ) : d. pteronyssinus and d. farinae . specific cord blood ige was measured by the enzyme - linked immunosorbent assay elisa in an allergopharma analyzer ( allergopharma , reinbek , germany ) , with the detection level of 0.35 iu / ml . data were expressed as means sd and were analyzed in relation to the median and mean levels of cord blood ige . concerning the former , children were subdivided into two groups , below and above the median level of ige ; concerning the latter , children were divided into 3 groups , depending on their total mean ige . the following environmental factors which might affect the total ige level were analyzed : family history of allergy , pregnancy and labor - associated events , environmental factors , and parental behavior in the prenatal period . associations between clinical and epidemiological data collected by a questionnaire and the allergy were analyzed by a chi - square test with yates 's correction and fisher 's test , according to a given contingency table . differences in immunoglobulin levels between the case and control groups were tested by a whitney - mann u test . moreover , spearman 's rank correlation coefficient was used to test associations between the studied factors . figure 1a shows the number of children with the ige below and above the median level born from normal and pathologically complicated pregnancies . in normal pregnancies , there were about twice more children with the ige below than above the median level . this ratio was reversed toward the preeminence of the above median level in pathological pregnancies . on the other hand , the numbers of children with the ige below and above its median level were about equal when there was no atopic rhinitis in history , whereas there were twice as many children with the ige above its median level in case of history of atopy ( figure 1b ) . umbilical cord blood ige concentration below and above the median level in relation to the normal or pathological pregnancy course ( panel a ) and to the presence of atopic rhinitis in history . the total ige level in umbilical blood plasma was in a range from 0.0 to 23.08 iu / ml ( mean 0.55 2.07 iu / ml ; median 0.16 iu / ml ) . the neonates were divided into 3 groups based on the cord blood ige level : the first group with the ige < 0.1 iu / ml consisted of 65 infants ( 37.5% ) , the second with the ige 0.1 - 0.5 iu / ml consisted of 63 infants ( 36.5% ) , and the third with the ige > 0.5 iu / ml consisted of 45 infants ( 26.0% ) . in the first group there were 24 boys and 41 girls , in the second 36 and 27 , and in the third 26 and 19 , respectively . overall , the total cord blood ige levels were significantly higher in boys compared with girls ( means 0.82 2.69 vs. 0.39 1.14 ; or = 2.2 ; p = 0.007 ) , in newborns with complicated as opposed to normal pregnancy ( means 0.75 2.37 vs. 0.17 0.26 ; or = 2.7 ; p = 0.003 ) and in newborns with family history of atopic rhinitis as opposed to the lack of such history ( means 1.45 4.84 vs. 0.48 1.26 ; or = 2.4 ; p = 0.03 ) . family size ( the number of siblings ) correlated with decreases of cord blood ige levels ( p < 0.02 ) . for the remaining variables ( allergic diseases in the mother , father , or siblings ) , no statistically significant differences were noted . no correlations were observed between infant 's exposure to allergy and the total ige level . overall , 519 assays for antigen - specific ige were performed ( 3 173 children ) . of the 40 positive tests , 20 were to grass and grain pollen , 11 to hdm , 9 to food . fifteen children had specific ige to grass , 8 to hdm , 5 to food , 2 to grass and hdm , 3 to grass and food , 1 to hdm and food . no correlations were noted between familial history of allergy and the presence of specific immunoglobulins . no statistically significant correlation was seen between antigen - specific ige in umbilical blood plasma and pregnancy- or labor - associated factors . the analyzed environmental factors included : place of residence ( urban or rural ) , living conditions and crowding ( number of people per room ) , the presence and kind of domestic animals during pregnancy , and environmental pollution . contact with cat 's allergens caused a higher prevalence of the specific ige for grass and grain pollen ( or = 3.24 ; p < 0.07 ) and children 's food ( or = 4.98 ; p < 0.04 ) . specific ige for grass and grain pollen were significantly lower in case of household contact with a dog during pregnancy ( or = 0.28 ; p < 0.05 ) . no significant correlations were found between the other environmental factors and the presence of ige in umbilical blood plasma . analysis of parental tobacco smoking habits during pregnancy revealed a significant association between the level of antigen - specific ige against domestic dust mites and maternal tobacco smoking . these antibodies were detected significantly more often in newborns of smoking mothers and this correlated with the umber of cigarettes smoked per day ( or = 4.7 ; p = 0.005 ) . a similar correlation ( p = 0.04 ) figure 1a shows the number of children with the ige below and above the median level born from normal and pathologically complicated pregnancies . in normal pregnancies , there were about twice more children with the ige below than above the median level . this ratio was reversed toward the preeminence of the above median level in pathological pregnancies . on the other hand , the numbers of children with the ige below and above its median level were about equal when there was no atopic rhinitis in history , whereas there were twice as many children with the ige above its median level in case of history of atopy ( figure 1b ) . umbilical cord blood ige concentration below and above the median level in relation to the normal or pathological pregnancy course ( panel a ) and to the presence of atopic rhinitis in history . the total ige level in umbilical blood plasma was in a range from 0.0 to 23.08 iu / ml ( mean 0.55 2.07 iu / ml ; median 0.16 iu / ml ) . the neonates were divided into 3 groups based on the cord blood ige level : the first group with the ige < 0.1 iu / ml consisted of 65 infants ( 37.5% ) , the second with the ige 0.1 - 0.5 iu / ml consisted of 63 infants ( 36.5% ) , and the third with the ige > 0.5 iu / ml consisted of 45 infants ( 26.0% ) . in the first group there were 24 boys and 41 girls , in the second 36 and 27 , and in the third 26 and 19 , respectively . overall , the total cord blood ige levels were significantly higher in boys compared with girls ( means 0.82 2.69 vs. 0.39 1.14 ; or = 2.2 ; p = 0.007 ) , in newborns with complicated as opposed to normal pregnancy ( means 0.75 2.37 vs. 0.17 0.26 ; or = 2.7 ; p = 0.003 ) and in newborns with family history of atopic rhinitis as opposed to the lack of such history ( means 1.45 4.84 vs. 0.48 1.26 ; or = 2.4 ; p = 0.03 ) . family size ( the number of siblings ) correlated with decreases of cord blood ige levels ( p < 0.02 ) . for the remaining variables ( allergic diseases in the mother , father , or siblings ) , no statistically significant differences were noted . no correlations were observed between infant 's exposure to allergy and the total ige level . overall , 519 assays for antigen - specific ige were performed ( 3 173 children ) . we detected specific ige in 34 newborns - 19.7% ( 6.6% positive tests ) . of the 40 positive tests , 20 were to grass and grain pollen , 11 to hdm , 9 to food . fifteen children had specific ige to grass , 8 to hdm , 5 to food , 2 to grass and hdm , 3 to grass and food , 1 to hdm and food . no correlations were noted between familial history of allergy and the presence of specific immunoglobulins . no statistically significant correlation was seen between antigen - specific ige in umbilical blood plasma and pregnancy- or labor - associated factors . the analyzed environmental factors included : place of residence ( urban or rural ) , living conditions and crowding ( number of people per room ) , the presence and kind of domestic animals during pregnancy , and environmental pollution . contact with cat 's allergens caused a higher prevalence of the specific ige for grass and grain pollen ( or = 3.24 ; p < 0.07 ) and children 's food ( or = 4.98 ; p < 0.04 ) . specific ige for grass and grain pollen were significantly lower in case of household contact with a dog during pregnancy ( or = 0.28 ; p < 0.05 ) . no significant correlations were found between the other environmental factors and the presence of ige in umbilical blood plasma . analysis of parental tobacco smoking habits during pregnancy revealed a significant association between the level of antigen - specific ige against domestic dust mites and maternal tobacco smoking . these antibodies were detected significantly more often in newborns of smoking mothers and this correlated with the umber of cigarettes smoked per day ( or = 4.7 ; p = 0.005 ) . a similar correlation ( p = 0.04 ) thinking about factors influencing the levels of total and antigen - specific ige in umbilical blood plasma , investigations focused on genetic predispositions , environmental factors and parental behavior . authors emphasize the influence of all these factors combined , although it is a matter of debate which one is the most important . a frequently studied issue is the influence of atopic diseases in the family on the level of total ige in umbilical blood . several reports suggest that atopy in parents , siblings , and 1st degree relatives is a presage of a significant increase in the ige level , which could be followed by increased incidence of atopic diseases in infancy [ 3 - 5 ] . most authors point to the presence of atopic diseases , particularly in the mother , as a key factor contributing to elevated levels of ige in umbilical blood . it has also been noticed that the presence of allergy in the father or in another relative is less important [ 6 - 8 ] . the presence of atopic rhinitis in the child 's family presages a significant increase in ige level . since the discovery of ige and the development of techniques for its assessment , there is a consistent finding of a higher level of ige in boys ' than in girls ' blood plasma [ 9 - 12 ] . most data obtained todate confirms also a higher level of ige in umbilical blood of males . some papers , where a different clinical methodology has been implemented , present this correlation as a higher proportion of boys in the groups of children who have a higher total ige level . a similar method was adopted by the authors of the present paper , confirming an association of male gender with a higher total ige level . factors associated with pregnancy and labor are seldom discussed in the literature with respect to their impact on the ige level in umbilical blood plasma . in the present paper , we found that maternal respiratory tract infections in the 1st and 2nd trimester were associated with increased total ige level in the neonates . the latter is concordant with the current knowledge concerning this problem . in spite of isolated publications discussing these issues , general opinion is that body height , apgar score , the number of past pregnancies , the number of miscarriages and parental age do not influence the ige level or the course of the allergization process . therefore , these factors can not be helpful in the assessment of the risk of the development of atopic diseases in children . studies published hitherto do not reveal any association between the total ige level and birth weight of the newborn . we did not find any correlations between parental socio - economic status , education or life - style and the ige concentration in umbilical blood ; the interpretation of the lack of correlation is , however , hindered by a fairly homogenous study group . a matter of much debate is the role of maternal and infant contact with domestic animals . some allergists consider that prolonged contact with domestic animals is a form of natural specific immunotherapy , effective particularly at the infant age , reducing hypersensivity to animal fur . this phenomenon may be a result of tolerance achieved during natural exposition to high concentration of allergens . other authors postulate that the presence of a pet may result in the development of allergy and exacerbation of its symptoms . . the issue of whether we should present a cat to a small child still remains unresolved . in the present study , maternal contact with a cat during pregnancy resulted in increased incidence of antigen - specific immunoglobulins e against grass , cereals , and foods . that may signal the role of cat allergens acting during pregnancy in the development of allergies . in contrast , maternal exposure to dog allergens during pregnancy was associated with decreased incidence of sige against grass and cereals ; a trend also seen regarding antibodies against domestic dust mites . gern et al . also demonstrate less frequent hypersensivity and lower incidence of atopic dermatitis in infants remaining in a continuous contact with a dog since birth . some suggest that maternal exposure to either type of smoking has a significant impact on ige levels in both maternal and fetal blood plasma and on the development of atopic diseases in early infancy ( 6 , 7 , 8) . bergman et al . showed a high ige level in children of smoking fathers and even higher levels of it in children of smoking mothers . this finding was explained by nonspecific stimulation of fetal ige production when exposed to low - intensity passive smoking and blockade thereof when faced with heavy exposure to tobacco smoke . however , many authors emphasize that no significant correlation between total ige level and smoking during pregnancy or maternal exposure to tobacco smoke has been found to - date . in the present study we did not find any correlation between parental smoking and total ige level either . nevertheless , it is worth pointing out that maternal or parental smoking was associated with higher incidence of sige against domestic dust mites . we may presume that women living in an environment of tobacco smoke had greater contact with domestic dust mite allergens , and that may result in higher incidence of atopic diseases in the future . in conclusion , in the present study we demonstrate that increased total ige level in umbilical blood was associated with male gender , pathological pregnancy course , and the presence of atopic rhinitis in the family ; while lower ige levels were found in children having numerous siblings . increased levels of specific ige against grass , cereals , and food stuffs were associated with maternal contact with a cat , but not a dog , and with parental smoking during pregnancy .

hepatitis c virus ( hcv ) , a worldwide blood borne infection with a global importance , is a significant cause of hepatic diseases , cirrhosis and hepatocellular carcinoma , after hepatitis b which its screening was introduced in blood banking laboratory methods ( 1 - 5 ) . chronic infection with hcv is a major cause of liver cancer , cirrhosis and end - stage liver in the world ( 6 - 8 ) . it has been estimated that more than 170 million people are infected worldwide , and approximately 280000 deaths are attributed to chronic hepatitis c virus ( hcv ) infection each year ( 9 ) . in the future prospect , deaths caused by hcv infection will be tripled over the next 20 years , especially in the developing countries ( 10 ) . the hcv is an enveloped , single - stranded rna virus and a member of the hepacivirus genus of the flaviviridae family ( 11 ) . analysis of the different hcv genomic sequences has ascertained that the virus nucleotide sequence can be different as much as 30% ( 14 ) . hcv virus has been divided into seven major genotypes ( hcv-1 to hcv-7 ) ( 15 ) . also , each genotype is divided into multiple subtypes ( e.g. 1a , 1b , etc . ) since the genotype is a critical baseline predictor of the response to hcv antiviral therapy , determination of such a genotype is very important ( 8 , 18 ) . genotyping is necessary to predict the response rate and duration of the treatment ( 10 , 19 ) . by improving hcv therapy , hcv genotyping has become very useful , because the patients who need more aggressive management are helped for screening ( 20 ) . prevalence and incidence of the virus vary depending on the area of conflict ( 17 , 21 ) . the rate of hcv infection in iran is high for its geographical situation and huge immigration from afghanistan and iraq ( 5 ) . recent findings have shown that genotyping pattern in iranian patients is comparable with western europe and north america , which constitutes genotypes 1 and 3 . despite the similarity of the distribution , these genotypes vary in different areas ( 22 ) . given the above - mentioned findings , hcv genotyping in each region is of great importance ; nonetheless , this study has not been performed in zanjan , a province in north west of iran . in this research , we evaluated the genotypes of the hcv in zanjan . identifying the most common type of genotype not only the cost of the treatment can be minimized , but also the length of the treatment can be optimized . the purpose of this study was to offer an applicable prospect in the therapeutic context by identifying hcv genotypes in zanjan province , the northwest of iran . from 2007 to 2013 , out of the patients who referred to the clinical laboratory for investigating anti - hcv antibodies , 136 patients with positive results were selected using anti hcv antibody elisa kit ( 4th generation ) ( anti hcv elisa , diasource , belgium ) for further investigations . about 5 ml of plasma ( edta ) for blood samples was stored in -20c for the following procedures . rna was extracted under standard conditions using cinnagen diagnostic kit ( cinnagen company , tehran , iran ) . in brief , cdna was synthesized as the template for pcr reactions in the first round of pcr . reagents with final volume of 45 l added to each tube on ice , containing 39 l mix i , 1 l rt enzyme , 0.3 l taq dna polymerase , and 40 l mineral oil . after mixing well , five microliter rna of each patient , positive control and depc ( diethylpyrocarbonate ) water were added to each tube for patients , positive and negative control , respectively . the mixture was centrifuged for 3 - 5 s and then placed on a cycle of 42c for 20 min , 94c for 2 min , 60c for 40 s and 72c for 40 s. next step comprised 20 cycles of 93c for 40 s , 60c for 40 s , 72c for 40 s based on the manufacturer 's instructions . the second pcr round included the expected fragment 216 bp amplification under the condition mentioned in the kit , including 35 cycles of 93c for 40 s , 60 c for 40 s and 72c for 40 s ( flex cycler - germany ) . final 216 bp of pcr product was diagnosed using agarose gel electrophoresis detection system on a 2% agarose gel . the samples were evaluated for hcv genotyping by using allele - specific pcr ( as - pcr ) ( 23 ) , and hcv genotype 2.0 line probe assay ( lipa ) ( versant hcv genotype 2.0 assay [ lipa ] , siemens , germany ) . from 2007 to 2013 , out of the patients who referred to the clinical laboratory for investigating anti - hcv antibodies , 136 patients with positive results were selected using anti hcv antibody elisa kit ( 4th generation ) ( anti hcv elisa , diasource , belgium ) for further investigations . about 5 ml of plasma ( edta ) for blood samples was stored in -20c for the following procedures . rna was extracted under standard conditions using cinnagen diagnostic kit ( cinnagen company , tehran , iran ) . in brief , cdna was synthesized as the template for pcr reactions in the first round of pcr . reagents with final volume of 45 l added to each tube on ice , containing 39 l mix i , 1 l rt enzyme , 0.3 l taq dna polymerase , and 40 l mineral oil . after mixing well , five microliter rna of each patient , positive control and depc ( diethylpyrocarbonate ) water were added to each tube for patients , positive and negative control , respectively . the mixture was centrifuged for 3 - 5 s and then placed on a cycle of 42c for 20 min , 94c for 2 min , 60c for 40 s and 72c for 40 s. next step comprised 20 cycles of 93c for 40 s , 60c for 40 s , 72c for 40 s based on the manufacturer 's instructions . the second pcr round included the expected fragment 216 bp amplification under the condition mentioned in the kit , including 35 cycles of 93c for 40 s , 60 c for 40 s and 72c for 40 s ( flex cycler - germany ) . final 216 bp of pcr product was diagnosed using agarose gel electrophoresis detection system on a 2% agarose gel . the samples were evaluated for hcv genotyping by using allele - specific pcr ( as - pcr ) ( 23 ) , and hcv genotype 2.0 line probe assay ( lipa ) ( versant hcv genotype 2.0 assay [ lipa ] , siemens , germany ) . result analysis of gel electrophoresis on pcr products , indicated a 216 bp band for each positive sample as shown in figure 1 . genotyping results demonstrated that subtype 3a ( 38.26% ) was the most prevalent hcv type in zanjan . prevalence of subtypes 1b , 1a , 2 , and 4 were 25.73% , 22.05% , 5.14% , and 4.41% , respectively . also , the frequency for unknown genotypes was 4.41% . 1 - 4 , four hcv positive patients ; 5 , negative control ; 6 , positive control . genotyping of hcv has been regarded as a fundamental factor for treatment , prognosis , and prediction in clinical management ( 24 ) , so that to begin the treatment and then follow up are in accordance with the virus genotype ( 25 ) . recent studies have shown that the distribution of hcv genotypes varies in different geographical regions . thus , determination of the predominant and less prevalent subtypes of hcv in various areas has a fundamental role . this study was the first report of hcv genotypes prevalence in zanjan ( the northwest of iran ) . the results indicated that subtype 3a with the frequency of 38.26% was the most current subtype in zanjan . this finding was consistent with a recent study performed in tehran ( a city near zanjan ) ( 22 ) . it was also comparable with the obtained results reported ( 25 ) from azerbaijani patients ( in west azerbaijan , northwest of iran ) with established chronic hepatitis c , who referred to some hospitals in tehran ( 26 ) . this similarity may be due to the proximity of zanjan with these cities . a study in shiraz , a city in southern part of iran estimated the prevalence of hcv genotype to be similar in the case of 3a type ( 8) . meanwhile , another comprehensive investigation performed in tehran with a wide range of infected patients referring from all over the country has introduced subtype 1a as the most prevalent subtype in iran ( 27 ) . additionally , in a study in isfahan ( iran ) and on the bases of other reports , high prevalence of subtypes 3a and 1a and very low frequency of genotype 2 in iran was demonstrated , which is different from europe , usa , and even some parts of asia ( 28 ) . hcv subtypes 3a and 1a were determined in the hemodialysis patients living in tehran as the prevalent subtypes ( 29 ) . this finding is in contrast with global hcv genotypes prevalence ( 30 ) and the high frequency of genotype hcv 1a/1b in addition to hcv 4 and hcv 2/2a plus hcv 4 in hemodialysis patients living in the neighboring countries such as bahrain and saudi arabia ( 31 ) , which have sea borders with khuzestan province in ahvaz . in addition , evaluation of chronic hepatic patients indicated 1a as the most prevalent subtype in hormozgan ( 32 ) . on the whole , genotype 3 was more prevalent in regions such as india , pakistan , and southern asia ( 33 - 35 ) . in both saudi arabia and egypt , in most parts of china , subtype 1b followed by 2a was found with high prevalence ( 36 , 37 ) . in a study in chile ( among blood donors ) , subtype 1b with the frequency of 46% was the dominant type ( 38 , 39 ) . a research in central italy gonzaga rm et al . investigated the distribution of hepatitis c virus genotypes in seropositive patients in brazil in 2008 ( 40 ) . the prevalence of different hcv genotypes in the world are as follows : subtype 1a is mostly reported in north america and europe . in southern and eastern europe , japan , and north africa , the mostly reported subtype is 1b . encountering subtype 3a from south east asia in europe and the usa may be transmitted by travelers . subtype 3a may be transmitted from south east asia in europe and the usa by travelers . genotype 4 is highly common in arab countries except jordan while in turkey and israel genotype 1 were prevalent . genotype 6a is restricted to hong kong and vietnam ( 41 ) . in conclusion , paying attention to high prevalence of a certain hcv genotype is essential in remedial terms of hcv . determining hcv genotypes could increase the chance of successful treatment . to obtain more precise information regarding the genotype of this virus

all of the subjects who were consecutively observed in the outpatient unit of the pediatric department of pozzuoli hospital in the period between 2004 and 2010 were included in the study . the majority of the children were referred by their general practitioners for allergy problems , overweight , and obesity , as described elsewhere ( 10 ) . after excluding subjects with gastrointestinal , liver , cardiac , renal , urinary , and infectious diseases , the sample consisted of 884 children ( 435 boys and 449 girls ) , with an age range of 616 years . of these subjects , 206 ( 23% ) were normal weight , 135 ( 15% ) were overweight , and 543 ( 61% ) were obese . all of the children were apparently healthy , and none of the children had a history of alcohol consumption or diabetes or were under pharmacological treatment . sexual maturity was evaluated by the tanner stage for pubic hair ( stages i v ) . blood pressure was measured according to the fourth report of the national high blood pressure education program on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents ( 11 ) . fasting plasma glucose ( fpg ) , insulin , total cholesterol , tgs , hdl - c , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and creatinine ( colorimetric compensated jaffe method ) were determined using a roche analyzer ( modular analytics serum work area , mannheim , germany ) . homocysteine was determined by the immunoenzymatic method ( abbott , abbott park , il ) . insulin resistance was evaluated by the homeostasis model assessment of insulin resistance ( homa - ir ) index using the following standard formula : fasting insulin ( units per liter ) fasting glucose ( millimoles per liter)/22.5 . estimated glomerular filtration rate ( egfr ) was calculated using the following updated schwartz formula : 0.413 height ( centimeters)/serum creatinine ( milligrams per deciliter ) . these children did not differ from the remainder of the children for age and sex distribution . standard echocardiograms were obtained by a commercially available echocardiographic system with tissue doppler imaging ( tdi ) capabilities ( artida / aplio ; toshiba ) . left ventricular mass ( lvm ) was calculated according to the penn convention and indexed for height ( lvmi ) . relative wall thickness ( rwt ) was calculated from lv posterior wall thickness ( lvpwt ) , interventricular septum thickness ( ivst ) , and lv diastolic diameter ( lvdd ) using the following formula : ( pwt + ivst)/lvdd . increased lvm was defined using age- and sex - specific quantiles as proposed by khoury et al . increased rwt was defined by a value > 0.375 ( 13 ) , corresponding to the 90th percentile of our nonobese children . lv geometry was defined as normal geometry ( normal lvmi and rwt ) , eccentric lv hypertrophy ( increased lvmi and normal rwt ) , concentric remodeling ( normal lvmi and increased rwt ) , and concentric lv hypertrophy ( increased lvmi and rwt ) . lv function was analyzed by conventional and tdi echocardiography , as described elsewhere ( 13 ) . transmitral peak rapid filling velocity ( e ) , peak atrial filling velocity ( a ) , the e - to - a ratio , and isovolumic relaxation time ( irt ) were obtained as measures of diastolic function . for tdi echocardiography , three major velocities were recorded at the annular site : peak positive systolic velocity and two peak negative velocities during the early ( ea ) and late ( aa ) phases of diastole . all echocardiographic readings were made online by the same investigator who was blinded to the metabolic status of the children . overweight and obesity were defined using the international obesity task force criteria ( 14 ) . impaired fasting glucose ( ifg ) was defined by an fpg 100 mg / dl . high blood pressure ( above the 90th percentile for age , sex , and height ) was defined according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents ( 11 ) . high waist circumference , high tgs , low hdl - c , and metabolic syndrome were defined using the cutoff proposed by cook et al . children and adolescents that intercept the cutoff values of each single component of metabolic syndrome ( adult treatment panel iii definition ) in adult life . insulin resistance was defined by the 95th percentile of the homa - ir values obtained in healthy nonobese italian children categorized according to tanner stage ( i.e. , homa - ir > 2.20 [ stage i ] , > 3.61 [ stages ii and iii ] , and > 3.64 [ stages iv and v ] ) , as described elsewhere ( 10 ) . high alt levels , as a surrogate of nonalcoholic fatty liver disease ( nafld ) , were defined using sex - related cut points ( i.e. , > 30 iu / l in boys and > 19 iu / l in girls ) , as described elsewhere ( 16 ) . high white blood cell ( wbc ) count was defined by a value 9.0 ( 10/l ) , corresponding to the 80th percentile of our population ( 10 ) . the study was approved by the local ethics committee , and informed consent was obtained from the parents of all participants . given the skewed distribution of homa - ir , tgs , plasma creatinine , alt , ast , and homocysteine , the statistical analysis of these variables was applied after log transformation and back transformation to natural units to allow presentation in the text and tables . or fisher exact tests , as appropriate , were used to compare proportions . to evaluate the cmr risk associated with a high tg - to - hdl - c ratio , we performed logistic regression analysis , controlled for age , sex , and pubertal stage , separately in nonobese and obese subjects . to assess the independent impact of some variables on high alt levels and concentric lv hypertrophy the statistical analysis was performed using spss for windows , version 13.0 ( spss , chicago , il ) . the determination of biochemical parameters was performed in the centralized laboratory of pozzuoli hospital . fasting plasma glucose ( fpg ) , insulin , total cholesterol , tgs , hdl - c , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and creatinine ( colorimetric compensated jaffe method ) were determined using a roche analyzer ( modular analytics serum work area , mannheim , germany ) . homocysteine was determined by the immunoenzymatic method ( abbott , abbott park , il ) . insulin resistance was evaluated by the homeostasis model assessment of insulin resistance ( homa - ir ) index using the following standard formula : fasting insulin ( units per liter ) fasting glucose ( millimoles per liter)/22.5 . estimated glomerular filtration rate ( egfr ) was calculated using the following updated schwartz formula : 0.413 height ( centimeters)/serum creatinine ( milligrams per deciliter ) . a total of 258 children underwent echocardiography . these children did not differ from the remainder of the children for age and sex distribution . standard echocardiograms were obtained by a commercially available echocardiographic system with tissue doppler imaging ( tdi ) capabilities ( artida / aplio ; toshiba ) . all measurements were analyzed according to the recommendations of the american society of echocardiography . left ventricular mass ( lvm ) relative wall thickness ( rwt ) was calculated from lv posterior wall thickness ( lvpwt ) , interventricular septum thickness ( ivst ) , and lv diastolic diameter ( lvdd ) using the following formula : ( pwt + ivst)/lvdd . increased lvm was defined using age- and sex - specific quantiles as proposed by khoury et al . increased rwt was defined by a value > 0.375 ( 13 ) , corresponding to the 90th percentile of our nonobese children . lv geometry was defined as normal geometry ( normal lvmi and rwt ) , eccentric lv hypertrophy ( increased lvmi and normal rwt ) , concentric remodeling ( normal lvmi and increased rwt ) , and concentric lv hypertrophy ( increased lvmi and rwt ) . lv function was analyzed by conventional and tdi echocardiography , as described elsewhere ( 13 ) . transmitral peak rapid filling velocity ( e ) , peak atrial filling velocity ( a ) , the e - to - a ratio , and isovolumic relaxation time ( irt ) were obtained as measures of diastolic function . for tdi echocardiography , three major velocities were recorded at the annular site : peak positive systolic velocity and two peak negative velocities during the early ( ea ) and late ( aa ) phases of diastole . all echocardiographic readings were made online by the same investigator who was blinded to the metabolic status of the children . overweight and obesity were defined using the international obesity task force criteria ( 14 ) . impaired fasting glucose ( ifg ) was defined by an fpg 100 mg / dl . high blood pressure ( above the 90th percentile for age , sex , and height ) was defined according to the fourth report on the diagnosis , evaluation , and treatment of high blood pressure in children and adolescents ( 11 ) . high waist circumference , high tgs , low hdl - c , and metabolic syndrome were defined using the cutoff proposed by cook et al . ( children and adolescents that intercept the cutoff values of each single component of metabolic syndrome ( adult treatment panel iii definition ) in adult life . insulin resistance was defined by the 95th percentile of the homa - ir values obtained in healthy nonobese italian children categorized according to tanner stage ( i.e. , homa - ir > 2.20 [ stage i ] , > 3.61 [ stages ii and iii ] , and > 3.64 [ stages iv and v ] ) , as described elsewhere ( 10 ) . high alt levels , as a surrogate of nonalcoholic fatty liver disease ( nafld ) , were defined using sex - related cut points ( i.e. , > 30 iu / l in boys and > 19 iu / l in girls ) , as described elsewhere ( 16 ) . high white blood cell ( wbc ) count was defined by a value 9.0 ( 10/l ) , corresponding to the 80th percentile of our population ( 10 ) . the study was approved by the local ethics committee , and informed consent was obtained from the parents of all participants . given the skewed distribution of homa - ir , tgs , plasma creatinine , alt , ast , and homocysteine , the statistical analysis of these variables was applied after log transformation and back transformation to natural units to allow presentation in the text and tables . or fisher exact tests , as appropriate , were used to compare proportions . to evaluate the cmr risk associated with a high tg - to - hdl - c ratio , we performed logistic regression analysis , controlled for age , sex , and pubertal stage , separately in nonobese and obese subjects . to assess the independent impact of some variables on high alt levels and concentric lv hypertrophy the statistical analysis was performed using spss for windows , version 13.0 ( spss , chicago , il ) . to evaluate the relationship between the tg - to - hdl - c ratio and cmr factors , all children were stratified into tertiles of the tg - to - hdl - c ratio . moving from the lowest to the highest tertile , we observed an increased percentage of pubertal stage ( p < 0.0001 ) , as well as increased levels of bmi , waist circumference , homa - ir , wbc count , alt , systolic blood pressure ( p < 0.0001 , for all ) , fpg ( p = 0.022 ) , total cholesterol ( p = 0.004 ) , creatinine ( p = 0.001 ) , ast ( p = 0.007 ) , and diastolic blood pressure ( p = 0.019 ) ( table 1 ) . no significant difference was seen between tertiles for age , sex , homocysteine , and egfr . features of children according to tertiles of the tg - to - hdl - c ratio a statistically significant difference across tertiles was found in rwt ( p = 0.009 ) and lvmi ( p < 0.0001 ) , whereas no difference was observed between tertiles of the tg - to - hdl - c ratio for both systolic and diastolic function , evaluated with conventional and tdi echocardiography ( table 2 ) . the prevalence of normal geometry , eccentric , lv hypertrophy , concentric remodeling , and concentric lv hypertrophy among children with a high tg - to - hdl - c ratio ( 2.0 ) was 24 , 39 , 33 , and 58% , respectively ( p = 0.001 ) . cardiac features of children according to tertiles of the tg - to - hdl - c ratio to analyze the cmr profile associated with a high tg - to - hdl - c ratio ( 2.0 ) independent of obesity , we performed a logistic regression analysis separately in nonobese and obese children . nonobese children with a high tg - to - hdl - c ratio showed a 3- to 58-fold increased risk of insulin resistance , high waist circumference , high blood pressure , ifg , high wbc , and metabolic syndrome compared with those with a tg - to - hdl - c ratio < 2.0 . in obese children , those with a high tg - to - hdl - c ratio showed a 1.5- to 10-fold increased risk of insulin resistance , high blood pressure , and metabolic syndrome compared with those with a tg - to - hdl - c ratio < 2.0 ( table 3 ) . odds ratios and 95% cis of cmr factors associated with a high tg - to - hdl - c ratio in nonobese and obese subjects elevated alt levels were predicted by high waist circumference , insulin resistance , and a high tg - to - hdl - c ratio . the independent predictors of concentric lv hypertrophy were a high tg - to - hdl - c ratio and high blood pressure ( table 4 ) . a statistically significant difference across tertiles was found in rwt ( p = 0.009 ) and lvmi ( p < 0.0001 ) , whereas no difference was observed between tertiles of the tg - to - hdl - c ratio for both systolic and diastolic function , evaluated with conventional and tdi echocardiography ( table 2 ) . the prevalence of normal geometry , eccentric , lv hypertrophy , concentric remodeling , and concentric lv hypertrophy among children with a high tg - to - hdl - c ratio ( 2.0 ) was 24 , 39 , 33 , and 58% , respectively ( p = 0.001 ) . cardiac features of children according to tertiles of the tg - to - hdl - c ratio to analyze the cmr profile associated with a high tg - to - hdl - c ratio ( 2.0 ) independent of obesity , we performed a logistic regression analysis separately in nonobese and obese children . nonobese children with a high tg - to - hdl - c ratio showed a 3- to 58-fold increased risk of insulin resistance , high waist circumference , high blood pressure , ifg , high wbc , and metabolic syndrome compared with those with a tg - to - hdl - c ratio < 2.0 . in obese children , those with a high tg - to - hdl - c ratio showed a 1.5- to 10-fold increased risk of insulin resistance , high blood pressure , and metabolic syndrome compared with those with a tg - to - hdl - c ratio < 2.0 ( table 3 ) . odds ratios and 95% cis of cmr factors associated with a high tg - to - hdl - c ratio in nonobese and obese subjects elevated alt levels were predicted by high waist circumference , insulin resistance , and a high tg - to - hdl - c ratio . the independent predictors of concentric lv hypertrophy were a high tg - to - hdl - c ratio and high blood pressure ( table 4 ) . this study provides evidence that in an outpatient , white pediatric population , a high tg - to - hdl - c ratio is associated with an unfavorable cmr profile . in particular , children with a tg - to - hdl - c ratio 2.0 showed an increased risk of subclinical signs of liver and cardiac abnormalities independent of high waist circumference , high blood pressure , and insulin resistance . in adults , there is growing interest in the tg - to - hdl - c ratio as a surrogate marker of atherogenic lipid abnormalities , namely small ldl cholesterol and small hdl - c ( 17 ) , and of insulin resistance and high cardiovascular risk ( 8) . in childhood , the clinical value of lipid ratios has been much less investigated . in a large sample of high school adolescents , musso et al . ( 18 ) demonstrated that the tg - to - hdl - c ratio and high - sensitivity c - reactive protein positively correlated with bmi and waist circumference . weiss et al . ( 9 ) , using a longitudinal design , demonstrated that the tg - to - hdl - c ratio in late adolescence predicts small ldl and hdl particles in adulthood independent of obesity and weight gain . in a recent report performed in a large multiethnic cohort of obese youths , giannini et al . ( 19 ) demonstrated that a high tg - to - hdl - c ratio is associated with insulin resistance in white girls and boys but not in african americans or hispanics . in the current study , we demonstrate that a high tg - to - hdl - c ratio is significantly associated with several cmr factors . in particular , in nonobese children , a tg - to - hdl - c ratio 2.0 is associated with a 3- to 58-fold increased risk of high waist circumference , high blood pressure , ifg , high wbc count , insulin resistance , and metabolic syndrome . in obese subjects a tg - to - hdl - c ratio 2.0 is associated with a 1.5- to 10-fold higher risk of insulin resistance , high waist circumference , and metabolic syndrome . these results emphasize the clinical usefulness of the tg - to - hdl - c ratio , which is greater in nonobese than in obese children in whom the excess adiposity may likely mask the role of the single cmr factors . a previous study performed in healthy korean adults demonstrated that fasting plasma insulin and tg - to - hdl - c ratio concentrations were associated with elevated alt and evidence of nafld ( 20 ) . of interest , cali et al . ( 21 ) used magnetic resonance imaging to demonstrate a close association between fatty liver and an atherogenic lipid profile characterized by large vldl , small dense ldl , and a decreased number of large hdl particles in obese children . our results expand this observation , demonstrating that a high tg - to - hdl - c ratio , visceral adiposity , and insulin resistance are all independently associated with elevated alt levels , which is recognized as a surrogate index of nafld . this finding also may have implications in terms of cardiovascular risk because high alt levels are considered to be a nontraditional marker of cardiovascular morbidity . there is in fact evidence that in overweight and obese children , nafld is associated with multiple cardiovascular risk factors ( 22 ) . the relationship between the tg - to - hdl - c ratio and lv geometry is unexplored . our study provides the novel finding that a high tg - to - hdl - c ratio , independently of visceral adiposity and high blood pressure , is associated with concentric lv hypertrophy , a well - known cardiovascular risk factor . although the pathophysiological significance of this association is not straightforward , some data from the literature may be helpful . first , in subjects with essential hypertension , low hdl levels are associated with lv hypertrophy ( 23 ) . ( 24 ) demonstrated accumulation of tgs in the myocardium of moderately obese subjects , which was more pronounced in those with elevated alt levels . collectively , these findings show that an atherogenic lipid phenotype ( low hdl and high tgs ) has a negative impact on lvm and that it is associated with lipid deposition in the liver and the heart . our finding of an independent association of a high tg - to - hdl - c ratio with liver and cardiac abnormalities supports the hypothesis that this lipid phenotype may represent a common substrate underlying both elevated alt and lv hypertrophy . the lack of clear cutoff values for the definition of dyslipidemia in childhood ( 6 ) may result in underdiagnosis and , consequently , undertreatment of children with lipid abnormalities who are at high risk for dyslipidemia in adulthood . we suggest that if confirmed in longitudinal studies , a high tg - to - hdl - c ratio might be used in clinical practice to identify children at risk for dyslipidemia and replace high tgs and low hdl in the definition of metabolic syndrome , also considering that the lipid cutoff levels in children still are debated . the strength of our study was the large sample size , the comprehensive analysis of several cmr factors , and the evaluation of preclinical signs of liver and cardiac abnormalities . the cross - sectional design precludes us to establish whether a high tg - to - hdl - c ratio has any causative effect on cardiac and liver abnormalities as well as on their progression in adulthood . in addition , our sample was composed of outpatient children , most of whom were overweight or obese , which may have limited the generalizability of our results . finally , alt levels were used as a surrogate index of nafld ; however , this proxy is widely accepted for epidemiological purposes , also considering that alt levels are a major predictor of fld ( 25 ) . in conclusion , our study demonstrates that a tg - to - hdl - c ratio 2.0 is associated with several cmr factors and proves to be useful in identifying children with a high risk of elevated alt levels and concentric lv hypertrophy . these results , obtained in outpatient , white children with high prevalence of overweight , need to be replicated in the general pediatric population , and longitudinal evaluations will clarify whether a high tg - to - hdl - c ratio may be a good predictor of cardiovascular disease in adult life .

nineteen healthy men ( mean sem age 23.2 0.6 years and bmi 23.5 0.3 kg / m ) who were free of medication participated in the experiments . they gave written informed consent to the study , which conformed to the declaration of helsinki and was approved by the local ethics committee . each subject participated in two conditions ( insulin and placebo ) spaced apart by at least 4 weeks . body weight and body composition ( bia 2000-m ; data input , frankfurt , germany ) did not differ between conditions . after a 12-h fast , experimental sessions started at 7:00 a.m. with baseline assessments of energy expenditure and blood parameters ( fig . , subjects rested in bed in a supine position in a quiet room of constant temperature ( 23c ) . at 9:10 a.m. , sixteen 0.1-ml puffs ( eight per nostril ) of insulin and placebo , respectively , were intranasally administered in 2-min intervals , amounting to a total dose of 1.6 ml insulin ( 160 iu insulin actrapid ; novo nordisk , mainz , germany ) or vehicle ( hoe 31 dilution buffer ; aventis pharma , bad soden , germany ) . insulin and placebo were administered using precision air pumps ( aero pump , hochheim , germany ) that fill the nostrils and the nasal cavity with aerosol , thus enabling the solution to effectively target the olfactory epithelium . the dose of intranasal insulin used here has previously been shown to be functionally effective in healthy humans ( 9,11 ) . following postinsulin administration measurements ( see below ) , subjects consumed 600 ml of a standard liquid meal ( fresubin energy drink , fresenius kabi , bad homburg , germany ) at a dose of 20 ml / min ( totaling 900 kcal , 33.6 g protein , 34.8 g fat , and 112.8 g carbohydrate ) from 10:1510:45:00 a.m. , and subsequently assessments were continued until 4:00 p.m. experimental schedule . nineteen healthy subjects who had fasted overnight spent the experimental day sitting in bed in a supine position . measurements of energy expenditure by 30-min periods of indirect calorimetry were performed during baseline ( 8:309:00 a.m. ) , immediately after intranasal insulin administration ( 9:4510:15:00 a.m. , 1.6 ml [ 160 iu ] insulin and placebo , respectively ; nose symbol ) , and five times following the standardized consumption of a predefined liquid meal of 900 kcal ( cup symbol ) . blood samplings for the determination of plasma glucose , serum insulin , c - peptide , and free fatty acids concentrations are indicated by syringe symbols . energy expenditure ( expressed as kcal / min ) was measured via indirect calorimetry using a ventilated - hood system ( deltatrac ii , mbm-200 metabolic monitor ; datex - engstrm deutschland , achim , germany ) . before each use , the device was calibrated with quick cale calibration gas to 5% co2 and 95% o2 . calorimetric measurements took place from 8:30 to 9:00 a.m. ( baseline ) , from 9:45 to 10:15:00 a.m. ( to assess effects of intranasal insulin alone ) , and five times between 10:45 and 3:15:00 p.m. , i.e. , after liquid food intake ( fig . the rise in energy expenditure between the fasting state ( baseline measurement from 8:45 to 9:15:00 a.m. ) and the postprandial state ( mean energy expenditure from 10:45 to 3:15:00 p.m. ) reflects diet - induced thermogenesis , i.e. , the energy that is emitted as heat during food metabolization and thus does not contribute to the production of atp . postprandial measurements were separated by 30-min breaks during which the ventilation hood was not worn but the subjects remained in bed . for the assessment of plasma glucose levels and serum concentrations of insulin , c - peptide , and free fatty acids ( ffas ) , blood was sampled twice during baseline ( 8:00 and 9:00 a.m. ) , immediately after intranasal insulin administration ( 9:40 a.m. ) , every 10 min during liquid food intake ( 10:2010:40 a.m. ) , and at 60-min intervals thereafter ( 11:30 a.m.2:30 p.m. ) with a final sample taken at 4:00 p.m. ( fig . plasma glucose levels were measured in fluoride plasma ( hexokinase method , aeroset ; abbott diagnostics , north chicago , il ) . serum concentrations of insulin and c - peptide were measured by an immulite analyzer ( siemens medical solutions diagnostics , los angeles , ca ) . statistical analyses were based on anova including the repeated - measures factors condition and time ( referring to the immediate posttreatment and postprandial periods ) . postprandial glucose and hormone concentrations ( 10:20 a.m.4:00 p.m. ) were expressed as areas under the curve ( aucs ) calculated according to the trapezoidal rule . each subject participated in two conditions ( insulin and placebo ) spaced apart by at least 4 weeks . body weight and body composition ( bia 2000-m ; data input , frankfurt , germany ) did not differ between conditions . after a 12-h fast , experimental sessions started at 7:00 a.m. with baseline assessments of energy expenditure and blood parameters ( fig . , subjects rested in bed in a supine position in a quiet room of constant temperature ( 23c ) . at 9:10 a.m. , sixteen 0.1-ml puffs ( eight per nostril ) of insulin and placebo , respectively , were intranasally administered in 2-min intervals , amounting to a total dose of 1.6 ml insulin ( 160 iu insulin actrapid ; novo nordisk , mainz , germany ) or vehicle ( hoe 31 dilution buffer ; aventis pharma , bad soden , germany ) . insulin and placebo were administered using precision air pumps ( aero pump , hochheim , germany ) that fill the nostrils and the nasal cavity with aerosol , thus enabling the solution to effectively target the olfactory epithelium . the dose of intranasal insulin used here has previously been shown to be functionally effective in healthy humans ( 9,11 ) . following postinsulin administration measurements ( see below ) , subjects consumed 600 ml of a standard liquid meal ( fresubin energy drink , fresenius kabi , bad homburg , germany ) at a dose of 20 ml / min ( totaling 900 kcal , 33.6 g protein , 34.8 g fat , and 112.8 g carbohydrate ) from 10:1510:45:00 a.m. , and subsequently assessments were continued until 4:00 p.m. experimental schedule . nineteen healthy subjects who had fasted overnight spent the experimental day sitting in bed in a supine position . measurements of energy expenditure by 30-min periods of indirect calorimetry were performed during baseline ( 8:309:00 a.m. ) , immediately after intranasal insulin administration ( 9:4510:15:00 a.m. , 1.6 ml [ 160 iu ] insulin and placebo , respectively ; nose symbol ) , and five times following the standardized consumption of a predefined liquid meal of 900 kcal ( cup symbol ) . blood samplings for the determination of plasma glucose , serum insulin , c - peptide , and free fatty acids concentrations are indicated by syringe symbols . energy expenditure ( expressed as kcal / min ) was measured via indirect calorimetry using a ventilated - hood system ( deltatrac ii , mbm-200 metabolic monitor ; datex - engstrm deutschland , achim , germany ) . before each use , the device was calibrated with quick cale calibration gas to 5% co2 and 95% o2 . calorimetric measurements took place from 8:30 to 9:00 a.m. ( baseline ) , from 9:45 to 10:15:00 a.m. ( to assess effects of intranasal insulin alone ) , and five times between 10:45 and 3:15:00 p.m. , i.e. , after liquid food intake ( fig . the rise in energy expenditure between the fasting state ( baseline measurement from 8:45 to 9:15:00 a.m. ) and the postprandial state ( mean energy expenditure from 10:45 to 3:15:00 p.m. ) reflects diet - induced thermogenesis , i.e. , the energy that is emitted as heat during food metabolization and thus does not contribute to the production of atp . postprandial measurements were separated by 30-min breaks during which the ventilation hood was not worn but the subjects remained in bed . for the assessment of plasma glucose levels and serum concentrations of insulin , c - peptide , and free fatty acids ( ffas ) , blood was sampled twice during baseline ( 8:00 and 9:00 a.m. ) , immediately after intranasal insulin administration ( 9:40 a.m. ) , every 10 min during liquid food intake ( 10:2010:40 a.m. ) , and at 60-min intervals thereafter ( 11:30 a.m.2:30 p.m. ) with a final sample taken at 4:00 p.m. ( fig . plasma glucose levels were measured in fluoride plasma ( hexokinase method , aeroset ; abbott diagnostics , north chicago , il ) . serum concentrations of insulin and c - peptide were measured by an immulite analyzer ( siemens medical solutions diagnostics , los angeles , ca ) . statistical analyses were based on anova including the repeated - measures factors condition and time ( referring to the immediate posttreatment and postprandial periods ) . postprandial glucose and hormone concentrations ( 10:20 a.m.4:00 p.m. ) were expressed as areas under the curve ( aucs ) calculated according to the trapezoidal rule . resting metabolic rates were comparable between conditions during baseline and immediately after insulin administration ( p > 0.19 for all comparisons ) ( fig . 2 ) . however , the increase in metabolic rate following liquid food intake was on average 17% greater in the insulin than in the placebo condition ( p < 0.05 for the insulin / placebo main effect ; fig . 2 ) , indicating an increase in postprandial energy expenditure , i.e. , diet - induced thermogenesis , due to intranasal insulin . intranasal insulin enhances postprandial energy expenditure . following baseline assessment of energy expenditure ( ee ) ( expressed per kcal / min ) , acute effects of intranasal administration ( nose symbol ) of insulin ( 160 iu ) ( ) , and placebo ( ) , respectively , on energy expenditure were frequently measured before and after ingestion of liquid food ( 900 kcal ; cup symbol ) for a total of 6.5 h ( left panel ) . the rise in energy expenditure between baseline ( 8:309:45:00 a.m. ) and the postprandial state ( 10:45:00 a.m.3:15:00 p.m. ) reflects the energy emitted mainly as heat during food metabolization ( diet - induced thermogenesis [ dit ] ) ( right panel ) . plasma glucose , hormonal , and ffa concentrations did not differ between conditions during baseline ( p > 0.25 ) . immediately after intranasal insulin administration , i.e. , at 9:40 a.m. , a slight and transient decrease in fasting plasma glucose was detected ( insulin vs. placebo 4.5 0.1 vs. 4.7 0.1 mmol / l ; p < 0.02 [ insulin / placebo main effect ] ) , but the subsequent postprandial increase in glucose concentrations did not differ between conditions ( auc , 10:20 a.m.4:00 p.m. , insulin vs. placebo 1,574 23 vs. 1,570 20 mmol / l / min ; p > 0.90 ) ( fig . 3a ) . in parallel with the slight postinsulin administration drop in plasma glucose , a small increase in serum insulin ( insulin vs. placebo 40.1 6.3 vs. 22.6 3.0 pmol / l ; p < 0.01 ) but not in c - peptide concentrations ( 0.34 0.02 vs. 0.37 0.03 nmol / l ; p > 0.31 ) emerged . following liquid food intake , the postprandial increase in both insulin and c - peptide concentrations was reduced by intranasal insulin in comparison with placebo ( auc , 10:20 a.m.4:00 p.m. , serum insulin 45,521 5,052 vs. 62,315 4,973 pmol / l / min ; serum c - peptide 409 30 vs. 487 33 nmol / l / min ; p < 0.001 and p < 0.02 , respectively ) ( fig . 3b and c ) . serum ffa concentrations showed a transient decrease during food intake in the insulin compared with the placebo condition ( p < 0.01 for condition time ) but did not differ between conditions during the postprandial period ( auc , 10:20 a.m.4:00 p.m. , insulin vs. placebo 53.7 4.6 vs. 56.6 4.2 mmol / l / min ; p < 0.60 ) ( fig . concentrations of plasma glucose ( a ) , serum insulin ( b ) , serum c - peptide ( c ) , and serum free fatty acids ( d ) before and after acute intranasal administration ( nose symbol ) of intranasal insulin ( 160 iu ; solid lines and black bars ) and placebo ( dashed lines and white bars ) followed by the standardized ingestion of 900 kcal of liquid food ( cup symbol ) . postprandial levels ( 10:20 a.m.4:00 p.m. ) were also expressed as aucs ( right panels ) . supplementary analyses revealed that the immediate effects of intranasal insulin on preprandial glucose and insulin levels as well as the prandial decrease in ffa levels were statistically unrelated to the treatment - induced increase in postprandial thermogenesis and the suppression in postprandial serum insulin concentrations ( pearson 's correlations ; p > 0.23 for all coefficients ) . we demonstrated in humans that acutely enhancing brain insulin signaling by intranasal administration of the hormone increases postprandial thermogenesis . the parallel treatment - induced reduction in postprandial serum insulin concentrations while plasma glucose levels were comparable between conditions indicates that following intranasal insulin administration to the brain , lower circulating levels of the hormone are sufficient to dispose of meal - related increases in plasma glucose . in line with findings in animals ( 4,5,13 ) , our results support the notion that brain insulin signaling in humans is involved in the control of whole - body energy homeostasis . in keeping with previous experiments ( 9,11 ) , intranasal administration of 160 iu insulin induced a transient and mild increase in serum insulin concentrations accompanied by a slight drop in prefood intake plasma glucose that clearly remained within the euglycemic range . due to the relatively high dose administered here compared with that in previous studies ( 6,8 ) , a small ratio of the hormone may have entered the circulation via the nasal mucosa . however , the transient nature and limited size of these immediate effects argues against an involvement of systemic uptake of intranasal insulin in its impact on postprandial thermogenesis and glucose metabolism . this conclusion is corroborated by the fact that immediate and postprandial effects were not statistically related . the balanced regulation of nutrient intake and energy expenditure relies on the hypothalamus as a major integrator of nutritional and hormonal signals from the body periphery , including glucose and insulin ( 1 ) . direct injections of insulin into the preoptic area of the hypothalamus induce a dose - dependent increase in core body temperature due to stimulation of brown adipose tissue thermogenesis that is assumed to be mediated by inhibitory insulinergic action on warm - sensitive hypothalamic neurons ( 3 ) . in our experiments , intranasal administration of the hormone to the brain did not affect resting energy expenditure but evoked a distinct increase in postprandial thermogenesis . increased postprandial energy expenditure due to enhanced brain insulin signaling adds to the reduction in food intake previously observed after intranasal administration of the hormone ( 9 ) , suggesting that the catabolic impact of central nervous insulin ( 10,14 ) is mediated not only by anorexigenic but also by thermogenic effects of the hormone . still , further studies on this issue are needed and should include measurements of body temperature , brown adipose tissue activity , and relevant vital signs like heart rate and blood pressure to elucidate the effect of brain insulin signaling on energy expenditure in humans . a most remarkable finding of our study is the intranasal insulin induced reduction in postprandial serum insulin concentrations while the food intake - induced rise in plasma glucose remained unaffected , suggesting that intranasal insulin improves postprandial insulin sensitivity . a regulatory effect of central nervous insulin on hepatic glucose metabolism has been indicated by animal studies showing that a selective decrease in hypothalamic insulin receptors reduces hepatic insulin sensitivity and results in marked increases in hepatic glucose production in the presence of plasma insulin concentrations equaling those of control animals ( 15 ) . fittingly , insulin hyperpolarizes glucose - responsive hypothalamic neurons by opening atp - sensitive k channels ( 16 ) which triggers a decrease in hepatic glucose production that is mediated by vagal efferences ( 5,15 ) . this pattern suggests that enhancing brain insulin signaling by intranasal administration of the hormone may act on glucose homeostasis in the body periphery by supporting hepatic insulin action . nevertheless , given that postprandial liver glucose production accounts for approximately one - fifth to one - half of fasting values ( 17 ) , improved insulin - dependent metabolization of ingested glucose may also have contributed to the intranasal insulin - induced decrease in postprandial serum insulin levels . such an effect could basically be supported by the observed decrease in prandial ffa levels due to intranasal insulin inasmuch as ffas are known to impair insulin - stimulated muscle uptake of glucose ( 18 ) . however , ffa effects on peripheral insulin - stimulated glucose uptake slowly develop over some hours ( 19 ) , which , in conjunction with the lack of a significant correlation between the decreases in prandial ffa and postprandial insulin concentrations , makes this view unlikely . furthermore , a contribution of enhanced noninsulin - mediated glucose disposal , i.e. , glucose effectiveness , to our effects can not be ruled out . although the present results suggest that insulin administration to the human brain enhances the efficiency of the glucoregulatory brain - liver axis in response to nutrient intake , our observations should be corroborated in future studies that rely on more refined measurements of insulin sensitivity , e.g. , euglycemic hyperinsulinemic clamps . it is also noteworthy that most recent animal data hint at divergent effects of hypothalamic insulinergic signaling on peripheral glucose homeostasis and energy expenditure depending on the involvement of agouti - related protein or proopiomelanocortin neuronal pathways ( 13 ) . in this regard , general enhancements in brain insulin signaling as performed in our study do not permit differentiations . taken together , our findings indicate that intranasal insulin acutely increases postprandial thermogenesis and improves the glucoregulatory response to food intake , suggesting that boosting brain insulin signaling in humans enhances the body 's ability to cope with calorie consumption ( 20,21 ) . against the background of studies indicating that obesity and peripheral insulin resistance are associated with reduced central nervous insulin sensitivity ( 2224 ) , enhancing brain insulin signaling may emerge as a useful approach in the therapeutic management of disorders hallmarked by disturbed glucose homeostasis ( 25 ) .

people with intellectual disabilities comprise an estimated five million persons in the eu 27 member states . evidence suggests that people in this group show negative disparities in health status and in access to optimal health care . they are more likely to incur secondary health conditions and thus report increased morbidity and poorer health status . higher rates of obesity , mental health disorders , and lower rates of cardiovascular fitness , vaccination levels , and preventative health screening have been identified among people with intellectual disabilities . epilepsy and mental health difficulties have a high rate of prevalence . to identify health disparities , measures to gather comparable health data are required . this presentation outlines the procedures adopted to develop and implement a set of health indicators for persons with intellectual disabilities in europe . with support from the dg - public health of the eu , the pomona group developed a set of 18 health indicators for persons with intellectual disabilities ( 20022004 ) . partners operationalized these indicators in a survey instrument , translated this into 14 languages , secured ethical approval , carried out a pilot study and gathered data among n=1269 participants . most ( 77% ) lived independently or semi - independently , with their families or in group homes with fewer than 16 residents . about one - half of participants were in the overweight or obese categories . results are discussed in the light of priorities for gathering and maintaining health information systems that can help to identify health needs of persons in this segment of the population , and efforts at european level to decrease health inequalities .

a seizure is a transient disturbance of the cerebral function secondary to abnormal paroxysms in the brain , which results in a sudden excessive disorderly discharge of the cerebral neurons . the discharge results in an almost instantaneous disturbance of sensation , loss of consciousness or psychic function , convulsive movements , or combinations of these.1,2 persons with epilepsy are at a risk of developing a variety of psychological problems , including depression and anxiety , and psychosis.3 previous studies show that the prevalence of active epilepsy in developing countries range from 5 to 10 per 1,000 people.4 however , the worldwide prevalence rate of epilepsy varies from 2.8 to 19.5 per 1,000 of the general population and is more prevalent among children.5 in nigeria , the estimated prevalence of epilepsy is 813 per 1,000 people.6 in developing countries , the disorder is to a significant degree associated with a host of parasitic and bacterial infectious diseases that are largely absent in industrialized countries.7 sociocultural attitudes continue to cause a negative impact on the management of epilepsy in many african nations.8 the disorder is enrobed in superstition , discrimination , and stigma in many of these states.9 religious and sociocultural beliefs influence the nature of treatment and maintenance received by people with epilepsy . many communities in africa and other developing countries believe that epilepsy results from witchcraft or possession by evil spirits , and hence , treatment should be through the use of herbs from traditional doctors , fetish priests , and religious leaders.9 persons with epilepsy are shunned and discriminated against in education , employment , and marriage in africa because epilepsy is seen as a highly contagious and shameful disease in the eyes of the public.10 study findings from ghana indicated that 45.3% of study participants did not know the cause of epilepsy ; besides , 37.6% of the participants did not know how to treat a patient with epilepsy . the study also shows that youngsters who have lower educational levels and single marital status did not have good knowledge of epilepsy.6 a study conducted in butajira regarding the attitude and practice of people related to mental disorders has shown that 41% of the informants preferred modern medicine for neuropsychiatry disorders.11 moreover , study results from rural community in jimma , ethiopia on the knowledge , attitude , and practice ( kap ) related to epilepsy reveal that poor knowledge as well as inappropriate attitude and practice were observed in the community.12 with several limitations , studies show that misconception , superstition , ignorance , and prejudice are still evident and contribute to the low levels of conventional epilepsy care and the treatment gap.10 though studies were conducted on kap related to epilepsy in various african countries , kap related to epilepsy has not yet adequately been studied at a community level in ethiopia . hence , this study tries to assess kap and its associated factors related to epilepsy in sululta woreda , oromia , ethiopia . this study aims to help improve the utilization of primary mental health services by bridging the treatment gap between the communities and health facilities . community - based cross - sectional study with the multistage sampling technique was conducted in oromia regional state , sululta woreda , ethiopia . the people belonging to the sululta community from all households were taken as source population . individuals of age over 18 years ( 357 males and 303 females ) with sound mental health were selected as the study population . the sample size was calculated using single population proportion formula to estimate the study sample by using margin of error 0.05 and proportion of event occurrence at 28% similar to studies conducted at northern nigeria13 at 95% of level of significance by adding a 10% nonrespondent rate . based on the above assumption , the minimum sample size required for the study was 618 . data were collected through interviews by using previously adopted standard questionnaire that was translated into local languages . in addition to providing a translation , the data quality was assured by daily checking to control the quality of data collected from the study subject by supervisors . sociodemographic data and the levels of kap of the study participants related to epilepsy were measured by the number of occurrences ( ie , frequencies ) for descriptive variables and binary logistic regression was undertaken to explore the factors associated with adherence ( p<0.05 ) to provide the odds ratio ( or ) at 95% confidence interval ( ci ) . bivariate association between dependent and independent variables was determined and covariates with p>0.2 with the outcome variable were dropped from further consideration in modeling by using multivariate analysis as an adjusted or ( aor ) . according to the current study , good knowledge is defined as when respondents scored 50% when answering questions related to epilepsy knowledge and poor knowledge is defined as when respondents scored < 50% . favorable for a score 50% and as unfavorable for a score < 50% . similarly , the study defines practice in epilepsy as safe for respondents who scored 50% , and as unsafe for those who scored < 50% . ethical clearance was obtained from oromia regional health bureau research and publication committee ( drpc ) . further , permission was obtained from oromia , finfine special zone , and woreda health office . of the 682 study participants , 660 completed the study with a 96.8% response rate . the median age of the respondents was 30 years and the range was 1855 years . a majority of the study participants ( 94.4% ) were of oromo ethnicity , and half ( 48.1% ) of them were women ; 26.1% of them were single girls . regarding prior source of information about epilepsy , 63.4% of the participants had the information from mass media ( tv / radio ) , whereas 22.1% , 12.6% , and 1.9% of the study subjects had the information from health facilities , health extension workers , and school , respectively . according to the study , 59.8% , 35.6% , and 33.5% of respondents had good knowledge , favorable attitude , and safe practice related to epilepsy , respectively , as shown in figure 1 . bivariate logistic regression was conducted to assess the association between the outcome variables and others variables of epilepsy . data collected show that the following factors had been associated with knowledge of epilepsy : respondents sex , family role , educational status , whether they lived in a rural or urban area , had heard about epilepsy , knew someone with epilepsy , had witnessed a seizure , or had epileptic family members . the following factors were found to be significantly associated with the attitude about epilepsy in bivariate analysis : respondent s age , family role , educational status , occupation , whether they had heard about epilepsy , where they heard about epilepsy , whether they knew someone with epilepsy , and the distance of their home from a health institution . likewise , in bivariate analysis , the following factors were shown to be associated with the practice related to epilepsy : respondents sex , age , family role , educational status , occupation , distance of home from health institution , whether they had heard about epilepsy , knew someone with epilepsy , had witnessed a seizure , or had epileptic family members . multivariate analysis shows that rural dwellers ( aor = 0.58 , 95% ci = 0.350.96 ) and participants living alone ( aor = 0.36 , 95% ci = 0.140.9 ) were less knowledgeable about epilepsy . there was a significant association between knowledge of epilepsy and the distance between the respondents home and health institutions : respondents who lived short distances from health institutions ( 3160 minutes walking time ) were likely to be more knowledgeable ( aor 95% ci , 0.47 [ 0.27 , 0.83 ] ) ; whereas respondents who lived further away from health institutions ( more than 61 minutes walking time ) were likely to be less knowledgeable . respondents with a higher educational status , for instance , secondary level ( aor 95% ci , 2.30 [ 1.18 , 4.480 ] ) and college level ( aor 95% ci , 2.43 [ 1.03 , 5.73 ] ) , have positive association with the level of knowledge about epilepsy . the results of this study showed that participants who had heard about epilepsy ( aor 95% ci , 0.13 [ 0.06 , 0.27 ] ) and knew someone who has epilepsy ( aor 95% ci , 0.57 [ 0.35 , 0.92 ] ) were more knowledgeable than those who had not heard and known ( table 2 ) . regarding factors associated with attitude toward epilepsy , residency being rural ( aor 95% ci , 0.12 [ 0.07 , 0.22 ] ) , had less attitude toward epilepsy , age group when 46 years ( aor 95% ci , 2.05 [ 1.04 , 4.04 ] ) had positive attitude than age group with early adolescence respondents with secondary and college levels of education had a favorable attitude about epilepsy compared with those who were illiterate : secondary education ( aor 95% ci , 3.89 [ 1.87,8.07 ] ) ; and college education ( aor 95% ci , 6.98 [ 2.67;18.31 ] ) . further , those who lived in an urban area ( aor 95% ci , 0.12 [ 0.07 , 0.22 ] ) were more likely to have had a positive attitude about epilepsy than those who lived in a rural area . moreover , respondents in the age group from 46 years to 55 years ( aor = 2.05 , 95% ci = 1.044.04 ) had positive attitude compared to those in the younger age group . respondents who had heard information about epilepsy had positive attitude ( aor 95% ci , 5.8 [ 1.2626.81 ] ) , compared with those who had not had information about epilepsy . meanwhile , respondents who knew someone with epilepsy were positively associated with attitude toward epilepsy ( aor 95% ci , 0.42 [ 0.26 , 0.67 ] ) than those who did not know someone who has epilepsy as shown in table 3 . according to the result of the study , with respect to the practice of the community related to the management of epilepsy , respondents in the age group ranging from 4655 years ( aor 95% ci , 2.41 [ 1.21 , 4.77 ] ) had an association with safe practice in the management of epilepsy . additionally , tertiary level of educational background ( aor 95% ci , 0.15 [ 0.05 , 0.45 ] ) , and individuals who had occupational history in labor and business areas , had a positive association with safe practice in management of epilepsy . having prior information about epilepsy ( aor 95% ci , 0.27 [ 0.10 , 0.690 ] ) had a positive association with safe practice of epilepsy management . having previous exposure / knowing epileptic patients , previous experience in the management of epilepsy , epilepsy in a family member , and safely managing epilepsy would be associated related to safe practice in epilepsy ( table 4 ) . of the 682 study participants , 660 completed the study with a 96.8% response rate . the median age of the respondents was 30 years and the range was 1855 years . a majority of the study participants ( 94.4% ) were of oromo ethnicity , and half ( 48.1% ) of them were women ; 26.1% of them were single girls . regarding prior source of information about epilepsy , 63.4% of the participants had the information from mass media ( tv / radio ) , whereas 22.1% , 12.6% , and 1.9% of the study subjects had the information from health facilities , health extension workers , and school , respectively . according to the study , 59.8% , 35.6% , and 33.5% of respondents had good knowledge , favorable attitude , and safe practice related to epilepsy , respectively , as shown in figure 1 . bivariate logistic regression was conducted to assess the association between the outcome variables and others variables of epilepsy . data collected show that the following factors had been associated with knowledge of epilepsy : respondents sex , family role , educational status , whether they lived in a rural or urban area , had heard about epilepsy , knew someone with epilepsy , had witnessed a seizure , or had epileptic family members . the following factors were found to be significantly associated with the attitude about epilepsy in bivariate analysis : respondent s age , family role , educational status , occupation , whether they had heard about epilepsy , where they heard about epilepsy , whether they knew someone with epilepsy , and the distance of their home from a health institution . likewise , in bivariate analysis , the following factors were shown to be associated with the practice related to epilepsy : respondents sex , age , family role , educational status , occupation , distance of home from health institution , whether they had heard about epilepsy , knew someone with epilepsy , had witnessed a seizure , or had epileptic family members . multivariate analysis shows that rural dwellers ( aor = 0.58 , 95% ci = 0.350.96 ) and participants living alone ( aor = 0.36 , 95% ci = 0.140.9 ) were less knowledgeable about epilepsy . there was a significant association between knowledge of epilepsy and the distance between the respondents home and health institutions : respondents who lived short distances from health institutions ( 3160 minutes walking time ) were likely to be more knowledgeable ( aor 95% ci , 0.47 [ 0.27 , 0.83 ] ) ; whereas respondents who lived further away from health institutions ( more than 61 minutes walking time ) were likely to be less knowledgeable . respondents with a higher educational status , for instance , secondary level ( aor 95% ci , 2.30 [ 1.18 , 4.480 ] ) and college level ( aor 95% ci , 2.43 [ 1.03 , 5.73 ] ) , have positive association with the level of knowledge about epilepsy . the results of this study showed that participants who had heard about epilepsy ( aor 95% ci , 0.13 [ 0.06 , 0.27 ] ) and knew someone who has epilepsy ( aor 95% ci , 0.57 [ 0.35 , 0.92 ] ) were more knowledgeable than those who had not heard and known ( table 2 ) . regarding factors associated with attitude toward epilepsy , residency being rural ( aor 95% ci , 0.12 [ 0.07 , 0.22 ] ) , had less attitude toward epilepsy , age group when 46 years ( aor 95% ci , 2.05 [ 1.04 , 4.04 ] ) had positive attitude than age group with early adolescence respondents with secondary and college levels of education had a favorable attitude about epilepsy compared with those who were illiterate : secondary education ( aor 95% ci , 3.89 [ 1.87,8.07 ] ) ; and college education ( aor 95% ci , 6.98 [ 2.67;18.31 ] ) . further , those who lived in an urban area ( aor 95% ci , 0.12 [ 0.07 , 0.22 ] ) were more likely to have had a positive attitude about epilepsy than those who lived in a rural area . moreover , respondents in the age group from 46 years to 55 years ( aor = 2.05 , 95% ci = 1.044.04 ) had positive attitude compared to those in the younger age group . respondents who had heard information about epilepsy had positive attitude ( aor 95% ci , 5.8 [ 1.2626.81 ] ) , compared with those who had not had information about epilepsy . meanwhile , respondents who knew someone with epilepsy were positively associated with attitude toward epilepsy ( aor 95% ci , 0.42 [ 0.26 , 0.67 ] ) than those who did not know someone who has epilepsy as shown in table 3 . according to the result of the study , with respect to the practice of the community related to the management of epilepsy , respondents in the age group ranging from 4655 years ( aor 95% ci , 2.41 [ 1.21 , 4.77 ] ) had an association with safe practice in the management of epilepsy . additionally , tertiary level of educational background ( aor 95% ci , 0.15 [ 0.05 , 0.45 ] ) , and individuals who had occupational history in labor and business areas , had a positive association with safe practice in management of epilepsy . having prior information about epilepsy ( aor 95% ci , 0.27 [ 0.10 , 0.690 ] ) had a positive association with safe practice of epilepsy management . having previous exposure / knowing epileptic patients , previous experience in the management of epilepsy , epilepsy in a family member , and safely managing epilepsy would be associated related to safe practice in epilepsy ( table 4 ) . in ethiopia , a few studies do suggest that epilepsy is a major problem within the country.14 this study tries to assess kap of epilepsy and associated factors of epilepsy among sululta woreda in oromia , ethiopia . according to the result of this study , approximately 59.8% of respondents were knowledgeable , 35.6% had favorable attitude , and 33.5% had safe practice toward epilepsy , respectively . this study shows less level of knowledge while it compares with studies conducted in karachi , pakistan ( 77.5%).15 moreover , our current study shows less improvement in knowledge of epilepsy compared with studies carried out among the general public in ethiopia ( 89%),16 italy ( 93.4%),17 cameroon ( 100% , 99.3%),18,19 croatia ( 97%),20 south korea ( 94%)21 and among school teachers in egypt ( 100%)22 and brazil ( 100%).23 the difference observed might be arising from differences in the methodologies adopted . regarding attitude , the current study shows that 35.6% of the community have a positive attitude towards epilepsy , and that there are still negative attitudes towards individuals who suffer from epilepsy . on the other hand , attitudes were more positive compared to that of the participants of a study in egypt ( 8%).24 the current study shows less favorable attitude of participants when compared to that of other studies carried out among students in cameroon ( 70.6% , 77.2%),25,26 india ( 77.7%),27 and trinidad and tobago ( 93%).28 this study finding regarding attitude is similar to the study finding in vietnam ( 33%).29 such differences are often considered to reflect national and cultural attitudes , but it can not be excluded that cultural differences in willingness to disclose what might be considered a politically incorrect opinion could contribute . as regards practice related to epilepsy management , this study shows a lower level of practice than studies done in jimma university specialized hospital ( 53.3%).30 this difference might be due to the methodological differences . regarding determinant factors , our current study shows that factors associated with knowledge of epilepsy were , formal secondary education and above , prior information on epilepsy , someone living within 1 hour travel time from the health institution and knowing someone with epilepsy had positively associated with the level of knowledge regarding to epilepsy . on the other hand , respondents who reside far away from health care institutions , rural dwellers , and those living alone were negatively associated with the level of knowledge about epilepsy . according to the study , variables that affect the outcome variables in the current studies are : being urban dwellers , being in the age group of 4655 years , having formal education above secondary level , having heard about epilepsy , having obtained information through colleges , and knowing someone with epilepsy . these variables had positive association with attitude toward epilepsy . on the contrary , being rural dwellers negatively associated to attitude toward epilepsy . studies done in mekele , ethiopia showed that factors associated with unfavorable attitude include refusal to marry people with epilepsy ( 44.8% ) , beliefs that epilepsy is caused by evil spirit ( 33.24% ) , is contagious ( 28.6% ) , or a kind of insanity ( 51.6%).13 hence , our current study includes other factors that positively and negatively affect but not covered in earlier studies . practices related to epilepsy were affected by age greater than 46 years , prior heard information related to epilepsy , being laborers or business men / women , knowing someone with epilepsy , witnessing epilepsy and having a family history of epilepsy had positive association with epilepsy , and distance negatively associated with practices related to epilepsy . on the other hand , a previous study related to epilepsy management showed that nearly 73% people believed that allopathy is a better option than ayurveda ( 25% ) . a very small proportion , ie , only 2% believed that holy treatment with worship is effective in treating epilepsy . approximately 40% felt that epilepsy can be cured , but almost 60% thought that a person with epilepsy has to take lifelong treatment . in response to first aid measures in response to epileptic seizures , 60% said that they would take the person to a hospital , 23% felt that they would put a shoe or onion on nose , and 15% would splash water over the face of the patient.31 the findings indicated that the sululta community is familiar with epilepsy , yet there is still problem with negative attitude and unsafe practice related to epilepsy ; but relatively promising knowledge about epilepsy . overall , 59.8% were knowledgeable about epilepsy , 35.6% had favorable attitude , and 33.5% of them observed safe practices related to epilepsy . living in rural locations , living alone , having education at the secondary or college level , having heard information about epilepsy , distance of health institution from the community , and witnessing epilepsy episodes had significant association to knowledge of epilepsy among respondents . there was an association for attitute in respondents who lived in a rural area , had secondary or college level education , were aged 46 to 55 years , had heard information about epilepsy , and had prior knowledge of epilepsy being more likely to have a favorable attitude towards epilepsy . the following factors had a positive association with practices related to epilepsy : study subjects aged 4655 years , college level of education , occupational history of being self - employed or a laborer , and had heard about epilepsy . additionally , the following factors had a positive association with epilepsy management : walking distance to health institution 31 minutes , knew someone with epilepsy , and had a family member with epilepsy . hence , we conclude that the current new findings might help initiate further studies because there is still a need for more widespread dissemination of information to improve general knowledge about epilepsy .

malignant liver tumors are considered to be one of the major public health problems in the world because of late diagnosis and failure of treatments . there are mainly three kinds of malignant liver tumors in adults : hepatocellular carcinoma ( hcc ) , cholangiocarcinoma ( cca ) , and metastatic liver tumors . approximately 500,000 people die of hcc each year , and more than 50% of the cases occur in china . it is the fifth leading cause of cancer death worldwide , and the asia - pacific region is a high - prevalence area . cca , the second most common primary hepatic malignancy , originates from the bile duct epithelial cells and accounts for 10% to 20% of the deaths from hepatobiliary malignancies . metastatic liver tumors are malignant tumors in the liver that have spread from other areas of the body . clinically , these malignant liver tumors are generally asymptomatic in early stages and easily lead to missed diagnosis . in addition , they also require differentiation from benign liver lesions to avoid unnecessary interventions . therefore , it is important to develop accurate diagnostic methods to distinguishing between benign and malignant liver tumors . at present , serologic markers , such as -fetoprotein ( afp ) and imaging technology , are the main methods to detect malignant liver tumors . however , serologic markers are limited by their low sensitivity , and imaging methods , such as computed tomography ( ct ) and magnetic resonance imaging ( mri ) , are limited by their high cost . ultrasound and elastography have been used as screening methods for liver tumors , but their accuracy was only about 70% [ 46 ] . in addition , these imaging methods have the risk of radiation exposure . in recent years , mass spectrometry ( ms ) has been used in clinical diagnosis and medical research fields , including discovery of cancer biomarkers , diagnosis of bacterial infection , and identification of mutations and genotypes of virus [ 711 ] . mass spectrometry technique is more precise , rapid , and cost - effective than the traditional methods . in this study , we applied matrix - assisted laser desorption time of flight mass spectrometry ( maldi - tof - ms ) combined with weak cation - exchange chromatography magnetic beads ( mb - wcx ) to establish classification models to distinguish benign and malignant liver tumors and identify the liver cancer - specific peptides by liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . subjects included 66 patients with malignant liver tumors ( 34 hcc , 22 cca , and 10 metastatic liver tumors ) and 79 patients with benign liver tumors ( 34 liver cirrhosis lesions , 30 liver hemangiomas , 11 hepatic cysts , 3 hepatic adenomas , and 1 focal nodular hyperplasia ) . all study subjects were first - visit outpatients who were newly diagnosed with focal liver lesions by ultrasound . the final diagnoses were made by liver histopathology or mri based on guidelines from the ministry of health of the people s republic of china and the guideline from the chinese society of hepatology and the chinese society of infectious diseases . some patients with the following conditions were excluded : other systemic disease such as diabetes and hypertension ; prior surgery , interventional therapy , radiotherapy , chemotherapy , and other invasive treatment ; and severe complications such as upper gastrointestinal bleeding and hepatic encephalopathy . the study procedures were approved by the ethics committee of the 302 military hospital of china , and written informed consent obtained from each subject . the serum samples were collected in a 5 ml vacuum blood collection tube without anticoagulant , then centrifuged for 5 min at 12,000 g at room temperature . mb - wcx ( bruker daltonik , germany ) kits were used according to the manufacturer s protocol . we added 10 l of mb - wcx beads and 10 l of binding solution ( bb ) in 200 l pcr tubes . then we added 5 l of serum samples and mixed thoroughly by pipetting up and down . subsequently , samples were incubated at room temperature for 5 min , and then we used a magnetic separator to collect the beads . the supernatant was removed , and the magnetic beads were washed three times using washing solution ( wb ) . finally , we added 5 l of elution solution ( eb ) and 4 l of stabilization buffer ( sb ) to elute the peptide fraction from the magnetic beads . we transferred 1 l of supernatant from the pcr tubes onto a 384 ground steel target plate , and then the samples were air - dried . every dried sample was mixed with 1 l of maldi matrix ( 60 mg of -cyano-4-hydroxycinnamic acid [ hcca ] dissolved in 1 ml of acetonitrile and 1 ml of 2% trifluoroacetic acid [ tfa ] ; bruker daltonik , germany ) . measurements were performed using an autoflex maldi - tof - ms ( bruker daltonik , germany ) instrument and flexcontrol software ( version 3.0 ; bruker ) . the parameter settings were as follows : positive linear mode ( lp ) ; mass range : 1000 da to 20,000 da ; laser power : 60% . the mass calibration was performed with the peptide calibration standard ( 7003500 da ; bruker daltonik ) and peptide calibration standard i ( 500020,000 da ; bruker daltonik ) . to evaluate the reproducibility of the mb - wcx bead kits and instrument , sera from eight normal subjects were treated by mb - wcx bead , and each of them was detected for 8 repeats . the mean value of the coefficient of variance ( cv ) for all peaks was calculated to evaluate intra- and inter - reproducibility . data analysis was performed with clinpro tools software and flex analysis software ( version 3.0 ; bruker ) . all the spectra were normalized , and baseline subtraction and smoothing were performed with flex analysis software . peaks with signal - to - noise ratio ( s / n ) > 5 were picked out , and statistical analysis was performed . data that were normally distributed were analyzed with student s t tests , and non - normally distributed data were analyzed by the wilcoxon test . in order to construct the diagnostic models to distinguish benign and malignant liver tumors , all samples were randomly divided into 2 subsets : training set and validation set . a total of 95 serum samples ( 43 malignant liver tumor patients and 52 benign liver lesion patients ) were used to construct classification models as training samples . three different machine - learning algorithms were used as follows : genetic algorithm ( ga ) , supervised neural network ( snn ) , and quick classifier ( qc ) . to confirm the clinical applicability of the models constructed , accuracy , sensitivity , specificity , positive and negative predictive values ( ppv and npv ) , and youden s index were calculated to assess the performances of the models . samples purified by mb - wcx beads were desalted using ziptipc18 pipette ( millipore , bedford , massachusetts , usa ) tips according to the procedure described below . then , a tfa - acidified sample was applied to the ziptip , followed by two washes with 10 l of 0.1% ( vol / vol ) tfa solution . bound proteins were eluted using a 50% ( vol / vol ) acetonitrile solution containing 0.1% ( vol / vol ) tfa . analysis of the eluted peptides was performed using a qtof synapt g2-s system ( waters , pittsburgh , pennsylvania , usa ) equipped with an esi source operated in the positive ion mode . the source temperature was set at 100c with a flow rate of 200 nl / min . the capillary and cone voltages were set to 3000 and 40 v. ms data were collected for m / z values in the range of 100 da and 2000 da with a scan time of 0.5 s , and ms / ms data were collected between 50 da and 1600 da with a scan time of 0.2 s. the ms / ms data were searched on mascot ( http://www.matrixscience.com ) . peptide mass tolerance was 10 ppm , fragment ion mass tolerance was 0.01 , and the mass type of the parent peptide and peptide fragment was at monoisotopic . subjects included 66 patients with malignant liver tumors ( 34 hcc , 22 cca , and 10 metastatic liver tumors ) and 79 patients with benign liver tumors ( 34 liver cirrhosis lesions , 30 liver hemangiomas , 11 hepatic cysts , 3 hepatic adenomas , and 1 focal nodular hyperplasia ) . all study subjects were first - visit outpatients who were newly diagnosed with focal liver lesions by ultrasound . the final diagnoses were made by liver histopathology or mri based on guidelines from the ministry of health of the people s republic of china and the guideline from the chinese society of hepatology and the chinese society of infectious diseases . some patients with the following conditions were excluded : other systemic disease such as diabetes and hypertension ; prior surgery , interventional therapy , radiotherapy , chemotherapy , and other invasive treatment ; and severe complications such as upper gastrointestinal bleeding and hepatic encephalopathy . the study procedures were approved by the ethics committee of the 302 military hospital of china , and written informed consent obtained from each subject . the serum samples were collected in a 5 ml vacuum blood collection tube without anticoagulant , then centrifuged for 5 min at 12,000 g at room temperature . mb - wcx ( bruker daltonik , germany ) kits were used according to the manufacturer s protocol . we added 10 l of mb - wcx beads and 10 l of binding solution ( bb ) in 200 l pcr tubes . then we added 5 l of serum samples and mixed thoroughly by pipetting up and down . subsequently , samples were incubated at room temperature for 5 min , and then we used a magnetic separator to collect the beads . the supernatant was removed , and the magnetic beads were washed three times using washing solution ( wb ) . finally , we added 5 l of elution solution ( eb ) and 4 l of stabilization buffer ( sb ) to elute the peptide fraction from the magnetic beads . we transferred 1 l of supernatant from the pcr tubes onto a 384 ground steel target plate , and then the samples were air - dried . every dried sample was mixed with 1 l of maldi matrix ( 60 mg of -cyano-4-hydroxycinnamic acid [ hcca ] dissolved in 1 ml of acetonitrile and 1 ml of 2% trifluoroacetic acid [ tfa ] ; bruker daltonik , germany ) . measurements were performed using an autoflex maldi - tof - ms ( bruker daltonik , germany ) instrument and flexcontrol software ( version 3.0 ; bruker ) . the parameter settings were as follows : positive linear mode ( lp ) ; mass range : 1000 da to 20,000 da ; laser power : 60% . for each spot , the mass calibration was performed with the peptide calibration standard ( 7003500 da ; bruker daltonik ) and peptide calibration standard i ( 500020,000 da ; bruker daltonik ) . to evaluate the reproducibility of the mb - wcx bead kits and instrument , sera from eight normal subjects were treated by mb - wcx bead , and each of them was detected for 8 repeats . the mean value of the coefficient of variance ( cv ) for all peaks was calculated to evaluate intra- and inter - reproducibility . data analysis was performed with clinpro tools software and flex analysis software ( version 3.0 ; bruker ) . all the spectra were normalized , and baseline subtraction and smoothing were performed with flex analysis software . peaks with signal - to - noise ratio ( s / n ) > 5 were picked out , and statistical analysis was performed . data that were normally distributed were analyzed with student s t tests , and non - normally distributed data were analyzed by the wilcoxon test . in order to construct the diagnostic models to distinguish benign and malignant liver tumors , all samples were randomly divided into 2 subsets : training set and validation set . a total of 95 serum samples ( 43 malignant liver tumor patients and 52 benign liver lesion patients ) were used to construct classification models as training samples . three different machine - learning algorithms were used as follows : genetic algorithm ( ga ) , supervised neural network ( snn ) , and quick classifier ( qc ) . to confirm the clinical applicability of the models constructed , accuracy , sensitivity , specificity , positive and negative predictive values ( ppv and npv ) , and youden s index were calculated to assess the performances of the models . samples purified by mb - wcx beads were desalted using ziptipc18 pipette ( millipore , bedford , massachusetts , usa ) tips according to the procedure described below . then , a tfa - acidified sample was applied to the ziptip , followed by two washes with 10 l of 0.1% ( vol / vol ) tfa solution . bound proteins were eluted using a 50% ( vol / vol ) acetonitrile solution containing 0.1% ( vol / vol ) tfa . analysis of the eluted peptides was performed using a qtof synapt g2-s system ( waters , pittsburgh , pennsylvania , usa ) equipped with an esi source operated in the positive ion mode . the source temperature was set at 100c with a flow rate of 200 nl / min . the capillary and cone voltages were set to 3000 and 40 v. ms data were collected for m / z values in the range of 100 da and 2000 da with a scan time of 0.5 s , and ms / ms data were collected between 50 da and 1600 da with a scan time of 0.2 s. the ms / ms data were searched on mascot ( http://www.matrixscience.com ) . peptide mass tolerance was 10 ppm , fragment ion mass tolerance was 0.01 , and the mass type of the parent peptide and peptide fragment was at monoisotopic . the cv of inter - reproducibility was 18.02% ( 4.46~32.16% ) , and that of intra - reproducibility was 14.97% ( 4.03~55.4% ) . after discrepancy analysis of serum peptidome fingerprints by using clinpro tools software , a total of 129 peaks were found and 27 of them were significantly different ( p<0.05 ) between the benign and malignant liver tumor groups . we also found that 16 peaks increased significantly in the malignant liver tumor group ; in contrast , the other 11 peaks were clearly reduced in the malignant liver tumor group . to demonstrate the differences visually , the simulated two - dimensional ( 2d ) gel electrophoresis map and mass spectra map are shown in figure 1 . the 2d peak distribution map of first 2 peaks showed that the 2 groups of peaks were separated completely , and we could discriminate the benign and malignant liver tumors easily ( figure 2 ) . we used the clinpro tools software to analyze the data and establish three identified models ( ga , snn , and qc models ) . results of identification of malignant liver tumors by the 3 models were as follows : the ga model had a cross - validation of 81.67% and a recognition capability of 100% , the qc model had a cross - validation of 86.11% and a recognition capability of 80.84% , and the snn model and a cross - validation of 81.11% and a recognition capability of 89.38% . from the results shown in table 3 we could infer that by using these algorithm models , we could achieve above an 80% accuracy rate for detecting malignant liver tumors . therefore , blinded validation study with more clinical samples should be done with the follow - up research . a total of 50 serum samples , including 23 malignant liver tumors and 27 benign liver tumors , were successfully analyzed as validation samples by maldi - tof - ms models . among these 50 samples , 39 ( 78% ) were correctly identified by the ga models with 4 false negatives and 7 false positives , giving a sensitivity of 82.61% and a specificity of 74.07% . a total of 42 ( 84% ) samples were correctly identified by both the snn and qc models with 5 false negatives and 3 false positives , giving a sensitivity of 78.26% and a specificity of 88.89% . a total of 27 differential peaks were found by using the peptidomic profiling analysis , but only 10 were identified ( table 5 ) . among them , peaks at m / z 2860.34 , 2881.54 , and 3155.67 were positively identified by a mascot database search . the peptide 2860.34 da was identified with high probability as a fragment of fibrinogen alpha chain with the amino acid sequence madeagseadhegthstkrghaksrpv ; the mascot score was 67 . the peptide 2881.54 da was a fragment of fibrinogen beta chain with the amino acid sequence ghrpldkkreeapslrpapppisgggy ; the mascot score was 65 . the peptide 3155.67 da was a fragment of inter - alpha - trypsin inhibitor heavy chain h4 with the amino acid sequence nvhsgstffkyylqgakipkpeasfspr ; the mascot score was 72 . the cv of inter - reproducibility was 18.02% ( 4.46~32.16% ) , and that of intra - reproducibility was 14.97% ( 4.03~55.4% ) . after discrepancy analysis of serum peptidome fingerprints by using clinpro tools software , a total of 129 peaks were found and 27 of them were significantly different ( p<0.05 ) between the benign and malignant liver tumor groups . we also found that 16 peaks increased significantly in the malignant liver tumor group ; in contrast , the other 11 peaks were clearly reduced in the malignant liver tumor group . to demonstrate the differences visually , the simulated two - dimensional ( 2d ) gel electrophoresis map and mass spectra map are shown in figure 1 . the 2d peak distribution map of first 2 peaks showed that the 2 groups of peaks were separated completely , and we could discriminate the benign and malignant liver tumors easily ( figure 2 ) . we used the clinpro tools software to analyze the data and establish three identified models ( ga , snn , and qc models ) . results of identification of malignant liver tumors by the 3 models were as follows : the ga model had a cross - validation of 81.67% and a recognition capability of 100% , the qc model had a cross - validation of 86.11% and a recognition capability of 80.84% , and the snn model and a cross - validation of 81.11% and a recognition capability of 89.38% . from the results shown in table 3 we could infer that by using these algorithm models , we could achieve above an 80% accuracy rate for detecting malignant liver tumors . therefore , blinded validation study with more clinical samples should be done with the follow - up research . a total of 50 serum samples , including 23 malignant liver tumors and 27 benign liver tumors , were successfully analyzed as validation samples by maldi - tof - ms models . among these 50 samples , 39 ( 78% ) were correctly identified by the ga models with 4 false negatives and 7 false positives , giving a sensitivity of 82.61% and a specificity of 74.07% . a total of 42 ( 84% ) samples were correctly identified by both the snn and qc models with 5 false negatives and 3 false positives , giving a sensitivity of 78.26% and a specificity of 88.89% . a total of 27 differential peaks were found by using the peptidomic profiling analysis , but only 10 were identified ( table 5 ) . among them , peaks at m / z 2860.34 , 2881.54 , and 3155.67 were positively identified by a mascot database search . the peptide 2860.34 da was identified with high probability as a fragment of fibrinogen alpha chain with the amino acid sequence madeagseadhegthstkrghaksrpv ; the mascot score was 67 . the peptide 2881.54 da was a fragment of fibrinogen beta chain with the amino acid sequence ghrpldkkreeapslrpapppisgggy ; the mascot score was 65 . the peptide 3155.67 da was a fragment of inter - alpha - trypsin inhibitor heavy chain h4 with the amino acid sequence nvhsgstffkyylqgakipkpeasfspr ; the mascot score was 72 . malignant liver tumors always present as focal lesions in liver , but not all focal lesions are malignant . most benign tumors also present similar clinical and imaging features , and they do not require aggressive treatment . therefore , it is very important for making a treatment plan to differentiate benign from malignant tumors . despite widespread current availability of advanced imaging techniques , in addition , advanced imaging techniques require complex procedures or high costs that many patients ca nt afford . liver biopsy was considered the reference method for the diagnosis of malignant liver tumors , but it has many limitations such as invasiveness , sampling error , and inter - observer variability . however , compared with imaging and histological methods , serum tests are more convenient and rapid with a relatively low cost , and they also can be used for dynamic monitoring . maldi - tof - ms is a powerful tool for the detection and identification of proteins , peptides , polysaccharides , nucleic acids , and other biological molecules , and it has been used in clinical diagnosis and medical research successfully . in the past few years , maldi - tof - ms has been used for the expression analysis of low - molecular - weight serum proteins and peptides . as we know , human serum contains many different peptides that are thought to be fragments of large proteins . some of these peptides may have the potential to be biomarkers for prognosis or diagnosis of diseases , because their presence / absence or relative abundances are correlated with health status of patients . mb - wcx were developed for enrichment and purification of low - mass peptides and proteins ( 120 kda ) directly from the biological samples prior to maldi - tof - ms analysis . many studies have shown that it is an effective strategy to increase the sensitivity and reproducibility of the mass spectra [ 1618 ] . there are many published reports that focused on the mass spectrometry technique in evaluation of liver diseases [ 1922 ] . established a neural network model to diagnose whether hcc patients had accompanying bone metastasis , and the sensitivity and specificity of this model were 85.29% and 85.71% , respectively . sandanayake et al . developed a predictive model to detect biliary tract cancer ( btc ) , and it had a sensitivity of 79.5% and a specificity of 83.9% in discriminating btc from benign biliary disease . scarlett et al . developed a classification model to distinguish cholangiocarcinoma ( cc ) serum samples from benign serum samples , and it had 65.0% sensitivity and 70.0% specificity . however , these studies were for diagnosis of a particular cancer and not for distinguishing malignant from benign liver tumors . our study indicated that maldi - tof - ms can be helpful in differentiation of malignant liver tumors from benign liver tumors . although the maldi - tof - ms method has greatly improved the possibility of finding new potential biomarkers for diseases , interference of protein produced due to a stress reaction or chronic disease is a well - known problem in biomarker research . for example , some patients with terminal malignant tumors always have accompanying severe complications or have undergone invasive treatment ; thus , their serum components have changed greatly . in order to rule out the influence of the patient s physical condition , some patients who had invasive treatment , other chronic disease , and severe complications were excluded in our study . in addition , we handled all the serum samples using mb - wcx beads and detected them on the same day to avoid day - to - day variation . in this study , we applied maldi - tof - ms technique combined with mb - wcx kits to analyze 95 serum samples from 43 patients with malignant liver tumors and 52 patients with benign liver lesions . a total of 27 differentially expressed peaks ( p<0.05 ) were found : 16 of them were over - expressed in patients with malignant liver tumors , whereas the others were under - expressed . three peptide peaks of m / z 2860.34 , 2881.54 , and 3155.67 were identified with high probability as a fragment of fibrinogen alpha chain , fibrinogen beta chain , and inter - alpha - trypsin inhibitor heavy chain h4 ( itih4 ) , respectively . fibrinogen , which participates in the process of blood clotting , is synthesized in the liver . many previous studies had shown that fragments of fibrinogen alpha or beta chains were associated with liver tumors . itih4 , an acute - phase glycoprotein produced primarily in the liver , is involved in liver development and stabilization of the extracellular matrix ( ecm ) , and its expression is altered in liver disease . confirmed that itih4 could be used as a diagnostic and prognostic indicator in patients with hepatitis b virus - associated hepatocellular carcinoma . in the present study , we have demonstrated that fragments of fibrinogen and itih4 have been obviously altered in the serum of malignant liver tumors patients , and they have the potential to be developed as ideal biomarkers for the identification of malignant liver tumors , which will be studied in our further work . the ga model obtained an 81.67% cross - validation rate and a 100% recognition rate . the qc model obtained an 86.11% cross - validation rate and a 80.84% recognition rate . the snn model obtained an 81.11% cross - validation rate and a 89.38% recognition rate . they can be used for distinguishing between benign and malignant liver tumors and also can meet clinical - application needs after further confirmation . the results of blinded validation are robust , since the accuracy rate of these models is around 80% . the ga model obtained 82.61% sensitivity and 74.07% specificity , which was better sensitivity and lower specificity compared with the qc and snn models . we believe that a combination of three models might be a more reliable approach for the screening of liver tumors . the numbers of samples tested in our study limited the reliability of the models , and the results of the blinded validation study are not yet reliable enough for routine clinical diagnosis . despite this , we found some specific peaks to distinguish benign and malignant liver tumors , and then we established three classification models . maldi - tof - ms can be helpful in differentiation of benign and malignant liver tumors . maldi - tof - ms combined with mb - wcx should be used as an additive method with conventional imaging methods . from the results of identification using the lc - ms / ms method , we found that fibrinogen and itih4 might be used as biomarkers for the diagnosis of malignant liver tumors .

economic evaluations are increasingly used to inform decisions regarding the allocation of scarce health care resources . they generally take the form of cost - utility analysis , in which incremental costs per gained qaly ( quality - adjusted life year ) are evaluated against some threshold to ascertain the intervention s value for money . in this procedure , qaly gains are ( implicitly ) valued equally irrespective of , for instance , the beneficiary or disease . such practice has been an issue of debate , however , because accumulating evidence shows that the public may prefer some qaly gains over others ( e.g. , young over old ) [ 13 ] , often relating to a more equitable distribution of health and health care . such notions of equity are normally not captured in economic evaluations where qalys are typically weighted equally and often remain implicit in subsequent policy decisions , if included at all . almost three decades ago , researchers recognized that equity concerns could be incorporated into allocation decisions in the health care sector by weighting qaly gains according to some agreed upon equity principle , such as giving more weight to gains in the severely ill . it seems that little has changed since schwappach s assertion that equity weighting , if considered at all , was at a developmental stage . however , given the growing pressure on health care budgets partly due to ( expensive ) new technologies and increased demand stemming from demographic changes we can expect that the process of allocating scarce health care resources will require increasing attention . since decision makers increasingly use economic evaluations to inform their decisions , the discrepancies between recommendations based on economic evaluation outcomes and actual or publicly desired decisions may become more evident , as experienced in the uk , netherlands , and australia [ 69 ] . one explanation for such discrepancies is the presence of equity concerns that are insufficiently reflected in current economic evaluations . therefore , we need a more explicit and systematic incorporation of equity weights to obtain sustainable decisions . recent developments in the uk with respect to the funding of costly life - prolonging drugs illustrate the attention to equity concerns [ 11 , 12 ] . in general , the national institute for health and clinical excellence ( nice ) appears to adhere to the principle that a qaly is a qaly, implying that all qaly gains should receive equal weight , but under that rule , it appears to have been difficult to come to sustainable decisions in the context of costly life - prolonging drugs . the appraisal committees thus now explicitly consider the magnitude of the additional weight that would need to be assigned to the qaly benefits for the cost - effectiveness of the technology to fall within the current threshold range . this guideline , specific in terms of applicable interventions and open in terms of what weights might be considered appropriate , might be a first step in defining more general rules regarding equity weights . in response to the equity problem , the dutch have developed a decision - making framework that uses equity weights in defining the basic benefits package . its primary criteria ( proposed in 1991 ) are necessity , effectiveness , and efficiency . the first refers to a notion of equity based on the need for medical intervention ; the latter two refer to the merits of the intervention itself . equity weights thus are intended to be an integral part of rather than exceptions to the rule . in this context , broad consensus appears to exist for using a particular operationalization of necessity as basis for equity weighting , i.e. , the concept of proportional shortfall [ 1619 ] . although choosing a specific equity principle may lead to a more systematic and transparent way of using equity weights to set priorities in the dutch health care system , it also requires justification . the dutch and uk experiences suggest that including equity concerns in practice is not straightforward . arriving at a systematic consideration raises two important questions : ( i ) which equity principle(s ) are used to base qaly weights on ? ( ii ) how can we derive practically applicable qaly weights that are in line with the chosen principle(s ) ? these difficult and inherently normative questions complicate the formalization of equity concerns . to our knowledge , the netherlands is the first country where decision makers and health economists have been involved in a joint effort to formulate an equity principle and develop a model for putting it into practice . although the concept of proportional shortfall is not yet firmly implemented , that does appear to be the ultimate goal . evaluating the proportional shortfall concept and the support for it in more detail is therefore directly relevant to the dutch situation as well as other countries currently struggling with the formalization of equity concerns for priority setting in health care . in this article , we will evaluate the proportional shortfall concept as used in the dutch health care context , with a focus on the above - mentioned research questions on qaly weighting within the context of a chosen principle . the common decision rule for economic evaluations is shown in eq . 1 : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{{{\text{qaly}}_{i } } } * \ , \updelta { \text{qaly}}_{i } - \ , \updelta \cos { \text{ts } } > \ , 0 , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{{{\text{qaly}}_{i } } } $ $ \end{document } denotes the monetary value society attaches to a qaly of type i and the subscript i is used to distinguish qaly gains according to some equity principle . qalyi represents the number of type i qalys gained ; costs are the associated costs . accordingly , the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{{{\text{qaly}}_{i } } } $ $ \end{document } * qalyi reflects the benefits related to the intervention . put simply , eq . 1 indicates that incremental benefits of the intervention need to outweigh its incremental costs to be eligible for funding . in common economic evaluations , however , the monetary value component in the benefits is not included in the equation . rather the focus is only on qalyi ( commonly without any distinction between qalys , following a qaly is a qaly no matter who gets it ) and costs . this means that common economic evaluations in the field of health care do not directly address \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{{{\text{qaly}}_{i } } } $ $ \end{document}. as such , these analyses are basically partial economic evaluations . 1:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{\updelta \cos { \text{ts}}}}{{\updelta { \text{qaly}}_{i } } } < v_{{{\text{qaly}}_{i } } } , $ $ \end{document}where the costs per qaly gained of type i have to be lower than the societal value attached to that particular type of qaly in order to be eligible for funding . only then a common cost - effectiveness or cost - utility analysis becomes a full economic evaluation as noted in eq . 1 . ( it should be noted that irrespective of how qalys , costs , and potentially the value of qalys are derived , the equivalence between a common cost - benefit analysis and the decision rule based on a cost - utility analysis may still be questioned [ 21 , 22 ] . also note , that a fuller discussion on decision rules , under different assumptions regarding decision contexts ( e.g. fixed budgets ) and goals ( e.g. focusing on health or the consumption value of health ) is provided by claxton et al . . commonly , one threshold is set for all qalys , regardless of the context in which they are gained . however , in line with eq . 2 , different cost - effectiveness ratios may be acceptable for different types of qalys gained if the value of a qaly is allowed to vary , for instance , on the basis of the disease or beneficiary characteristics . if so , rather than having one threshold value for all qalys , we have a range whose endpoints are defined by the lowest and highest possible values attached to a gained qaly in a particular context . it should be noted that such a practice is equal to keeping the threshold value constant but attaching equity weights to the qaly gains on the left - hand side of eq . this implicitly ensures the use of an appropriate threshold value since the equity weights may simply be regarded as the relative values of different qalys . in the dutch context , the former approach is taken , i.e. , different threshold values are used when the burden of illness is high ( e.g. , acute life - threatening diseases ) or low ( e.g. , toenail fungus ) . the latter approach appears to have been adopted for life - prolonging drugs in the uk , i.e. , keeping the threshold fixed but weighting qaly gains . to illustrate that one can either vary the threshold values or attach equity weights to qalys , we use a hypothetical example in which the qaly value is allowed to vary between young and old . for children , the value is highest , say 1qaly = 100,000 . for people aged 90 and over , the value is lowest , say 1qaly = 5,000 . let the reference value of a qaly be that of a 40-year - old , or 40,000 . one way to judge a ce ratio of gains in children is to compare the ce ratio to the threshold line running from 5,000 to 100,000 , where for this intervention the high endpoint is relevant . equity weights can be used to adjust the ce ratio itself , which can be judged against the common threshold of 40,000 . in this example , the equity weight of qalys for children relative to the 40-year - old reference group is 2.5 ( vchildren / v40 year olds = 100,000/40,000 ) . thus , in order to use equity weights appropriately , we ( implicitly ) compare the relative values of different qaly gains to some reference group with the standard threshold . subsequently , such equity weights can be multiplied with the qaly gains in the ce ratio ( i.e. , c/2.5 * e ) , which indirectly corrects the threshold value used . the above demonstrates how using a flexible threshold is basically equal to attaching appropriate equity weights to different qalys and comparing them to a single , relevant threshold . in that sense , the dutch and uk approaches are similar , albeit the uk approach currently seems to be used in only a few specific circumstances . a crucial question , however , is which equity principle should guide the derivation or evaluation of equity weights ( research question ( i ) ) . to this end , we next discuss the well - known principles of fair innings and prospective health , followed by discussion of the principle selected in the dutch context , proportional shortfall . which ( combination of ) equity concept(s ) is most suitable for equity weighting ? several have been proposed even more equity concepts are imaginable [ 2426 ] . a problem is that improved equality with one particular definition of equity may be ( necessarily ) accompanied by greater inequalities in the context of a different definition . selecting one ( or more ) principle(s ) to guide the derivation or evaluation of equity weights is thus important and not straightforward . fair innings , roughly speaking , strives for equity in lifetime health , while prospective health is more concerned with people s health expectations , regardless of experienced health . an aspect both principles share is that their basis is found within the health domain , i.e. , they both focus on health characteristics of beneficiaries , not on aspects like gender or income . the principles differ , however , in that one strives to equalize lifetime health and the other prospective health . the fair innings approach , advocated by williams , is based on the assumption that everyone is entitled to some anyone failing to achieve this has in some sense been disadvantaged in terms of lifetime health , while anyone getting more than this is living on borrowed time . this assumption implies that qaly gains in people who have had their fair innings should be valued lower than qaly gains in people who are expected to get less than their fair innings . thus , the equity weights depend on the expected lifetime qaly total , therefore also considering past health losses and age is a key element ( as proxy for lifetime health achievement ) , resulting in higher weights for qaly gains in relatively young persons and lower ones for those in relatively older persons . in contrast , the principle of prospective health bases equity weights on the expected qaly profile of a person in the case of no treatment . this aligns with an alternative definition of need , namely , expected ill health over the remaining years of life . prospective health considers the expected health ( including death ) in future years in the case of non - intervention and distributes qaly gains initially to those with the worst prognosis if left untreated . the approach appears to be related to the rule of rescue , which implies that rescuing identifiable individuals facing avoidable death should have priority over other types of care [ 29 , 30 ] . while prospective health incorporates non - identifiable individuals and non - life - threatening conditions , both prioritize people with poor health prospects . the different perspectives of fair innings and prospective health obviously result in different equity weights . as seen in table 1 , group a faces immediate death and group b has one remaining qaly . however , group b consists of younger persons who , consequently , have enjoyed fewer qalys than persons in group a. adhering to the equity principle of prospective health , group a would have priority because they face immediate death . the fair innings principle , on the other hand , would prioritize group b since it comprises younger people , or , put more precisely , it has a lower lifetime quality - adjusted life expectancy [ 28 , 31 , 32].table 1illustrating fair innings and prospective healthpatient groupqaly consumedqalys remaining ( prospective health)expected qaly total ( fair innings)a ( immediate death)60060b ( younger)40141 illustrating fair innings and prospective health a number of empirical studies have found at least some public support for both the principles of fair innings and prospective health [ 1 , 2 , 33 ] , although it may depend on the context of the decision . as mentioned , age is important to the fair innings principle . schwappach have found in their reviews that the majority of studies reveal support for giving less weight to health gains in older people , but schwappach argues that age preferences vary across countries , study designs , and context . additionally , both reviews note that age weighting may reflect underlying rationales other than the fair innings principle . for example , people may prefer health gains in young people because they expect them to last longer . separation of the different rationales places specific requirements on the design of studies , which are not always met . furthermore , shah s recent review indicates that the public prefers to prioritize individuals in poorer health rather than those in better health without treatment , even if it results in lower overall health gains [ 1 , 2 ] . shah , however , emphasized that the strength of the support should be estimated more precisely to gain a true reflection of it . there appears to be little evidence showing that either equity principle reflects the distributional preferences of society completely or one fully lacks support . in that regard , we should note that people may in fact adhere to both principles : someone may feel that ( holding other things constant , i.e. , health prospects ) young people should receive priority over older people and , at the same time , feel that ( holding other things constant , i.e. , age ) people with worse health prospects should receive priority over those with better health prospects . in the dutch context , therefore , the equity principle proposed was a measure of severity labelled proportional shortfall , which contained elements of both fair innings and prospective health . we should note that the dutch decision , while deliberatively taken and explicitly justified , should be seen as a first , pragmatic attempt to find an equity principle that is practically applicable and supported by the public . the concept of proportional shortfall adopts the normative viewpoint that priority should be given to those patients who lose the greatest proportion of their remaining health expectancy due to some illness if the illness remains untreated . in other words , measurements of inequalities in health should concentrate on the fraction of qalys lost due to illness , relative to remaining life expectancy . proportional shortfall ( ps ) can be measured on a scale from 0 ( no health loss ) to 1 ( complete loss of remaining health ) using the following formula:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{ps } } = \frac{{{\text{disease}}\;{\text{related}}\,{\text { qaly}}\,{\text { loss}}}}{{{\text{remaining}}\,{\text { qaly}}\,{\text { expectation}}\,{\text { in}}\,{\text { absence}}\,{\text { of } } \,{\text{the}}\,{\text { disease } } } } , $ $ \end{document}where the denominator reflects the remaining qaly expectation in normal health , which could , for example , be determined on the basis of the age and gender . the numerator presents the qaly loss , which is determined by deducting a patient s qaly expectancy given the disease without treatment from the remaining qaly expectancy in absence of the disease . the proportional shortfall is 1 for all patients who face a threat of immediate death , irrespective of their age . since they will lose 100% of their remaining life expectancy , they all receive equal weight . likewise , if a young patient with a normal qaly expectation of 40 loses 20 qalys , he or she will get the same equity weight as an older patient with a qaly expectation of 2 who stands to lose 1 qaly : both patients lose 50% of their remaining life expectancy . since proportional shortfall is a relative measure , both younger and older individuals can experience a low or high proportional shortfall . for another example , a 30-year - old losing 1 of 40 remaining qalys would receive low treatment priority ( 1/40 = 0.025 ) , while a 70-year - old losing 1 of 5 remaining qalys would receive higher priority ( 1/5 = 0.2 ) . whereas fair innings and prospective health equalize absolute health outcomes in terms of total and future health , proportional shortfall proposes to equalize relative attainments . by doing so , the concept combines elements of both fair innings and prospective health . in accordance with fair innings , proportional shortfall is concerned with disease - related qaly loss ; at the same time , in accordance with prospective health , it takes the remaining qaly expectation without treatment into account . therefore , the principle may be perceived as an intermediate position between fair innings and prospective health . a limitation to normative justification is that unlike , say , fair innings , the concept is not derived from a particular theory about distributive justice . instead , normative arguments for both prospective health and fair innings are assumed to be compelling ; thus , given that both principles result in different prioritizations , a reconciliation of or trade - off between the two is required . while balancing two principles that appear to have some normative and empirical support may intuitively make sense , whether the resulting combination has the same ( or even a better ) moral status than the individual principles themselves remains questionable . one convincing argument for this ( and thus proportional shortfall ) may be whether the latter reflects societal preferences better than either of its underlying principles . evidence is thus far inconclusive , as is the only head - to - head comparison of all three concepts in which stolk et al . performed an experiment in a sample of dutch health policy makers , researchers , and students to explore support for the individual principles . observed rank orders of ten conditions while both fair innings and proportional shortfall were highly correlated with the observed rank order , rank orderings based on prospective health showed only a moderate correlation coefficient with the respondents ranking . fair innings seemed to slightly outperform proportional shortfall , but neither offered a fully accurate description of societal preferences : there were always cases where the predicted rank ordering diverged from the observed . it was therefore concluded that more ( elaborate ) research was required before firm conclusions could be drawn about which concept best reflects societal preferences . despite the limited empirical and theoretical evidence to support proportional shortfall , the choice was partly pragmatic , subject to possible adjustment according to incoming evidence and ongoing experience . from the outset , it was obvious that the adoption of any equity principle would not be without problems . to avoid perfection becoming the enemy of the good , however , efforts were made to operationalize proportional shortfall for use in practice . we highlight this in the following section , while addressing the issue of how practically applicable qaly weights can be derived in line with the chosen principle and how they can subsequently be used in decision making on the allocation of scarce health care resources . as indicated , three criteria have been particularly important in the delineation of the basic benefits package in the dutch context : necessity , effectiveness , and efficiency . while proposed and well received about two decades ago , adopting them for practical use has proven difficult and controversial . effectiveness , which has always been a dominant criterion in the health care sector , was least controversial but not efficiency , which is nonetheless increasingly used and accepted . it has been operationalized primarily through cost - effectiveness and cost - utility analyses , and most widely applied in the context of new pharmaceuticals . necessity, while intuitively important , eluded definition and measurement and thus remained barely applied as a ( systematic ) selection criterion in practice until 2002 . attempts to exclude services based on the criterion of necessity commonly provoked political or societal debate . proportional shortfall was introduced to provide a more systematic and quantitative definition of necessity , which also solved its problem of dichotomy : interventions were deemed either necessary or unnecessary with no in - between . this proved virtually impossible as a proper definition of necessity , and a clear and universal cutoff point between unnecessary and necessary ( or , for that matter , cost - effective and cost - ineffective ) care was lacking . mostly , there would be ( groups of ) patients for whom it would be difficult to label the intervention as unnecessary or cost - ineffective . the introduction of proportional shortfall made it possible to quantify the necessity criterion and to integrate it with or relate it to results regarding ( cost-)effectiveness in the decision - making framework . basically , the threshold of cost per qaly ( i.e. , the value of the qaly ) was allowed to vary with the necessity of the intervention , creating a decision framework in line with eq . the idea was that society is willing to pay more for an intervention , given the underlying proportional shortfall , considered more necessary . put differently , a less favorable cost - effectiveness ratio is acceptable for an intervention in the context of a greater proportional shortfall , i.e. , when the treatment is deemed to be more necessary , and unacceptable when the associated proportional shortfall is low . 1.fig . 1cost - effectiveness threshold varying with proportional shortfall cost - effectiveness threshold varying with proportional shortfall while the framework is increasingly supported [ 17 , 19 ] , it is unclear how it has affected decisions and it is likely that it is currently used predominantly as a conceptual framework . one reason may be that it is relatively unclear how the threshold of costs per qaly gained varies with proportional shortfall . the equity weights placed on the different qaly gains ( the value of gained qalys relative to the underlying proportional shortfall ) remain uncertain , although existing evidence may inform policy makers to some extent ( see for example ) . in that sense , recently , a maximum threshold height of some 80,000 has been suggested [ 17 , 39 ] , although the choice lacks sound basis . furthermore , it has been suggested that social willingness to pay for qaly gains in the case of a very low burden of disease ( proportional shortfall less than 10% ) might not exist at all in the context of a collectively funded health insurance scheme ( which explains the dotted threshold line near the origin of the graph in fig . 1).1 the shape of the curve would ultimately be based on adequately derived dutch evidence regarding the relative valuations of qaly gains in different proportional shortfall contexts . a linear relationship has thus been proposed as a pragmatic starting point but , even under this assumption , we need to be able to adequately judge the proportional shortfall in different circumstances to use the framework in practice . supporting this may be the fact that the shortcomings of proportional shortfall have barely been discussed . as highlighted by the fair innings and prospective health principles , not all consequences of the proportional shortfall principle may be in line with common conceptions of an equitable distribution of health care . perhaps the most counterintuitive implication of the principle of proportional shortfall is that anyone facing imminent death should receive the maximum necessity score of 1 , since all remaining health will be lost . indeed , proportional shortfall assigns a necessity score of 1 when all remaining health is lost , regardless of the absolute number of life - years lost . the principle is indifferent to whether a 3-year - old is losing 80 years or an 80-year - old is losing 3 years ( when both expected to become 83 otherwise ) , but in practice , many people judge intervention to be more necessary in the former situation , making it conceivable that the result conflicts with society s equity principles . likewise , since women have a higher life expectancy than men , an absolute qaly loss at a certain age will have more weight for men than women ( for instance when comparing breast cancer and prostate cancer in certain age groups ) , and it is unclear whether this would be judged equitable [ 35 , 42 , 43 ] . moreover , it became apparent that the operationalization of proportional shortfall required numerous normative choices that have important impacts on final outcomes . coming to practically applicable proportional shortfall scores that are in line with public preferences is therefore not straightforward . for instance , the calculation of proportional shortfall in preventive treatments requires clear normative choices . considering that many who receive preventive treatment will never experience the negative health effects precluded by the intervention , which group is relevant ? while the treated group may be relevant in a cost - utility analysis of a preventive intervention , it may not be when calculating the proportional shortfall of the underlying disease . calculating proportional shortfall over the entire group would result in a very low average proportional shortfall since only a small percentage of the treated group would actually experience a health loss . this in turn results in low priority for ( primary ) preventive action , but the very aim of the intervention is to avoid health loss in those who would experience it without the preventive intervention . then , it seems reasonable to calculate the proportional shortfall in the subgroup only , resulting in a higher proportional shortfall and threshold value . consequently , normative choices are necessary in defining the relevant group in which to determine proportional shortfall , which does not necessarily coincide with the population involved in the economic evaluation . should the onset of preventive treatment be the starting point from which to calculate proportional shortfall ? or should it be the moment at which the negative health effects would have actually occurred ? obviously , the shorter timeframe will increase the proportional shortfall . consider , for example , that a preventive intervention reduces a risk factor that , left untreated , results in death 20 years hence , reducing lifespan by 10 years . until the moment of death , patients are without health loss . calculating proportional shortfall starting from treatment time means that the first 20 years are in health and only the final 10 years are lost , resulting in a proportional shortfall of 33% . calculating from the moment of illness , the proportional shortfall is 100% ( since then 10 of the remaining 10 life - years are lost ) . in the netherlands , it has been argued that since society is likely to feel quite different about acute death than about a predicted ( or certain ) death in 20 years , calculating proportional shortfall from the moment of treatment would be more appropriate . an average proportional shortfall of 0.04 can result from a stable yet mild condition that causes a loss of 0.04 per day as well as from a disease that is primarily latent ( no health loss for 350 days per year ) , but leaves the patient in agony during the episode ( a loss of 1.0 during the remainder of the year ) . in the current operationalization of proportional shortfall , such episodes are simply averaged over the full year ( as in normal qaly calculations ) . however , the appropriateness and justification of such a simple method of transforming health profiles into proportional shortfall scores can be disputed . can we really conclude on such a basis that a certain illness has a modest severity ? it appears that the variation over time may be important here too ; yet , how could or should this be included in the calculation of proportional shortfall ? the above illustrates that not just the choice of an equity principle is normative ; putting whatever it is into practice requires additional normative choices . whatever the chosen principle , it appears inevitable that counterintuitive prioritizations may result in certain circumstances . clearly , therefore , decision makers should be aware of additional and potentially conflicting equity considerations . explicit inclusion of equity weights in the decision - making framework for allocation decisions in the health care sector has become increasingly important . a pragmatic start has been made in the netherlands with the principle of proportional shortfall , which adopts the normative viewpoint that when people stand to lose relatively more of their remaining health , a higher cost per qaly threshold is appropriate . this article highlights that the approach is not without problems . both the normative basis and empirical support warrant further study . of particular concern for example , it seems hard to defend that avoiding a full loss of all remaining health would be equally important when the choice concerns either a very large or small absolute qaly loss , i.e. , young and old people , respectively . whether proportional shortfall adequately reflects societal preferences in such cases is uncertain and . we also highlighted that operationalizing proportional shortfall ( or of any equity principle for that matter ) involves normative choices that can have a profound effect on outcomes , like in the case of preventive interventions . it is crucial that these normative choices are as widely discussed as those embedded in cost - effectiveness analyses . the highlighted shortcomings of proportional shortfall clearly should not be misinterpreted as a plea to replace it with a different equity concept such as fair innings . indeed , whatever principle is chosen , similar shortcomings and normative choices will arise in transitioning from principle to practice . since different notions of equity all of which have some support in some instances will always conflict in certain circumstances , conflicts with societal preferences will be inevitable . at this moment , there is no conclusive evidence that another equity principle reflects the distributional preferences of society better than proportional shortfall . it seems more appropriate , given the relatively strong ( political ) support for the equity concept of proportional shortfall in the dutch context , to further test , develop , and refine the principle and its operationalization . for example , one might consider incorporating age weights in proportional shortfall in order to better reflect societal preferences.2 obviously , this requires more and detailed research on relevant societal preferences as well as public debate . the current situation , while perhaps far from perfect , may be seen as an important and perhaps essential step in the development of an appropriate set of equity weights in the netherlands . the associated quantification of necessity should improve consistency and transparency in the decision - making process . meanwhile , experience from the systematic use of proportional shortfall will potentially improve the principle and its practical use . besides proportional shortfall or health profiles of beneficiaries in general , many other factors may be relevant for decision making in relation to equity considerations . reviews by dolan et al . and schwappach have identified numerous factors besides health attainments or prospects that appear to influence the relative valuation of qalys , such as prior health consumption , culpability , age , having dependents , and socioeconomic status . more recent studies have added to this field [ 33 , 44 ] , but so far most involve small and unrepresentative samples , the studies are quite context specific , and findings are sometimes contradictory . it seems difficult at this stage to be conclusive regarding the relative weights given to these considerations in an empirical sense . while it may be interesting and helpful to analyze such additional equity concerns in relation to proportional shortfall , such empirical work should coincide with normative debates regarding whether such additional ( or alternative ) concerns should be included in the decision - making process . for instance , even if the public ( on average ) considers culpability important in fair allocation of health care resources , wanting to institutionalize such sentiments is questionable if only because dutch legislation prohibits it . currently , the qaly value is unclear , and how it varies with different proportional shortfall percentages and which equity weights should be placed on various qaly gains needs to be investigated further . to use the decision - making framework in practice , the ( relative ) values assigned to qaly gains for different levels of proportional shortfall have to be elicited , for instance , from the public . different methods have been used such as willingness to pay , person trade - off , or discrete choice analysis . which technique best captures the preferences of society may depend on the research question and whether relative weights of various equity concerns will be investigated simultaneously in combined trade - offs . in conclusion , although proportional shortfall provides important information for decision makers by acceptably quantifying the necessity of treatment in the dutch context , it clearly does not perfectly capture societal preferences . sufficient room should be left in the decision - making framework and process to judge whether the equity weights accurately reflect the public preferences in particular circumstances and to improve on principles and practice if so indicated . more generally , the dutch experience with equity considerations in relation to economic evaluations has given insight into the difficulties related to the choice for and operationalization of an equity concept for the allocation of scarce health care resources . although the dutch experiences are based on the concept of proportional shortfall , similar issues are likely to occur when opting for other equity concepts . therefore , the dutch experiences can provide helpful lessons for countries currently struggling with the important issue of formalization of equity concerns in priority setting in health care .

since , this gamma radiation is potentially harmful to the body , it is essential that minimum ( as low as reasonably achievable [ alara ] ) radiopharmaceutical dose be administered . another important requirement for a diagnostic radioisotope imaging is that the patient remains static during data acquisition . however , this requirement demands injection of relatively larger doses of radiopharmaceuticals , so that good counts are acquired in short acquisition time . it has been observed that after a certain threshold of acquired counts , even if we increase the counts in the image , the diagnostic information remains same ; however , the image becomes visually more appealing . as most of the times , radioisotope imaging reports are based on visual analysis ; high count image raises observer confidence , while interpreting the scan and is thus preferred . unfortunately , the extra counts acquired after a certain threshold to obtain more visually appealing images is usually acquired at the cost of additional radiation burden to the patients . many methods have been previously explored to reduce the administered radiopharmaceutical dose without losing the diagnostic information . these methods ranged from development of newer radiopharmaceuticals , development of image enhancement techniques , newer image reconstruction method , hardware development such as collimators for specific studies , sensitive electronic , time of flight acquisition , etc . there are reports available in the literature that noise can play a constructive role in detecting weak periodic or aperiodic signals by a mechanism known as stochastic resonance . stochastic resonance is a statistical phenomenon where when noise is added to the original signal , it increases the probability of detection of the signal amplitude where the resonance occurs . a study previously conducted at our center explored the role of stochastic resonance in image enhancement . the authors generated noise based on the image itself and explored many methods to generate this noise such as the bit - slice plane , white random noise ( not based on the information in the image ) and using poisson random variable . with all the methods , they showed the possibility of enhancement of the image quality and observed phenomenon of stochastic resonance . recently , gonzalez et al . described a noise model to corrupt the image with poisson noise generated on the basis on the image pixel values . this model of noise generation and its addition to the image for image enhancement have not been explored . if this method is effective , then it can serve as a basis for image processing and thereby indirectly reduce the dose of radiopharmaceutical administered ( i.e. radiation burden ) . furthermore , the low information density images can be processed by this technique and will make available feature in the image more obvious to the nuclear medicine physician . therefore , in this study , we have investigated the effect of addition of image noise to the positron emission tomography - computed tomography ( pet - ct ) image itself as an image enhancement technique , once the image has achieved the minimum counts threshold . clinical pet - ct images of patients acquired on a dedicated pet - ct scanner ( biograph 2 , siemens , erlangen , germany ) were included for evaluation . to simulate the routine clinical scenario , low , medium , and high count density images were included in this study . in the algorithm to calculate structural similarity index ( ssim ) and feature similarity index ( fsim ) , entropy as an image quality quantitative index , we converted rgb data into gray scale that is from 0 to 255 . at a scale of 0 - 255 , we designated the low count density image as one in which mean pixel counts was < 50 , medium count density images in which mean pixel counts was from 51 to 150 , and high count density image in which mean pixel count image was more than 150 . altogether , images of 15 patients ( f-18 florodeoxyglucose pet - ct [ n = 13 ] and f-18 fluoride pet - ct [ n = 2 ] ) were analyzed . the processing algorithm was implemented using matrix laboratory ( matlab ) r2013b ( the mathworks , inc . , we generated six different poisson noise matrix ( equal in image matrix sizes ) having amplitude reduced by the factor 1 , 2 , 4 , 8 , 16 , and 32 from the original given image . for each image , six noise matrices were added to the image matrix to produce six different resultant output image . for eight sample input images , 48 ( 8 6 = 48 ) output images were generated . these images were assessed for image quality quantitatively . the matlab function imnoise ( f , poisson ) was used to generate the poisson noise . it generates poisson noise from the data instead of adding artificial noise to the data . input pixel values were interpreted as mean of poisson distributions and then the corresponding output pixel were generated from a poisson distribution with mean as input pixel values . for accepting or rejecting the generated random number , first , where pixels intensities are < 50 units , monte - carlo rejection method have been used . second , for pixel intensity 50 , the poisson probability distribution function becomes very similar to a gaussian probability distribution function of mean and variance equals to the local pixel intensities . three well - known image quality assessment matrices namely ssim , fsim and entropy were used . ssim is based on the assumption that human visual perception is highly adapted for extracting structural information from a scene and assesses the image quality based on the degradation of structural information . fsim is based on the fact that human visual system understands an image mainly according to its low level features . phase congruency ( a measure of the significance of a local structure ) is used as the primary feature and image gradient magnitude is the secondary feature in fsim . respectively and they have also developed the matlab function to calculate these image quality metric and demonstrated the usefulness of these metric . the respective matlab functions and associated published articles are freely available for download and usage . entropy provides the measure of the information content , a measure of randomness in the image . more the value of entropy more is the randomness , which means more the uncertainty involved with the data of image and more information available in the image . if no uncertainty is associated with the event ; no information would be transferred by communicating that the event has occurred . another set of 15 pet - ct images were processed using the six different generated poisson noise matrix ( equal in image matrix sizes ) having amplitude reduced by the factor 1 , 2 , 4 , 8 , 16 , and 32 from the original given image and given to nuclear medicine physician for qualitative evaluation . first , all images including the original image were randomly arranged and scored based on image quality . the score varied between 1 and 10 , with 10 being the best image quality and one being the worst . second , the reviewers were shown the processed image side by side the original image . the reviewers commented whether the processed images were superior ( score 3 ) or same ( score 2 ) or inferior ( score 1 ) to original unprocessed image . clinical pet - ct images of patients acquired on a dedicated pet - ct scanner ( biograph 2 , siemens , erlangen , germany ) were included for evaluation . to simulate the routine clinical scenario , low , medium , and high count density images were included in this study . in the algorithm to calculate structural similarity index ( ssim ) and feature similarity index ( fsim ) , entropy as an image quality quantitative index , we converted rgb data into gray scale that is from 0 to 255 . at a scale of 0 - 255 , we designated the low count density image as one in which mean pixel counts was < 50 , medium count density images in which mean pixel counts was from 51 to 150 , and high count density image in which mean pixel count image was more than 150 . altogether , images of 15 patients ( f-18 florodeoxyglucose pet - ct [ n = 13 ] and f-18 fluoride pet - ct [ n = 2 ] ) were analyzed . the processing algorithm was implemented using matrix laboratory ( matlab ) r2013b ( the mathworks , inc . , we generated six different poisson noise matrix ( equal in image matrix sizes ) having amplitude reduced by the factor 1 , 2 , 4 , 8 , 16 , and 32 from the original given image . for each image , six noise matrices were added to the image matrix to produce six different resultant output image . for eight sample input images , 48 ( the matlab function imnoise ( f , poisson ) was used to generate the poisson noise . it generates poisson noise from the data instead of adding artificial noise to the data . input pixel values were interpreted as mean of poisson distributions and then the corresponding output pixel were generated from a poisson distribution with mean as input pixel values . for accepting or rejecting the generated random number , first , where pixels intensities are < 50 units , monte - carlo rejection method have been used . second , for pixel intensity 50 , the poisson probability distribution function becomes very similar to a gaussian probability distribution function of mean and variance equals to the local pixel intensities . three well - known image quality assessment matrices namely ssim , fsim and entropy were used . ssim is based on the assumption that human visual perception is highly adapted for extracting structural information from a scene and assesses the image quality based on the degradation of structural information . fsim is based on the fact that human visual system understands an image mainly according to its low level features . phase congruency ( a measure of the significance of a local structure ) is used as the primary feature and image gradient magnitude is the secondary feature in fsim . ssim and fsim index were developed by wang et al . and zhang et al . respectively and they have also developed the matlab function to calculate these image quality metric and demonstrated the usefulness of these metric . the respective matlab functions and associated published articles are freely available for download and usage . entropy provides the measure of the information content , a measure of randomness in the image . more the value of entropy more is the randomness , which means more the uncertainty involved with the data of image and more information available in the image . if no uncertainty is associated with the event ; no information would be transferred by communicating that the event has occurred . another set of 15 pet - ct images were processed using the six different generated poisson noise matrix ( equal in image matrix sizes ) having amplitude reduced by the factor 1 , 2 , 4 , 8 , 16 , and 32 from the original given image and given to nuclear medicine physician for qualitative evaluation . first , all images including the original image were randomly arranged and scored based on image quality . the score varied between 1 and 10 , with 10 being the best image quality and one being the worst . second , the reviewers were shown the processed image side by side the original image . the reviewers commented whether the processed images were superior ( score 3 ) or same ( score 2 ) or inferior ( score 1 ) to original unprocessed image . the noisy image created using imnoise function was taken as the highest amplitude of the noise . the various grading of amplitude of noise were created by dividing the each pixel value by 1 , 2 , 4 , 8 , 16 , and 32 to have six different amplitudes of noisy images . these were added to the image and the corresponding resultant images were subjected for analysis and the value of entropy , ssim and fsim were recorded . it clearly demonstrates that ssim index values increases with the increasing value of noise amplitude and attains a peak and then it starts decreasing . ssim index attains maximum value at noise amplitude of 4 . at noise amplitude 4 , the ssim index value is 0.88 ; meaning that structural similarity after processing the image by this method is 88% with the input image . plot showing change in structural similarity index ( ssim ) with noise for the processed images . initially , the ssim index increases with noise then it declines the relationship among the fsim index and noise amplitude is shown in figure 2 . it is clear from the figure that , fsim initially increases to the maximum value up to the noise amplitude of 4 and then decreases rapidly with further increase in the value of noise amplitude . still at the value of noise amplitude 4 , the value of fsim is more than 0.95 , that is , after the addition of noise amplitude 4 , the resultant image is 95% similar to the input image as far as this feature is concerned . plot showing change in feature similarity index ( fsim ) with noise for the processed images . initially , the fsim index increases with noise then it declines the relationship among the entropy and noise amplitude is shown in figure 3 . for all the eight images , the entropy of the processed image was more than that of the entropy of the input image even at the lowest value of noise amplitude . as the noise amplitude increases the value of the entropy increased for the entire image up to the noise amplitude of 4 . initially , the entropy increases with noise then it declines in our study , the addition of noise improved the image quality on quantitative analysis . we found that the amplitude of the noise to be added has a great role to play . the optimum amount of noise , which will definitely improve the image , depends on the value of the count density available in the image . higher count density images improved with the addition of the low amplitude noise and lower count density images improved with high amplitude noise . the eight sample images that have maximum value of difference in entropy before and after processing , ssim and fsim are shown in table 1 . from table 1 , it is clear that the entropy of the resultant images was more than the original in seven out of eight images and ssim and fsim value was above 0.84 in all eight images . this means that the noise matrix having amplitude value 4 is optimum because it improves the information content and simultaneously retains the structure and feature of the original image . these images are not only visually more appealing , but also do not deteriorate the original information of the image . it increases the information density content of all images without sacrificing the original structure in the input image . the amplitude of the noise was given symbolic representation as 0 , 1 , 2 , 3 , 4 , and 5 , respectively , 0 being the lowest amplitude and 5 the maximum the scores of the original and processed pet - ct images are shown in table 2 . an important observation was that there was no deterioration in the image quality after addition of noise . the highest scores were seen for processed image 1 in figure 4 and processed image 2 in figure 5 , even better than original images . all images had a score of 2 or more , thereby implying that the quality of images was either superior or same as that of the original image . the highest mean score was 2.6 ( median score 3 ) for the processed image 1 . score of the original and processed images at clinical evaluation an original low count positron emission tomography - computed tomography image along with six processed images with their mean score given by nuclear medicine physician , amplitude of noise added , structural similarity index , feature similarity index and entropy . based on clinical judgment processed 1 image scored maximum and was superior than original an original high count positron emission tomography - computed tomography image along with six processed images with their mean score given by nuclear medicine physician , amplitude of noise added , structural similarity index , feature similarity index and entropy . based on clinical judgment processed 2 image scored maximum and was superior than original comparative scores of different processed images relative to original image the noisy image created using imnoise function was taken as the highest amplitude of the noise . the various grading of amplitude of noise were created by dividing the each pixel value by 1 , 2 , 4 , 8 , 16 , and 32 to have six different amplitudes of noisy images . these were added to the image and the corresponding resultant images were subjected for analysis and the value of entropy , ssim and fsim were recorded . it clearly demonstrates that ssim index values increases with the increasing value of noise amplitude and attains a peak and then it starts decreasing . ssim index attains maximum value at noise amplitude of 4 . at noise amplitude 4 , the ssim index value is 0.88 ; meaning that structural similarity after processing the image by this method is 88% with the input image . plot showing change in structural similarity index ( ssim ) with noise for the processed images . initially , the ssim index increases with noise then it declines the relationship among the fsim index and noise amplitude is shown in figure 2 . it is clear from the figure that , fsim initially increases to the maximum value up to the noise amplitude of 4 and then decreases rapidly with further increase in the value of noise amplitude . still at the value of noise amplitude 4 , the value of fsim is more than 0.95 , that is , after the addition of noise amplitude 4 , the resultant image is 95% similar to the input image as far as this feature is concerned . plot showing change in feature similarity index ( fsim ) with noise for the processed images . initially , the fsim index increases with noise then it declines the relationship among the entropy and noise amplitude is shown in figure 3 . for all the eight images , the entropy of the processed image was more than that of the entropy of the input image even at the lowest value of noise amplitude . as the noise amplitude increases the value of the entropy increased for the entire image up to the noise amplitude of 4 . initially , the entropy increases with noise then it declines in our study , the addition of noise improved the image quality on quantitative analysis . we found that the amplitude of the noise to be added has a great role to play . the optimum amount of noise , which will definitely improve the image , depends on the value of the count density available in the image . higher count density images improved with the addition of the low amplitude noise and lower count density images improved with high amplitude noise . the eight sample images that have maximum value of difference in entropy before and after processing , ssim and fsim are shown in table 1 . from table 1 , it is clear that the entropy of the resultant images was more than the original in seven out of eight images and ssim and fsim value was above 0.84 in all eight images . this means that the noise matrix having amplitude value 4 is optimum because it improves the information content and simultaneously retains the structure and feature of the original image . these images are not only visually more appealing , but also do not deteriorate the original information of the image . it increases the information density content of all images without sacrificing the original structure in the input image . the amplitude of the noise was given symbolic representation as 0 , 1 , 2 , 3 , 4 , and 5 , respectively , 0 being the lowest amplitude and 5 the maximum the scores of the original and processed pet - ct images are shown in table 2 . an important observation was that there was no deterioration in the image quality after addition of noise . the highest scores were seen for processed image 1 in figure 4 and processed image 2 in figure 5 , even better than original images . all images had a score of 2 or more , thereby implying that the quality of images was either superior or same as that of the original image . the highest mean score was 2.6 ( median score 3 ) for the processed image 1 . score of the original and processed images at clinical evaluation an original low count positron emission tomography - computed tomography image along with six processed images with their mean score given by nuclear medicine physician , amplitude of noise added , structural similarity index , feature similarity index and entropy . based on clinical judgment processed 1 image scored maximum and was superior than original an original high count positron emission tomography - computed tomography image along with six processed images with their mean score given by nuclear medicine physician , amplitude of noise added , structural similarity index , feature similarity index and entropy . based on clinical judgment processed 2 image scored maximum and was superior than original comparative scores of different processed images relative to original image given the possible long - term harmful effects of radiation , the minimum radiopharmaceutical dose ( alara ) should be administered to each patient . diagnostic information does not improve after the acquisition of a certain threshold of counts ; however , image becomes visually more appealing as counts in the image increases , and therefore is more preferred . this study was conducted to develop a postprocessing method to improve the visual perception of the image having required threshold counts . this will help in reduction of radiopharmaceutical dose as well as radiation dose to the patient . this will also reduce the public exposure because patient becomes unsealed source of radiation after administration of radiopharmaceuticals . the image information is conveyed by a finite number of photons , these photons follows poisson distribution in time and space and results in image graininess . hence , it is important to guard against false alarms ( spurious visual pattern that may arise from the random character of the photon distribution and not from the original content of radionuclide itself ) . the only way to guard against the false alarms is to increase the per pixel count in the images . in this study , we have increased the count per pixel in the pet - ct image by mathematical processing ( adding poisson noise corrupted image to the image itself ) . since the two data sets happen to come from the same distribution function , the numerical value at any sample point would resonant ( influence the intensity value of that pixel ) . it is obvious that this technique is different from multiplying each pixel value by a constant since there would be no such single constant common to all the sample points . the value by which each pixel is changed is derived from the inherent attribute of the underlying population . this technique was called stochastic resonance since it was taken from both stochastic process ( poisson process ) and numerical resonance ( under algebraic manipulation ) . the present technique is based on stochastic resonance as we are utilizing the principle of stochastic resonance in which we are adding generated noise based on image data to the image itself . in this study as the amplitude of noise increases , the image improves and after a certain threshold , the image starts deteriorating . in this respect , our result is similar to that reported by other investigators working on the principle of stochastic resonance . they also have observed that the amplitude of the noise is very important and they have adjusted this parameter in such a way that it improves signal to noise ratio . in the process of stochastic resonance the signal gets enhanced when noise was added to it , because resonance occurs at the matching frequency of noise and signal . input pixel value are taken as mean of poisson distribution for the noise to be generated . the generated noise pixel value will be comparable to the input pixel value and after addition to the input pixel value ; some of the pixel value , which was already above the threshold may attain maximum intensity . and since the gray scale value ranges from 0 to 255 , so , with the addition of high amplitude noise , the maximum number of pixel may attain counts equal to 255 , resulting in low information and the number of gray level in the input image will be reduced ( low value of entropy ) . similarly , if the input image has good counts statistics then it will require the addition of low amplitude noise for better image quality . qinghua et al . has shown an image enhancement method using stochastic resonance in sonar image processing . their stochastic resonance model is based on overdamped motion of a browanian particle in a bistable potential in the presence of noise and periodic forcing . they have shown an image of a rectangle imbedded in the very high noise and recovered that rectangle by adding gaussian white noise . similar model have been used by other authors in the enhancement of computed tomography and magnetic resonance images . they had added gaussian noise with zero mean and standard deviation equal to the pixel value in the image for improving image quality . in this study , we have added poisson noise corrupted image to the image data . here , it adds poisson random variable ( noise pixel value ) proportional to the image pixel value to every pixel value , and increases maxima and minima point both proportional to the pixel value in the image data , so the each pixel values are scaled by random number as if the patient had activity and the time period of data acquisition would have more . this study was conducted on patient pet - ct images and is unique in this regard . processing by this method improves the image quality of both low as well as high count images . although it may not improve the image quality for visual analysis , there is definitely no deterioration of image quality . we have not included phantom images in this study . in phantom study , it is possible to embed weak signal in noise and enhancement of that weak signal also needs to be verified with this technique . however , our objective was to make the information available in the image more obvious to the nuclear medicine physician reporting the pet - ct . detection of weak signal embedded in the noise have been shown by using stochastic resonance model as bistable potential well and adding white noise gaussian noise with mean zero . in this study , it has been investigated on the pet - ct images where generally we have good counts images , even though in this study , we have selected only those images where the reader confidence was low at the time of reporting the scan . our future plan is to apply the same technique on very low count density images such as i-131 whole body imaging or 131 i - metaiodobenzylguanidine imaging , etc . this method of stochastic resonance can be used to improve the visual perception of the pet - ct image .

the kidney is an organ for metabolism and elimination of ths and a target of some of the iodothyronines actions . thyroid dysfunction may effect on glomerular and tubular functions and electrolyte and water ( h2o ) homeostasis . it may cause a reduction in glomerular filtration , hyponatremia , and h2o excretion alteration . many studies have shown that patients on regular hemodialysis ( hd ) tolerate a chronic illness which was not involved the thyroid , but these patients indicated low serum thyroxin ( t4 ) and triiodothyronine ( t3 ) levels . some studies revealed decreased t3 and t4 levels while the thyroid - stimulating hormone ( tsh ) levels are not elevated . there may be an association between ths and the oxidative and antioxidative status in the body . it has been reported that thyroid dysfunction such as hypothyroidism reduces oxidant production which may not protect the organism against oxidant damage . different antioxidant enzymes may protect oxidative effects of free radicals on lipids , proteins , and dna . lipid peroxidation which express as malondialdehyde ( mda ) may change in different age and ethnic groups , gender and different kinds of diseases . hd is the most effective method in balancing the metabolic abnormalities related to renal oxidative stress that causes to morbidity in hd patients . chronic renal failure ( crf ) patients who are on hd therapy show a high percentage of goiter and thyroid dysfunction and cardiovascular ( cv ) disorders . free radicals have been revealed to change the activity of some membrane - bound tissue enzymes . end - stage renal disease can only be overcome by hemodialysis which can be considered as crucial therapeutic regiments for such kidney diseases and for those subjects which are a candidate for kidney transplantation . the production of reactive oxygen species ( ros ) which are the byproducts of hemodialysis therapy is the main obstacle and complications in hemodialysis application for the end stage renal failure . the ths abnormality may accompany with various disorders , including systemic acidosis , the hemodialysis duration , and endothelial injuries and inflammation . the aim of this study focused on serum mda levels which is an indicator of lipid peroxidation and erythrocyte sod and cat activities in hemodialysis patients and compared with age - matched control groups in gorgan . forty - five hemodialyzed patients with mean age 51.88 15.90 years who were referred to the hd unit at 5 azar education hospital of gorgan faculty of medicine , golestan university of medical sciences in gorgan , iran in 2015 . patients were compared with 45 age - matched control groups ( mean age 48.68 15.60 ) . this study was approved by the research deputy ethics committee of the golestan university of medical sciences . patients with diabetes mellitus , liver disease , endocrinal diseases , acute or chronic illness , thyroid disorders and any drug that affect th were excluded from the study . a 10 ml blood samples were provided after an overnight fast of 12 h. biochemical tests including serum creatinine and urea , ths ( tsh , t3 , t4 , free triiodothyronine [ ft3 ] and free thyroxin [ ft4 ] ) and erythrocyte antioxidant enzymes ( sod [ code number : 706002 , cayman chemical commercial kit , colorimetric method , usa ] and cat [ code number : 707002 , cayman chemical commercial kit , colorimetric method , usa ] ) were determined . the hormonal analysis was done by monobind kit ( code number : 1225 - 300 , lake forest ca : 92630 , usa ) and enzyme immunoassay ( elisa ) method . serum creatinine and urea levels were determined with commercial kits by spectrophotometer techniques ( model jenway 6105 uv / vis ) in the metabolic disorders research center ( gorgan faculty of medicine ) . weight was measured , while subjects were minimally clothed without shoes , using digital scales . height was measured in standing position using tape meter while the shoulder was in a normal position . body mass index ( bmi ) was defined as weight in kilograms divided by height in meters squared . overweight was defined as bmi 25.029.9 kg / m and obese as bmi 30 kg / m . , chicago , il , usa ) software was used to calculate the statistical analysis . the evaluation of data was done by using independent sample t - test and pearson correlation test . there was no difference in serum tsh , age , and bmi among the studied groups . there were well - significant differences in serum t3 , t4 , ft3 and ft4 , mda , sod , and cat compared with control groups . hd patients showed higher levels of mda than those control groups ( p < 0.01 ) , but the levels of t3 , ft3 and ft4 , sod and cat were low in hd patients ( p < 0.01 ) . spearman correlation analysis showed that the serum t3 , ft3 , and ft4 levels were significantly negative correlated with mda [ table 2 ] . clinical data of study subjects correlation analyses between oxidative stress biomarkers and thyroid hormones in hemodialysis subjects in the present study , we revealed that the thyroid function differed among the hd group compared to the healthy controls . serum t3 , ft3 , and ft4 levels were lower ; tsh was slightly higher , and t4 was unchanged among those with hd . serum lipid peroxidation and antioxidant enzyme activities were significantly high and low among the hd group compared to the healthy controls , respectively . some studies have shown a low serum level of t3 in patients with end - stage renal disease ( esrd ) . reported a reduced t3 level and decreased conversion of t4 to t3 in uremic patients . it is also revealed low serum t4 or ft4 levels in patients undergoing maintenance hd . the serum tsh level in patients with crf had been reported to be almost normal . our study showed that the serum tsh level in the hd patients slightly increased and serum ft3 and ft4 level significantly decreased which is in agreement with other studies . in our study , slightly increase of tsh level in hemodialyzed patients in comparison to control groups was not in accordance with others studies . the low serum ft4 and ft3 levels may be responsible for the low metabolism of the body . studies on a uremic rat model have shown that there may be a difference in the th metabolism or action in the peripheral system and in the central nervous system in the esrd patients . showed that crf patients revealed a significant decrease in t3 ( in accordance with our study ) and t4 ( not in accordance with our study ) levels in comparison to control groups , but serum tsh levels indicated no significant difference in patients and control groups which was in agreement with other findings . the movement of th into and out of the extravascular space may be a possible mechanism of th changes related to hd . there are different findings on variations in serum lipid peroxidation level and erythrocyte antioxidant enzyme activities due to hd . some studies indicated an increase while some show a decrease in the levels . in our study , we determined the level of serum mda of hd patients . mda uses as a specific and sensitive biomarker for the estimation of the lipid peroxidation status in different diseases , including in patients under chronic hd treatment . our results show a significant increase of serum mda in the hd group when compared with the control group . oxidative damage may depend on different risk factors , but it can be caused by the imbalance between the productions of free radicals and different anti - oxidant enzymes . lipid peroxidation disrupts the structural integrity of the lipid bilayer , leading to elevated membrane permeability ( the release of hydrolytic enzymes and increasing cell damage ) and subsequent impaired electron transport for oxidative phosphorylation in mitochondria and increased lysosomal permeability . the results of present study indicate that significant difference of anti - oxidant enzyme activities between hd and control group may be related with the loss of antioxidant enzymes , and the reduction of antioxidant enzymes may be related to elevation of lipid peroxidation in hemodialyzed patients which is in agreement with findings of other studies . these results show that oxidants and anti - oxidants imbalance may play an important role in the pathogenesis of some diseases . some findings showed that the elevated mda level may act as a metabolic signal for kidney damage and protein leakage including ths . it is reported that there is a significant increase of the blood sod and cat activity in plasma and red blood cells of patients undergoing hemodialysis . some other studies also indicated that the activity of erythrocyte sod and cat increased significantly while another finding showed a decrease of antioxidant enzymes in erythrocyte of crf patients . study on the animal model kidney of the cytoplasmic sod has revealed the decrease of the sod protein abundance in the kidney tissues of the crf animals which is in accordance with findings that reported the decrease of sod enzymatic activity in the erythrocytes of patients with crf and in our results . we found significantly positive correlation between t3 , ft3 , and ft4 and mda , which is not in agreement with other studies . this correlation between mda and above mentioned th levels indicate that the alteration of these hormone levels in hemodialyzed patients may depend on variations of lipid peroxidation in these patients . reduced serum ft3 and t3 may also relate to impaired extrathyroidal changing of t4 to t3 . the exact mechanisms for the decrease of t3 are not well known , but some studies have shown that the decreased t3 level correlates with poor cardiac prognosis and a strong predictor of death in cardiac patients . decreased ft3 is not only important in crf patients but also in acute and chronic infections ; diabetes ; and different cv diseases . studies have shown that t3 and ft3 are as survival markers in patients with ckd and in hd patients . some researchers have suggested that there is a relationship between t3 levels and thyroid dysfunction and risk of mortality in these patients . because of the high prevalence of thyroid dysfunction in hd patients , it suggests that screening of thyroid function should be needed .

in contrast to the common finding of mucin production in non - endocrine epithelial tumors or mucinous metaplasia , in endocrine tumors it is an unexpected rare finding.1 mucin is rarely encountered in the primary thyroid neoplasm and only occasionally has mucin production been reported in medullary carcinoma or poorly differentiated carcinoma of the thyroid , which subsequently may lead to diagnostic difficulties , especially in cytologic specimens.25 most of these tumors produce variable amounts of intracytoplasmic mucin similar to epithelial tumors.6,7 several cases of stromal mucin - producing thyroid neoplasms have also been reported.816 we report here on two additional cases of follicular adenoma with thick capsule and extensive stromal mucin deposits . importantly , this is the first time that braf gene mutation analysis is performed in these types of lesions . case 1 is a 58-year - old korean man who had undergone radioactive iodine therapy at another hospital for thyrotoxicosis twelve years prior to analysis . the neck ultrasound scan taken at the health care center revealed a small nodule ( 0.6 cm ) at the lower pole of the right lobe near the isthmus . at that time , the free t4 level was within normal limits ( 1.15 ng / dl ) and there was a slightly elevated tsh level ( 5.98 mciu / ml ) . the fna taken from the right lobe showed many singly scattered discohesive cells and occasional clusters had scanty to abundant granular cytoplasm in a mixed sticky mucin and colloidal background ( fig . although nuclear grooves were not found , the cytologic diagnosis was a thyroid neoplasm with a possibility of papillary carcinoma . surgical removal was recommended . the excised right lobe measured 5.0 2.7 0.8 cm and weighed 7.5 grams . the external surface was smooth tan and the cut surface showed a well - circumscribed dark brown , oval shaped , muddy , mucoid mass at the lower pole of the right lobe near the isthmus measuring 0.6 0.5 0.4 cm ( fig . the tumor was composed of microfollicles in the background of bluish mucin pools ; no papillary structures were seen . there were rare follicular structures , and the microfollicles contained some colloid or mucin ( fig . 2c ) . case 2 is a 47-year - old caucasian american woman who had a previous history of slowly growing masses in the thyroid with clinical symptoms of hypothyroidism . a 1.5 cm mass with a cystic surface containing clear yellow fluid and slightly calcified areas and covered by a thick capsule was located in the right lobe . microscopically , the tumor was composed of anastomosing cords and trabeculae of polygonal cells with intervening amyloid - like extracellular eosinophilic stroma and bluish mucinous material ( fig . some areas of the first case s lesion showed nuclear irregularities with frequent inclusions , whereas case 2 showed nuclear irregularities with occasional intranuclear inclusions ( fig . all the tumor of both samples including the surrounding capsule were entirely submitted for histologic diagnosis . in addition to the abundant extracellular mucin pools , occasional intracytoplasmic mucin vacuoles were found . immunohistochemically , the tumor cells in both cases were positive for thyroglobulin ( 1d4 ; dako , glostrup , denmark , prediluted ) and thyroid transcription factor-1 ( ttf-1 ) ( 8g7g3/1 ; dako , 1:50 dilution ) , whereas they were negative for cyclin d-1 ( sp4 ; dako , prediluted ) , cam5.2 ( dako , prediluted ) , cd56 ( 123c3 ; dako , 1:100 dilution ) , p53 ( do-7 ; dako , 1:100 dilution ) , calcitonin ( dako , 1:100 dilution ) , ck19 ( rck108 ; dako , 1:200 dilution ) , galectin-3 ( 9c4 ; novocastra , newcastle upon tyne , uk , 1:100 dilution ) , hbme-1 ( m3505 ; dako , 1:50 dilution ) , synaptophysin ( dako , 1:200 dilution ) , chromogranin ( dako , 1:200 dilution ) , carcinoembryonic antigen ( cea ) ( dako , prediluted ) , high molecular weight cytokeratin ( 34e12 ; dako , prediluted ) , and smooth muscle actin ( 1a4 ; dako , prediluted ) . mutation status in the braf gene was tested in these two cases using two different methods : ( 1 ) allele - specific polymerase chain reaction ( pcr ) for v600e mutation , and ( 2 ) braf mutation targeted for exon 15 of the braf gene at the rna and dna levels . each case was diagnosed as a follicular adenoma due to a lack of capsular and vascular invasion . after the surgical procedures , the patients are alive and well after 28 months ( case 1 ) and 27 months ( case 2 ) of follow up . the most characteristic pathologic findings of our two cases were thick capsule , extracellular mucin deposition , and frequent nuclear inclusions . the main differential diagnosis included the mucin - producing papillary carcinoma , medullary carcinoma , and mucinous carcinoma . 8,12,17,18 mucin - producing medullary carcinoma can be distinguished from the present two cases based on the absence of amyloid , the positivity for thyroglobulin , and negativity for cea and calcitonin . however , the cytological overlapping features such as abundant mucin , frequent nuclear inclusions , and intravacuolar globules with violaceous - colored magenta bodies of the target - like cytoplasmic vacuoles under diff - quik stain , prompted a differential diagnosis with mucin - producing papillary carcinoma.2,19,20 intranuclear inclusion is usually considered the most important feature and is a characteristic but not a specific cytologic finding of papillary carcinoma in up to 90% of cases . it can be observed in a variety of thyroid lesions such as follicular adenoma , hyalinizing trabecular adenoma , and even colloid goiter , as well as lesions mentioned above.21 cytology of case 1 did not show nuclear grooves but frequent nuclear inclusions in mucinous background and the cytologic diagnosis was challenging . the possibility of papillary carcinoma in our cases was finally excluded based on the histological absence of papillae or nuclear grooves and a lack of the braf mutation , along with immunonegativity for galectin-3 , ck19 , and hbme-1.22 on histologic slides , our cases were finally diagnosed as follicular adenomas with extensive extracellular mucin deposition , which according to the latest who classification can belong to the mucinous variant of follicular adenoma , albeit the term does not fully describe the pathologic characteristics of our two cases.13,16.23 these atypical nuclear changes and extensive mucin spillage into the thick capsule may also be described as follicular neoplasm of uncertain malignant potential or atypical follicular adenoma , although these terms are not fully accepted and they generally pursue benign courses contrary to follicular carcinoma.24 mucin can be seen in a wide spectrum of primary thyroid tumors including follicular neoplasms , medullary carcinoma , poorly differentiated carcinoma , papillary and undifferentiated carcinomas , and mucoepidermoid and primary mucinous carcinomas.116,19,25,26 a mucin - containing thyroglossal duct cyst in the thyroid has also been described in the literature.27 most of the mucin of these tumors are of intracellular location and a large amount of mucin can cause a signet - ring appearance in signet - ring cell adenoma of the thyroid gland.6,2831 the presence of mucosubstances in the normal thyroid gland is well known because the complex carbohydrate type of mucosubstances is essential for thyroglobulin metabolism . as a result , one of the possible pathogeneses of the mucin - materials present in a thyroid neoplasm is due to protein - degradation of thyroglobulin , suggesting that the mucin is originally produced in follicular cells . in recent years , mucin genes such as muc1 and muc4 have been detected in papillary carcinoma.32,33 the epithelial mucin found in the mucinous change of papillary carcinoma can be explained by this theory . dual differentiation of the tumor cells that allows a combination of mucin secretion and endocrine function may be another mechanism considered.7 other possible explanations of mucin production include an origin from the ultimobranchial gland,34 solid cell nests,35 intrathyroidal embryonal nests of salivary gland , or a thyroglossal duct cyst.27 the presence of stromal mucin , like those found in the present cases , may be produced by stromal fibroblasts or myofibroblasts.11 extracellular mucin differs from epithelial mucin and is composed of uniformly sulphated acid mucin , which is stained with mucicarmine and alcian blue but not with pas , with or without diastase treatment.36 we retrieved 9 cases of primary thyroid neoplasms with extensive extracellular mucin deposition found in the literature.816 of 11 cases of primary thyroid neoplasms with extensive extracellular mucin deposition , including our two cases , there were 3 cases of follicular carcinomas , 4 cases of mucinous carcinoma , 1 case of mucoepidermoid carcinoma and 3 cases of the mucinous variant of follicular adenoma . in only three previously reported cases , aspiration cytologic findings were described;12,13,16 case 1 of current cases and another case of follicular carcinoma reported by kuma et al12 described nuclear grooves ( not in case 1 ) , inclusions , and pleomorphism , but no nuclear clearing , requiring the lesion to be distinguished from papillary carcinoma . careful search for mucin in the background as well as the intracytoplasmic portion may be a clue to these uncommon thyroid neoplasm . to our knowledge , papillary carcinoma with extensive extracellular mucin deposition has not yet been reported . despite these sharing fna findings with those of papillary carcinomas , the possibility of papillary carcinoma could be excluded by lack of the braf mutation in the present cases . papillary thyroid carcinomas harbor the braf mutation , whereas follicular carcinomas , medullary carcinomas , and benign neoplasm are hardly associated with the braf mutation.23,37 these thyroid tumors with stromal mucin deposits are summarized in table 1 . given the rarity and uncertainty of these mucin - producing thyroid tumors , it is unknown whether extensive mucin production in benign and malignant thyroid neoplasms confers a different biologic behavior . mucinous changes are one of several clear cell changes occurring in thyroid neoplasms of various microscopic types and as a consequence of variable mechanisms and subsequent accumulation of mucin , glycogen , lipid or thyroglobulin . in general , mucins have become molecules of interest as prognostic factors for various cancers . however , treatment and clinical prognosis of the thyroid neoplasm producing extracellular mucin deposits are not different to that of their non - mucinous counterparts . the clinical course and treatment of these tumors is determined by their basic nature rather than the presence of cytoplasmic clearing.38,39 it is unclear whether it belongs to an existing category of thyroid neoplasm with mucin production or it is truly a new tumor variant . furthermore , pathologists should pay attention to avoid misdiagnosis of this variant of follicular neoplasm that shows overlapping cytology with that of papillary carcinoma .

cleft palate is a frequent congenital defect , with an incidence of 1 per 700 to 1,000 live births . the patients experience several problems in speech , feeding , dentition , maxillofacial growth , health status of the middle ear , and social relationships . the paramount issues aimed at in the treatment of the cleft palate are to achieve reconstruction of normal anatomy and normal speech without confronting maxillofacial growth and to improve the function of the eustachian tube to minimize hearing loss and middle ear infections . current surgical methods comprise suturing palatal mucosal flaps over the cleft without repair of the bony cleft . these techniques , especially if not performed properly , heal with scar contraction , resulting in impairment of later growth and consequently , an unfavorable appearance and dental malocclusion . in addition , when the tension resulting from the mucosal flaps combines with the lack of bony closure , the risk of dehiscence and oronasal fistula increases . the incidence of oronasal fistula after primary palatoplasty has been reported to be 11 to 25% . the oronasal fistula after palatal surgical repair undoubtedly compromises the above goals and brings about a serious challenge for the treatment team . thus , any effort made to preserve normal facial growth would reduce the need for subsequent reconstructive interventions . at present recently , the induction of new bone formation and soft tissue migration in the area has gained considerable attention . the main superiority of do is the lengthening of the soft tissue over the bone . mandibular distraction , expansion of the hard palate , midfacial advancement , and closure of the alveolar cleft are some applications of do in the craniofacial region . it could be assumed that cleft palate repair maybe improved by the application of do , and do could be an effective means to at least reduce wide clefts . do for lengthening of the hard palate in canine models to introduce a new approach in the treatment of velopharyngeal incompetence . there are also reports of do application in closure of the palatal cleft in animal models . liu et al . carried out a study on 45 dogs to investigate the possibility of suture expansion osteogenesis in the management of bony palatal defects . assessed the utility of do in the closure of hard palate clefts in canine models . successfully applied do for the closure of palatal bone defects in 15 cats . in a finite element study , wang et al . explained the biomechanical aspects of rapid maxillary expansion in a cleft lip and palate model and reported the differences in tissue reactions of normal and cleft palate patients . launched a similar finite element study to investigate the skeletal and dental effects of rapid maxillary expansion in a patient with unilateral cleft . pichelmayer and zemann investigated pitfalls of alveolar cleft closure by osseodistraction . up to now , we have found no reports of applying do in the human hard palate cleft . it is very thin , with a distinctive curve and we did not know how it would react to do . our hypothesis was that the hard palate cleft can be closed or reduced successfully with do without any unfavorable outcome . to investigate the biomechanical aspects of this hypothesis , the location of the do screw is the most important issue in this regard that can influence the overall healing process . the purpose of this study was to evaluate stress distribution in the intact side and affected side of the cleft and the movement pattern when the do device is placed anteriorly or posteriorly in the palate . in the present study , do simulation was applied to reduce the size of a modeled cleft palate . a three - dimensional model ( 3d ) of the maxilla with a unilateral hard palate cleft was created . the 3d model of the maxilla was developed from the computerized tomography ( ct ) data of a patient with neurological problems and no craniofacial deformity which had been scanned with a 2.0 mm slice thickness . as ct scans just propose the co - ordinate data of cloudy scatter points in the material boundaries , these discrete points may not be proper to produce finite element models directly . thus , different data - fitting techniques have to be employed to make an approximate mathematical model . the maxilla was modeled through a ct scan image processing of the anatomic data by an image control system ( mimics , materialise interactive medical image control system , leuven , belgium ) . the external geometry obtained from mimics was exported in stereolithography ( stl ) format . as the stl file could not be processed further to a 3d model due to cad / cae ( cad : computer - aided design , cae : computer - aided engineering ) inconsistency , solidview ( solidview / pro3.53 , solid concepts inc . ) and solidworks ( solidworks corp , concord , mass ) were used with care so as not to lose any important geometric information . regarding the higher incidence of left side clefts in previous reports , a unilateral cleft was created on the left side by removing the oral mucosa and bone from the anterior aspect of the alveolar process through the posterior edge of the hard palate , assuming the left lateral incisor to be missed . the created cleft was about 6 mm wide and 50 mm long [ figure 1 ] . the 3d model of the created cleft for the position of osteotomy lines , two different models were assumed , one with the osteotomy line and the free bone island ( about 8 mm wide and 37 mm long ) on the affected side , referred to as model a , and the other with the osteotomy line and bony island ( about 13 mm wide and 40 mm long ) on the intact side , referred to as model b [ figure 2 ] . in both cases , the osteotomy line was created with approximately 5 mm distance from the alveolar crest . subsequently , do was applied to reduce the cleft size . in each model , do screws were assumed to be positioned on two different locations , one anteriorly and the other posteriorly . the four created models : a ) a1 ( osteotomy on the affected side , anterior do screw ) , b ) a2 ( osteotomy on the affected side , posterior do screw ) , c ) b1 ( osteotomy on the intact side , anterior do screw ) , d ) b2 ( osteotomy on the intact side , posterior do screw ) in our study , mesh generation and data processing were carried out by the 3d finite element analysis package ( abaqus v6.7 - 1 ; simulia corp . , providence , usa ) . all parts of the entire model were treated as homogeneous , isotropic , and linear elastic materials ; the properties of these were adapted from the literature [ table 1 ] . to the best of our knowledge , tissue properties in cleft palate patients have not been documented . therefore , in the present study , the material properties were assumed to be similar to those of normal tissues . mechanical properties of materials the nodes at the zygomaticotemporal suture and superior surface of the maxilla were fixed in all directions as boundary conditions . a linear static analysis with five steps was performed on the prepared 3d solid models . as the bone segments are separated by 1 mm / day in do , in this virtual study , the segments were displaced 1 mm in each step . to evaluate the course of the cleft closure , the mentioned procedure was repeated five times to achieve 5 mm displacement at screw sites . the movement was accomplished by closing the open screws which were placed at midline to avoid injury to tooth buds . displacement pattern of the bony island in each of the four finite element models was achieved . the bony island in model a1 ( osteotomy on the affected side with do screw on the anterior position ) moved almost 2.41 mm on the anterior part and 3.4 mm on the posterior , but the maximum movement took place in the middle of the bony island , where the appliance was fixed [ figure 3a ] . the bony island in model a2 ( osteotomy on the affected side with screw on the posterior position ) moved 6.44 mm on the anterior part and 2.26 mm on the posterior part [ figure 3b ] . displacement patterns of the bony island : a ) model a1 , b ) model a2 ; the transparent object shows the initial position of the bony island the bony island in model b1 ( osteotomy on the intact side with screw on the anterior position ) moved 5.76 mm on the anterior part and 2.44 mm on the posterior [ figure 4a ] . the bony island in model b2 ( osteotomy on the intact side with screw on the posterior position ) moved 4.87 mm on the anterior part and 4.4 mm on the posterior , but the minimum movement was 3.32 mm and took place on the midpalatal region [ figure 4b ] . displacement pattern of the bony island : a ) model b1 , b ) model b2 ; the transparent object shows the initial position of the island the movement of the bony island in all models , except model a1 , showed that the rotation center of the bony island was located beyond the posterior edge of the hard palate . the bony island in model b2 shifted more linearly and was expected to achieve a more complete bone closure of the hard palate cleft [ figure 5 ] . the bony island in this model demonstrated a curve - shaped pattern of movement from the anterior to the posterior . displacement pattern in different models reaction forces at the assumed do locations were estimated for each model . the diagram of force variations versus steps is shown for all models in figure 6 . the minimum force belongs to models a1 and b2 and it increases dramatically in models a2 and b1 . maximum reaction force in the first step maximum reaction force in different models estimation of von mises stress on the bony islands was also done [ figure 7 ] . as shown , maximum von mises stress value was found around the bone edge and the area in which the appliances were fixed . von mises stress on the bony islands : a ) model a1 , b ) model a2 , c ) model b1 , d ) model b2 von mises stress on the bone edges recently , researchers have focused on presurgical manipulation of surrounding tissues to decrease the need for extensive surgery in cleft palate patients . the well - known orthopedic procedure , do , can potentially be helpful for this aim . with the help of the method examined in the present study , the two segments of the hard palate might be brought slowly into closer approximation to completely close or reduce the size of the cleft . the gradual tension on the soft tissue stimulates its lengthening and steadily accommodates the new bone . therefore , achievement of a tension - free closure would potentially decrease the incidence of persistent oronasal fistula and impairment of midfacial growth . previous investigators have addressed retruded midface and alveolar clefts with do techniques in reconstructive procedures of cleft palate patients . some animal studies applied do for lengthening of the hard palate to introduce a new approach in the treatment of velopharyngeal incompetence . there are also reports of do application in closure of the palatal cleft in animal models . to the best of our knowledge , the mechanical aspects of they discussed about the placement of the distraction device and concluded that presurgical planning and the construction of the distraction devices maybe modified to reduce problems in such procedures . therefore , the present study was designed with the aim of evaluating mechanical aspects of do application and determining the potentially best locations for the osteotomy line and do screws for closure of the palatal cleft . the most favorable pattern was observed in the b2 model , in which the osteotomy line was located on the intact side and the screws were placed posteriorly . in model a1 , the bony island moved on the posterior part more than the anterior , with the maximum movement in the middle of the bony island , where the screws were fixed . displacement of the bony island in model a1 demonstrated a curved pattern which was completely different from other models . the maximum movement of the bony island in models a2 and b1 took place on the anterior part , with a progressive descending trend toward the posterior . the only model with roughly even antero - posterior displacement pattern along the bony island was b2 , which showed a slightly greater movement on the anterior area . this pattern ensures a more complete bone closure of the cleft . although the osteotomy line was placed on the cleft side , wang et al . also they reported that , interestingly , at the end of active distraction , the thin fissure between the two parts disappeared automatically . a complex of reaction forces of support and joint areas , in addition to the applied force on the bony island , caused deformation and movements . it should also be noted that due to the presence of deformable parts in the model , the rules of free body diagram used for rigid bodies is not applicable . in a1 , do screws were positioned close to the center of the mass of both the maxilla and the island . although the island displaces together with pulling the soft tissue , the body of the maxilla resists uniformly against movement as a result of the presence of zygoma . therefore , more displacement occurs in the middle part of the island , where the force is applied , and the entire alveolar process demonstrates movement , although a minor one [ figure 3 ] . given that the maxilla is thin in posterior region , the island slightly moves along the z axis , with the maximum movement in the posteromedial edge [ figure 9a ] . movement of the island along z axis : a ) model a1 , b ) model a2 in a2 , the applied force to the bony island first displaces the soft tissue . in the posterior region , as a consequence of the minor mass of the maxilla that causes this part of the maxilla to move more freely , a part of the total force moves the maxilla along the x axis and the island demonstrates less movement , individually [ figure 3 ] . the movement along the z axis is larger in the posterolateral edge and the island shows a curve - shaped deformation in the z - y plane [ figure 9b ] . on the contrary , in the anterior region , the amount of movement of the the bony island is greater , because the larger size of the anterior maxilla and its connection to upper structures do not allow the maxilla to move freely . in this region , greater thickness of the bony island , especially at the lateral border , leads to an insignificant displacement along the z axis . when the bony island is located in the intact side ( b1 , b2 ) , the frontal cross - section of the island shows a u - shaped profile that leads to a more uniform movement [ figure 4 ] . in b1 , the amount of movement of the bony island decreases linearly toward the posterior area such that the maximum movement can be detected in the anterior region [ figure 4 ] . in fact , the island rotates around a center of rotation beyond the posterior palatal edge . this pattern of displacement results in minimum deformation of the internal geometry of the island . figure 10a demonstrates that the maximum movement along the z axis occurs at the first molar area , whereas the minimum movement happens in the anterior maxilla at the central incisor area . movement of the island along z axis : a ) model b1 , b ) model b2 in model b2 , the island moves almost evenly along the x axis [ figure 4 ] . analysis of the movement along the z axis reveals that the amount of movement is different on the medial and lateral areas , and shows a rotation around the y axis , with the maximum on the lateral side [ figure 10b ] . although b1 and b2 show different patterns of movement along the z axis , the amount of displacement is almost the same . the stress observed in the bony islands results from the resistance against displacement , which is different among the assumed models . as seen in figure 7 , maximum stress levels were detected around the do screws and the bone edges ; the more the thickness of bone around the do screws , the less the stress concentration around it . as seen in the models with posterior do location ( a2 , b2 ) , the stress concentration around the do screws increases because of the lesser thickness of bone in the posterior region of the palate . apart from the stress concentration around the do screws , von mises stress distribution is almost similar in a1 and a2 , as well as b1 and b2 models due to the geometry of the island . in b1 , as the screws were placed in the region with a u - shaped profile , stress distribution was more uniform . in this study , a complex of reaction forces of support and joint areas , in addition to the applied force on the bony island , caused deformation and movements . it should also be noted that due to the presence of deformable parts in the model , the rules of free body diagram used for rigid bodies is not applicable . in a1 , do screws were positioned close to the center of the mass of both the maxilla and the island . although the island displaces together with pulling the soft tissue , the body of the maxilla resists uniformly against movement as a result of the presence of zygoma . therefore , more displacement occurs in the middle part of the island , where the force is applied , and the entire alveolar process demonstrates movement , although a minor one [ figure 3 ] . given that the maxilla is thin in posterior region , the island slightly moves along the z axis , with the maximum movement in the posteromedial edge [ figure 9a ] . movement of the island along z axis : a ) model a1 , b ) model a2 in a2 , the applied force to the bony island first displaces the soft tissue . in the posterior region , as a consequence of the minor mass of the maxilla that causes this part of the maxilla to move more freely , a part of the total force moves the maxilla along the x axis and the island demonstrates less movement , individually [ figure 3 ] . the movement along the z axis is larger in the posterolateral edge and the island shows a curve - shaped deformation in the z - y plane [ figure 9b ] . on the contrary , in the anterior region , the amount of movement of the the bony island is greater , because the larger size of the anterior maxilla and its connection to upper structures do not allow the maxilla to move freely . in this region , greater thickness of the bony island , especially at the lateral border , leads to an insignificant displacement along the z axis . when the bony island is located in the intact side ( b1 , b2 ) , the frontal cross - section of the island shows a u - shaped profile that leads to a more uniform movement [ figure 4 ] . in b1 , the amount of movement of the bony island decreases linearly toward the posterior area such that the maximum movement can be detected in the anterior region [ figure 4 ] . in fact , the island rotates around a center of rotation beyond the posterior palatal edge . this pattern of displacement results in minimum deformation of the internal geometry of the island . figure 10a demonstrates that the maximum movement along the z axis occurs at the first molar area , whereas the minimum movement happens in the anterior maxilla at the central incisor area . movement of the island along z axis : a ) model b1 , b ) model b2 in model b2 , the island moves almost evenly along the x axis [ figure 4 ] . analysis of the movement along the z axis reveals that the amount of movement is different on the medial and lateral areas , and shows a rotation around the y axis , with the maximum on the lateral side [ figure 10b ] . although b1 and b2 show different patterns of movement along the z axis , the amount of displacement is almost the same . the stress observed in the bony islands results from the resistance against displacement , which is different among the assumed models . as seen in figure 7 , maximum stress levels were detected around the do screws and the bone edges ; the more the thickness of bone around the do screws , the less the stress concentration around it . as seen in the models with posterior do location ( a2 , b2 ) , the stress concentration around the do screws increases because of the lesser thickness of bone in the posterior region of the palate . apart from the stress concentration around the do screws , von mises stress distribution is almost similar in a1 and a2 , as well as b1 and b2 models due to the geometry of the island . in b1 , as the screws were placed in the region with a u - shaped profile , stress distribution was more uniform . the results of the present study suggest that the achievement of complete closure of the palatal cleft by do is possible by positioning of do screws posteriorly in the molar region and the osteotomy line on the intact side . the above - mentioned design showed the best pattern of movement of the bony island during the virtual distraction . it should also be borne in mind that patients with different cleft types may respond in different ways ; therefore , clinicians should always consider the need for customization of the therapy for every patient .

world health organization estimated that 17.5 million of people die from cardiovascular diseases ( cvd ) each year constituting 31% of all deaths worldwide . among the cvd deaths , the evolution of treatment modalities and the intense public health preventive efforts has reduced the influence of smoking , untreated hypertension , and hypercholesterolaemia . however , sedentary lifestyle and overconsumption of processed and energy dense food products of poor nutritional values are on the increasing trend . although past studies have reported the role of obesity in the occurrence of cvd , recent studies have focused on body fat itself which give more distinct impact on cvd . however , the relationship between body fat and the cvd risk factors is complicated by the dynamic influence and the mediating roles of the lipid parameters triglycerides , hdl , and ldl . malaysia , with its rapid urbanization and changing lifestyle , is facing an increase in noncommunicable diseases ( ncd ) which resulted in the rise of mortality due to cvd from 15.7% in 1996 to 25.4% in 2006 [ 4 , 5 ] . a study in a rural community in sarawak among three major ethnic groups ( malay , bidayuh , and iban ) reported the prevalence of overweight and obesity to be 39.6% and 11.9% , respectively . it further reported that , overall , 13% had hypertension and 1.5% had unhealthy random blood sugar reading above the national guideline level of 11.1 mmol / l . previous studies have identified the effect of gender , race / ethnicity on lipid profile , and body fat where both gender and ethnicity have been linked to the differences in body composition and lipid profiles among the females . another study has shown that there were significant differences in the muscle mass , fat distribution , bone mass , and modulation of lipid profiles between non - hispanic black and white women and hispanic and black women tend to accumulate body fat more than their white counterpart . the differences in fat composition and lipid profile among major ethnic groups in malaysia were reported by rampal et al . where indigenous groups in sarawak have the highest prevalence in raised triglycerides and blood pressure and the lowest in hdl cholesterol . however the findings only reported indigenous groups in sarawak as a whole , in which there are more than 40 subethnic groups with different languages , culture , and lifestyle . the major ethnic groups residing in rural areas of sarawak are the iban , the bidayuh , and the malay . this study aimed to determine whether gender and ethnic differences have an effect on cardiovascular risk factors among these three ethnic overweight and obese adults in sarawak . this was a cross - sectional study which included rural communities in kuching and samarahan division . only these two divisions were chosen as majority of the bidayuhs reside in these two divisions . kuching division consists of three districts ( kuching , lundu , and bau ) , whereas samarahan division consists of four districts ( samarahan , asajaya , serian , and simunjan ) . based on the list obtained from the state district office , three villages with the largest population from each division were randomly selected . with the help of the respective sarawak administration officers , informed consent forms were sent out to the head of each village to invite all the villagers . the inclusion criteria for respondents were age between 18 to 65 years ; either overweight ( body mass index [ bmi ] 25 to 29.9 kg / m ) or obese ( bmi 30 kg / m ) , physically capable , and not intellectually challenged . sample size was calculated based on prevalence of obesity 14.3 , with 95% confidence rate and 10% nonresponse ; estimated minimum sample size needed was 204 . after screening all the villagers , 190 respondents fulfilled the inclusion criteria . the data were collected using a set of questionnaire that asked for sociodemographic data , level of physical activity , measurement of blood pressure ( bp ) , body mass index ( bmi ) , body fat percentage , fasting blood sugar , and lipid profile . blood pressure was measured using an accoson mercury sphygmomanometer ( ac cossor & son [ surgical ] ltd . , level of physical activity was assessed using a validated translated malay version of international physical activity questionnaire ( ipaq ) long form . the classification of physical activity level is based on computation of met - minutes according to ipaq procedure . respondents were asked to stand on the body meter without footwear , and the measuring beam was adjusted to rest on top the respondent 's head . tanita body composition analyser ( sc-204 ) was used to obtain weight and body fat percentage . body fat consists of both essential fat which is stored in small amounts to protect the body and adipose tissue that provides cushion and insulation for the whole body . in this study , body fat percentage refers to the amount of body fat mass in regard to the total body weight . blood samples were collected by trained laboratory assisted and analysed in a certified private laboratory hired under the grant . calculation of atherogenic index of plasma ( aip ) was based on 10 log of the ratio of the concentration of triglyceride ( tg ) to high - density lipoprotein cholesterol ( hdl - c ) , where each concentration is expressed in mmol / l . aip values of 0.1 and below are associated with low and above 0.1 with medium and high cardiovascular risk . classification of fasting total cholesterol and glucose is based on ncep atpiii and the malaysian diabetes mellitus guideline 2009 . descriptive analyses such as mean and standard deviations , frequency , and percentages for all variables were presented . binary logistic regression analyses were performed to identify gender and race associated with cardiovascular risk factors . a confidence interval of 95% and ethical approval was granted from the human research ethics committee of universiti sains malaysia ( ref . : usmkk / ppp / jepem[246.3(6 ) ] ) and conformed to the requirements for ethical procedures for research in malaysia . a total of 155 respondents participated in the study with the highest proportion being the bidayuh ( response rate of 81.6% ) . a preliminary univariate analysis was performed to determine the effect size of the study where cohen 's d was reported to be 0.7 and effect size of 0.33 , indicating a medium to large effect size . table 2 showed the health profile of the respondents and the differences between gender and three ethnic groups . physical activity level , bmi status , body fat , hypercholesterolemia , and hyperglycemia did not differ across the three ethnic groups , as well as between male and female respondents . however , the iban had significant higher mean aip compared to malay and bidayuh ( p = 0.004 ) . the malays were found to have the highest percentage of hypertension compared to other races ( p = 0.02 ) . between male and female respondents , male respondents have higher percentage of intermediate and high aip ( p = 0.019 ) . logistic regression was used to determine the prevalence of cvd risk factors by race , age , and gender ( refer to table 3 ) . all the full models containing the cvd risk factors ( aip , body fat , physical activity , hypertension , hypercholesterolemia , and hyperglycemia ) were statistically significant with p < 0.001 , indicating that the models were able to distinguish between respondents with cvd risk factors and those without . the odds ratio or exp(b ) value for bidayuh was 0.3 , indicating bidayuh respondents were 0.3 times more likely to have intermediate and high aip than malay respondents . similarly for iban respondents the odds ratio or exp(b ) value was 0.29 , indicating iban respondents were 0.29 times more likely to have intermediate and high aip than malay respondents . the wald values of the independents variables indicated that race was significant predictor of aip . consistent with bmi , body fat percentage among respondents in this study was found to be at very unhealthy level with the mean percentage above 36% for all ethnic groups . similarly for gender more than 94% of both males and females were found to be overfat . the mean fasting cholesterol , ldl , and triglyceride were also above the normal limits , indicating higher disease risk . excess body fat has been shown to be related to diabetes , cardiovascular disease , metabolic syndrome , and other noncommunicable chronic diseases . such finding is alarming , particularly in rural communities where most of the respondents are involved in farming activities . there is a significant age - adjusted racial difference in the prevalence of intermediate and high aip among the respondents . bidayuh and iban respondents had higher prevalence of aip compared to malay respondents . based on the descriptive results ( table 2 ) , malay respondents may have the highest mean bmi , body fat percentage , and lipid profile ( except triglyceride ) compared to iban and bidayuh respondents , but their aip level was the lowest , indicating higher aip might not be associated with higher bmi , body fat percentage , and other lipid indicators . it is important to note that although individual lipid profile reflects cardiovascular risk , it is not as accurate as aip level which has a stronger sensitivity that reflects the interaction between atherogenic and protective lipoprotein . the mathematical formula involved the logarithmic transformation of triglyceride / hdl which is closely associated with ldl which is found to be a strong indicator in atherogenic lipoprotein phenotype . this could explain why iban have seemingly lower cholesterol and ldl and were at highest risk of cardiovascular disease based on aip . another possible explanation is that this community might not consume the right type of fat in their diet . this phenomenon is common in rural community where consumption of food is based on availability and affordability . a focus group study carried out among the overweight and obese iban community reported that among the underlying cause of overweight and obesity was lack of knowledge on proper eating . a study carried out among the reproductive - aged women showed significant ethnic differences between body fat distribution and serum lipid profiles . it was further argued that such differences could be due to difference in adipose tissue metabolism or lipid - clearance capacity and production . genetics and environmental factors may have also played a role in determining the differences in aip . although cardiovascular risk factors such as lipid profile and body fat remain the major cause of death for both women and men in the world , literatures had showed that there are significant differences in gender differences in the prevalence of cardiovascular risk factors . it is important to note that statistics had also showed that more men are living with and dying of cardiovascular related diseases than women but such pattern is only observed when at the age of 75 years . being the gender that has longer life expectancy , women would have lower mortality and morbidity rate than men . it is assumed that the endogenous oestrogen during the fertile period of female life delays the manifestation of atherosclerosis . however , what is alarming now is that the rate of cardiovascular related diseases among younger women aged 35 to 44 years has increased significantly on an average of 1.3% annually over a period of five years between 1997 and 2002 . the findings of this study indicated otherwise that no significant difference was found between male and female respondents in all cardiovascular risk factors . the limitation of this study was the use of a cross - sectional study design , which could not explain the causal relationship between variables . nevertheless , the findings of this study indicated the need to address obesity pandemic more seriously , particularly in the rural communities where detection of disease is still lacking . aip can serve as a reliable indicator for higher risk of cardiovascular disease when the individual lipoproteins seem normal but with elevated triglyceride concentration . the relationship between lipid profiles and body fat varies according to gender and different ethnic groups . a better understanding of these differences in their health profile would help in the design and implementation of intervention programme in the prevention of cardiovascular disease . for future research , a bigger sample size that covers more divisions in sarawak together with the adoption of more analytical study design such as prospective cohort or case - control study this will help to generate a clearer picture on the potential risk factors that are peculiar with the sarawak indigenous populations .

recently , there has been a shift in focus from individuals deficits to exploration of their strengths . a study of the history of nursing shows that human strengths have been addressed in some nursing theories . however , these theories are based on a deficit model rather than on improving the strengths and well - being . the belief that human beings have the strength for goodness is the base of humanistic theories . according to these theories , through therapy , they claim that strengths are buffers against mental disorders . focusing nursing care on a person 's strengths , as a complement to the traditional approach of deficit - based nursing , research has revealed a number of important links between strengths and valued outcomes ( e.g. life satisfaction and achievement ) . it has shown that strength - based reports result in better academic , social , and overall outcomes for clients with mental health disorders , as compared to traditional socio - emotive reports that focused on the deficits that students were facing . studies also indicate an association between strengths and hope for recovery , a productive and satisfying life , the ability to buffer suicide risk , survivorship , and resilience . the outcomes of developing and identifying strengths are change in the client 's self - image and improvement of self - esteem . worldwide studies showed that half to two - thirds of people diagnosed with schizophrenia and other major mental disorders significantly improve or recover . however , much of the research on clients strengths has been conducted on disciplines other than nursing . furthermore , the participants recruited were survivors , children , families , students , and women . in iran , rahimian and asgharnejad found that resilience and ego - resiliency were significantly related to mental well - being . higher levels of resiliency had positive outcomes in adult survivors of the earthquake of bam , iran . in this study , samples consisted of both men and women . results of other studies also provided support for the hypothesis that subjective well - being and strengths are related . nursing researchers have studied inner strength in healthy women , women with breast cancer , and men with aids . although these studies were not exactly the same as the present study a conceptual analysis of health assets revealed the five core dimensions of mobilization , motivational , relational , volitional , and protective strengths . described the nurses perceptions and experiences of cancer patients health assets . in this qualitative study , 26 expert nurses in cancer care participated in focus group interviews . when the data were subjected to thematic analysis , three new core dimensions of cognitive , emotional , and physical strengths were revealed . eloranta et al . , in their study , describe the personal resources of old clients in finland . a sense of control over one 's life and a determination to remain active were the resources of these clients . factors enhancing older people 's resources were their involvement in leisure activities and social networks . the results revealed that professionals still do not have sufficient skills and abilities to identify and support older people 's existing resources . in addition to having access to necessary resources , it is also crucial that people know how to use them . resiliency resources can be a valuable mechanism for maintaining the well - being and understanding differential resistance to , and recovery from ill - health in later life . therefore , there is little known about the strengths of psychiatric patients . understanding and supporting psychiatric patients in using their repertoire of capacities to help them manage their illness , findings of this research may enrich our understanding of psychiatric patients strengths and how they are used to improve their well - being . the present study seeks to understand how psychiatric patients describe their experiences and perception of the strengths needed or used by them or supported by their care providers . we used descriptive and exploratory approach because it best fits the aim of this study to uncover all aspects of psychiatric patients strengths and to gain understanding of their strengths . data were collected using individual , semi - structured interviews with psychiatric patients , and expert panel . we chose these methods because interview is one of the most commonly used strategies for collecting qualitative data , and expert panel is an effective tool to validate the results . moreover , mainly to assess the quality and significance of data which support the validity of the results , we used expert panel approach as an effective and inexpensive tool . they were asked to examine all the data , and then , to highlight consensus on the conclusions , particularly on the answers to evaluative questions . the clinical context chosen for for gathering qualitative data , we used purposive sampling to seek participants ( patients ) according to two criteria , experiential fit and good informant . samples included individuals of both sexes with psychiatric illness . participants were recruited by some criterion such as age , sex , and diagnosis that ensured variation in the sample . inclusion criteria were that the patient had a current or previous mental disorder diagnosis , was older than 20 years , was able to communicate and understand persian , and was willing to describe his / her experiences from his / her personal strengths in her / his life with mental illness and healing process . we formed two expert panels from two clinical fields at the psychiatric clinics and psychology departments of two university hospitals . each session lasted for 2 h. each panel had 57 participants , for a total of 12 experts , including men and women . the other members had 25 years of clinical teaching experience in psychiatric - mental health nursing practice . the second panel had five experts : one clinical psychologist with over 20 years of experience in psychiatric clinics , one associate professor in psychology , two phds in nursing with 6 and 25 years of experience in nursing , respectively , and two psychiatric ( msn ) nurses with 25 years of experience in psychiatric nursing . the present study was approved by the medical ethics committee , isfahan university of medical sciences . the researchers worked collaboratively with the personnel of psychiatric clinics to recruit people with psychiatric disorders and conduct individual interviews with ( participants ) patients from july 2012 to december 2012 . interviews were conducted face - to - face in the education room of psychiatric wards . it was hoped that interviewing participants in a familiar , comfortable , and convenient environment would reduce anxiety and produce more informative interviews . the interviews lasted for approximately 45 min to 1 h , were audio - taped , and later transcribed verbatim . before the interview began , the aim of the study , the voluntary quality of participating , protection of confidentiality , and participants rights to leave the interview were explained for each participant . the following questions guided the interviews : what you think are your important personal strengths?can you give experiences from your life describing your strengths?can you talk about experiences in which your significant other talked about your strengths ? the panels were convened in the nursing and midwifery council room , where the heads of departments usually meet . the researchers welcomed the experts , and presented the aim of the study and the results of interview analysis . the panels were guided by this question : what are the most important strengths of people with mental disorders based on the data ? the panel discussions were audio - taped , imaged , and transcribed verbatim . to improve the accuracy of transcripts , researchers completely checked a subset of all completed discussions . the panel discussions lasted for approximately 2 h. data analysis was performed concurrently with data collection . we chose the widely used method of content analysis . to analyze the text transcribed from the individual interviews , three steps were followed . first , in this study , the first author read the transcripts several times to obtain a sense of the whole . the analysis started by identifying the units of meaning that could be extracted from the statements . then , the analysis proceeded with line - by - line coding , and noting initial explicit and implicit results for each interview . second , the initial findings were reviewed to develop initial codes , which were then systematically applied to the data set to identify all data extracts related to these codes . then , the codes were compared and unified . codes with similar meanings finally , the emerged theme was given a final definition and a name that reflects its content . the themes gained from analysis of interviews and the most important strengths from review of literature were presented to expert panels . we looked for a critical , common sense understanding of psychiatric patients experiences of their personal strengths . furthermore , the text resulting from expert panel discussions and the notes were read several times , and an overall impression was recorded . credibility was established through member checking , peer checking , and prolonged engagement . through prolonged engagement with the subject matter , and returning a full transcript of their coded interview and a summary of the emergent categories to the informants to see whether they recognized the findings of the study , we increased the credibility of the findings . in addition , two expert supervisors and one doctoral student of nursing carried out the peer - checking . to achieve conformability , we recorded the analytic process including the evidence and thought processes . using multiple strategies for gathering data , we tried to improve the objectivity of the study , to confirm the results , and to gain a more holistic description of the patients strengths . finally , after analyzing the data , we presented the results to a committee composed of three independent researchers of nursing school to obtain their comments and discuss the results . we conducted 13 individual interviews with 7 women and 6 men with an average age of 37 ( range : 2447 ) years and 2 expert panel groups ( table 1 for additional background information ) . in total , 21 strengths were identified . themes and quotations were translated by an english - speaking nursing teacher . for brevity , we discussed the main strengths identified . these strengths were shown to be important to all those interviewed , mental health professionals , and expert panel groups . based on the results , the four main strengths consisted of finding a meaning in daily life , work as enduring strength , entertaining activities , and positive relationship . background information on participants ( n=13 ) many participants frequently highlighted the impact of becoming involved in an activity as a powerful source of energy . sometimes participants identified valued activities that gave their lives a sense of direction , meaning , and purpose . these useful activities raised the self - esteem and helped to reduce symptoms , avoid hospitalization , and/ or spending time . for instance a participant found that : through participation in some activities , we are entertained and we may earn income . 3 , male participant ) . finding a meaning in daily life was accepted as a main strengthening factor . another participant pointed out thus : in doing the everyday action of life , i feel i am serving my family and reaching goals . almost all the participants raised the issue of work as the essence of human dignity . for one research participant , work not only made life hectic but also filled her life with the eternal asset : work is a recreation . but since i m working and i m busy , i do not think and i ve not been sick . go work ; do nt ask what is work? ( persian poetry ) work is something eternal . my husband too tried to help us and we have shown adaptability and , thus , made our lives . ( no . all the research participants stated that some activities may lead to gaining personal strengths and promote wellness . many activities , such as following diet recommendations , doing artwork , bathing , exercising , spending time with family , having intimate relationships , fishing , reading newspapers , completing crossword tables , shopping , being with friends , sleeping , walking along the river , and talking on the phone , were mentioned by participants . when i exercise , my mood is better , my sleep is regulated , i can establish a better relationship with others , and will continue to live with greater power . ( no . if i do nt work and have no entertainment , i feel under the weather . 11 , male participant ) . activities , as strength , had earned a high score and were chosen by the expert panel groups as a major strengthening factor . this included positive regard and trust in significant others , being seen and accepted by friends and family members , and fellowship with peers . my family and friends listen to me and express their concerns about events affecting me. . another participant said : if you do not have a relationship with other people , you will be corrupted . there is no life without relationship with others . when you have a relationship with siblings and relatives , you will be happy . furthermore , the positive relationship component was chosen by the expert panel as a major strength , and its effect on mental health of clients was emphasized . many participants frequently highlighted the impact of becoming involved in an activity as a powerful source of energy . sometimes participants identified valued activities that gave their lives a sense of direction , meaning , and purpose . these useful activities raised the self - esteem and helped to reduce symptoms , avoid hospitalization , and/ or spending time . for instance a participant found that : through participation in some activities , we are entertained and we may earn income . 3 , male participant ) . finding a meaning in daily life was accepted as a main strengthening factor . another participant pointed out thus : in doing the everyday action of life , i feel i am serving my family and reaching goals . almost all the participants raised the issue of work as the essence of human dignity . for one research participant , work not only made life hectic but also filled her life with the eternal asset : work is a recreation . but since i m working and i m busy , i do not think and i ve not been sick . go work ; do nt ask what is work? ( persian poetry ) work is something eternal . my husband too tried to help us and we have shown adaptability and , thus , made our lives . ( no . all the research participants stated that some activities may lead to gaining personal strengths and promote wellness . many activities , such as following diet recommendations , doing artwork , bathing , exercising , spending time with family , having intimate relationships , fishing , reading newspapers , completing crossword tables , shopping , being with friends , sleeping , walking along the river , and talking on the phone , were mentioned by participants . when i exercise , my mood is better , my sleep is regulated , i can establish a better relationship with others , and will continue to live with greater power . another participant stated : if i do nt work and have no entertainment , i feel under the weather . activities , as strength , had earned a high score and were chosen by the expert panel groups as a major strengthening factor . this included positive regard and trust in significant others , being seen and accepted by friends and family members , and fellowship with peers . my family and friends listen to me and express their concerns about events affecting me. . another participant said : if you do not have a relationship with other people , you will be corrupted . there is no life without relationship with others . when you have a relationship with siblings and relatives , you will be happy . furthermore , the positive relationship component was chosen by the expert panel as a major strength , and its effect on mental health of clients was emphasized . the aim of the present study was to explore psychiatric patients experiences and perception of their strengths , and mental health professionals views about these strengths . the results revealed that psychiatric patients possess a repertoire of strengths . some of these strengths were actively used and others were considered as important during their illness and healing . studying the strengths of individuals with psychiatric disorders as represented in their first - person accounts is the positive aspect of this study . they consist of finding a meaning in daily life , work as enduring strength , entertaining activities , and positive relationships . the varied emphasis that the participants placed on these four areas indicates the critical role of strengths in patients life journey . the findings from this study highlight the positive impact of assessing the capabilities of psychiatric patients , evident from their eagerness to talk about their abilities instead of deficits . overall , the present study both adds support and is supported by the literature on existing strengths , although the current study indicates that there are some more novel aspects to psychiatric patients strengths . these findings are largely consistent with the results of the study by young and ending . working hard , having courage , being honest with self and others , being able to take care of oneself , exercising , connecting with others ( including friends , family , and community ) , searching for meaning or purpose in life , and positive focus were the main components of the recovering model . finding a meaning in daily life , work , entertainingactivities , and positive relationships can help patients increase their well - being . meaning in life is gained when people perceive that they can effectively manage their lives so that they feel they have control over their lives . meaning as an independent component of well - being is associated with life satisfaction and happiness , and a lack of meaning is predictive of depression and disengagement . , the participants attached themselves to the work environment because it strengthens their sense of control . findings of a study indicated that mental well - being is associated with activity , better health , and mobility status , which should become targets for preventive measures . making use of meaningful work is a way to overcome self - harm and suicidal impulses in mental health clients . generally , the emerging strengths from this study were partly similar to the components of other studies about the strengths of patients that were identified by researchers in different regions across the world . for instance , the existing literature has described positive relationships , trust , hope and optimism , commitment to worthy goals , and protection as strengths . to rebuild networks and relationships with loved ones was important to psychotic patients . another study showed that empowerment , connections to others , meaning of work , and vocational future are the main components of recovery . based on the aforementioned findings , it can be concluded that psychiatric patients in different areas of the world have a similar perception of their strengths with almost similar components . although the strengths , finding a meaning , work , entertaining activities , and positive relationships , that emerged in the present study were mostly found in other studies , it is noted that the data provided by the participants of this study provided unique and specific ideas in regard to these strengths . in addition , our study found several new aspects of strengths in psychiatric patients not previously identified in the literature . review of literature showed that the majority of researches on strengths have been conducted in women , children , and adolescents or in a public health context . however , our study focused on the experiences and strengths of adult psychiatric patients of both sexes . our study on psychiatric patients , therefore , found strengths not previously mentioned and is particularly relevant for mental health care . however , an important finding in this study was that the clients seem unaware of , and do not focus on , their own strengths in their care . the experiences of study participants showed that professionals did not investigate , discuss , or use their strengths . the few studies that have focused on strengths in mental health care have mainly used patients with schizophrenic , depressive , and bipolar mood disorder ( bmd ) , or substance abuse disorders as participants . our study included patients of both sexes with schizophrenia , bmd , obsessive compulsive disorder ( ocd ) , depression , substance abuse , and schizoaffective disorder . moreover , we conducted expert panels to ensure that the study reflected the diverse views on patients capabilities , thus providing richer insights into mentally ill patients perceptions of internal strengths across different groups . furthermore , our study indicates that there are individual differences and that sex differences may exist , findings that need to be explored further in future research . these included the importance of promoting and encouraging personal strengths by patients and professionals who are able to engage in identifying patients strengths . the following part of the essay provides suggestions for ways of addressing strengths , and how practitioners can actively facilitate the strengths perspective . the concept of strengths might be a helpful way to create a new strategy in caring for these groups of patients . power statements in order to convey the importance of potentials to health care practitioners . mental health care providers should consider this when working with the mentally ill by working more holistically and being mindful of the importance of strengths . the present study demonstrates a need for greater attention to patients capabilities and greater access to alternative options of care based on these strengths . this could include enabling professionals , patients , and families to assess the strengths , and the interventions that might best suit their strengths . it should be noted that this study was carried out with a limited number of participants in isfahan . therefore , the wide transferability of the results needs further investigation by future studies with a large group of participants , and if necessary , with quantitative studies . the present study reaffirms that psychiatric patients possess a repertoire of strengths that should be assessed and used in care plans . this study gives further evidence that providing insight into the strengths of patients will facilitate the process of care and healing in this population . in order to identify the strengths and enhance patients quality of care this point has particular significance for psychiatric nursing and other mental health care professionals as they are well placed to assess and offer patients with specific information and encouragement on a variety of strengths and design care strategies to help the development of the care process . more emphasis could be placed on the possibility of strengths , generally , offering the patients a much more hopeful care . this emphasis will be of great value in enhancing the quality of care . in this regard , it is suggested that strengths be included in nursing courses and that nursing school curricula incorporate the theoretical and practical aspects of patients strengths . hopefully , the benefits of strengths will become more widely accepted and produce changes in service delivery , and this will cut to the heart of issues that are important to psychiatric patients , dispelling the preconceived beliefs of mental health professionals .

electron paramagnetic resonance ( epr ) spectroscopy , when combined with detailed computer modeling , is a powerful and valuable tool for oligomeric protein complex structure determination and can be used to probe structural details for these protein assemblies in the many situations where x - ray crystallography or nmr spectroscopy are not practical or possible . appropriate epr experiments for spin - labeled protein complexes can provide distance measurements over a range of 580 , making epr an ideal technique to study the structures of multiprotein complexes . epr studies also require markedly smaller sample amounts than nmr experiments or crystallization trials and can be applied routinely to samples in various physical environments , including cell membranes . epr techniques may be particularly useful when protein complex formation involves highly flexible structures . the measurements obtained from epr double electron electron resonance ( deer ) experiments can be analyzed in terms of gaussian distributions of the distances between the unpaired electrons in the paramagnetic spin labels as given by1where p is the relative probability of observing a particular distance r , r0 is the center of the gaussian , and describes the width of the gaussian . this distance distribution reflects an ensemble of conformations and is determined by the complex interplay of global protein conformation , spin - label flexibility , and spin - label interaction with neighboring side chains . as a result , interpretation of these distance distributions based simply on side - chain cc distances may introduce errors as large as 1214 . however , deer distance measurements are typically more precise . thus , it should be advantageous to model spin - label side chains explicitly during three - dimensional model construction or assessment . we have previously developed the tagdock software package to treat rigid body docking problems . here , we describe an enhanced version of the tagdock software suite , which now includes an mtssl side - chain rotamer library to facilitate explicit spin label modeling in the protein docking calculations . to illustrate the utility of these tagdock enhancements , we present results for detailed structural refinement of an important human erythrocyte cytoskeletal complex formed between ankyrin - r and the cytosolic domain of anion exchange protein ae1 , or band 3 ( cdb3 ) , using epr pair distance measurements and spin - label accessibility data to filter candidate complex models . human erythrocytes are unique among cells in the body due to their unusual biconcave discoidal shape , high structural integrity , and elasticity . these unique properties are essential for long - term survival under the turbulent conditions often present in the circulatory system . these unique structural and mechanical properties are due to the extensive membrane skeleton , composed primarily of actin and spectrin , and the connections formed between the membrane skeleton and integral membrane proteins . one major connection type is composed of ankyrin - r and protein 4.2 , which form a complex with cdb3 . defects in this connection complex , sometimes due to mutations in ankyrin - r , can result in spherical erythrocytes with reduced elasticity and diminished structural integrity . these deformed erythrocytes are more easily ruptured , leading to a class of clinical conditions known collectively as hereditary spherocytosis , manifested by hemolytic anemia and associated problems . a detailed structure for the ankyrin - r : cdb3 complex would help us better understand how various ankyrin or cdb3 mutations alter the skeletal membrane connections and produce spherocytosis . however , as is the case with many heterodimeric protein complexes , there is no high - resolution crystal structure yet for ankyrin - r : cdb3 and we only have high - resolution structures for individual protein components of this connection network . we reported recently a detailed model for the complex formed between the cytoplasmic domain of erythrocyte band 3 ( cdb3 ; pdb code 1hyn ) and repeats 1324 in the membrane binding domain of ankyrin - r ( ankyrin - r ; pdb code 1n11 ) , using epr distance measurements to guide a combined automated molecular docking and manual molecular model building exercise . here we describe a refined ankyrin - cdb3 complex model , generated using the tagdock toolkit , that exhibits much improved agreement with the biophysical data while retaining the general features reported for our previous model . the computational tools and protocols that we present here are applicable to any macromolecular dimer modeling task , when limited biophysical data such as distance measurements are available from epr , fluorescence resonance energy transfer ( fret ) , solid - state nmr spectroscopy , or other biophysical techniques . as described previously , tagdock is a software toolkit that produces structures for macromolecular complexes by generating randomly posed docked pairs ( decoys ) , starting from rigid structures for each monomer . the program first generates all geometrically possible docking poses and then filters all docking solutions using a penalty function that determines the agreement for each pose with available biophysical data , most typically a set of intermonomer distance measurements obtained from , e.g. , epr , nmr , or fret experiments . in the current work , we extend tagdock to include explicit mtssl spin labels ( figure 1 ) at specified amino acid positions , subject to steric constraint considerations . with this option , tagdock computes interresidue distances as boltzmann - weighted averages of internitroxide spin - label distances determined from all spin - label conformers that can be accommodated at each site . a histogram of distance density , in 0.1 bins , can be created , for qualitative comparison to experimentally determined distance distributions . a rotamer library was constructed using molecular mechanics methods in amber v10 , to determine a boltzmann - weighted set of sterically allowed mtssl spin - label rotamers at each site . while there is considerable precedence for rotamer libraries , a general - purpose library for docking calculations derived from molecular dynamics is quite distinct . we used molecular dynamics simulations to observe accessible rotamer conformations for surface - exposed mtssl side chains in the context of a globular protein structure . we modified the t4l structure ( pdb entry 2ou8 ) by replacing native side chains at positions 5 , 16 , 38 , 53 , 65 , 89 , 109 , 135 , and 144 with mtssl side chains . these positions were carefully selected such that ( a ) each position corresponds to a residue site that has been labeled successfully in previous experimental studies , so we know an mtssl side chain can be accommodated ; ( b ) the side chain was located at the surface of the protein , with the cc bond vector oriented away from the center of mass of the protein ; and ( c ) no spin - label position was close enough to any other modified position such that any two mtssl side chains could interact directly with each other and thus bias the conformational sampling , allowing us to run simulations with all nine labels included . we used previously published molecular mechanics force field parameters for mtssl to run these calculations . the 3 dihedral centered on the disulfide linkage favors values of 90 and is well - known to have a large energy barrier for interconversion between the two low - energy conformers . therefore , we ran two simulations , one with 3 set initially at + 90 , and another with 3 set at 90 , so as not to bias the rotamer states produced by our simulations due to the starting conformations for 3 . both starting structures ( 3 = + 90 ; 3 = 90 ) were solvated in a truncated octahedral box of simple point charge ( spc ) waters with a 12.2 solvent layer in all directions , producing a total system size of 42,300 atoms . the protein atoms were then held fixed and the solvent was relaxed with 100 steps of steepest descent minimization , followed by 1900 steps of conjugate gradient minimization . next , the solvent was held fixed and the protein was minimized and finally , the whole system was minimized without restraints . the system was then heated to 298 k and equilibrated in a 310 ps procedure consisting of six separate molecular dynamics ( md ) runs of between 10 and 80 ps each , using the anderson temperature coupling option . at the beginning of the procedure , solute atoms were restrained to their starting positions with a force constant of 16 kcal / mol , and the md step size was set to 0.5 fs . restraints were iteratively decreased and the md step size was iteratively increased in subsequent runs until the solute atoms were unrestrained and the step size was 1.5 fs . constant volume dynamics were run for the first 70 ps , switching to constant pressure for the remainder of the procedure . the equilibrated 3 = 90 and 3 = + 90 systems were then each simulated for 200 ns using constant pressure md with isotropic position scaling . a trajectory snapshot was saved every 1 ps , yielding 400,000 individual snapshots for each of the nine mtssl side chains . the resulting 3.6 million mtssl conformations were extracted from the trajectories for subsequent conformational analysis . the observed values for each of the 15 mtssl dihedral angles over the two 200 ns md simulations are depicted as polar probability plots in figure 2 . the values for the 3 = 90 and 3 = + 90 simulations are overlaid in the right - hand panels , illustrating how the 3 conformation impacts the probability of observing each of the three low - energy states for the other side - chain dihedral angles . the left panels illustrate the overall probabilities for each dihedral value , summed over both simulations . as these data show clearly , our observed populations for 15 are nearly identical to similar analysis reported by polyhach et al . the gauche+ population for 1 is more prevalent in jeschke s simulations compared to our results , but this is the only observable difference in the two studies . this difference is presumably because their simulations were performed for an isolated mtssl residue while we simulated the spin labels in an intact protein . a gauche+ conformation at 1 can incur steric clashes with the protein backbone , which is why this conformation is less prevalent in our simulations . since these two libraries were derived using quite different simulation protocols , the overall excellent agreement between these two independent analyses lends an added measure of confidence to our rotamer libraries ( i.e. , the rotamer states observed do not appear to be strongly dependent on the exact conformational sampling procedures , potential functions , and so on ) . polar probability plots for 15 rotamers of the mtssl spin label , determined from molecular dynamics simulations . on the right side , probabilities for our simulations with 3 initialized at + 90 ( red ) and 90 ( black ) are superimposed . on the left side , the two are summed to give the probability for each dihedral taken from all snapshots of both simulations . each of the 3.6 million mtssl side - chain conformations captured during our md simulations were classified into one of 162 possible rotamer states , defined by binning the measured values for the 1 , 2 , 4 , and 5 dihedral angles ( figure 1 ) into g+ ( 0120 ) , g ( 1200 ) , or trans ( 120240 ) conformations , and binning 3 into 90 conformations . then , for each rotamer state , each of the five values were averaged over all of the conformers that were classified to that state and stored as the values that define that rotamer conformation . we observed 155 of the possible 162 states , with the most frequent state representing 5.5% of the total population ( 198,000 total observations ) , while the three least frequent rotamer states were observed only 4 times each . a cutoff was then applied such that all observed rotamer states with a probability density of at least 0.0005% were retained in our model , producing a library of the most frequently observed 146 unique rotamers ( supporting information table s1 ) . we also used the same procedure to create rotamer libraries classified using only four dihedrals ( 14 ) or three dihedrals ( 13 ) , which produced 51 and 18 rotamer states , respectively ( supporting information tables s2 and s3 ) . the 4 and 5 dihedral angles that were not directly used in the classification of the three- and four - dihedral rotamer libraries are still reported as the corresponding averages in those libraries . these simplified rotamer libraries could be used to further enhance computational efficiency but were not used in this study . the rotamer library is input to tagdock as a simple text file , with each line specifying one of our observed mtssl rotamers by listing its five dihedral angles and the probability density for that rotamer . in the present work , 146 rotamers , ranging in probability from 5.5% to 0.0005% , each mtssl rotamer is inserted at each spin - label position in both isolated protein monomers prior to the initial docking step . tagdock then filters each rotamer candidate by applying one of four user - selectable algorithms . the user can elect to simply incorporate all 146 rotamer states at each position ( i.e. , no bump - checking ) , or else employ a bump - check algorithm to eliminate rotamers that form unfavorable steric interactions with either backbone ( n , c , ca , o ) atoms and/or neighboring side - chain heavy atoms . the bump - check algorithm itself is simple and efficient : when the distance between a pair of heavy atoms ( i.e. , non - hydrogen atoms ) is less than 85% of the sum of their van der waals radii , a steric clash is noted and the spin - label rotamer candidate is eliminated on that basis . rotamers that pass a backbone bump check , but fail when side - chain clashes are considered , can often be accommodated with minor rearrangements of neighboring protein side chains , and we use the scwrl 4.0 software package to make these adjustments objectively and quickly . scwrl 4.0 leverages a backbone - dependent rotamer library to globally sample a protein s energetically accessible side - chain conformations . though scwrl 4.0 is most frequently utilized for in - silico mutagenesis experiments , it can also be used to repack side chains in response to user - defined , occlusive quasi - atom hard spheres . scwrl 4.0 takes fixed protein backbone atom coordinates and generates new side - chain heavy atom coordinates , which globally minimize potential energy as calculated from a combination of an anisotropic hydrogen bonding term and van der waals contacts . sophisticated computational algorithms in scwrl 4.0 efficiently manage the geometric and combinatorial challenges of rapid side - chain sampling . even with large proteins , scwrl 4.0 typically returns repacked structures within seconds . for each mtssl rotamer state we consider , we generate a pdb file that contains fixed protein backbone coordinates and the mtssl side - chain atoms defined as a collection of hard spheres . we then use scwrl to globally repack all remaining protein side chains around this hard - sphere site . the resulting repacked protein structure is again bump - checked by tagdock , and all mtssl rotamer states that pass this second side - chain atom bump - check step are added to the set of accessible rotamers associated with that spin - label position . using this protocol to iterate over all mtssl rotamer states that fail the initial side - chain bump check , tagdock identifies the largest plausible set of mttsl rotamer conformations at each spin - label position . all mtssl rotamer conformations selected for each monomer partner are retained in all subsequent docking trials , with no further adjustment or reevaluation during the docking pose calculations . for each decoy pose generated , interresidue distances are calculated for all accepted spin - label rotamer states at each position , using the n o bond vector midpoint for each spin - label residue . this calculation yields a probability - weighted distance distribution for each residue pair ( i.e. , a boltzmann - weighted distance distribution , since our rotamer state populations are derived from long md trajectories ) . we emphasize that these distance distributions will not necessarily reproduce the fine detail of an experimental deer distribution profile , as we do not at present include local backbone dynamics in these calculations , but backbone motion will make a significant contribution to the distribution profile in some cases . we have shown in previous studies for a large number of test examples that we can almost always obtain good docking results without inclusion of backbone dynamics , provided we generate a sufficiently large ensemble of docking poses during the coarse - docking phase so that the intermonomer distances populate the full distance distribution ranges observed in the deer experiments . structure refinement protocols that utilize distance data require a metric that can efficiently and objectively evaluate structures , both for ranking the relative quality of structures ( i.e. , how well do the structures fit the experimental data ) and for automatic filtering of large numbers of structures ( i.e. , data reduction via automatic elimination of structures that are inconsistent with the experimental data ) . each experimental distance restraint is typically represented as a distance distribution or possibly a distance range , rather than a discrete distance value , so we need a scoring function that is designed specifically to handle these types of distance restraint data . the distance data generated in nmr noe experiments likewise are represented as distance ranges , since the nmr experiments also sample an ensemble of conformations that each have unique values for any specific interatomic distance . the harmonic penalty function we used previously for filtering structures is not well suited to these types of distance data sets , since a harmonic function emphasizes the median distance value , while penalizing distances that deviate from the median but still fall within the experimental distance range . therefore , we have implemented a flat - bottomed harmonic scoring function , similar to those used widely by the nmr structure refinement community . this scoring function has the form2where n is the number of individual restraints and vi is the individual penalty score,3where riexp is an experimental distance , ridecoy is the corresponding distance in the decoy being scored , is the associated width of the distance distribution , and hrange is the width of the range over which the scoring function is harmonic . the default value we use for hrange is 4 , but it is a user - tunable parameter . outside of the range defined in eq 3 , ( riexp i hrange ) < ridecoy < ( riexp + i + hrange ) , the scoring function becomes linear with a slope equal to that at the point ( riexp + i + hrange ) or ( riexp i hrange ) , illustrated in figure 3 . the utility of this function for structure selection and/or refinement using experimental distance range data is well - documented . illustration of the flat - bottomed harmonic scoring function with riexp = 30 , = 1 , and hrange = 4 . the flat - bottomed harmonic score is calculated as 0 for r within of riexp , harmonic between and + hrange from riexp , and linear outside this region . the value of 1 for is similar to the experimental values of found in supporting informaton table s4 . we have also implemented a feature to compute nitroxide spin - label surface accessibility at labeled positions . this option is accomplished with a simple script that exports a pdb structure file and then uses an external program to calculate solvent accessibility . we use the msms program for this operation , but the user can specify any suitable surface area calculation software for this calculation via simple modification of the control script . as with inter - residue distance measurements , residue surface accessibility data are not used as restraints during the docking procedure , but rather used in the filtering step to determine which docking poses are consistent with the experimental accessibility data . using the published experimental distances and distributions ( table s4 , supporting information ) , we first generated structures for the cdb3 homodimer . we followed the computational protocol reported in previous test calculations . ideally , we would begin docking calculations with the crystal structures for the independent protein monomers , but there are neither x - ray nor solution - phase structures for cdb3 monomer . since the individual cdb3 monomer chains in the homodimer crystal have similar , but unique , conformations , designated p and q , a docking calculation that begins with these unique structures would clearly be biased and would not provide a meaningful assessment of program performance . we therefore performed a 10 ns molecular dynamics simulation with a generalized born continuum solvent model using amber . we applied weak positional restraints to maintain backbone atoms near the crystallographic coordinates for p and q monomers , while allowing side - chain atoms to move freely , and then used the final configurations for p and q monomers from the dynamics trajectory to begin docking calculations . the second molecule of the dimer complex was randomly and repeatedly rotated and translated onto the surface of a large virtual sphere centered on the first molecule . next , the shortest intermonomer c distance was calculated , and the second molecule was translated toward the first molecule by the distance , with no specific tests for unreasonable physical overlaps at this stage . we generated 5 10 random docking poses and then selected the 200 poses that best satisfied the experimental distance measurements . we next performed higher resolution docking searches , using these 200 poses as starting structures . the high - resolution docking phase entailed a series of more focused docking attempts , restrained by progressively smaller translation and rotation angle values , all driven by a monte carlo optimization procedure . during the first phase of high - resolution docking , translation moves were restricted to a range [ 0.03.0 ] and rotation about each axis to angle values from the range of 0.015.0. these search ranges were tightened , or focused during subsequent iterations until the translation moves were restricted to the range of 0.01.0 and rotations to the range of 0.01.0. we retained the 200 top docking poses , i.e. , those poses that gave the best agreement with the experimental distance measurements , for final analysis . we performed two sets of test calculations : one with simple cc distance filtering reported previously , and a second calculation using explicit all - atom spin labels and our new rotamer library ( together with the scwrl option as described in methods ) . in both cases , we generated 500,000 decoys using individual monomers from the homodimer crystal structure and performed focused refinement on the best 200 decoys , scoring with the flat - bottom harmonic function . we then plotted the penalty score for each of the 200 refined docking poses versus the c root mean square deviation ( rmsd ) for that pose relative to the homodimer crystal structure , as shown in figure 4 . the plot shows that both filtering strategies yield two sets of docking poses that cluster tightly around two different penalty score values ( we should note that there is no statistically significant difference between the two scores for either filtering method ) . however , the filtering calculation based on explicit spin - label side - chain modeling also produced a third penalty score cluster with smaller rmsd values relative to the crystal structure . specifically , filtering based on simple c distances yields an optimal pose with a 4.0 rmsd relative to the crystal structure , while the optimal pose we generate when we use explicit spin - label side chains and the rotamer library for conformational sampling has a 2.2 rmsd relative to the crystal structure , as shown in figure 5 . we should note that the c filtering results reported here are based on the new flat - bottom harmonic scoring function , so the results differ slightly from those reported previously . c rmsd with respect to the crystal structure of cdb3 for the top 200 tagdock results from the rigid c algorithm previously published ( red ) and incorporating the mtssl rotamer library ( blue ) . in both cases , we see similar degeneracy for both algorithms ; however , the incorporation of the spin - label rotamer library yields a third , lower rmsd cluster . in both cases c backbone stereoplots for tagdock results shown in figure 4 , overlaid on the crystal structure of the cdb3 homodimer ( gray , pdb 1hyn ) . panel a displays the best structure ( red ) from the c distance filtering method ( c rmsd 4.0 ) . panel b shows the best structure ( blue ) from the explicit spin - label rotamer sampling method ( c rmsd 2.2 ) . we next generated structures for the ankyrin - cdb3 complex , starting with crystal structures for the isolated ankyrin - r fragment ( repeats 1324 ) ( pdb code 1n11 ) and cdb3 ( pdb code 1hyn ) , and used both the side - chain c approximation and explicit spin - label side - chain modeling for distance measurements . we used the experimental distances and distributions published previously to filter results from both calculations with our new flat - bottomed harmonic scoring function . in figure 6 , we show the docking pose closest to the geometric mean of the top 200 candidates for each filtering protocol , as well as the structure closest to the geometric mean of the top 30 poses generated previously with rosettadock and manual modeling building . the top 30 models reported previously have scores ranging from 285 to 470 , while the geometric mean score is 53 for the cc method and 22 for the explicit spin - label rotamer method , where lower scores indicate better agreement with the experimental distance data . three structures are compared , with the ankyrin subunits ( dark gray ) superimposed . in gray , we show the docking result that is closest to the mean from a previously published rosettadock calculation after applying a nonautomated filtering procedure . in red , we show the closest to mean tagdock result from the rigid c algorithm . in blue , , we used the 20 experimental distances reported previously to filter our docking pose solutions . we also generated cdb3-ankyrin complex structures using subsets of the deer distance data , to assess solution convergence as a function of total interresidue distances used in the filtering step . each spin - label position in one partner that had distances measured to multiple positions in the other partner became a restraint set ; e.g. , cdb3 70ank 707 and cdb3 70ank 722 is one distance subset of the full data set . table 1 shows the c rmsd for the structure closest to the geometric mean of the top 200 refined tagdock poses computed with various distance data subsets , compared to the structure closest to the geometric mean of the top 200 refined tagdock poses generated when all experimental distances are used for solution filtering . in some cases , a distance subset contains as few as two interresidue distances for filtering , so it is not surprising that the c rmsd values for these subsets are quite large . for the limited - distance data subsets only , we also computed spin - label solvent accessibility for each docking pose and compared these results to the experimental accessibility measurements as an additional filter , to test if solvent accessible surface area data can be used effectively to improve the filtering process . when we used the solvent accessibility filter together with limited - distance data subsets to eliminate docking poses that were inconsistent with the experimental data , we observed improved c rmsd values for the mean structures in most cases , as well as dramatically smaller pairwise c rmsd values for all remaining poses that passed both the distance and accessibility filters ( i.e. , the docking pose solution set is more tightly clustered about a mean structure ) , suggesting solution convergence within the limits of the available experimental data . the closest structure to the mean of each ensemble is compared to the reference structure obtained by refining the cdb3-ankyrin structure with all available restraints . the average pairwise rmsd for each ensemble is shown in parentheses . for reference , the pairwise c rmsd for the ensemble generated when all experimental distances are used is 0.4 . in most cases , reducing the total number of distances used for the filtering step yields docking poses that deviate significantly from the results obtained when all 20 distances are used for filtering , and the structural variation among the top 200 docking poses is considerable . when sasa data are included in the filtering step ( column 4 ) , the docking poses obtained are generally much closer to the solution generated with the full 20-distance restraint set , illustrating the potential utility of these data to help select the best docking models . we emphasize that the goal of our refinement procedure is not to precisely match a given distance , nor should it be . the results obtained in the epr experiments are interpreted as gaussian distributions of the distances from an ensemble of structures , and this fact greatly influenced our choice of a flat - bottomed harmonic scoring function . penalize any structure that falls within the gaussian distribution , unlike a simple harmonic function that favors the mean value of the distribution . we do not expect a single structure to simultaneously match the center of the gaussians for all 20 distances measured for the ankyrin - cdb3 complex , but we do expect that a small ensemble of structures will reproduce the experimental distance distributions , as has been reported in a previous study of a synaptotagmin - snare complex by lai et al . in our previous study with a large database of protein heterodimer complexes , we rarely observed any docking models with low penalty scores that exhibit any notable distance violations . in only a few cases have we obtained a docking solution with a good penalty score , i.e. , excellent overall agreement with experimental distance data , which nonetheless displayed one pair distance that deviated significantly from the experimental value . for one of these cases , the pair - distance discrepancy was due to an experimental error , and the experimental result was subsequently retracted . in the remaining cases , we strongly suspect the isolated pair - distance discrepancies are likely due to our rigid backbone docking protocol . proteins are inherently flexible , and at a minimum , minor structural fluctuations are to be expected . our earlier studies with a large data set of protein heterodimer complexes suggests that we can generally obtain good docking models without explicit inclusion of backbone flexibility . however , in cases where large conformational changes occur during complex formation , it will likely be important to include the impact of local backbone flexibility on spin - label distance distributions to achieve better agreement with epr deer experiments . it was not necessary to include backbone flexibility for the cdb3 homodimer docking calculations reported here because of the computational protocol we used to generate the isolated cdb3 monomer structures . the large size and inherent flexibility of the mtssl spin labels can also produce local structural fluctuations that could easily alter simulated distances by several angstroms , and this was our primary motivation to incorporate an mtssl rotamer library for explicit side - chain conformational sampling in the tagdock toolkit . our results for the cdb3 homodimer show clearly that explicit consideration of mtssl side - chain conformations during the solution filtering step can yield much better docking poses than a simple cc distance assessment , in this case , cdb3 homodimer models that are considerably closer to the reference crystal structure . we emphasize that the mtssl rotamer library must be coupled with an efficient method to repack protein side chains adjacent to the spin - label site , and we use the scrwl application for this task . at some residue positions , we do not identify any allowable spin - label side - chain rotamers until we have performed extensive repacking for all neighboring amino acid side chains . failure to identify and consider all possible spin - label rotamers at each labeled site will almost certainly reduce the number of candidate docking poses that pass the experimental distance distribution filtering step , and lead to the inappropriate exclusion of viable docking pose solutions . the tagdock toolkit is not intrinsically limited to experimental distance measurements for solution filtering . other metrics , such as side - chain solvent accessible surface area , can be used for the filtering step as well . our ankyrin : cdb3 docking results , using limited distance measurements combined with spin - label solvent accessibility measurements , illustrate the potential power and effectiveness of combined filtering metrics . when solvent accessibility data are used together with limited distance measurements for filtering , we achieve in many cases a docking solution ensemble that is closer to the result obtained with the full distance restraint data set than we could achieve with limited distance data alone . this result is significant , because distance measurement based on site - directed spin labeling is a labor - intensive procedure , and any strategy that might yield comparably good docking results with fewer experimental measurements has obvious appeal . of course , solvent accessibility is a useful filtering metric only when the specific residue positions are impacted by the protein docking interface . otherwise , the solvent accessibility profiles provide little or no additional useful information . the refined ankyrin - r : cbd3 complex reported here exhibits much better agreement with the deer distance and spin - label accessibility measurements , compared to our earlier model . these improved results are directly attributable to the tagdock toolkit design and implementation . since tagdock calculations are fully automated and require no user intervention for model generation or scoring , we avoid potential biases that might be introduced with any manual model building or model selection procedures , unlike our previously published work . furthermore , since tagdock exhaustively considers all geometrically possible docking poses during the initial docking step , we are far more likely to identify all solutions that satisfy the experimental restraints . as reported previously , solutions sets typically converge to a well - defined consensus structure as additional experimental restraints are added in the filtering step , providing a convenient metric to determine the optimal solution ( within the limits of the experimental data used for the filtering process ) . an alternate model reported previously for the ankyrin - cdb3 complex places cdb3 more directly along the concave surface region of the ankyrin - r fragment . while our automated docking protocol sampled this region extensively , it did not identify any docking poses in the region that provide good agreement with the deer distances or solvent accessibility measurements . we should note that our previous rosettadock calculations , when performed without distance restraints , did generate docking poses in the ankyrin - r concave surface region that scored well , but these poses failed to satisfy the deer distance restraints . using a combination of epr distance measurements and rigid docking simulations , we have defined a fast and efficient method for determining and refining the structure of a complex between two proteins based on epr distance data . for our specific test cases , we studied the cdb3 homodimer and the complex between cdb3 and ankyrin - r . we show that a small distance restraint data set such as that used in this study is sufficient to reliably dock two rigid proteins . furthermore , sampling spin - label conformations with our new custom mtssl rotamer library improves agreement with the crystal structure of the cdb3 homodimer complex , compared to calculations based on a simple cc distance approximation . in cases where one or more of the individual protein partners undergoes significant conformational changes , this strategy will likely not be sufficient to refine an atomic resolution model . in these cases , a simple rigid body docking strategy to generate initial model candidates will likely be inadequate , and efficient strategies for flexible protein docking calculations will be needed . we are currently evaluating several computational schemes for this purpose , using structurally well - characterized protein protein complexes as test systems . local protein backbone conformational sampling will be crucial for these situations , and we are testing several modified md simulation methods that are more computationally efficient than traditional equilibrium md simulations , such as the accelerated md method of hamelberg et al . there are of course many good protein docking programs available now , as we reviewed in a previous publication . however , there are only a few protein docking programs or tools that model spin - label side chains explicitly when utilizing epr data . rosettaepr has this capability , as does the mtsslsuite available in the pymol graphics package . however , the spin - label rotamer libraries employed in these tools are derived primarily from existing crystal structures . at the moment , there are not enough crystal structures with mtssl side chains to derive rotamer libraries that yield the same statistical confidence levels as , e.g. , the scrwl amino acid rotamer libraries . as discussed above , this was our primary motivation to develop a boltzmann - weighted mtssl rotamer library from extensive simulation data . the mmm software package is the only other tool that utilizes an mtssl rotamer library similar to ours and that can incorporate explicit spin - label rotamer sampling along with sampling of neighboring side - chain rotamers , in protein docking calculations . however , tagdock differs substantially from all other protein docking programs in that it incorporates automated distance - difference matrix analysis to analyze results and suggest additional experimental measurements . this capability , coupled with the computational performance ( tagdock can generate 1 10 docking poses in 1 h on a single - processor linux workstation ) , makes tagdock particularly useful for rapid docking model generation and assessment . for these reasons , we promote the package primarily as a tool to facilitate experiment planning , rather than a replacement for other capable protein docking programs . of course , in cases where the rigid body docking protocol is reasonable ( i.e. , cases where the individual monomers do not undergo significant conformational changes during complex formation ) , tagdock is also an effective and efficient protein docking program . the software extensions reported here that enable us to better utilize epr distance data further enhance tagdock s utility as an experiment planning tool . the results presented in this work and in an earlier publication for the ankyrin - cdb3 complex demonstrate clearly that detailed , chemically reasonable three - dimensional structural models can be generated for protein protein complexes using a small number of accurate , long - range distance measurements and a relatively simple , inexpensive computational refinement strategy , provided there is sufficient information to define the internal three - dimensional structures for the individual protein partners used as starting structures for the automated docking calculations . the experimental data used in this study for solution filtering ( deer distance and spin - label solvent accessibility measurements ) can be generated for many other protein complexes , and the tagdock toolkit can be used for any oligomer docking project where reliable three - dimensional structures are available for the individual monomers , so the procedures and strategy presented here should be widely applicable for many biomolecular oligomer structure refinement problems .

one of the goals of the program is to support research projects that include one of the following objectives : identify health risks including those affecting vulnerable peoples , analyse the risks to health , and/or conduct a health risk assessment , and examine exposure and/or modelling data collection . proposals must also incorporate local / traditional knowledge and must include the development of adaptation approaches to climate change impacts , as well as a plan for communicating results back to the community or communities involved . in order to ensure successful implementation of the program , a series of community visits , 15 in total , as well as 3 capacity - building workshops were held for communities and organizations to become familiar with climate change impacts , their potential effects on health , as well as methods to develop good research proposals and budgets that would meet the requirements of the funding agreements . these workshops were delivered in partnership with aboriginal organizations and were held in whitehorse , yukon ; yellowknife , northwest territories ; and ottawa , ontario . during the workshops , participants were invited to share their perceptions on what kind of changes they were experiencing and their concerns about these observed changes . it was then jointly determined which of the changes they mentioned were linked to climate effects and in turn how these could affect the health and well - being of affected communities . as participants became more familiar with climate change and health , they were invited to think about what kinds of projects they could undertake to reduce these effects . on the second day of the workshops , the participants , either in small groups or together , designed one or several research projects and developed a budget that had relevance to their own communities . over the last 3 years , 50% of the proposals came as a direct result of visits and workshops ( 9 proposals from communities visited and 10 from the capacity - building workshops ) . also , to ensure all eligible communities were aware of the program and because not all communities could attend the capacity - building workshops , posters about the program and the application process were widely distributed . plain - language application guides were developed that included step - by step procedures to develop a proposal to meet health canada 's requirements . these guides were mailed to interested applicants and organizations and were made available on northern and aboriginal websites . the application guides included the following topics : description of the climate change program and program goals , potential fields of study , funding amounts and deadline for submission , eligible candidates / communities , required elements of the proposals , collection and storage of data , proposal review and selection process , timelines and contact information , elements for health canada 's ethics review board approval process , a summary check list , and a cd with further information and templates ( e.g. proposal , budget , consent forms ) that could be used for developing the proposals . required elements of the proposals included a cover page , plain language summary , community background , introduction , project description ( background , objectives , rationale , methodology , activities / outcomes , partners , capacity building , and traditional knowledge ) , work plan and timelines , budget , project evaluation , communication and/or results , reporting plan , background information on team members , consent forms etc . this pre - review helped to ensure that the applications met program criteria , and that proposals had clear objectives , goals and realistic timelines . this application procedure follows the world health organization recommendations ( 4 ) to ensure strong community - based participatory research . in order to have an equitable and transparent review process for proposals , 2 selection committees were set up , one for first nations and a second for inuit . members of the committees include climate change and health experts , northern experts , members of aboriginal organizations and government staff who were responsible for approving budgets and implementing other aspects of the program . committee members reviewed proposals and make final decisions on which communities / organizations would receive funding . the workshops , community visits and other outreach activities generated considerable interest in the program . ten community - based participatory health research projects from across canada 's north were funded in 20082009 , 10 projects in 20092010 and 16 projects in 20102011 for a total of 36 ( fig . 1 ) . funding ranged from $ 30,000 to a maximum of $ 200,000 per year . each year the requests for funding increased and were always greater than the funding available . the research examined a range of topics including loss of traditional foods ; water quality and safety , erosion / loss of permafrost , changes in traditional medicines ; relationship with ice through ice monitoring ; landslides ; and numerous climate change and health research and education projects . number and types of projects are listed in table i. areas of research for the climate change and health adaptation program for northern first nations and inuit communities film : caribou , culture and climate change film : our changing homelands , our changing lives 2 publications in food security and gender emergency food storage unit built 20102011 booklet on polar bear : wabusk of wasaho : a part of history and everyday life , results of project were incorporated into iqaluit nunavut 's public works division water education and stewardship program developed for youth film : the students of moose kerr school presents : a documentary about climate change , blog site : http://www.ournorthourfuture.blogspot.com/ film : qapirangajuq : inuit knowledge and climate change 3-day health research methods training workshop and photovoice film : inuit women 's perspectives on climate change : impacts on inuit women 's health , media research lab established literature reviews and media articles gis and traditional knowledge databases , on the land women 's retreat youth and elder on the land workshops traditional medicine workshops and wilderness camps 2 books : climate change and traditional medicine herbarium established ice monitoring stations built training in ice monitoring book : life with ice real time monitoring for travel safety promotional products for water safety many of the projects enabled youth to re - connect with their elders and gain climate change and health awareness . in some cases , youth spent time in traditional wilderness camps learning about how to observe the land and its changes , about how to hunt and respect the land and wildlife , which helped to develop valuable attributes such as patience , respect , self - sufficiency , self - esteem and traditional knowledge on how their natural world is changing . most importantly , youth gained a strong sense of purpose because they were a part , and in many cases , leading valuable climate change research projects . youth created videos and blogs about their experiences and their observations of how climate change is affecting their health . these research videos are being used as education tools for youth in other regions across the north . as one of the 20082009 youth - driven projects points out ( 11 ) , this type of research created community conversations , discussions and awareness about climate change and its impact on health , and created many linkages between the often talked about belief that the health of the land is the health of the people and the reality of climate change . in many ways , research , in that it remains largely a process and concept defined outside the community , and much of the challenge of community - based research is to create a way of coming- to- know that is respectful of and at the pace of the people involved . as a number of the participants noted , these types of projects are creating strong voices that promote important messages about climate change and health not only for individual communities , but at a larger national and circumpolar level . some of the community - based research projects have been showcased internationally at events such as the 16th annual meeting of the united nations framework convention on climate change conference of the parties ( cop 16 ) held 29 november to 10 december 2010 in mexico where nations met to assess progress in dealing with climate change and to negotiate climate change mitigation and adaptation measures . two of the youth - led projects were invited to present their work at cop17 held 28 november to 9 december 2011 in south africa . one of the films produced is the first - ever film about climate change impacts solely in inuktitut ( 12 ) . in february 2011 , a pan - arctic results workshop was held in ottawa to bring northern communities , who received funding under the program , to showcase their research and results through photo - voice , videos , powerpoint presentations , posters , and discussions . it was an opportunity for community researchers to meet others , share their project results and gain insight into what others are doing across the north . over 150 attendees , mostly northerners but also government and non - government representatives , scientists , policy - makers , members of parliament , aboriginal leaders and a few international experts actively participated in the workshop . there were some participants that had never left their communities but travelled to ottawa to share the results of their work . their participation is a testament to the importance that communities hold for their concern about effects of climate change for their own research and for sharing information with others in the hopes of creating further awareness and policy change . during the workshop , participants were invited to fill in evaluation forms in order for the program to gain further perspective on how to meet the needs of community - based research and whether or not this style of workshop was beneficial for the dissemination of information . the evaluation form asked questions including : what did you like about workshop , what would have made it better , what was learned , what would you like to see as next steps for the program ? with a response rate of 70% , a typical response is the following : the program has accommodated communities at a variety of stages in understanding climate change and health issues and research . however , there remains a lot of work to be done in developing indigenous research methodologies . many of the participants stated how important it was to have a space provided where communities can share their knowledge , their research , and their plans of how to deal with the changes through adaptation plans . participants also mentioned that the workshop was an opportunity to connect with and receive support from other researchers across the north where distances between communities are great and the isolation from research support is a challenge . participants also stated that the workshop provided a forum for them to witness how their work was feeding into the larger picture of northern community research . one of the participant 's responses when asked what did you learn at this workshop ? summed up the effect of the workshop by saying : i can make a difference . the results from all the research projects demonstrated the strong voices that represented community - based participatory research in canada 's north voices that are creating impetus to deal with the many changes that they are experiencing . over the coming years , the results of this program will be used to develop innovative human health risk management plans and tools , including culturally - sensitive educational and awareness materials , to improve decision - making regarding local response plans / health adaptation measures and to raise awareness of potential vulnerabilities at community , regional , national and international levels . an interim evaluation of the climate change program was conducted a year and a half after its inception on relevance , design and delivery , success , and immediate outcomes . the initial evidence showed that the program has been successful in all aspects including its 3 intended outcomes , namely : increasing access to climate change tools and information ; building capacity ; and promoting collaboration . longer - term outcomes , namely increased use of adaptation information and products ; increased capacity of communities to adapt to climate change and finally reduced vulnerabilities and risks to communities will be assessed in a subsequent evaluation . integral to the program 's success has been the level of community engagement throughout the years of program implementation . communities played an active role in developing and managing research projects that were meaningful and beneficial to their communities . they determined their research needs and carried out the projects with the assistance of experts where and when they determined their need . the success of this program has been reported not only by individual communities who have benefitted from the program but also by first nations and inuit organizations and selection committee members . during the capacity - building workshops , northern organizations and communities gained a clear understanding of the program 's purpose , goals , scope , and an awareness of climate change and health links . participation in the workshops was also an opportunity for government staff to listen to and gain a greater understanding of the many climate change and related challenges that northern communities are currently facing on a day - to - day basis . following the workshops , program staff have given numerous presentations to government policy and program staff on these understandings and challenges . another successful aspect of the program was to have northerners participate in the selection committees to determine who would receive funding . their experience and understanding of northern issues and challenges provided significant input to the selection committees during their deliberations . they also played a vital role in making suggestions on how to improve the program , its communication tools and the application guides by suggesting tools to include such as proposal and reporting templates . one challenge with implementation was the location of the program , which is based in the south , in ottawa . distance from the north made building relationships more difficult when trying to support northern communities in their research . the program has been able to alleviate this challenge by visiting a number of the communities where staff presented information about the program and learned about local challenges . the program , however , has only been able to visit a portion of eligible communities . to illustrate , after the first call for proposals in 2009 , program staff focussed on visiting the yukon where the participation rate was the lowest . staff worked with the council of yukon first nations and met with chiefs and councils , health directors , and lands and resource directors in 8 communities to discuss the program and how communities could apply for funding . the following year , yukon had the highest number of community participants and diversity in research topics . when a community receives approval and funding , it is essential that the program works directly with the communities . this relationship ensures that there is a person available to support each community through the various research stages including interim reporting , research ethics board reviews , budget reporting , evaluations , and final reporting . it is imperative to build these relationships , to make connections , to be flexible in order to ensure that each community feels comfortable and that research projects are successful and beneficial to the community . this relationship also ensures achievement of 2 other essential steps of community - based participatory research as outlined by the who ( 4 ) , namely : mechanisms ensure regular and effective liaison and communication between the community and research organization.course of action to be followed by both parties if the research is stopped due to an unforeseen inability to reach its objectives , or as a result of a collective decision by the community that they no longer wish to or can participate . course of action to be followed by both parties if the research is stopped due to an unforeseen inability to reach its objectives , or as a result of a collective decision by the community that they no longer wish to or can participate . a significant challenge in this community - based participatory research is the duration of funding . to date although applications are reviewed and approved 3 months before the beginning of the year , there are always delays in release of funds to communities who can not start the research project without the funding , and should not commence research until all approvals and funding are in place . when the delays extend into the field season , important information can not be collected and the quality of research is compromised . this delay has been noted by most if not all communities and the program is looking at different funding models , such as multi - year funding , to try and address the issues . another challenge is the requirements for ethics review , which for the most part require the same standards as western - based laboratory or medical research and do not consider the unique nature of community - based participatory research and the incorporation of traditional knowledge , which must be protected . these challenges are being worked on to ensure the ethics review can accommodate the unique nature of this research . although this model of community - based participatory research has been developed in consultation with northern communities and to fit health canada 's requirements for approvals and funding , other comparable models have been developed such as the community adaptation and vulnerability in arctic regions ( caviar ) methodology ( 13 ) . the caviar group got its beginnings , during the 20072008 international polar year ( ipy ) , with partners from arctic countries who came together to respond to the need for systematic assessment of community vulnerabilities and adaptations across the arctic . this methodology has been applied in 7 arctic countries including russia , norway , sweden , finland , greenland , canada and alaska who have used this framework and its multi - disciplinary methodology to describe their communities social and environmental conditions that have created exposure sensitivities and that require adaptation measures . this methodology does not limit itself to climate change and health but is an excellent approach to provide a framework for communities to be an active leader in determining their specific vulnerabilities and solutions . although community - based participatory research may be considered as grassroots - based , it is important to promote this research to empower communities to understand and be able to make decisions on the environmental changes that affect their health and livelihood . it is also important for the program to inform governments and other organizations on its achievements to support ground - up and top - down practices required to create the evidence and impetus that are needed to address the numerous climate change and health issues that northerners face including input that will contribute to understanding and awareness in the circumpolar world . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

the online version of this article ( doi:10.1007/s00228 - 014 - 1660 - 7 ) contains supplementary material , which is available to authorized users . given limited sample sizes , short duration , and lack of generalizability of preapproval clinical trials , a drug s safety profile at the time of regulatory approval is often incomplete . therefore , postmarketing safety surveillance is an essential exercise in detecting and evaluating adverse drug reactions ( adrs ) of a given drug . the primary source of postmarket safety surveillance data is voluntary reporting by clinicians and consumers . although now computerized , the spontaneous reporting system has been in place in the united states since the 1950s . strengths of the system include the potential to encapsulate rich clinical details on a single case and the ability to detect rare adrs ( < 1:1,000 ) compared with clinical trials . however , limitations of the system are well recognized and include incomplete reporting , the presence of reporting biases that may result in both under- and overascertainment , the lack of a denominator for balanced comparisons , and the inability to determine incidence rates ( irs ) from spontaneous reports . in this paper , we present an example of how disproportionate reporting of pancreatitis cases associated with the postmarket surveillance of mmx multi matrix system1(mmx ) mesalazine , a 5-aminosalicylic acid ( 5-asa ) formulation , was further investigated using a pharmacoepidemiology study . treatment with 5-asa is considered first - line therapy for patients with active mild to moderate ulcerative colitis ( uc ) . mmx mesalazine is a once - daily formulation of 5-asa administered orally that is effective in induction and maintenance of uc remission [ 58 ] . mesalazine is also available in the united states in several other formulations ( i.e. , controlled - release mesalazine , delayed - release mesalazine , extended - release mesalazine , balsalazide disodium , olsalazine sodium , sulfasalazine , or sulfasalazine delayed release ) . acute pancreatitis is an inflammatory disease of the pancreas characterized by severe acute upper abdominal pain and elevated blood levels of serum amylase and/or lipase . annual incidence of acute pancreatitis is estimated to be 32 cases per 100,000 persons in the general population . although rare , pancreatitis is an event associated with uc as well as with mesalazine treatment of uc [ 11 , 12 ] . as such , pancreatitis events were observed during clinical development and postmarketing experience of mmx mesalazine , and this class effect is listed in its us package insert . analysis of reports received by the manufacturer and entered into the postmarketing surveillance database revealed a higher rate of reporting for pancreatitis with mmx mesalazine ( 21.0 cases per 100,000 person - years of exposure ) compared with controlled - release mesalazine ( 1.3 cases per 100,000 person - years ) [ data on file ] . in addition , the us food and drug administration ( fda ) adverse event reporting system database also revealed this pattern of disproportionate reporting . however , due to the aforementioned reporting biases , the large differences in the time these drugs have been on the us market ( mmx mesalazine launch in 2007 , controlled - release mesalazine in 1993 , and delayed - release mesalazine in 1992 ) and other limitations in spontaneous reporting systems , further investigation was undertaken to determine whether the reporting data indicated a true association of pancreatitis with mmx mesalazine compared with other mesalazine formulations . a summary of the findings from the postmarketing spontaneous reporting data and the associated limitations in interpretation is presented in table 1.table 1postmarketing spontaneous reporting data for pancreatitis with mmx mesalazinefindings from spontaneous reporting systems shire global safety system [ data on file ] higher rate of reporting for pancreatitis with mmx mesalazine ( 21.0 cases per 100,000 person - years of exposure ) compared with another marketed controlled - release mesalazine formulation ( controlled - release mesalazine ; 1.3 cases per 100,000 person - years ) us fda adverse event reporting system ( aers ) database disproportionate reporting of pancreatitis for mmx mesalazine compared with controlled - release mesalazine and delayed - release mesalazine [ lower bound of the 95 % confidence interval of the empirical bayesian geometric mean ( eb05 ) of pancreatitis cases : 5.78 for mmx mesalazine compared with 2.05 for controlled - release mesalazine and 1.92 for delayed - release mesalazine]limitations in interpretation [ 2 , 3 ] reporting biases due to the difference in time on the us market of the compared drugs ( mmx mesalazine launch in 2007 , controlled - release mesalazine in 1993 , and delayed - release mesalazine in 1992 ) underreporting overall and differential underascertainment between drugs source population ( denominator ) is unknown , resulting in inability to calculate true incidence rates incomplete reporting of patient covariates required to adjust for confounding incomplete supporting data to validate or adjudicate endpoints fda food and drug administration fully validated safety database utilized for data entry , storage , and analysis of adverse event information received from sources including but not limited to healthcare practitioners , regulatory authorities , clinical trials , consumers , and the published medical literature . the database allows for aggregate report production and reporting of individual - case safety reports to regulatory authorities postmarketing spontaneous reporting data for pancreatitis with mmx mesalazine fda food and drug administration fully validated safety database utilized for data entry , storage , and analysis of adverse event information received from sources including but not limited to healthcare practitioners , regulatory authorities , clinical trials , consumers , and the published medical literature . the database allows for aggregate report production and reporting of individual - case safety reports to regulatory authorities one strategy to investigate whether spontaneous adr reports reflect a true association in the rate of pancreatitis with mmx mesalazine is to conduct a pharmacoepidemiology study with healthcare claims data . the use of electronic healthcare data to monitor drug safety has evolved in recent years , as evidenced by the fda s sentinel initiative , a long - term program designed to build and implement a national electronic system for monitoring the safety of fda - approved drugs . the purpose of this paper is to describe a pharmacoepidemiology study designed to evaluate a signal observed in spontaneous reporting data for acute pancreatitis in patients with uc using mmx mesalazine , and to report on the methods and findings of this study and how it overcame the limitations of the postmarketing reports . this retrospective cohort study analyzed us pharmacy and medical claims data from the thomson reuters ( truven health ) marketscan commercial and medicare supplemental claims databases . they are fully integrated patient - level databases covering healthcare delivery from the inpatient , outpatient , drug , and laboratory settings , as well as health risk assessment , and benefit design information from us commercially insured and medicare supplemental populations . the marketscan databases are compliant with the health insurance portability and accountability act of 1996 . patient data provided by truven health were de - identified ; therefore , per international society for pharmacoepidemiology guidelines on data privacy , medical record confidentiality , and research in the interest of public health , no internal review board approval or patient authorization was required . using these databases , patient and treatment information was obtained for all patients with any diagnosis of uc [ international classification of diseases , 9th revision , ( icd-9 ) 556 , 556.0 , 556.1 , 556.5 , 556.6 , 556.8 , and 556.9 ] between 1 july 2007 and 1 december 2010 . given the objective of this study , mmx mesalazine was considered the treatment of primary interest . comparator drugs consisted of all other branded orally administered mesalazine - containing medications indicated for uc . comparator 5-asa uc drugs were combined into one category , which was subdivided into categories according to the specific mechanism of 5-asa release : moisture dependent ( controlled - release mesalazine ) , ph - dependent ( delayed- and extended - release mesalazine ) , and azoreductase dependent ( balsalazide disodium , olsalazine sodium , sulfasalazine , and sulfasalazine delayed release ) . the comparator drugs were restricted to branded , orally administered uc medications ; suppositories and generic forms were not used as comparators . the rationale for these restrictions was to minimize potential bias when comparing mmx mesalazine ( available as branded and for oral administration only ) due to channelling of patients to older and less expensive uc drugs . the baseline period was defined as the 6-month interval prior to the initial dispensing of one of the study drugs . follow - up began on the index date , defined as the first day of initial dispensing of a study drug after the 6-month drug - free interval , and continued until treatment discontinuation of index mesalazine treatment , date of first pancreatitis event , disenrollment from the database , or the end of the study period ( 31 december 2010 ) . the main study outcome was acute pancreatitis ( icd-9 : 577.0 ) occurring within the exposure episode , which was defined as the period from the index date through 30 days following the days supply of the last prescription . discontinuation was defined by determining the days between each consecutive pair of prescriptions ; if this timeframe was longer than the days supply plus 30 days , then the patient was considered to have discontinued the index drug . additionally , after 30 days from the days supply of the last prescription of the index drug , the patient was also defined as discontinued . as patient follow - up was censored in parallel with index drug discontinuation , there was no risk of any patient having exposure to multiple study drugs during follow - up . per algorithms applied in prior database studies , acute pancreatitis must have been associated with a hospital / emergency room ( er ) visit [ 17 , 18 ] . based on the claims - based algorithm in another study that produced the highest positive predictive value ( 82.2 % ) for acute pancreatitis , if both chronic and acute pancreatitis were recorded in the same admission / er visit , the event was assumed to be chronic pancreatitis . eligible participants in this study were adults aged 18 years diagnosed with uc within 6 months prior to receiving one of the study drugs . patients were required to be new users of mmx mesalazine or a comparator 5-asa uc treatment and were categorized by the first mesalazine drug received . new users were defined as patients with one prescription or more for a study drug , with no prior use of mmx mesalazine or any of the comparator uc treatments in the previous 6 months , ensuring that the patient drug cohorts were mutually exclusive . the inclusion criteria used to identify patients were aligned with patient selection criteria in prior retrospective uc database studies [ 1922 ] . patient eligibility criteria for this analysis are provided in the supplementary table 1 . to control for confounding by indication , analyses were also conducted on a propensity - matched set , wherein each patient from the mmx mesalazine cohort was matched one to one with a comparator patient by decile of propensity scores ( i.e. , the probability of being prescribed mmx mesalazine versus comparator , regardless of actual drug received ) . mmx mesalazine users not matched to a comparator were excluded from the propensity - matched set . patient identification and the majority of analyses were conducted using sfetyworks software ( ubc corporation , harrisburg , pa , usa ) . data were processed into a common data model format prior to being loaded into the sfetyworks environment . because of limitations inherent in the sfetyworks software , certain analyses ( e.g. , calculations and model building involving dose , conducted only for mmx mesalazine patients and comparators remaining after propensity - score matching ) were conducted outside of the sfetyworks platform using sas , version 9.2 ( sas institute inc . , unadjusted irs were calculated as the number of acute pancreatitis cases per 1,000 person - years of follow - up ; unadjusted ir ratios ( irrs ) were calculated as the ratio of the mmx mesalazine ir to the comparator ir . as a measure of relative risk , an irr < 1.0 depicts lower incidence among patients receiving mmx mesalazine . mmx mesalazine irs and related irrs were further stratified individually by age and sex categories . prior to propensity - score matching , several sensitivity analyses were conducted by varying both the gap between refills ( i.e. , drug - persistence gap ) and length of the risk period after the last treatment episode ( interval at the end of the supply of the last prescription , within which a pancreatitis event would still be included in the study ) . these analyses were performed at 15 , 30 , and 60 days to evaluate the impact of assumptions about continuous treatment and the risk period to the unadjusted irs . adjusted irrs were estimated from the propensity - matched data set using a poisson regression model , with the log of follow - up time as an offset parameter . to achieve balance in baseline risk , each patient in the mmx mesalazine cohort was matched to a comparator by propensity scores within the same decile of the propensity - score - distribution . after matching , irr analyses comparing mmx mesalazine to comparators were conducted without further adjustment ( i.e. , propensity - score matched only ) and also using two further adjusted models ( to control for possible residual confounding ) . the first adjusted model incorporated age group ( 1829 , 3039 , 4049 , 50 + ) , gender , and continuous average daily dose measure . the second adjusted model also incorporated risk factors of acute pancreatitis , consistent with frossard et al . . of note , not all variables were included in the final model ; ones that were weakly associated in unadjusted models ( p > 0.5 ) and collinear variables were excluded . propensity - matched sets were used for all adjusted analyses , whereas the entire study sample was used for unadjusted ( crude ) analyses . for mmx mesalazine and comparators , the dose of each prescription was calculated on a per - patient basis as the number of pills dispensed multiplied by the medication strength in the propensity - matched set only . mmx mesalazine and comparators delayed - release , controlled - release , and extended - release mesalazine , as well as olsalazine , were considered to be in equivalent units of mesalazine . mesalazine conversion factors were used to equate doses of balsalazide and sulfasalazine with the other mesalazines : 6,750 mg balsalazide and 6,200 mg sulfasalazine were considered roughly equivalent to 2,400 mg mesalazine [ 24 , 25 ] . although it was later discovered that the conversion for sulfasalazine should have been 6,000 mg ( not 6,200 mg ) as equivalent to 2,400 mg mesalazine , this small error affected 4 % of all comparators and was deemed not to have had a meaningful impact on dose classification . after application of the mesalazine conversion factors , the average daily dose per treatment course for each patient was calculated as the cumulative dose divided by the total days of supply , and average daily doses were categorized as low ( < 1,500 mg / day ) , medium ( 1,5004,800 mg / day ) , or high ( > 4,800 mg / day ) . this retrospective cohort study analyzed us pharmacy and medical claims data from the thomson reuters ( truven health ) marketscan commercial and medicare supplemental claims databases . they are fully integrated patient - level databases covering healthcare delivery from the inpatient , outpatient , drug , and laboratory settings , as well as health risk assessment , and benefit design information from us commercially insured and medicare supplemental populations . the marketscan databases are compliant with the health insurance portability and accountability act of 1996 . patient data provided by truven health were de - identified ; therefore , per international society for pharmacoepidemiology guidelines on data privacy , medical record confidentiality , and research in the interest of public health , no internal review board approval or patient authorization was required . using these databases , patient and treatment information was obtained for all patients with any diagnosis of uc [ international classification of diseases , 9th revision , ( icd-9 ) 556 , 556.0 , 556.1 , 556.5 , 556.6 , 556.8 , and 556.9 ] between 1 july 2007 and 1 december 2010 . given the objective of this study , mmx mesalazine was considered the treatment of primary interest . comparator drugs consisted of all other branded orally administered mesalazine - containing medications indicated for uc . comparator 5-asa uc drugs were combined into one category , which was subdivided into categories according to the specific mechanism of 5-asa release : moisture dependent ( controlled - release mesalazine ) , ph - dependent ( delayed- and extended - release mesalazine ) , and azoreductase dependent ( balsalazide disodium , olsalazine sodium , sulfasalazine , and sulfasalazine delayed release ) . the comparator drugs were restricted to branded , orally administered uc medications ; suppositories and generic forms were not used as comparators . the rationale for these restrictions was to minimize potential bias when comparing mmx mesalazine ( available as branded and for oral administration only ) due to channelling of patients to older and less expensive uc drugs . the baseline period was defined as the 6-month interval prior to the initial dispensing of one of the study drugs . follow - up began on the index date , defined as the first day of initial dispensing of a study drug after the 6-month drug - free interval , and continued until treatment discontinuation of index mesalazine treatment , date of first pancreatitis event , disenrollment from the database , or the end of the study period ( 31 december 2010 ) . the main study outcome was acute pancreatitis ( icd-9 : 577.0 ) occurring within the exposure episode , which was defined as the period from the index date through 30 days following the days supply of the last prescription . discontinuation was defined by determining the days between each consecutive pair of prescriptions ; if this timeframe was longer than the days supply plus 30 days , then the patient was considered to have discontinued the index drug . additionally , after 30 days from the days supply of the last prescription of the index drug , the patient was also defined as discontinued . as patient follow - up was censored in parallel with index drug discontinuation , there was no risk of any patient having exposure to multiple study drugs during follow - up . per algorithms applied in prior database studies , acute pancreatitis must have been associated with a hospital / emergency room ( er ) visit [ 17 , 18 ] . based on the claims - based algorithm in another study that produced the highest positive predictive value ( 82.2 % ) for acute pancreatitis , if both chronic and acute pancreatitis were recorded in the same admission / er visit , the event was assumed to be chronic pancreatitis . eligible participants in this study were adults aged 18 years diagnosed with uc within 6 months prior to receiving one of the study drugs . patients were required to be new users of mmx mesalazine or a comparator 5-asa uc treatment and were categorized by the first mesalazine drug received . new users were defined as patients with one prescription or more for a study drug , with no prior use of mmx mesalazine or any of the comparator uc treatments in the previous 6 months , ensuring that the patient drug cohorts were mutually exclusive . the inclusion criteria used to identify patients were aligned with patient selection criteria in prior retrospective uc database studies [ 1922 ] . patient eligibility criteria for this analysis are provided in the supplementary table 1 . to control for confounding by indication , analyses were also conducted on a propensity - matched set , wherein each patient from the mmx mesalazine cohort was matched one to one with a comparator patient by decile of propensity scores ( i.e. , the probability of being prescribed mmx mesalazine versus comparator , regardless of actual drug received ) . mmx mesalazine users not matched to a comparator were excluded from the propensity - matched set . patient identification and the majority of analyses were conducted using sfetyworks software ( ubc corporation , harrisburg , pa , usa ) . data were processed into a common data model format prior to being loaded into the sfetyworks environment . because of limitations inherent in the sfetyworks software , certain analyses ( e.g. , calculations and model building involving dose , conducted only for mmx mesalazine patients and comparators remaining after propensity - score matching ) were conducted outside of the sfetyworks platform using sas , version 9.2 ( sas institute inc . , cary , nc , usa ) . unadjusted irs were calculated as the number of acute pancreatitis cases per 1,000 person - years of follow - up ; unadjusted ir ratios ( irrs ) were calculated as the ratio of the mmx mesalazine ir to the comparator ir . as a measure of relative risk , mmx mesalazine irs and related irrs were further stratified individually by age and sex categories . prior to propensity - score matching , several sensitivity analyses were conducted by varying both the gap between refills ( i.e. , drug - persistence gap ) and length of the risk period after the last treatment episode ( interval at the end of the supply of the last prescription , within which a pancreatitis event would still be included in the study ) . these analyses were performed at 15 , 30 , and 60 days to evaluate the impact of assumptions about continuous treatment and the risk period to the unadjusted irs . adjusted irrs were estimated from the propensity - matched data set using a poisson regression model , with the log of follow - up time as an offset parameter . to achieve balance in baseline risk , each patient in the mmx mesalazine cohort was matched to a comparator by propensity scores within the same decile of the propensity - score - distribution . after matching , irr analyses comparing mmx mesalazine to comparators were conducted without further adjustment ( i.e. , propensity - score matched only ) and also using two further adjusted models ( to control for possible residual confounding ) . the first adjusted model incorporated age group ( 1829 , 3039 , 4049 , 50 + ) , gender , and continuous average daily dose measure . the second adjusted model also incorporated risk factors of acute pancreatitis , consistent with frossard et al . . of note , not all variables were included in the final model ; ones that were weakly associated in unadjusted models ( p > 0.5 ) and collinear variables were excluded . propensity - matched sets were used for all adjusted analyses , whereas the entire study sample was used for unadjusted ( crude ) analyses . for mmx mesalazine and comparators , the dose of each prescription was calculated on a per - patient basis as the number of pills dispensed multiplied by the medication strength in the propensity - matched set only . mmx mesalazine and comparators delayed - release , controlled - release , and extended - release mesalazine , as well as olsalazine , were considered to be in equivalent units of mesalazine . mesalazine conversion factors were used to equate doses of balsalazide and sulfasalazine with the other mesalazines : 6,750 mg balsalazide and 6,200 mg sulfasalazine were considered roughly equivalent to 2,400 mg mesalazine [ 24 , 25 ] . although it was later discovered that the conversion for sulfasalazine should have been 6,000 mg ( not 6,200 mg ) as equivalent to 2,400 mg mesalazine , this small error affected 4 % of all comparators and was deemed not to have had a meaningful impact on dose classification . after application of the mesalazine conversion factors , the average daily dose per treatment course for each patient was calculated as the cumulative dose divided by the total days of supply , and average daily doses were categorized as low ( < 1,500 mg / day ) , medium ( 1,5004,800 mg / day ) , or high ( > 4,800 mg / day ) . a total of 14,936 patients ( 54 % male ) were identified in the study . nine ( 0.06 % ) patients were given prescriptions for both mmx mesalazine and a comparator mesalazine on their index date . these patients were included in the total cohort but excluded from both the mmx mesalazine and all - comparator subgroups . more than 10 % of patients with uc in the mmx mesalazine and 5-asa comparator groups were receiving other types of medication at baseline , including acetaminophen , potassium chloride , hydrocodone , metronidazole , other mesalazines , ciprofloxacin , and sodium bicarbonate . the most common preindex medical conditions present in patients at baseline were gastrointestinal inflammation , diarrhea , abdominal pain , rectal hemorrhage , essential hypertension , and hematochezia , each occurring in > 15% of patients in both the mmx mesalazine and comparator groups.table 2patient demographic and baseline characteristicspatient characteristicmmx mesalazine ( n = 4,751)all comparators ( n = 10,176)total ( n = 14,936)mean age ( median ) , years44.38 ( 44.0)47.44 ( 47.0)46.46 ( 46.0)age categories , n ( % ) 1819130 ( 2.7)187 ( 1.8)318 ( 2.1 ) 2029740 ( 15.6)1,367 ( 13.4)2,108 ( 14.1 ) 30391,031 ( 21.7)1,829 ( 18.0)2,862 ( 19.2 ) 40491,075 ( 22.6)2,172 ( 21.3)3,249 ( 21.8 ) 50591,013 ( 21.3)2,307 ( 22.7)3,322 ( 22.2 ) 6069532 ( 11.2)1,451 ( 14.3)1,984 ( 13.3 ) 7079164 ( 3.5)534 ( 5.2)698 ( 4.7 ) 8066 ( 1.4)329 ( 3.2)395 ( 2.6)gender , n ( % ) female2,528 ( 53.2)5,527 ( 54.3)8,060 ( 54.0 ) male2,223 ( 46.8)4,649 ( 45.7)6,876 ( 46.0)index date , n ( % ) year 20081,589 ( 33.4)3,958 ( 38.9)5,549 ( 37.2 ) year 20091,953 ( 41.1)3,608 ( 35.5)5,566 ( 37.3 ) year 20101,209 ( 25.4)2,610 ( 25.6)3,821 ( 25.6)common baseline pharmacotherapy ( > 10% of patients ) , n ( % ) acetaminophen904 ( 19.0)2,106 ( 20.7)3,010 ( 20.2 ) potassium chloride1,049 ( 22.1)1,827 ( 18.0)2,877 ( 19.3 ) hydrocodone650 ( 13.7)1,596 ( 15.7)2,246 ( 15.0 ) metronidazole627 ( 13.2)1,536 ( 15.1)2,165 ( 14.5 ) other mesalazine857 ( 18.0)1,035 ( 10.2)1,893 ( 12.7 ) ciprofloxacin546 ( 11.5)1,290 ( 12.7)1,839 ( 12.3 ) sodium bicarbonate560 ( 11.8)948 ( 9.3)1,508 ( 10.1)common pre - index medical conditions ( > 10% of patients ) , n ( % ) gastrointestinal inflammation1,851 ( 39.0)4,035 ( 39.7)5,891 ( 39.4 ) diarrhoea1,559 ( 32.8)3,353 ( 33.0)4,913 ( 32.9 ) abdominal pain1,110 ( 23.4)2,797 ( 27.5)3,910 ( 26.2 ) rectal hemorrhage1,111 ( 23.4)2,131 ( 20.9)3,245 ( 21.7 ) essential hypertension742 ( 15.6)2,047 ( 20.1)2,790 ( 18.7 ) hematochezia866 ( 18.2)1,666 ( 16.4)2,534 ( 17.0 ) gastrointestinal disorder584 ( 12.3)1,107 ( 10.9)1,691 ( 11.3 ) hyperlipidemia447 ( 9.4)1,166 ( 11.5)1,613 ( 10.8 ) hemorrhoids483 ( 10.2)1,007 ( 9.9)1,491 ( 10.0 ) benign colonic neoplasm485 ( 10.2)976 ( 9.6)1,463 ( 9.8 ) patient demographic and baseline characteristics the primary outcome measured was the occurrence of acute pancreatitis in patients receiving mmx mesalazine compared with those receiving comparator mesalazines . results from this analysis are provided in table 3 . from study - drug index date through 30 days postdiscontinuation , 20 and 46 acute pancreatitis cases associated with a hospital / er visit were identified for the mmx mesalazine and comparator groups , respectively , with 2,338 and 4,576 person - years of exposure , respectively ( table 3).table 3acute pancreatitis incidence rates ( ir ) and incident rate ratios ( irr ; crude and adjusted)characteristicmmx mesalazineall comparatorsirr ( 95 % ci ) crude rates events , n 2046 person - time , years2,338.24,576.1 ir ( 95 % ci ) 8.55 ( 5.5413.21)10.05 ( 7.5413.41)0.85 ( 0.481.47)age categories , years ir ( 95 % ci ) 182920.03 ( 10.1539.53)29.70 ( 18.7846.94)0.67 ( 0.251.63 ) 303910.68 ( 4.5625.00)10.27 ( 5.2020.27)1.04 ( 0.273.60 ) 40495.52 ( 1.8816.22)4.32 ( 1.6811.11)1.28 ( 0.197.55 ) 504.32 ( 1.6811.10)7.06 ( 4.3511.47)0.61 ( 0.151.89)gender , ir ( 95 % ci ) female10.91 ( 6.3718.66)8.94 ( 5.8413.66)1.22 ( 0.562.55 ) male6.11 ( 2.9612.61)11.23 ( 7.6116.58)0.54 ( 0.201.29)adjusted rates events , n 2021 person - time , years2,220.071,958.90 propensity - matched ir ( 95 % ci ) 9.01 ( 5.8113.96)10.72 ( 6.9916.44)0.84 ( 0.461.55 ) adjusted 1 ir ( 95 % ci ) 4.40 ( 1.949.98)5.62 ( 2.5712.31)0.78 ( 0.421.46 ) adjusted 2 ir ( 95 % ci ) 2.37 ( 0.916.15)3.10 ( 1.247.75)0.76 ( 0.411.43 ) age categories , years 1829 propensity - matched ir ( 95 % ci ) 21.43 ( 10.7242.86)26.77 ( 13.9351.45)0.80 ( 0.312.08 ) adjusted 1 ir ( 95 % ci ) 25.17 ( 10.5060.33)32.76 ( 15.1570.83)0.77 ( 0.292.01 ) adjusted 2 ir ( 95 % ci ) 22.77 ( 8.5260.86)30.66 ( 12.8972.96)0.74 ( 0.281.95 ) 3039 propensity - matched ir ( 95 % ci ) 11.10 ( 4.6226.67)12.16 ( 5.0629.22)0.91 ( 0.263.15 ) adjusted 1 ir ( 95 % ci ) 9.06 ( 1.7846.10)10.89 ( 3.0139.38)0.83 ( 0.213.23 ) adjusted 2 ir ( 95 % ci ) 3.61 ( 0.4230.63)4.35 ( 0.7824.29)0.83 ( 0.203.44 ) 4049 propensity - matched ir ( 95 % ci ) 5.80 ( 1.8717.99)4.80 ( 1.2019.17)1.21 ( 0.207.24 ) adjusted 1 ir ( 95 % ci ) 2.13 ( 0.2617.66)1.89 ( 0.2017.76)1.13 ( 0.186.99 ) adjusted 2 ir ( 95 % ci ) 1.42 ( 0.1315.19)1.25 ( 0.1015.80)1.13 ( 0.187.00 ) 50 propensity - matched ir ( 95 % ci ) 4.55 ( 1.7112.12)6.29 ( 2.6215.12)0.72 ( 0.192.69 ) adjusted 1 ir ( 95 % ci ) 3.56 ( 0.3932.19)4.60 ( 0.9921.25)0.77 ( 0.163.80 ) adjusted 2 ir ( 95 % ci ) 1.80 ( 0.1719.15)2.26 ( 0.3713.86)0.80 ( 0.163.97 ) gender female propensity - matched ir ( 95 % ci ) 11.52 ( 6.6919.83)6.95 ( 3.3214.59)1.66 ( 0.664.15 ) adjusted 1 ir ( 95 % ci ) 7.72 ( 3.0519.55)4.94 ( 1.8113.51)1.56 ( 0.614.00 ) adjusted 2 ir ( 95 % ci ) 3.58 ( 1.1211.46)2.35 ( 0.707.83)1.52 ( 0.593.92 ) male propensity - matched ir ( 95 % ci ) 6.41 ( 3.0613.46)14.70 ( 8.7124.82)0.44 ( 0.181.08 ) adjusted 1 ir ( 95 % ci ) 2.00 ( 0.547.45)4.70 ( 1.4315.38)0.43 ( 0.171.07 ) adjusted 2 ir ( 95 % ci ) 1.05 ( 0.225.10)2.52 ( 0.5810.98)0.42 ( 0.171.04 ) ci confidence interval mmx mesalazine relative to all comparators per 1000 person - years adjustment 1 , adjusted for age category , gender , and daily dose adjustment 2 , adjusted for age category , gender , daily dose , steroids , enalapril , viral herpes , furosemide , sulfasalazine , and post - surgery procedures acute pancreatitis incidence rates ( ir ) and incident rate ratios ( irr ; crude and adjusted ) ci confidence interval mmx mesalazine relative to all comparators per 1000 person - years adjustment 1 , adjusted for age category , gender , and daily dose adjustment 2 , adjusted for age category , gender , daily dose , steroids , enalapril , viral herpes , furosemide , sulfasalazine , and post - surgery procedures the overall crude ir [ 95 % confidence interval ( ci ) ] of acute pancreatitis in the mmx mesalazine group was 8.55 ( 5.5413.21 ) per 1,000 person - years compared with 10.05 ( 7.5413.41 ) per 1,000 person - years in the all comparators group . in regards to relative risk in the overall population , the crude irr ( 95 % ci ) was 0.85 ( 0.481.47 ) , suggesting that no difference in pancreatitis risk existed prior to adjustment between the mmx mesalazine and all comparator groups ( table 3 ) . among the comparator subgroups by 5-asa release mechanism , crude pancreatitis irs ( 95 % ci ) were lowest in the azoreductase - dependent group [ 5.11 ( 0.9028.92 ) per 1,000 person - years ] and highest in the moisture - dependent group [ 10.88 ( 2.9839.68 ) per 1,000 person - years ] . sensitivity analyses varying both the drug persistence gap and risk period between 15 , 30 , and 60 days had little influence on the overall incidence of acute pancreatitis in the mmx mesalazine group . similarly , the irrs of the mmx mesalazine and comparator group were relatively stable ( range 0.780.89 ; data not shown ) . of the 4,751 patients in the mmx mesalazine group , 4,499 ( 94.7 % ) patients were propensity - score matched to comparators . 20 acute pancreatitis cases were identified in the mmx mesalazine group , and 21 cases were identified in the all comparators group . prior to matching , propensity scores for the mmx mesalazine and the all - comparator groups were 0.341 and 0.305 , respectively , reflecting a 12 % difference . after matching , the all - comparator group had a mean propensity score of 0.340 , essentially unchanged for the mmx mesalazine group . of the 4,499 mmx mesalazine and 4,499 comparator propensity - score matched patients , 16 mmx mesalazine and 11 comparator patients were missing information needed to calculate average daily dose ; thus , the following adjusted incidence analyses were conducted on 4,483 mmx mesalazine and 4,488 comparator patients . the propensity - matched ir ( 95 % ci ) of acute pancreatitis among patients on mmx mesalazine [ 9.01 ( 5.8113.96 ) per 1,000 person - years ] was comparable with that of comparator 5-asa formulations [ 10.72 ( 6.9916.44 ) per 1,000 person - years ] . all propensity - score matched analyses yielded comparable irrs to the crude irr . from the model that incorporated propensity - score matching only , the irr ( 95 % ci ) for mmx mesalazine relative to all comparators was 0.84 ( 0.461.55 ) . propensity - score matched models further adjusted ( age , gender , dose ; adjusted 1 ) and fully adjusted ( steroids , enalapril , viral herpes , furosemide , sulfasalazine , and postsurgery procedures added ; adjusted 2 ) yielded irrs of 0.78 ( 0.421.46 ) and 0.76 ( 0.411.43 ) , respectively ( table 3 ) . comparisons between mmx mesalazine and comparator subgroups defined by 5-asa release mechanism were not possible in the adjusted analyses due to the limited number of events . mmx mesalazine users were almost always prescribed a medium average daily dose , whereas nearly one in five comparator drug users were prescribed a low daily dose . average daily dose was entered into the two further adjusted models that estimated relative risk . a total of 14,936 patients ( 54 % male ) were identified in the study . nine ( 0.06 % ) patients were given prescriptions for both mmx mesalazine and a comparator mesalazine on their index date . these patients were included in the total cohort but excluded from both the mmx mesalazine and all - comparator subgroups . more than 10 % of patients with uc in the mmx mesalazine and 5-asa comparator groups were receiving other types of medication at baseline , including acetaminophen , potassium chloride , hydrocodone , metronidazole , other mesalazines , ciprofloxacin , and sodium bicarbonate . the most common preindex medical conditions present in patients at baseline were gastrointestinal inflammation , diarrhea , abdominal pain , rectal hemorrhage , essential hypertension , and hematochezia , each occurring in > 15% of patients in both the mmx mesalazine and comparator groups.table 2patient demographic and baseline characteristicspatient characteristicmmx mesalazine ( n = 4,751)all comparators ( n = 10,176)total ( n = 14,936)mean age ( median ) , years44.38 ( 44.0)47.44 ( 47.0)46.46 ( 46.0)age categories , n ( % ) 1819130 ( 2.7)187 ( 1.8)318 ( 2.1 ) 2029740 ( 15.6)1,367 ( 13.4)2,108 ( 14.1 ) 30391,031 ( 21.7)1,829 ( 18.0)2,862 ( 19.2 ) 40491,075 ( 22.6)2,172 ( 21.3)3,249 ( 21.8 ) 50591,013 ( 21.3)2,307 ( 22.7)3,322 ( 22.2 ) 6069532 ( 11.2)1,451 ( 14.3)1,984 ( 13.3 ) 7079164 ( 3.5)534 ( 5.2)698 ( 4.7 ) 8066 ( 1.4)329 ( 3.2)395 ( 2.6)gender , n ( % ) female2,528 ( 53.2)5,527 ( 54.3)8,060 ( 54.0 ) male2,223 ( 46.8)4,649 ( 45.7)6,876 ( 46.0)index date , n ( % ) year 20081,589 ( 33.4)3,958 ( 38.9)5,549 ( 37.2 ) year 20091,953 ( 41.1)3,608 ( 35.5)5,566 ( 37.3 ) year 20101,209 ( 25.4)2,610 ( 25.6)3,821 ( 25.6)common baseline pharmacotherapy ( > 10% of patients ) , n ( % ) acetaminophen904 ( 19.0)2,106 ( 20.7)3,010 ( 20.2 ) potassium chloride1,049 ( 22.1)1,827 ( 18.0)2,877 ( 19.3 ) hydrocodone650 ( 13.7)1,596 ( 15.7)2,246 ( 15.0 ) metronidazole627 ( 13.2)1,536 ( 15.1)2,165 ( 14.5 ) other mesalazine857 ( 18.0)1,035 ( 10.2)1,893 ( 12.7 ) ciprofloxacin546 ( 11.5)1,290 ( 12.7)1,839 ( 12.3 ) sodium bicarbonate560 ( 11.8)948 ( 9.3)1,508 ( 10.1)common pre - index medical conditions ( > 10% of patients ) , n ( % ) gastrointestinal inflammation1,851 ( 39.0)4,035 ( 39.7)5,891 ( 39.4 ) diarrhoea1,559 ( 32.8)3,353 ( 33.0)4,913 ( 32.9 ) abdominal pain1,110 ( 23.4)2,797 ( 27.5)3,910 ( 26.2 ) rectal hemorrhage1,111 ( 23.4)2,131 ( 20.9)3,245 ( 21.7 ) essential hypertension742 ( 15.6)2,047 ( 20.1)2,790 ( 18.7 ) hematochezia866 ( 18.2)1,666 ( 16.4)2,534 ( 17.0 ) gastrointestinal disorder584 ( 12.3)1,107 ( 10.9)1,691 ( 11.3 ) hyperlipidemia447 ( 9.4)1,166 ( 11.5)1,613 ( 10.8 ) hemorrhoids483 ( 10.2)1,007 ( 9.9)1,491 ( 10.0 ) benign colonic neoplasm485 ( 10.2)976 ( 9.6)1,463 ( 9.8 ) patient demographic and baseline characteristics the primary outcome measured was the occurrence of acute pancreatitis in patients receiving mmx mesalazine compared with those receiving comparator mesalazines . results from this analysis are provided in table 3 . from study - drug index date through 30 days postdiscontinuation , 20 and 46 acute pancreatitis cases associated with a hospital / er visit were identified for the mmx mesalazine and comparator groups , respectively , with 2,338 and 4,576 person - years of exposure , respectively ( table 3).table 3acute pancreatitis incidence rates ( ir ) and incident rate ratios ( irr ; crude and adjusted)characteristicmmx mesalazineall comparatorsirr ( 95 % ci ) crude rates events , n 2046 person - time , years2,338.24,576.1 ir ( 95 % ci ) 8.55 ( 5.5413.21)10.05 ( 7.5413.41)0.85 ( 0.481.47)age categories , years ir ( 95 % ci ) 182920.03 ( 10.1539.53)29.70 ( 18.7846.94)0.67 ( 0.251.63 ) 303910.68 ( 4.5625.00)10.27 ( 5.2020.27)1.04 ( 0.273.60 ) 40495.52 ( 1.8816.22)4.32 ( 1.6811.11)1.28 ( 0.197.55 ) 504.32 ( 1.6811.10)7.06 ( 4.3511.47)0.61 ( 0.151.89)gender , ir ( 95 % ci ) female10.91 ( 6.3718.66)8.94 ( 5.8413.66)1.22 ( 0.562.55 ) male6.11 ( 2.9612.61)11.23 ( 7.6116.58)0.54 ( 0.201.29)adjusted rates events , n 2021 person - time , years2,220.071,958.90 propensity - matched ir ( 95 % ci ) 9.01 ( 5.8113.96)10.72 ( 6.9916.44)0.84 ( 0.461.55 ) adjusted 1 ir ( 95 % ci ) 4.40 ( 1.949.98)5.62 ( 2.5712.31)0.78 ( 0.421.46 ) adjusted 2 ir ( 95 % ci ) 2.37 ( 0.916.15)3.10 ( 1.247.75)0.76 ( 0.411.43 ) age categories , years 1829 propensity - matched ir ( 95 % ci ) 21.43 ( 10.7242.86)26.77 ( 13.9351.45)0.80 ( 0.312.08 ) adjusted 1 ir ( 95 % ci ) 25.17 ( 10.5060.33)32.76 ( 15.1570.83)0.77 ( 0.292.01 ) adjusted 2 ir ( 95 % ci ) 22.77 ( 8.5260.86)30.66 ( 12.8972.96)0.74 ( 0.281.95 ) 3039 propensity - matched ir ( 95 % ci ) 11.10 ( 4.6226.67)12.16 ( 5.0629.22)0.91 ( 0.263.15 ) adjusted 1 ir ( 95 % ci ) 9.06 ( 1.7846.10)10.89 ( 3.0139.38)0.83 ( 0.213.23 ) adjusted 2 ir ( 95 % ci ) 3.61 ( 0.4230.63)4.35 ( 0.7824.29)0.83 ( 0.203.44 ) 4049 propensity - matched ir ( 95 % ci ) 5.80 ( 1.8717.99)4.80 ( 1.2019.17)1.21 ( 0.207.24 ) adjusted 1 ir ( 95 % ci ) 2.13 ( 0.2617.66)1.89 ( 0.2017.76)1.13 ( 0.186.99 ) adjusted 2 ir ( 95 % ci ) 1.42 ( 0.1315.19)1.25 ( 0.1015.80)1.13 ( 0.187.00 ) 50 propensity - matched ir ( 95 % ci ) 4.55 ( 1.7112.12)6.29 ( 2.6215.12)0.72 ( 0.192.69 ) adjusted 1 ir ( 95 % ci ) 3.56 ( 0.3932.19)4.60 ( 0.9921.25)0.77 ( 0.163.80 ) adjusted 2 ir ( 95 % ci ) 1.80 ( 0.1719.15)2.26 ( 0.3713.86)0.80 ( 0.163.97 ) gender female propensity - matched ir ( 95 % ci ) 11.52 ( 6.6919.83)6.95 ( 3.3214.59)1.66 ( 0.664.15 ) adjusted 1 ir ( 95 % ci ) 7.72 ( 3.0519.55)4.94 ( 1.8113.51)1.56 ( 0.614.00 ) adjusted 2 ir ( 95 % ci ) 3.58 ( 1.1211.46)2.35 ( 0.707.83)1.52 ( 0.593.92 ) male propensity - matched ir ( 95 % ci ) 6.41 ( 3.0613.46)14.70 ( 8.7124.82)0.44 ( 0.181.08 ) adjusted 1 ir ( 95 % ci ) 2.00 ( 0.547.45)4.70 ( 1.4315.38)0.43 ( 0.171.07 ) adjusted 2 ir ( 95 % ci ) 1.05 ( 0.225.10)2.52 ( 0.5810.98)0.42 ( 0.171.04 ) ci confidence interval mmx mesalazine relative to all comparators per 1000 person - years adjustment 1 , adjusted for age category , gender , and daily dose adjustment 2 , adjusted for age category , gender , daily dose , steroids , enalapril , viral herpes , furosemide , sulfasalazine , and post - surgery procedures acute pancreatitis incidence rates ( ir ) and incident rate ratios ( irr ; crude and adjusted ) ci confidence interval mmx mesalazine relative to all comparators per 1000 person - years adjustment 1 , adjusted for age category , gender , and daily dose adjustment 2 , adjusted for age category , gender , daily dose , steroids , enalapril , viral herpes , furosemide , sulfasalazine , and post - surgery procedures the overall crude ir [ 95 % confidence interval ( ci ) ] of acute pancreatitis in the mmx mesalazine group was 8.55 ( 5.5413.21 ) per 1,000 person - years compared with 10.05 ( 7.5413.41 ) per 1,000 person - years in the all comparators group . in regards to relative risk in the overall population , the crude irr ( 95 % ci ) was 0.85 ( 0.481.47 ) , suggesting that no difference in pancreatitis risk existed prior to adjustment between the mmx mesalazine and all comparator groups ( table 3 ) . among the comparator subgroups by 5-asa release mechanism , crude pancreatitis irs ( 95 % ci ) were lowest in the azoreductase - dependent group [ 5.11 ( 0.9028.92 ) per 1,000 person - years ] and highest in the moisture - dependent group [ 10.88 ( 2.9839.68 ) per 1,000 person - years ] . sensitivity analyses varying both the drug persistence gap and risk period between 15 , 30 , and 60 days had little influence on the overall incidence of acute pancreatitis in the mmx mesalazine group . similarly , the irrs of the mmx mesalazine and comparator group were relatively stable ( range 0.780.89 ; data not shown ) . of the 4,751 patients in the mmx mesalazine group , 4,499 ( 94.7 % ) patients were propensity - score matched to comparators . 20 acute pancreatitis cases were identified in the mmx mesalazine group , and 21 cases were identified in the all comparators group . prior to matching , propensity scores for the mmx mesalazine and the all - comparator groups were 0.341 and 0.305 , respectively , reflecting a 12 % difference . after matching , the all - comparator group had a mean propensity score of 0.340 , essentially unchanged for the mmx mesalazine group . of the 4,499 mmx mesalazine and 4,499 comparator propensity - score matched patients , 16 mmx mesalazine and 11 comparator patients were missing information needed to calculate average daily dose ; thus , the following adjusted incidence analyses were conducted on 4,483 mmx mesalazine and 4,488 comparator patients . the propensity - matched ir ( 95 % ci ) of acute pancreatitis among patients on mmx mesalazine [ 9.01 ( 5.8113.96 ) per 1,000 person - years ] was comparable with that of comparator 5-asa formulations [ 10.72 ( 6.9916.44 ) per 1,000 person - years ] . all propensity - score matched analyses yielded comparable irrs to the crude irr . from the model that incorporated propensity - score matching only , the irr ( 95 % ci ) for mmx mesalazine relative to all comparators was 0.84 ( 0.461.55 ) . propensity - score matched models further adjusted ( age , gender , dose ; adjusted 1 ) and fully adjusted ( steroids , enalapril , viral herpes , furosemide , sulfasalazine , and postsurgery procedures added ; adjusted 2 ) yielded irrs of 0.78 ( 0.421.46 ) and 0.76 ( 0.411.43 ) , respectively ( table 3 ) . comparisons between mmx mesalazine and comparator subgroups defined by 5-asa release mechanism were not possible in the adjusted analyses due to the limited number of events . mmx mesalazine users were almost always prescribed a medium average daily dose , whereas nearly one in five comparator drug users were prescribed a low daily dose . average daily dose was entered into the two further adjusted models that estimated relative risk . the impetus for conducting this pharmacoepidemiology study was a signal of disproportionate reporting of postmarketing pancreatitis cases , suggesting a potentially elevated risk of pancreatitis for mmx mesalazine compared with other mesalazine formulations used to treat uc . because spontaneous reporting data lack complete case ascertainment and defined patient populations in which to compute irs and are thus subject to reporting biases , further evaluation of this possible relationship was warranted . the primary objective of this pharmacoepidemiology study was to formally evaluate whether the risk of pancreatitis among patients with uc using mmx mesalazine differed from the risk among similar patients using other branded orally administered mesalazine drugs . results from this analysis demonstrated that the adjusted irr was 0.76 ( 0.411.43 ) , which suggests that the risk for pancreatitis is not elevated for mmx mesalazine . in estimating this relative risk , confounding factors controlled for in the pharmacoepidemiology study , but not in the reporting data , included the calendar period under comparison , age and gender of the patient , and dose , as well as baseline comorbidity and concomitant medication . acute pancreatitis is a multifactorial condition with many known causes or combination of causes , such as gallstones , alcohol use , hypertriglyceridemia , and > 500 implicated medications . even for suspected drug - induced pancreatitis , it is proposed that drugs are most likely a trigger for pancreatitis in patients with other risk factors . therefore , given the nonspecificity of mesalazine as a cause of pancreatitis , and that this study was comparing mmx mesalazine to other mesalazine drugs to determine whether mmx mesalazine was associated with a further elevation in risk , it was extremely important to control for the multitude of competing risk factors , most prominently , concomitant medications . the spontaneous reports did not allow for this control , whereas in the pharmacoepidemiology study , some of these factors were used to adjust relative risk estimates . the other prominent bias in the spontaneous reporting data for mmx mesalazine and pancreatitis was reporting bias for mmx mesalazine due to the webber effect . this bias was likely present due to the different lengths of time on the us market between mmx mesalazine ( 2007 ) and the comparator drugs controlled - release mesalazine ( 1993 ) and delayed - release mesalazine ( 1992 ) . it has been well established that newer drugs are monitored more closely for adverse effects ( aes ) and that those aes are more likely to be reported than medications that have been in long - term use . whereas healthcare claims data sources are subject to selection biases related to the population recorded , there is no reporting bias affecting the recording of an adverse event . this pharmacoepidemiology study was an effective means for eliminating that hurdle , which was likely present in the spontaneous reporting data . despite the methodological advantages of using a pharmacoepidemiologic study to evaluate a signal generated via spontaneous reporting pancreatitis is a rare event , so despite the inclusion of > 4,000 patients in each treatment group , adjusted effect estimates were somewhat imprecise . however , the fully adjusted model revealed an irr point estimate of 0.76 , with an upper bound of the 95 % ci of < 1.5 , which , if valid , suggests that any increase in risk is very likely to be small . another potential limitation is that the accuracy of icd-9 codes used to detect cases of acute pancreatitis may be questioned , as case status was not validated in hospital records . however , prior research has demonstrated that the ability to properly identify acute pancreatitis using administrative claims data is good ; the icd-9-cm 557.0 codes for acute pancreatitis have demonstrated high sensitivity ( 93 % ) and good specificity ( 72 % ) when recorded as the primary diagnosis . the algorithm we used to define an acute pancreatitis event was similar , suggesting that diagnostic miscoding was likely minimal . however , because alcohol consumption may not be validly or reliably recorded in healthcare claims data sources , it was excluded from our analysis so as not to introduce bias due to differences in ascertainment . because alcohol consumption is not a contraindication for mmx mesalazine or comparator drugs , it was assumed that the groups were fairly balanced on this risk factor ; however , without data , this assumption can not be confirmed . the signal of disproportionate reporting of pancreatitis for patients treated with mmx mesalazine in relation to other mesalazine medications was not confirmed by a formal pharmacoepidemiology study . by using healthcare claims data ( that capture healthcare encounters regardless of whether they were reported as adverse events ) , the study eliminated potential spontaneous reporting bias due to time between launch of mmx mesalazine relative to comparator drugs . in addition , the study controlled confounding due to risk factors for pancreatitis ( e.g. , disease under treatment , concomitant medications , and comorbid conditions ) by propensity - score matching and multivariate modelling . these findings demonstrate the value of pharmacoepidemiology studies for evaluating a drug s postmarket safety profile when confronted with spontaneous reporting data suggestive of a safety issue . lr contributed to the conception and design of this study , collaborated on analysis and data interpretation , and actively contributed to and approved the final manuscript version . gs contributed to the day - to - day management of the project , including conceptualization of study methods , preparation of study protocol / statistical analysis plan , conduction of analysis , interpretation / review of analysis , and preparation and review of the manuscript . mhg contributed to the study rationale , background data on spontaneous reporting , uc , pancreatitis , and drug - induced pancreatitis ; and identification of covariates . ps supported the concept development , contributed to study design , endpoint development , and interpretation of results for the mmx mesalazine / pancreatitis evaluation . sr contributed to the study conception and data review and was involved in revising the article . authors leo russo , margarita gardiner , paul streck , and susan rosen are employees of shire and hold stock and/or stock options in shire . authors gary schneider and stephan lanes , respectively , are current and former employees of evidera , which received funding from shire for assistance with study design , data analysis , and preparation of the study report and manuscript . although the sponsor was involved in the design , collection , analysis , interpretation , and fact checking of information , the content of this manuscript , the ultimate interpretation , and the decision to submit it for publication in european journal of clinical pharmacology was made by the authors independently .

, we will discuss the experience of diagnosis and treatment of ovarian cyst in infants at 3 months . the approval for the study was obtained from the institutional review board of the hospital of wuhan medical and health center for women and children . a retrospective analysis of 20 infants who suffered from ovarian cyst and received surgical treatment in our hospital from january 2009 to december 2012was done [ as shown in table 1 ] . all the patients underwent b - ultrasonic , computed tomography / magnetic resonance imaging ( ct / mri ) , and tumor marker examination . according to the medical history , all these lesions were preliminarily identified as benign , while the origin can not be determined . preoperative imaging examinations of seven cases indicated simple cyst [ as shown in figure 1a and b ] . the inspection was achieved by laparotomy with an abdominal transverse cleavage line incision in the hypogastric region in four cases . and in the remaining three cases , the inspection was achieved by three - port laparoscopy . the selection of the surgical procedure depends mainly on the choice made by parents as well as the factors , such as size of cyst , operational space of the abdominal cavity , and the general conditions of the patients . as soon as a simple ovarian cyst was confirmed during the operation [ as shown in figure 1a and b ] , fenestration was conducted to drain the cyst fluid . all the seven cases were diagnosed with ovarian follicular cyst on the pathological examination [ as shown in figure 1c and d ] . according to the conventional b - ultrasonic examination , at 1 month , 3 months , and 1 year postoperatively , ovary on the affected side developed normally in all the seven cases . preoperative mri / ct examination indicated quasi - circular cystic low - density lump image , and it could not be distinguished from intestinal duplication and mesenteric cyst ( a and b ) that is proved to be an ovarian cyst during the surgery . at an early stage , laparotomy and fenestration were conducted for the cases ; at the late period , the surgery was completed under trans - umbilical double - port laparoscope for the cases ( a and b ) . the postoperative pathological result indicated simple ovarian cyst ( c and d ) preoperative imaging examinations of 13 cases indicated solid cystic ingredient . for seven cases , inspections were done by making incision of abdominal transverse cleavage line in the hypogastric region . for six cases , the inspections were achieved by three - port laparoscopy . during the surgery , it was discovered that all the cysts showed pedicle torsion with varying degrees ( 360-1,080 ) . ovarian necrosis was revealed in four cases [ figure 2a c , d and f ] . two cases had intracystic hemorrhage , only obsolete hematocele drained out when the cystic wall was cut open [ figure 2b and e ] , but no tumor constituent was found . ovariectomy was performed in the four patients who had ovarian necrosis . as for the rest of the patients , the cystic wall was cut open , followed by embrocating with carbolic acid , alcohol , and normal saline . without resection of the cystic wall , the ovarian tissue was retained as far as possible . as per pathological examination after the surgery , the ovarian cyst was accompanied by obsolete hemorrhage , calcification , and granuloma [ as shown in figure 2 g and h ] . according to the findings of conventional b - ultrasonic examination , at 1 month , 3 months , and 1 year after the surgery , no ovary was seen on the affected side in six cases , blood supply on the affected side was worse than that on the opposite side in two cases , and the ovary on the affected side of the other five cases developed almost normally . at the follow - up , preoperative mri / ct indicated the following symptoms : mixed ingredients in the cyst and hemorrhage ( a ) , calcification ( c , d , and f ) , a large number of sediments ( b , d , and e ) , and separations in the cyst ( d ) . this was a complicated cyst that should be distinguished from the intestinal duplication , mesenteric cyst , and teratoma . it is proved to be an ovarian cyst during the surgery . at an early stage , laparotomy and enucleation were conducted for the cases ; at the late period , the surgery was performed by three - port laparoscopy technique in abdomen for the cases [ figure 1a f ] . the postoperative pathological result ( 2 g and h ) indicated ovarian cyst disease accompanied by obsolete hemorrhage , calcification , and granuloma during the follow - up , no ovary was seen on the affected side in six cases . according to the preoperative imaging examinations [ figure 2a f ] of the infants ( mri / ct examination ) , the cyst that was often accompanied by bleeding , calcification , necrosis , cyst wall thickening , and a large number of sediments and separations in the cyst was a complicated cyst . d and f ] in the surgery , and cyst bleeding appeared in two cases [ figure 2b and e ] . the formation mechanism of ovarian cyst is still unknown , and most scholars consider that it originates from mature follicle . when the mother suffers from gestational diabetes mellitus , blood poisoning , and rhesus ( rh ) isoimmunization , the placenta tissue might secrete excessive -human chorionic gonadotropin ( hcg ) that will result in ovarian cyst in the fetus . most ovarian cysts can be alleviated automatically with the reduction of estrogen level after the babies are born . the harm caused by ovarian cyst to human body is mainly triggered by complications , and the incidence rate of complications is 36 - 71% . the common complications include pedicle torsion and cyst bleeding , and necrosis of ovary and fallopian tube will be caused under severe cases . pedicle torsion of ovarian cyst is a common complication that can occur easily to cyst larger than 50 mm . some literature report that pedicle torsion rate of ovarian cyst is 25% . in this study , the incidence rate of ovarian cyst accompanied by pedicle torsion was 59% ( 13/22 ) . this might be related to the conservative therapeutic method adopted at an early stage as well as the poor compliance of infants at the time of follow - up visit . clinically , b - ultrasonic examination is the first choice for ovarian cyst . once there is a possibility of torsion , mri / ct examination is adopted . it is very important to judge the cyst nature and pedicle torsion . in this study , among the cases that lost ovary on the affected side , the following symptoms were noted on preoperative mri / ct examination : mixed ingredients in the cyst , hemorrhage , calcification , or necrosis . the torsional ovary is often not restored on time during the pedicle torsion and the inducement of causing ovarian torsion is not removed and these may lead to poor prognosis of ovarian cyst . nevertheless , most scholars consider that surgical treatment is required as long as there is a symptom , infants have no self - expression ability , so even pedicle torsion and ovarian necrosis can hardly be discovered . other scholars believe that there is a possibility for ovarian cyst to be relieved automatically and the operation wound is severe , so only asymptomatic cyst larger than 40 mm or 50 mm needs surgery . in this study , the ovarian cysts of two cases that are smaller than 21 mm still underwent intracystic hemorrhage and pedicle torsion , and necrosis of the ovary was detected during surgery . the observation method was conducted in 10 infants with ovarian cyst , while no surgical treatment was performed . as a result , four cases lost ovary on one side and one case lost ovary on both sides . by contrast , fenestration was conducted in seven infants with simple ovarian cyst , which helped in avoiding severe bad consequences , such as ovarian necrosis , caused by cyst bleeding and pedicle torsion . with the development of laparoscopy in pediatric surgery , some scholars have reported successful treatment for ovarian cyst under laparoscopy . at the late period , this study also adopted three - port laparoscopy , and three small incisions at 3 mm and 5 mm around the navel as well as 5 mm in the hypogastric region were taken . compared with traditional laparotomy , laparoscopic surgery has small trauma and the postoperative scar is not obvious . once the surgical treatment is decided for ovarian cyst , individualized treatment is selected for different types cysts . seven cases of this study suffered from simple ovarian cyst , so an incision of 5 mm 5 mm was taken at the top for pathological examination . there are some difficulties in operation for ovarian cyst accompanied by cyst bleeding , large tumor volume , and mixed cyst fluid ingredients . in our study , puncture needle was used to puncture and suck some cyst fluids on the abdominal wall near the cyst by avoiding blood vessels on the abdominal wall under the vision of laparoscope , and then the cyst was stripped . after the cyst wall was completely stripped , there was no need to completely strip the ovary side and the ovarian tissue should be retained as far as possible , so as to avoid influence on the infant 's hormone level in the future . the hemorrhagic spot was examined carefully and electric coagulation hemostasis was carried out with ultrasonic scalpel . in conclusion , a positive intervention should be adopted for the treatment of ovarian cyst in infants , and laparoscope can be used as an effective method . based on our results , such a guideline for the management of ovarian cyst in infants this study has some shortcomings ( the number of cases is less , the follow - up time is short , etc . ) , which we will overcome in the next research . algorithm for the management of ovarian cyst in infants there are no conflicts of interest . yxq , znn , yl performed the data analyses and wrote the manuscript.yjun , dxf , qxk helped perform the analysis with constructive discussions .

nitrous oxide ( n2o ) is the thirdmostimportant , longlived greenhouse gas , after carbon dioxide and methane [ myhre et al . , these topthree greenhouse gases have a mixture of natural and anthropogenic sources , and the challenge has always been to quantify the human perturbation to their budgets [ prather et al . , 2012 ; ciais et al . , surface emissions of n2o have an average atmospheric residence time of a century or more [ ko et al . , 1991 ; world meteorological organization ( wmo ) , 2014 ] , and thus , the current imbalance between emissions and atmospheric loss , as quantified by the observed growth rate of 3.8 tg n / yr ( 0.8 ppb / yr ) , is expected to lead to further increases in n2o abundances over this century , even if anthropogenic emissions are stabilized . anthropogenic emissions of n2o are driven primarily by fertilizer use and the handling of animal wastes , sources difficult both to control and to quantify [ galloway et al . , 2008 ; estimates of n2o emissions from summing individual sources have greater than factor of 2 uncertainties [ prather et al . , 2009 ; ciais et al . , 2013 ] , and atmospheric inversions rely on the accuracy of stratospheric losses and emission patterns [ thompson et al . , scenarios for future n2o levels make assumptions about both presentday and preindustrial n2o lifetimes to determine the anthropogenicnatural balance in emissions levels [ meinshausen et al . , 2011 ; kirtman et al . , thus , the uncertainties in the current budget and 21st century projections of n2o are its lifetime and the feedback that an n2o perturbation has on its lifetime [ prather , 1998 ] . assessment of the lifetime and feedback has been notably absent from climate and ozone assessments [ intergovernmental panel on climate change ( ipcc ) , 2007 ; wmo , 2011 ; ipcc , 2013a ] but has received more attention in the past couple years [ stratospheric processes and their role in climate ( sparc ) , 2013 ; chipperfield et al . , 2014 ; wmo , 2014 ] . in this paper we combine satellite measurements of n2o , o3 , and temperature ( t ) from the aura microwave limb sounder ( mls ) instrument [ lambert et al . , 2007 ; 2008 ] with global chemistry models to derive a semiempirical ( measured ) lifetime for n2o . previous studies have been done with less extensive satellite data [ ko et al . , 1991 ; minschwaner et al . , 1998 , 2013 ] . this lifetime is constrained by the observation of all quantities controlling n2o loss , is independent of transport uncertainties , and includes seasonal and yeartoyear variability . the only chemical modeling involves photolysis rates for n2o and o3 and the kinetics of the o(d ) reactions . uncertainties in this semiempirical lifetime are due only to possible errors in these chemical rates and in the measured profiles of n2o , o3 , and t in the critical region ( 330 hpa in the tropics ) . four chemistrytransport models ( ctms ) using meteorological data for the aura period are compared with monthly mls observations of stratospheric n2o and o3 , the n2o losses derived therefrom , and stratospheric noy from the environmental satellite michelson interferometer for passive atmospheric sounding ( mipas ) [ funke et al . , 2014 ] in order to understand the cause of model errors in n2o lifetime . in addition , two chemistryclimate models ( ccms , one 3d and one 2d ) with climatedriven circulations are also evaluated against the average mlsderived lifetime . the models evaluated here overestimate the semiempirical lifetime , 116 9 years , by a wide range , but agree on the negative sensitivity of lifetime to burden of 0.065 0.010 ( not constrained by observations ) that results in a lower residence time for n2o perturbations of 109 years . a best estimate for preindustrial conditions gives a longer lifetime of 123 years due primarily to reduced photochemical rates for n2o loss because of increased midstratospheric and upper stratospheric o3 caused by reduced levels of chlorine and n2o . results from the mls data and the constrained photochemical models are described in section 2 . the ctms , their calculation of n2o lifetimes , and their comparison with stratospheric constituents and the constrained n2o loss are presented in section 3 . in section 4 , four models are used to estimate the reduction in lifetime when n2o increases and the change in lifetime since the preindustrial era . an overview of issues related to n2o lifetime is discussed in section 5 . the mls instrument on the nasa aura satellite provides highly accurate , wellresolved latitudebyaltitude colocated profiles of n2o , o3 , and t throughout the stratosphere [ lambert et al . we use monthly mean mls data sets from august 2004 to december 2010 ( version 2.2 for o3 and version 3.3 for n2o and t ) . profiles are averaged longitudinally and within 5 latitude bins from 85s80s to 80n85n ( 34 in total ; mls observations are limited to 82s82n ) . for global budgets , the 8085 latitude bins were extended to 90. there are six standard pressure levels per decade in pressure ( i.e. , 100 , 68 , 46 , 32 , 22 , 15 , 10 , up to 0.10 hpa ) . these data sets are very similar to the monthly mean data records produced for the global ozone chemistry and related trace gas data records for the stratosphere project [ froidevaux et al . , the vertical profiles of o3 and t define all the conditions needed to calculate the profiles of n2o loss and noy production from the following reactions ( ( r1)(r5 ) ) . the rates of these reactions respond primarily to overhead o3 column and t. o3+hv=>o1d+o2 o1d+n2,o2=>o+n2,o2 n2o+hv=>n2+o1d n2o+o1d=>n2+o2 n2o+o1d=>no+no we use two independent photochemical submodels , one from nasa goddard space flight center ( gsfc , embedded in the g2d model ) and one from university of california , irvine ( embedded in the uci ctm ; see table 1 ) . these submodels solve the 1d radiative transfer problem to calculate jn2o ( reaction ( r3 ) ) and o(d ) densities ( reactions ( ( r1 ) and ( r2 ) ) and are documented in photolysis studies [ olson et al . , 1997 ; photocomp , 2010 ] . while the methodologies and numerical approaches for both are independent , they ( and all the chemistrytransport models here ) adopt standard values for the cross sections ( o2 , o3 , and n2o ) and rate coefficients controlling these reactions [ sander et al . the uci version ( mlsu ) uses the standard mls levels ( 2.7 km ) and integrates monthly , latituderesolved losses from 0.10 hpa to 100 hpa . the gsfc version ( mlsg ) interpolates and fills the mls data and integrates at 1 km resolution through the atmosphere . given the linear n2o profile in the dominant loss region ( figures 1a and 2a ) , the integrated loss should not be sensitive to interpolation methods . from mlsg we find that about 2% of n2o loss occurs at altitudes below 100 hpa . from previous studies with the uci ctm [ prather and hsu , 2010 ] we found thus , n2o losses from mlsu are corrected upward by 2% , and those from the uci ctm ( running here without tropospheric chemistry ) are corrected upward by 1% . measurements and models multiyear annual average loss of n2o plotted against ( a ) pressure altitude ( z * km , units tg n / yr / km ) and ( b ) latitude ( degrees , units tg results are shown for mlsg ( black solid line ) and mlsu ( black vertical bars or diamonds ) . mlsu primary production of noy ( times 20 ) is also shown in figure 1a ( red dashed vertical bars ) . n2o loss from the uci ctm is shown for ifs cycles 29 ( blue ) and 36 ( green ) . tropical ( 30s30n ) profiles of ( a ) n2o ( ppb ) and ( b ) o3 ( ppm ) as a function of pressure altitude ( z * km ) average over 20052010 where available . mlsg ( solid black ) is interpolated to 1 km resolution , and mlsu ( black dots ) shows the original mls resolution . also shown are the modeled profiles for gmi ( orange ) , g2dm ( red ) , ucic29 ( blue ) , ucic36 ( green ) , and g2d ( gray ) . the critical region for n2o loss is the tropical middle stratosphere as shown in figures 1a and 1b . photochemical loss ( dominated by r3 ) peaks at the equator near 32 km pressure altitude . both models mlsg and mlsu agree on the integrated loss averaged over years 20052010 , although coarse vertical layering for mlsu ( vertical black bars ) obscures the peak loss . in terms of total loss , 81% occurs between 24 and 40 km including all latitudes , while 76% occurs between 30s and 30n including all heights . polar regions produce only 4% of the loss , and that occurs at high altitudes , so that ozone depletion below 24 km has minimal direct photochemical impact on the n2o lifetime . ozone depletion from the clo catalytic cycle ( 4050 km altitudes ) does impact the n2o lifetime : see discussion of preindustrial lifetime ( section 4 ) . figure 2a shows the vertical profile of mls n2o from 16 to 48 km in pressure altitude averaged over 30s to 30n for years 20052010 , and figure 2b , that of mls o3 . note that the peak loss ( figure 1a ) centers on the 50% ( ~160 ppb ) level of n2o at about 10 hpa ( 32 km ) . the critical region for modeling the n2o lifetime thus centers on the region 30s30n and 24 km to 40 km . the mls observations show that monthly zonalmean n2o abundances at 10 hpa have large seasonal and interannual variations ( figure 3a ) . the upwelling region with highest n2o levels , and hence greatest loss , occurs in the tropics ( 20s20n ; white lines in figures 3a3e ) , where the mls monthly average at 10 hpa ranges from 147 to 206 ppb . contours of n2o at 10 hpa plotted as function of latitude and month from january 2004 to december 2011 . results are shown for ( a ) mls , ( b ) ucic29 , ( c ) ucic36 , ( d ) g2dm , and ( e ) gmi . with this variability in the critical loss region the 5 year averaged monthly mean loss rates are shown in figure 4 for mlsg ( black solid + dots ) and mlsu ( black dashed ) . not unexpectedly , both chemical submodels give nearly identical results with a mean loss rate of 13.2 tg n / yr and peaktopeak amplitude of 25% . the peak losses occur with the peak upwelling ( januaryfebruarymarch ) seen in figure 3 . the interannual variation in n2o lifetime is calculated as 12 month running averages of the burden ( tg n = 4.79 times lower tropospheric mean abundance in ppb ) divided by the 12 month accumulated loss ( tg n / yr ) and is plotted for mlsg and mlsu from 1 february 2005 to 1 july 2010 in figure 5 . the calendar mean ( january december ) lifetimes for mlsg are shown as dots . the mean burden over the mls period is calculated as 1539 tg n , and the mean loss is 13.2 tg n / yr , giving a lifetime of ~116.5 years for both photochemical submodels ( see table 1 ) . a quasibiennial signal is clear over the 5 years of mls data , and the minimumtomaximum amplitude is about 10% . monthly n2o loss rate ( units of tg n / yr ) for the mls period of 20052010 or best equivalent period from mlsg ( black solid + dots ) , mlsu ( black dashed ) , gmi ( orange ) , g2dm ( red ) , g2d ( gray ) , ucic29 ( dark blue ) , osloc29 ( light blue ) , ucic36 ( dark green ) , and osloc36 ( light green ) . values are derived from 12 month average burden ( tg n ) and loss rate ( tg n / yr ) centered on each month the n2o lifetime varies with solar activity , and mlsg and mlsu evaluated this by changing the solar spectrum and thus the photolysis rates , which are summarized in table 2 . mlsg has the more accurate treatment , and its 20052010 lifetime value uses the actual solar activity of the time period ( mostly solar minimum ) . mlsg then repeated the calculation using constant minimum and maximum solar activities , giving a span of 7% ( 109 years at solar maximum to 117 years at solar minimum ) . this range uses the fixed mls observations of o3 and t , which are more suitable to solar minimum conditions , and thus , background atmospheric changes expected at solar maximum ( including the n2o ) are not included . mlsu uses a solar spectrum that averages minimum and high ( but not maximum ) solar activity . thus , the standard 20052010 mlsu lifetime represents higher solar activity than occurred for the period , and the agreement with mlsg is fortuitous since the solarminimum calculation gives a slightly longer lifetime of 119 years . both models show a similar response to changes in the solar spectrum when the difference in minimummaximum ranges is considered . the 20052010 period covers an extended solar minimum period ; the solar activity of the current cycle 24 is weaker than average and expected to reach maximum in 2015 [ pesnell , 2014 ] . presentday n2o lifetime and other model comparisonsa note that stratospheric n2o is multiyear average at 10 hpa , 20s20n . ln(lifetime)/ln(burden ) is related to the feedback factor as ff = 1/(1 s ) [ prather , 2007 ] . lifetimes are calculated as the annual average burden ( tg n ) divided by the accumulated annual loss ( tg n / yr ) , and all are based on a transient n2o burden increasing at 0.25%/yr . oslo and uci models ran with fixed n2o burden and , based on a ucic29 test case , have their lifetimes increase by 0.4% . because models have different atmospheric dryair masses , all burdens are calculated from the lower tropospheric mean n2o abundance ( ppb ) using the relationship 4.79 tg n / ppb [ prather et al . , mlsg standard simulation used transient solar activity for the period of mls observations , while mlsu used the average of two solar conditions ( solar ultraviolet spectral irradiance monitor 11 november 1994(low ) and 29 march 1992(high ) ) . mlsg solar maximum conditions are representative , while the mlsu solar maximum simulation is high , but not maximum . the mlsu lifetime has been corrected for missing 2% of loss below 100 hpa ( the lower limit of mls data ) . ucic29/36 model is a stratosphereonly chemistry model , and its lifetimes are corrected by 1% to account for lack of tropospheric o(d ) loss . both c29 results stop in year 2007 , but correction is made because the mlsu lifetime derived for 20052007 is almost identical to that over the full period of 20052010 . likewise , comparisons of the period 2004/2007 versus period 2004/2010 using cycle 36 and merra fields show little difference ( 0.9 years ) . g2d has reduced variability using yearly repeating steady state transport . combining interannual variability and other uncertainties , we derive the n2o lifetime of 116 9 years for the period of 20052010 . comparison with modeled values follows , and the 1 sigma uncertainty of 8% is derived in section 4 . four ctms used meteorological data for the mls period and before , calculating n2o lifetimes to compare with the mlsg / u results . see table 1 . two of these ( nasa global modeling initiative ctm ( gmi ) and gsfc 2d model ( g2dm ) ) used the modernera retrospective analysis for research and applications ( merra ) goddard earth observing system version 5(geos5 ) assimilation met data ( designated gmi and g2dm , respectively ) . the gsfc 2d model with interactive transport ( g2d ) also ran a standard simulation and sensitivity studies for steady state , yearly repeating 2010 conditions . the gsfc goddard earth observing system chemistry climate model ( geosccm ) is a freerunning atmospheric climate model driven by prescribed sea surface temperatures . uc irvine and university of oslo ctms used the european centre for mediumrange weather forecasts ' ( ecmwf ) integrated forecast system ( ifs ) to generate pieced forecasts , and two different cycles of the ifs generated met data that partially overlap ( designated ucic29 , ucic36 , osloc29 , and osloc36 according to which ifs cycle was used ) . the oslo and uci models used identical met data and tracertransport models , but oslo has full stratospheretroposphere chemistry ( like gmi ) , whereas uci uses a linearized stratospheric chemistry model ( o3 , n2o , noy , ch4 , and h2o [ see hsu and prather , 2010 ] ) . globally integrated n2o loss versus altitude from ucic29 and ucic36 are compared with mlsg / u in figure 1a . both uci models match the mls shape but calculate losses that are smaller by 10% ( c29 ) and 20% ( c36 ) with largest differences near the peak loss at 10 hpa . these results are explained for the most part by the lower average tropical abundances of the modeled n2o between 24 and 40 km , see figure 2a . the difference between two ifs versions are clear in figure 2 : ucic29 has more rapid upwelling and higher n2o below 30 km , whereas ucic36 has slower upwelling ( or more rapid horizontal dispersion ) from 20 to 40 km . the best matches to the mls n2o profiles are from gmi , ucic29 and osloc29 ( not shown ) . g2dm ( using 2d monthly mean residual circulation fields derived from the same merra fields used directly in gmi ) and g2d ( using a steady state yearly repeating transport ) are similar to ucic36 , and all have consistently lower n2o abundances than mls . a more quantitative comparison of the 10 hpa n2o abundances is given in table 2 , where the mean and standard deviation of the monthly mean 20s20n abundances are given under this statistic emphasizes the core tropics more than in figure 2 , and the spread across models widens . three models ( gmi , ucic29 , and osloc29 ) are about 10% below mls values , and the other models ( g2dm , g2d , ucic36 , and osloc36 ) are about 20% below . mls observations of n2o show largescale , quasibiennial variability within the tropical upwelling region ( 20s20n ) at the critical altitude of 10 hpa on a monthly zonalmean basis . as noted before , ucic29 ( and osloc29 , nearly identical and not shown ) as well as gmi are similar in magnitude to mls data but down one contour level ( 20 ppb ) , while ucic36 ( osloc36 ) and g2dm are reduced by two or more contour levels . the ucic36 n2o variability is less well correlated with observations than is that of ucic29 . overall , both the ecmwf and merra meteorology produce a modeled 10 hpa n2o surface that is biased but highly correlated with the mls observations , indicating that transport variations in the tropical midstratosphere on a monthbylatitude basis are well represented . the other major controlling factor for n2o lifetimes is the profile of overhead o3 . differences in the o3 profiles ( figure 2b ) can help explain why the lifetimes listed in table 2 are not inversely related to the 10 hpa n2o abundances . ucic29 matches the mls tropical o3 profiles with gmi being slightly smaller and ucic36 being larger . the greater o3 in ucic36 absorbs more solar ultraviolet , thus reducing the n2o loss even more than expected from the smaller n2o abundances . both gsfc 2d models ( g2dm and g2d ) have nearly identical o3 profiles that in the 3040 km region are almost 10% less than mls . these g2d offsets cancel in terms of total n2o loss with more solar ultraviolet enhancing the loss of the reduced levels of n2o . thus , both g2d lifetimes are smaller than the gmi lifetime , even though their n2o abundances are 1020% smaller . all models match the monthly seasonality of n2o loss in mlsg / u as shown in figure 4 . the oslo / ucic29 results are not so smooth since there are only three overlapping years . other models show different levels of flattening ( or reduction ) of the octobernovemberdecember month period , but the overall cycles with the februarymarch peak and junejuly trough are in agreement . the greatest difference in figure 4 is the overall magnitude of the loss , and this large range in modeled n2o lifetime is summarized in table 2 . for models using met data back to 1997 , we find that the 19972010 average lifetime was about 2 years shorter than the 20052010 mls period lifetime , indicating more rapid loss of n2o in the earlier period . the pre2005 period thus has a systematically different circulation in the tropical middle stratosphere leading to more rapid upwelling of n2o , which might be a response to the last solar maximum that occurred during this preaura period or to a change in the observing system used to generate the met data . it can not be due to photochemical changes driven by the solar cycle since several models did not include this . the models using ifs forecast or merraassimilated met data show realistic variations when compared with the mlsg / u results . the steady state g2d results are shown as a gray bar for the year 2010 . in terms of correlations , mlsg and mlsu are nearly identical ( r = 0.98 ) . in terms of model correlations with mlsg , the g2dm , osloc29 , and ucic29 models are excellent ( 0.850.90 ) , but gmi , osloc36 , and ucic36 models do not match as well ( ~0.78 ) . it is not clear why the gmi and g2dm should not be more closely correlated with each other . this may be due to small differences caused in converting the merra met fields to 2d transport ( via the heating rates and eddy fluxes ) for g2dm as opposed to using the direct merra 3d wind field in gmi . a critical part of the stratospheric nitrogen chemistry involves production via reaction ( r5 ) of odd nitrogen ( noy = no + no2 + hno3 + others ) , which in turn drives catalytic o3 loss centered about 35 km [ crutzen , 1970 ; johnston , 1971 ] . for mlsu , 5.7% of the n2o loss produces noy , and this primary production ( figure 1a ) has a lower center of mass than the total n2o loss because o(d ) reactions become relatively more important than photolysis in the lower stratosphere . consequently , the n2onoy tracer relationship [ murphy and fahey , 1994 ] is curved in the lower stratosphere . about 30% of primary noy produced is lost following photolysis of no ( jno ) in the upper stratosphere and mesosphere [ olsen et al . , while this photochemical process is included in the ctms , it can not be derived simply from the mls data , and modeling of jno remains highly uncertain ( 30% or more [ see photocomp , 2010 ] ) . stratospheric noy , however , is the key link between n2o and o3 chemistries that causes an n2o perturbation to decay more rapidly than indicated by the atmospheric lifetime [ prather , 1998 ] , and thus , we seek an observational test of the modeled noy to understand differences in the modeled lifetime sensitivity to burden , s ln(lifetime)/ln(burden ) , and the resulting lifetime feedback factor , ff = 1/(1 s ) ( see table 4.5 of prather et al . ) . we focus on the tropics where most n2o is lost : values of noy derived from satellite observations show a broad peak of about 16 ppb near 40 km that varies with season and year and that falls off rapidly at lower altitudes [ brohede et al . , 2008 ; jones et al . , 2011 ] . as a robust measure of the total amount of stratospheric noy , we integrate the 30s30n column of noy above 100 hpa ( expressed in dobson unit ( du ) , 2.687 10 molecules / cm ) and compare with mipas observations ( figure 6 ) . the observations have a mean column of 0.39 du with a march minimum and july maximum and with a minimumtomaximum amplitude of ~12% . the g2d model is an exception with a very weak seasonal cycle in noy columns . stratospheric tropical noy columns ( du , p < 100 hpa , 30s30n ) by month showing ( a ) absolute columns and ( b ) monthly standard deviations over the mls period . the n2o lifetimes from the mls data and the ctms are summarized in table 2 . all the models in this study have too little n2o loss and hence longer lifetimes . numerous small , percentlevel corrections to the lifetimes have been made due to the different chemistries and methods of calculation . many are noted above and summarized in the table notes . for example , the results here consist of both transient ( mlsg / u , gmi , and g2dm ) and steady state calculations ( uci and oslo ) , and the difference between these two approaches has been discussed [ sparc , 2013 ; wmo , 2014 ] . in the transient calculations , the n2o lower tropospheric abundance increases at the observed rate of about 0.8 ppb / yr ( 0.25%/yr ) . given that the lag for the mixed tropospheric abundance to reach the tropical midstratosphere is a couple of years , the troposphere accumulates an additional burden . in transient versus steady state simulations with ucic29 , we find that transient lifetimes are about 0.4% larger than steady state ones , and we have so corrected the oslo and uci lifetimes in table 2 . our semiempirical estimate of the current lifetime , 116 9 years , is based on the mlsg / u calculations and has a 1 sigma uncertainty of 8% . the only sources of uncertainty here are those in the mlsmeasured profiles ( n2o , o3 , and t ) and those in the photochemical submodels ( jn2o and o(d ) ) . we assume that uncertainties in the o3 and t profiles ( affecting the photochemical model uncertainty ) are negligible since these measurements have a longer history of satellite measurement and calibration . we identify the primary measurement uncertainty with the n2o profile in the 303 hpa altitude range ( n2o~40240 ppb ) . the possible bias in the mls n2o measurements near 10 hpa has been assessed as being less than 10% [ lambert et al . , 2007 ] , and taking this as a 90% confidence level , we place a generous 1 sigma uncertainty of 7% on the n2o abundances which translates directly to the n2o loss . uncertainty in the photochemical data leads to a 1 sigma uncertainty of 3% in the photochemically modeled lifetime ( see chapter 3 of sparc ) . the uncertainty in the n2o burden is negligible because the mass of the atmosphere is well known and the falloff of n2o in the stratosphere is well characterized [ prather et al . , 2012 ] . combining the two major uncertainties , we get 7.6% and adopt 8% as a symmetric 1 sigma uncertainty in the lifetime . note that the dominant factor is the n2o abundance and that adding another 3% uncertainty ( e.g. , from o3 and t ) would not change this value . geosccm is the only model here whose lifetime fits within this uncertainty ; osloc29 , ucic29 , and both g2d models lie just above the upper range ; and osloc36 , ucic36 , and gmi models have lifetimes more than 2 sigma above . some but not all of the model range in lifetimes can be attributed to the low n2o abundances near 10 hpa in the tropics . our uncertainty is notably less than previous semiempirical estimates based on the premls satellite data [ ko et al . , 1991 ; minschwaner et al . , 1998 ] , in part because of the much denser , more accurate set of mls colocated observations of n2o , o3 , and t that resolve the quasibiennial cycle . the separately modeled and satellitederived n2o lifetimes updated in sparc are based on premls data and have essentially the same lifetime as our best estimate , especially when one considers the large interannual variability ( 6 years ) not resolved in the premls data ( see figure 5 ) . the stratospheric processes and their role in climate ( sparc ) overall best estimate , based on models , satellite data , and tracertracer lifetimes , is slightly higher with greater uncertainty , 123 years ( range of 104152 years ) , because of the inclusion of a 144 year lifetime determined from tracertracer correlations . tracertracer correlations in the lower stratosphere are used to derive relative lifetimes [ volk et al . , 1997 ; 2013 ] , but this approach is not useful here because the uncertainties are much larger ( e.g. , they propagate from the reference gas lifetime , and even modeled two gas tracer correlations like n2ocfcl3 are difficult to interpret as a single slope [ avallone and prather , 1997 ] ) . our estimate of the lifetime sensitivity of n2o to its burden is s = 0.065 0.010 , where the 0.010 is a 1 sigma uncertainty as it includes almost the entire range of models except for g2d . thus , the feedback factor is ff = 0.94 0.01 , and the effective residence time of an n2o perturbation is 109 10 years [ e.g. , prather , 2007 ; prather and holmes , 2013 ] . unfortunately , there is no semiempirical method to derive this sensitivity using mls observations , but given the limited range for this value since first reported in 1998 , we feel that 15% 1 sigma uncertainty is a conservative choice . all chemistrytransport models here are consistent with this range , except for g2d , whose sensitivity is nearly twice as large ( table 2 ) . both g2d and g2dm have identical chemistries and similar n2o lifetimes and tropical noy columns but very different sensitivities . analysis shows that with a greater burden of n2o , g2d 's interactive circulation is responding as slightly reduced o3 ( 2% ) , heating rates , and temperatures ( 0.3 k ) ; enhanced upwelling with age of air changed by 0.05 years ; and consequently more n2o loss . while the freerunning geosccm shows similar reductions in o3 and temperature , it does not simulate a circulation enhancement . at this time we do not include the 0.11 feedback factor in g2d in our uncertainty range but must recognize it as presenting an open question . there is also no simple relationship found in table 2 between the lifetime sensitivity and the noy column , in contrast to what was expected . an interesting finding from the 14 year ucic36 calculation was that the lifetime sensitivity took more than 7 years to stabilize following a sudden 10% increase at the lower boundary , even though the lifetime appeared to stabilize more rapidly . surprisingly , our best estimates of lifetime and feedback factor have not changed much since the first multimodel estimates were made in 2001 ( see table 4.5 of prather et al . ) . analysis of the preindustrial ( pi ) lifetime of n2o was made with g2d , osloc29 , and ucic29 , with results shown in table 3 . the pi simulations assumed n2o tropospheric abundance of 270 ppb ( versus 321 ppb ) , ch4 abundance of 722 ppb ( versus 1785 ppb ) , co2 abundance of 278 ppm ( versus 384 ppm , used in g2d only ) , and zero anthropogenic ozonedepleting substances ( odss ) [ ipcc , 2013b ] . changes in lifetime , calculated as the natural log of pi lifetime divided by presentday lifetime , are estimated at + 1 to + 2% due to the lower levels of n2o alone . the other major change , a + 3 to + 5% increase due to the lack of anthropogenic ods , is caused by the higher levels of pi 4050 km tropical o3 , leading to reduced penetration of solar radiation to 32 km , and hence reduced losses of n2o . the full pi lifetime is estimated from these three models to be + 4% to + 8% larger than present day , with the upper range including co2 dynamical effects ( g2d only ) . as long as the uncertainty in this change ( 2% ) is independent of other errors , then the 1 sigma range remains at about 8% . preindustrial ( pi ) n2o lifetime changes ( approximately % ) separated by key factors , shown as ln [ lifetime(pi)/lifetime(present day)]a note that pi changes include n2o dropping from 321 ppb ( 20042010 ) to 270 ppb , anthropogenic ozonedepleting substances ( ods ) dropping to zero , ch4 dropping from 1785 ppb to 722 ppb , and climate change driven by co2 dropping from 384 ppm to 278 ppm . individual models chose slightly , but insignificantly different ranges for the pi , and the values from ipcc [ 2013b ] are quoted here . g2d has a stratospheric circulation that responds dynamically to heating changes from o3 and co2 ( in full pi ) ; see fleming et al . . this study has rigorously tested four global chemistrytransport models ' ability to simulate the observed distribution and loss patterns of n2o based on 5 years of mls observations . these tests address seasonal and interannual variability showing that the models have high fidelity in reproducing the measurements on a monthbymonth basis in spite of having lifetimes that are biased high by 9% to 26% , outside of the 8% uncertainty range . most biases can be understood in terms of lower model n2o abundances in the middle stratosphere or ( in some cases ) o3 profiles that are notably different from mls data . the geosccm , a freerunning climate model not expected to reproduce the observed variations in n2o lifetime ( e.g. , figure 5 ) , was added to check if the n2o lifetime sensitivity was significantly perturbed by induced circulation changes . geosccm has the most accurate simulation of multiyear average n2o abundances in the middle tropical stratosphere among all the models here , and it has a lifetime within the uncertainty range of the mlsderived value . overall , this points to the importance of circulation and net tracer transport in the middle tropical stratosphere in determining n2o lifetime . what is the best estimate for the current n2o lifetime , one that includes the steady slow increase ( 0.25%/yr ) in burden ? the sparc assessment 's recommendation ( see table 6.1 of sparc ) is 123 years with a 1 sigma range of 113 to 148 years based on a range of models , observations , and tracer correlations . our 1 sigma range of 109 to 125 years overlaps with but is limited to the lower half of the sparc range . ; also chapter 5 from sparc ] averaged the results from six chemistryclimate models to estimate a steady state lifetime of 115 years with a model variance of 8% . to correct this to current observations ( the transient as recorded in mls data ) would add about 0.5 years , and thus , this result is essentially identical to the semiempirical mls lifetime derived here . what is unusual is that the models in this study ( ccm and ctms ) calculate much longer lifetimes , 120 to 146 years , almost outside the range of the sparc models , 105 to 127 years . geosccm participated in both studies ( 120 years in this study versus 117 years in sparc ) as did g2d ( 130 years versus 125 years ) . the differences are not greatly significant and may depend on the scaling of tropospheric abundance ( ppb ) to burden ( tg n ) , but both models are at the high end of the sparc models and the low end of the models here . geosccm used here and in sparc is closely related in terms of chemistry to gmi but has very different lifetimes , 120 versus 137 years . the merra fields are known to have excessive subtropical mixing in the lower stratosphere , which reduces n2o in the tropics , thus reducing its flux to the tropical midstratosphere . similarly , the ifs fields for cycle 36 appear to have more stagnant vertical transport in the midstratosphere than cycle 29 . the n2o lifetime is a good diagnostic of the circulation and mixing below the middle stratosphere [ strahan et al . , how does this new lifetime change our estimates of n2o emissions , both preindustrial and presentday anthropogenic ? the evaluation of emissions based on observed abundances and lifetime ( both pi and present ) gave pi emissions of 9.1 1.0 tg n / yr and present anthropogenic emissions of 6.5 1.3 tg n / yr ( assuming that pi emissions , but not lifetime , remain constant ) [ prather et al . , those values were based on the thenmostrecent model study with a lifetime of 131 years [ fleming et al . , 2011 ] . correcting to our current lifetime recommendation increases the pi natural sources to 10.5 tg n / yr but hardly changes the anthropogenic source , 6.6 tg n / yr . uncertainties do not significantly change . how will the n2o lifetime change in the future ? the sparc model 's year 2100 simulations showed a wide range of changes in lifetime from 6 years to + 6 years [ chipperfield et al . , if a 5% uncertainty in the future lifetime was a true bound , then the climate and ozone impacts of future n2o emissions can be evaluated with some confidence . however , the seemingly random shifts in future lifetime need to be evaluated in terms of the key factors driving them , as was attempted for the pi lifetime in table 3 here . were able to identify the primary factors causing the wide range of future ozone projections in ccms by separating their responses to increasing greenhouse gases from their temperature and chemistry responses to decreasing odss . if the goal is to understand future changes in the lifetimes of gases controlled by stratospheric chemistry , then a more rigorous protocol of model studies is needed to assess separately , e.g. , the decline in ods , future stratospheric temperatures , and the role of changes in transport .

approximately 15 - 20% of the population will have urticaria at least once during their lifetime . this is referred to as chronic idiopathic urticaria or more recently , as chronic spontaneous urticaria ( csu ) . in recent years , 30 - 50% of patients with csu have been shown to have an autoimmune basis . this autoimmune subgroup referred to as chronic autoimmune urticaria ( cau ) has autoantibodies directed at the fcr1 receptor located on mast cells and basophils or less commonly against immunoglobulin e ( ige ) . it is often not possible to distinguish cau from those without autoantibodies either clinically or histologically . the gold standard diagnostic test for detecting clinically relevant autoantibodies to fcr1 is the functional in - vitro donor basophil histamine assay . however , this bioassay is difficult to standardize because it requires fresh basophils from healthy donors and is time consuming . other tests like western blot , elisa , flow cytometry may be useful for screening in the future , but need to be validated . autologous serum skin test ( asst ) is a simple in - vivo clinical test for the detection of basophil histamine releasing activity . in 1986 , grattan et al . ( the term chronic idiopathic urticaria is now referred to as chronic spontaneous urticaria ) . it may be used as a reasonably accurate predictive clinical test to indicate the presence of functional circulating autoantibodies . various practical considerations influence the outcome of the test , including the use of antihistamines , immunosuppresants and antidepressants . in the majority of studies antihistamines were withdrawn at least 2 - 3 days prior to asst and immunosuppressants were discontinued at least two months before testing . leukotriene receptor antagonists probably do not need to be discontinued before testing , since the early phase of asst response is thought to be histamine mediated . diagnosing these patients is also important as they may need high doses of antihistamines and systemic corticosteroids during acute exacerbations and immunomodulatory drugs may have therapeutic benefit in them . the percentage of patients with cau among csu patients varies widely in different studies conducted both in india and abroad . a study by kulthanan et al . from thailand found the percentage of patients with autoimmunity to be 24.7% . a study by godse from mumbai found the incidence of autoimmune urticaria to be 26.6% which is similar to reports from western countries . the few studies conducted in south india using asst among patients with csu show widely varying asst positivity ranging from 34% to 50% . therefore , we conducted a study using asst as an indicator of cau because of the varying asst positivity as well as possible differences is management and course of the disease . this was a cross - sectional study , done between march 2011 and june 2012 , after obtaining approval from the institutional ethics committee . during the study period , 48 patients with csu who attended the department of dermatology were included in the study after obtaining informed consent . patients who were < 18 years of age , pregnant / lactating females , patients taking long - term corticosteroids and antidepressants were excluded from the study . all patients were asked to stop antihistamines 2 days before the test . a detailed history was taken and thorough physical examination was done for all the patients . routine investigations ( hemoglobin , total leucocyte count , differential leucocyte count , erythrocyte sedimentation rate , routine urine examination and random blood sugar examination ) were done for all patients and recorded . further tests such as liver and renal function tests , anti - nuclear antibody ( ana ) , thyroid stimulating hormone , antithyroid antibodies were done on those patients indicated by history and clinical examination . autologous serum skin test - about 5 ml of venous blood was collected in a sterile vacutainer and allowed to clot at room temperature for 30 min . serum was centrifuged at 2000 rpm for 15 min and 0.05 ml of autologous serum was injected intradermally , in uninvolved skin , using a 1 ml insulin syringe ( 30 gauge needle ) into the right forearm 2 cm below the cubital fossa . similarly , 0.05 ml of 0.9% sterile normal saline ( negative control ) was injected intradermally proximally into the left forearm , and at a distance of at least 5 cm , 0.05 ml of histamine ( 10 g / ml ) was injected distally into the left forearm as a positive control . a serum induced erythematous weal with a diameter of 1.5 mm more than the saline induced response within 30 min was taken as positive . statistical analysis was performed using chi - square tests , student 's t - test , and mann - whitney u - test . the age range was 18 to 75 years with a mean age of 36.7 years ( standard deviation [ sd ] : 15.1 years ) . as many as 36 out of 48 patients were found to be in the age group between 21 and 50 years . total duration of the disease ranged from 2 months to 10 years with a median of 1 year ( interquartile range [ iqr]-0.5 years , 2 years ) . age distribution of asst positive and negative individuals after taking a detailed history 36 ( 75% ) patients gave history of large weals ( > 3 cm in diameter ) and the rest gave history of small weals . classifying the patients on the basis of shape of weals , 20 ( 41.6% ) of them gave history of linear weals and 15 ( 31.25% ) of them gave history of round weals . the duration of weals ranged from 10 min to 6 h with a median of 1 h ( iqr-0.5 h , 3 h ) . all four patients who had undergone ana tests were found to be negative . in this study , 20 patients ( 41.6% ) were asst positive [ figure 1 ] while the remaining 28 ( 58% ) were asst negative [ figure 2 ] . females had a higher percentage ( 46.4% ) of asst positivity than males ( 35% ) , but this was not statistically significant ( p = 0.43 ) . correlating asst positivity with the age distribution of our patients it was found that the mean age of asst positive patients was 36 years ( sd : 17.3 years ) and asst negative patients was 37 years ( sd : 13.4 years ) . ten ( 62.5% ) out of 16 patients in the third decade turned out to be asst positive , a much higher percentage than all other groups . the median duration of disease in both asst positive and negative patients was 1 year ( iqr in asst positive patients-0.5 years , 2 years and iqr in asst negative patients-0.5 years , 2 years ) . autologous serum skin test positive autologous serum skin test negative autologous serum skin test positivity was low among patients with history of large weals . fourteen out of 36 patients with large weals were found to be asst positive ( 38.8% ) and 6 out of 12 patients ( 50% ) with small weals found to be asst positive . asst positivity was higher ( 66.6% ) among patients with a history of round weals , though not statistically significant ( p = 0.06 ) compared with patients with linear weals ( 30% ) and patients with mixed morphology of weals ( 30.7% ) . the proportion of asst negativity in patients present with linear weals was higher , but not statistically significant . median duration of weals in asst positive patients was 1 h ( iqr-0.5 h , 2.87 h ) , whereas in asst negative patients it was 0.5 h ( 0.25 h , 3 h ) . of the 21 atopic patients only 9 were found to be asst positive ( 42.8% ) . asst positivity was seen in 5 ( 71.4% ) out of seven patients with systemic involvement ( such as nausea , abdominal pain , and vomiting ) , again not statistically significant ( p = 0.084 ) . our study showed a female predominance , similar to previous reports . in our study , ages ranged from 18 to 75 years with a mean age of 36.7 years . other studies showed the mean age of presentation varying from 34 to 45 years [ table 2 ] . the results of our study showed 36 out of 48 patients to be in the age group between 21 and 50 years similar to a study by george et al . who reported 81 out of 100 patients in the above - mentioned age group . comparison of our study with previous studies various studies from abroad showed asst positivity ranging from 24% to 67% whereas studies from india by george et al . , krupa shankar et al . , vohra et al . no significant difference was found between asst positive and negative patients regarding total duration and frequency of disease , matching results obtained by nettis et al . and kulthanan et al . showed that both duration and frequency of disease was significantly more in asst positive patients , whereas george et al . as in many other studies our study did not show any difference between asst positive and negative patients regarding duration of weals . george et al . however , found weals lasted significantly longer in asst positive patients . our study showed no significant difference between asst positive and asst negative patients regarding mean age and sex distribution . where there was no correlation between disease severity and asst positivity [ table 2 ] . there was no relationship between asst positive and negative patients in terms of large weals ( > 3 cm in diameter ) and small weals which was similar to other studies by kulthanan et al . and seven out of 48 patients had systemic involvement and asst positivity was seen in five out of those seven patients . a study from sweden showed that asst positive patients had significant systemic involvement such as gastrointestinal symptoms and flushing . regarding atopy , there was no significant difference between serum positive and serum negative groups . it is expected that the atopic subjects would belong mostly to a less severe disease subgroup because it has been demonstrated that the atopic subject 's trend toward high level of ige can prevent the binding of anti - fcr1 antibodies to the receptor , already saturated by immunoglobulin . in our study , 66% of the patients presented with a history of angioedema and 50% of them were asst positive . in other studies , there was no significant difference between patients with or without autoantibodies with regard to angioedema and asst positivity ranged from 7% to 87% . autoimmune diseases such as thyroid disease , vitiligo , diabetes mellitus , pernicious anemia and rheumatoid arthritis were reported more commonly in autoimmune urticaria . although higher frequency of autoimmune diseases has been reported in patients with autoimmune urticaria by odonnell et al . , we did not find any significant association between autoimmune diseases and asst positive patients which was consistent with some other studies . the association of chronic urticaria with thyroid autoimmunity has been studied by leznoff and sussman . they postulated that thyroid autoimmunity may play a role in pathogenesis of chronic urticaria and angioedema . we found abnormal thyroid function test ( tft ) value and thyroid autoantibodies in two patients . in our study , tft and thyroid autoantibodies were measured only if indicated , hence the significance of the finding can not be commented upon . however , the possibility that a larger study may reach statistically significant results can not be ruled out .

lung cancer is one of the leading cancers worldwide , with an estimated 1.8 million new cases recorded in 2012 . according to the world health organization ( who ) , lung cancer accounted for approximately 1.59 million cases of the 8.2 million cancer - related deaths reported in 2012 ; as such , lung cancer is one of the most significant causes of deaths worldwide . lung cancer is also the leading cancer among men and the third most common cancer type among women . the incidence rate of lung cancer worldwide varies between 0.8 and 53.5 cases per 100,000 population ; the rate is high in central europe , eastern europe , and eastern asia and low in the middle and west africa . global estimates show that the age - standardized incidence rates of lung cancer are the highest in central and eastern europe and eastern asia , with 53.5 and 50.4 cases per 100,000 people , respectively . low age - standardized incidence rates of lung cancer among men ( 2.0 and 1.7 per 100,000 , respectively ) and women ( 1.1 and 0.8 per 100,000 , respectively ) were reported in middle and west africa . epidemiologic studies suggest that lung cancer is the fifth most common cancer in australia and the most common cause of cancer - attributable death among people aged 45 - 64 years . in the philippines , lung cancer is the most common cause of cancer - related mortality among men and the third most common cause of mortality and morbidity . in singapore , lung cancer is the second most common cancer in males and the third most common cancer in females , with reported lung cancer incidence rates of 16.2% in men and 7.7% among all cancer cases . cancers account for the highest economic loss among the 15 leading causes of death worldwide . the total global economic loss incurred because of cancer burden was estimated to be $ 895 billion in 2008 , which is 1.5% of the gross domestic product ( gdp ) worldwide . cancers of the lung , bronchus , and trachea conferred the major economic burden , accounting for nearly $ 180 billion on a global scale in 2008 . the numbers are expected given that smokers die 15 years earlier than non - smokers on an average . the majority of lung cancer cases are reported in the middle aged and elderly population as a result of the increasing prevalence of smoking among young adults in the asia - pacific region . cancer symptoms require 10 - 30 years to develop ; as such , the years of life lost or loss of productivity caused by cancer is a huge drain not only to the individual but also to the economy of these societies . despite epidemiologic studies suggesting that the incidence of lung cancer is relatively lower in the asia - pacific than that in the north america and europe , recent studies show that the burden imposed by lung cancer on the health systems is enormous in the former . furthermore , epidemiologic studies evaluating the true burden of lung cancer are insufficient in the asia - pacific region . this paper aims to understand the burden of lung cancer in three asia - pacific countries : australia , singapore , and the philippines , and the burden of lung cancer is evaluated by understanding the economic loss caused by premature mortality and morbidity . to estimate the burden of lung cancer in australia , the philippines , and singapore , we measure disability adjusted life year ( daly ) , which is calculated using years of life lost ( yll ) and years lost due to disability ( yld ) . one daly loss indicates the loss of 1 year of healthy life ; thus , the total daly in the entire population indicates the difference between the ideal healthy status without disease or disability and the current state . daly is a sum of yll caused by premature death and yld . in simple terms , yll represents the total number of lives lost prematurely because of early death caused by the disease . yld represents the years lost due to disability and considers disability weightage , which reflects the severity of the disease on a scale of 0 ( perfect health ) to 1 ( dead ) . yll and yld were calculated using the formula developed by lopez and murray , as follows : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \begin{array}{l } { \rm{ylls}}\left [ { r , k,\beta } \right ] = \frac{{kc{e^{ra}}}}{{{{\left ( { r + \beta } \right)}^2}}}\left\ { { { e^ { - \left ( { r + \beta } \right)\left ( { l + a } \right)}}\left [ { - \left ( { r + \beta } \right)\left ( { l + a } \right ) - 1 } \right ] } \right.\\ \left . - { e^ { - \left ( { r + \beta } \right)a } } { \left [ { - \left ( { r + \beta } \right)a - 1 } \right ] } \right\ } + \frac{{1 - k}}{r}\left ( { 1 - { e^ { - rl } } } \right ) \end{array}$\end{document } k = age weighting modulation factor ; c = constant ; r = discount rate ; a = age of death ; b = parameter from the age weighting function ; l = standard expectation of life at age a. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \begin{array}{l } { \rm{ylds } } \left [ { r , k,\beta } \right ] = d \left\ { { \frac{{kc{e^{ra}}}}{{{{\left ( { r + \beta } \right)}^2 } } } } \right.\left . \left\ { { { e^ { - \left ( { r + \beta } \right)\left ( { l + a } \right)}}\left [ { - \left ( { r + \beta } \right)\left ( { l + a } \right ) - 1 } \right ] } \right.\\ - { e^ { - \left ( { r + \beta } \right)a } } { \left [ { - \left ( { r + \beta } \right)a - 1 } \right ] } + \frac{{1 - k}}{r } \left({1 - { e^ { - rl } } } \right ) \right\ } \end{array}$ \end{document } k = age weighting modulation factor ; c = constant ; r = discount rate ; a = age of onset of disability ; b = parameter from the age weighting function ; l = duration of disability ; d = disability weight . the same formula is represented in the global burden of disease ( gbd ) template provided by the who . this formula uses the values defined in the who template , where the international standard discount rate is 0.03,k - values are 0 when no age weights are used and 1 when age weights are used ; the standard age weights use a beta of 0.04 and a constant of 0.1658 . however , thek - value was set to 0 because no age weights were used in the present study . the value ofd was considered as 0.146 according to the who statistics and information system . although recent gbd studies did not apply age weights or discounts , the authors decided to use daly calculations with discount rates ; the us panel on cost - effectiveness in health and medicine recommended the use of a 3% real discount rate to adjust both costs and health outcomes in economic analysis of health . moreover , numerous studies used discounts to calculate dalys considering that the who reported time - discounted and age - weighted dalys as their standards . therefore , in the present study , the authors decided to compute dalys with and without discounts so the results could be compared with those of other international studies . the ci5plus database , which provides age- and sex - specific incidence data of cancer , was used to source diagnosed incident cases of lung cancer . the ci5plus database , which was established by the international agency for research on cancer , contains the updated annual incidence rates for all cancers up to 2007 in 118 selected populations worldwide . the database is considered the gold - standard source for international cancer data and statistics because of its ability to provide validated annual incidence data , which can be used to compare accessible cancer incidence data among countries worldwide ( table 1 ) . first , age- and sex - specific incidence rates for lung cancer from 2003 to 2007 were extracted . regression analysis was then applied to the 5-year historical data obtained from ci5plus to calculate age- and sex - specific incidence rates until 2015 . in the absence of reliable evidence for change in trends in a specific age - group or in instances where researchers reported relatively stable trends , the incidence rate reported in the most recent year was kept constant throughout the forecast period . when regression analysis showed a decreasing / increasing trend that resulted in negative values or steep decline / incline in the incidence rate over the forecast period , the incidence rate reported / calculated in the most recent year was maintained constant throughout the forecast period . sources for lung cancer incidence data in australia , the philippines and singapore we obtained incidence data from cancer registries and population - based studies published in peer - reviewed journals . the absolute number of lung cancer incident cases in each country were obtained by multiplying the age- and sex - specific incidence rates of lung cancer with the corresponding age and sex - specific population estimates . the calculated age - specific incident cases among men and women in each country were added to obtain the total diagnosed incident cases of lung cancer among men and women in each country . population - based studies or country - specific cancer registries were used to collect 5-year survival rates for lung cancer in each country ( table 2 ) . a report by the australian institute of health and welfare canberra was used to determine lung cancer survival rates for australia . for singapore , lung cancer survival data for 1 , 3 , and 5 years- were obtained from cancer survival data in africa , asia , the caribbean , and central america . similarly , lung cancer survival data for 1 , 3 , and 5 years in the philippines were obtained from a journal article titled " cancer and the philippine cancer control program . moreover , 2- and 4-year survival rates for singapore and the philippines were calculated by taking the average of the 1- and 3-year as well as 3- and 5-year data , respectively . the 5-year survival rates obtained in the most recent year were kept constant throughout the forecast period because of lack of sufficient historical or contemporary data in each country . the survival rates for lung cancer in these countries the 5-year prevalent cases in each country were calculated using incident case data and relative survival data through a life - table method . the 1-year relative survival rate was applied to the total number of diagnosed incident cases for a specific year to obtain the expected number of cases that survived within that year . the 2-year relative survival rate was applied to the total number of diagnosed incident cases that were diagnosed 1-year ago to determine the expected number of cases that survived until year two . the same process was repeated to obtain 3- , 4- , and 5-year survival rates . thus , the obtained 1- to 5-year prevalent cases in each year were added to obtain the 5-year prevalent cases of lung cancer in each country . the 5-year prevalence of lung cancer in each year was determined by dividing the calculated 5-year prevalent cases with the population estimates for that year . considering that the 5-year survival rate varies by stages of lung cancer and data on the stage - specific survival rates of lung cancer for each year are not available from epidemiologic studies worldwide , researchers calculated the duration of disability based on incidence and prevalence rates to maintain uniform methodology across the countries : prevalence=(incidence rate)(average duration of disease ) the duration of lung cancer in each year was obtained by dividing the 5-year prevalence in each year with the incidence rate for that year . life expectancy is a critical aspect for calculation of ylds ; hence , constant life expectancy rates of 82.5 years at birth for women and 80 years at birth for men were maintained in each of the three countries . researchers obtained the age- specific crude mortality rates for each country from country - specific sources because yll calculation requires age - specific crude mortality cases ( table 3 ) . regression was then applied to the historical data to forecast the mortality cases of lung cancer in australia during the forecast years . however , for singapore and the philippines , the studies provided overall mortality rates and not age - specific mortality rates for 2009 - 2013 and 2010 , respectively . to calculate the age - specific mortality rates in singapore and the philippines , researchers obtained the weights of age - specific mortality rates to the overall mortality rate in australia . researchers then applied these weights to the overall mortality rate in singapore and the philippines to obtain the age - specific mortality rates in these countries . the ci5plus database , which provides age- and sex - specific incidence data of cancer , was used to source diagnosed incident cases of lung cancer . the ci5plus database , which was established by the international agency for research on cancer , contains the updated annual incidence rates for all cancers up to 2007 in 118 selected populations worldwide . the database is considered the gold - standard source for international cancer data and statistics because of its ability to provide validated annual incidence data , which can be used to compare accessible cancer incidence data among countries worldwide ( table 1 ) . first , age- and sex - specific incidence rates for lung cancer from 2003 to 2007 were extracted . regression analysis was then applied to the 5-year historical data obtained from ci5plus to calculate age- and sex - specific incidence rates until 2015 . in the absence of reliable evidence for change in trends in a specific age - group or in instances where researchers reported relatively stable trends , the incidence rate reported in the most recent year was kept constant throughout the forecast period . when regression analysis showed a decreasing / increasing trend that resulted in negative values or steep decline / incline in the incidence rate over the forecast period , the incidence rate reported / calculated in the most recent year was maintained constant throughout the forecast period . sources for lung cancer incidence data in australia , the philippines and singapore we obtained incidence data from cancer registries and population - based studies published in peer - reviewed journals . the absolute number of lung cancer incident cases in each country were obtained by multiplying the age- and sex - specific incidence rates of lung cancer with the corresponding age and sex - specific population estimates . the calculated age - specific incident cases among men and women in each country were added to obtain the total diagnosed incident cases of lung cancer among men and women in each country . population - based studies or country - specific cancer registries were used to collect 5-year survival rates for lung cancer in each country ( table 2 ) . a report by the australian institute of health and welfare canberra was used to determine lung cancer survival rates for australia . for singapore , lung cancer survival data for 1 , 3 , and 5 years- were obtained from cancer survival data in africa , asia , the caribbean , and central america . similarly , lung cancer survival data for 1 , 3 , and 5 years in the philippines were obtained from a journal article titled " cancer and the philippine cancer control program . moreover , 2- and 4-year survival rates for singapore and the philippines were calculated by taking the average of the 1- and 3-year as well as 3- and 5-year data , respectively . the 5-year survival rates obtained in the most recent year were kept constant throughout the forecast period because of lack of sufficient historical or contemporary data in each country . the survival rates for lung cancer in these countries were assumed not to have undergone any significant changes . the 5-year prevalent cases in each country were calculated using incident case data and relative survival data through a life - table method . the 1-year relative survival rate was applied to the total number of diagnosed incident cases for a specific year to obtain the expected number of cases that survived within that year . the 2-year relative survival rate was applied to the total number of diagnosed incident cases that were diagnosed 1-year ago to determine the expected number of cases that survived until year two . the same process was repeated to obtain 3- , 4- , and 5-year survival rates . thus , the obtained 1- to 5-year prevalent cases in each year were added to obtain the 5-year prevalent cases of lung cancer in each country . the 5-year prevalence of lung cancer in each year was determined by dividing the calculated 5-year prevalent cases with the population estimates for that year . considering that the 5-year survival rate varies by stages of lung cancer and data on the stage - specific survival rates of lung cancer for each year are not available from epidemiologic studies worldwide , researchers calculated the duration of disability based on incidence and prevalence rates to maintain uniform methodology across the countries : prevalence=(incidence rate)(average duration of disease ) the duration of lung cancer in each year was obtained by dividing the 5-year prevalence in each year with the incidence rate for that year . life expectancy is a critical aspect for calculation of ylds ; hence , constant life expectancy rates of 82.5 years at birth for women and 80 years at birth for men were maintained in each of the three countries . researchers obtained the age- specific crude mortality rates for each country from country - specific sources because yll calculation requires age - specific crude mortality cases ( table 3 ) . regression was then applied to the historical data to forecast the mortality cases of lung cancer in australia during the forecast years . however , for singapore and the philippines , the studies provided overall mortality rates and not age - specific mortality rates for 2009 - 2013 and 2010 , respectively . to calculate the age - specific mortality rates in singapore and the philippines , researchers obtained the weights of age - specific mortality rates to the overall mortality rate in australia . researchers then applied these weights to the overall mortality rate in singapore and the philippines to obtain the age - specific mortality rates in these countries . the values of the burden of disease caused by lung cancer in 2015 in australia , the philippines , and singapore were 91,695 ; 38,584 ; and 12,435 dalys , respectively , as per a discount rate of 3% ( table 4 ) . the daly rate in australia was 4.0 dalys per 1,000 person - years , with the calculated burden of disease being the highest among people aged 45 - 79 years . individuals aged 45 - 59 , 60 - 69 , and 70 - 79 years had 4.4 , 12.6 , and 18.5 dalys per 1,000 person - years , respectively . this trend of increase in dalys with increase in age from 45 - 79 years was also evident in the philippines and singapore . although the philippines presented the second highest calculated burden of lung cancer , the daly rate was higher in singapore ( 2.2 dalys per 1,000 person - years ) than that in the philippines ( 0.4 dalys per 1,000 person - years ) ( figures 1 - 3 ) . when researchers calculated dalys without a discount rate , the burden of disease increased substantially in these countries , with 117,438 dalys in australia , 50,977 in the philippines , and 16,379 in singapore ( table 5 ) . australia demonstrated the highest burden of disease caused by lung cancer even when the dalys were calculated without using a discount rate . the daly rate in australia was 5.2 dalys per 1,000 person - years , with the calculated burden of disease being the highest among people aged 70 - 79 years ( 21.8 dalys per 1,000 person - years ) . a similar trend was evident in the philippines and singapore , where people aged 70 - 79 years presented the highest calculated burden of lung cancer ( the philippines : 4.7 dalys per 1,000 person - years and singapore : 16.8 dalys per 1,000 person - years ) ( figures 1 - 3 ) . the age - specific rates of dalys among men in australia , the philippines , and singapore ( 2015 ) when a discount rate of 3% was used and when no discount rate was used . ( a ) age - specific dalys rate per 1,000 person - years among men in australia , the philippines and singapore ( using a discount rate of 3% ) . ( b ) age - specific dalys rate per 1,000 person - years among men in australia , the philippines and singapore ( without using a discount rate ) . the age - specific rates of dalys among women in australia , the philippines , and singapore ( 2015 ) when a discount rate of 3% was used and when no discount rate was used . ( a ) age - specific dalys rate per 1,000 person - years among women in australia , the philippines and singapore ( using a discount rate of 3% ) . ( b ) age - specific dalys rate per 1,000 person - years among women in australia , the philippines and singapore ( without using a discount rate ) . the age - specific rates of dalys among men and women in australia , the philippines , and singapore ( 2015 ) when a discount rate of 3% was used and when no discount rate was used . ( a ) age - specific dalys rate per 1,000 person - years among men and women in australia , the philippines and singapore ( using a discount rate of 3% ) . ( b ) age - specific dalys rate per 1,000 person - years among men and women in australia , the philippines and singapore ( without using a discount rate ) . almost 98% of dalys in australia , 96% in the philippines , and 98% in singapore were due to yll . gender distribution showed that both yll and yld were higher in males than those in females . the contribution of males to yll , yld , and daly ranged from 52%-57% in australia , 70%-72% in the philippines , and 60%-62% in singapore , respectively . an age - wise distribution of dalys showed that people aged 60 - 69 years in all three countries contributed to the highest number of dalys ( table s1 totable s6 in the supplementary materials , available with the full text of this article at www.cancerbiomed.org ) . however , people aged 60 - 69 years contributed almost 31% of all dalys in their respective countries , with the highest per - capita dalys detected in people aged 70 - 79 years in the three countries . researchers of studies on burden of disease can not always precisely predict the true burden posed by a disease , considering that analyses that involve estimates are often subject to errors . different researchers may not arrive at the same conclusions because of their own distinctive and complex methods for estimating disease burden . therefore , an intuitive and transparent methodology was adapted in the present study to present a comprehensible methodology for research peers . national level and country - specific estimates on lung cancer incidence , mortality , and survival were obtained to calculate the true burden of lung cancer in australia , the philippines , and singapore . although the burden of disease studies no longer use discounts and age - weightage , dalys were calculated with and without discount rates to compare findings with country - specific burden of disease studies . according to the australian institute of health and welfare ( aihw ) , lung cancer was predicted to be the leading cause of burden of disease due to cancer among men and the second highest burden of disease among women in australia during 2012 . the total number of people diagnosed with lung cancer in australia in 2012 was 11,280 cases , which include 6,620 males and 4,650 females . during the same period , the estimated number of deaths from lung cancer were 8,410 cases , which include 5,070 males and 3,340 females . the aihw study showed that the burden of lung cancer in 2012 was 57,300 dalys among men ( 19% of the total cancer burden ) and 43,400 dalys among women ( 17% of the total cancer burden among women ) , with a discount rate of 3% . the present study showed that the burden of lung cancer was 53,158 dalys in men and 38,537 dalys in women , with a discount rate of 3% . although the dalys calculated by the research team are lower than the aihw results , the differences are minimal and can be attributed to the use of the australian specific life expectancy tables and disability weights and not to the who standards . in addition , the aihw study used population estimates from the australian bureau of statistics , whereas the present research group used census data from the international database of the united states census bureau . furthermore , the differences in estimates may be due to regression analysis of the historical incidence and mortality data . when the dalys were calculated without discount rates , the results considerably differed from the aihw estimates , with the burden of lung cancer increasing to 66,977 dalys in men and 50,461 dalys in women in 2015 . although a difference in the daly estimates was expected by the researcher group when no discount rate was used , the difference was remarkable . the causes of this difference remain unclear but could be partially attributed to the use of different population estimates and increasing trend of lung cancer incidence . similarly , in singapore and the philippines , lung cancer is the commonly diagnosed and one of the leading causes of mortality among cancers in the asean region . singapore presents high age standardized ( asr ) mortality due to lung cancer in this region , whereas the philippines has a low age - standardized mortality rate among males and females combined . according to the singapore burden of diseases study ( sbod ) , cardiovascular diseases and cancers accounted for 40% of the total dalys in 2010 , of which 80% was due to mortality . in singapore , lung cancer contributed to 3.4% or 13,589 of the total dalys ( total daly 399,675 ) in 2010 . the findings from this study , which was conducted using a methodology similar to that used in the sbod study , showed that lung cancer burden in singapore was 12,435 dalys . moreover , the sbod study showed that lung cancer accounted for 4.4% ( 9,252 ) and 2.4% ( 4,546 ) of the total 210,267 dalys among males and 189,408 dalys among females , respectively . the present study found these values to be 7,861 and 4,574 dalys among males and females , respectively . the difference in dalys between the sbod study in 2010 and those calculated by the researchers with a discount rate of 3% could be due to the use of singapore - specific standard life expectancy tables in the sbod study with increasing burden of lung cancer in singapore . although no other studies have evaluated the burden of lung cancer in singapore without using a discount rate , the present study found the burden to be 16,379 dalys . in the philippines , as per a previous study that estimated lung cancer burden without using a discount rate , however , the value was found to be 50,977 dalys in this study , which is less than half of the dalys reported in a 2008 philippines study . although both studies were conducted using the same methodology , the differences in dalys can be attributed to the use of dismod ii for assessment of disease epidemiology rather than obtaining estimates from the ci5plus database as well as to the assumptions and projections made by the authors . when a discount rate of 3% was used , the dalys decreased further to 38,584 in 2015 . the philippines is the only country that showed significant differences in dalys among the three countries of interest . this finding is consistent with the findings from previous studies , where 99% of dalys was due to yll in the philippines and 93% of dalys were due to yll in singapore . the high incidence and mortality rates and poor survival rates for lung cancer among older individuals can be attributed to the strong effect of ylls among dalys in these countries . furthermore , the incidence , prevalence and mortality estimates made by the researchers are approximately similar to the projections made by globocan . notwithstanding the positives that arise out of this study , limitations were also present . although the estimated dalys are believed to reflect the actual lung cancer burden in these countries , the burden may considerably vary if varying prevalence of causal agents or other risk factors are considered when estimating dalys . despite the major achievements in the field of oncology , the prognosis for lung cancer remains extremely poor and has only improved marginally over the decades . data from the past three decades in australia show that the 5-year survival rates marginally increased between 1982 and 2010 . the 5-year survival was 8% for males and 10% for females during 1982 - 1987 and 13% for males and 17% for females during 2006 - 2010 . at the same time , morbidity due to lung cancer has increased over the years . the number of hospitalizations for lung cancer in australia increased by 29% between 2000 - 2001 and 2008 - 2009 . considering the high mortality rates caused by lung cancer worldwide , appropriate strategies must be developed to manage the disease . cancer burden can be controlled and reduced by identifying and developing strategies that focus on prevention , early diagnosis , and treatment . initiatives such as the who framework convention on tobacco control can significantly reduce economic loss due to lung cancer . tobacco , alcohol , obesity , unhealthy diet , physical inactivity , pollution , radiation , and infections are considered the main cancer risk factors worldwide . given that smoking tobacco is one of the leading risk factors accounting for almost 70% of lung cancer deaths globally , the main focus should be on preventive strategies that reduce smoking . studies have shown that interventions reduce the prevalence of smoking and have proven to be cost - effective . although numerous countries have already formulated and enforced regulations to restrict tobacco usage , most health care systems have failed in giving priority to smoking interventions . in the philippines , the lung cancer control program utilizes primary and tertiary prevention at special medical centers and rehabilitation activities to control smoking . awareness messages have been spread through collaboration with department of education , culture , and sports and bureau of secondary education in schools . smoking is prohibited in workplaces , schools , gyms , parks , hospitals , elevators , and stairwells of all buildings , buses and bus depots , restaurants , and government facilities . singapore implemented the legislation of smoke - free public places in 1970 and was the first asian country to ban tobacco advertisements in 1971 . currently , singapore is implementing the point of sale display ban to reduce exposure of non - smokers to smoke . moreover , the national cancer center of singapore currently treats 60%-70% of all subsidized patients and 50% of all cancer patients in the country . the cancer council australia develops cancer control policy resources , including prevention , early detection , clinical practice ( treatment ) , and supportive care . regulations such as reducing the affordability of tobacco products , strengthening mass media campaigns , eliminating advertising and promotion , and promoting smoke free zones are being encouraged . to add to its many initiatives , australia became the first nation to implement the plain packaging of tobacco products in 2012 . since the implementation of the plain packaging act , tobacco consumption decreased by 2.9% in a quarter and 12.2% over the year , at 2 years after the inception of the legislation . these efforts include advocacy , providing information for policy , health promotion , surveillance , prevention , and management .

cation diffusion facilitator ca - sensitive cross - complementer 1 inositol 1 , 4 , 5-trisphosphate receptor vacuolar iron transporter vacuolar transporter chaperone acidocalcisomes were first identified in trypanosomes and later found from bacteria to human cells . although originally described in bacteria as metachromatic or volutin granules they were though to lack a surrounding membrane . the discovery that in some bacteria they possess a proton pump and are able to accumulate protons ( h ) and calcium ions ( ca ) established them as real organelles and led to the recognition that , together with lipid droplets , they are the only organelles present in both prokaryotes and eukaryotes . acidocalcisomes belong to the group of lysosome - related organelles and their acidification is driven by either a vacuolar h - atpase or a vacuolar h - pyrophosphatase or , in the case of trypanosomes , by both . however , a definitive demonstration of the co - localization of these proton pumps in t. brucei was not established until our recent work . in this regard , acidocalcisomes of t. brucei share the property of being acidified by 2 proton pumps with the plant vacuole , the plant - like vacuole of toxoplasma gondii , and the digestive food vacuole of the malaria parasites . an acidic acidocalcisome ph is required to maintain polyp in its polymeric state because their alkalinization results in polyp hydrolysis . polyp is a polyanion of 3 to thousands of orthophosphate monomers and binding with organic ( basic amino acids , polyamines ) or inorganic ( calcium , magnesium , sodium , potassium , zinc , iron ) cations neutralizes its negative charges . here , we discuss our recent proteomic analysis of acidocalcisomes of t. brucei to further clarify their important role in phosphate and cation homeostasis , and ca signaling . of the 2 best - studied life stages of this organism , the procyclic stages found in the vector , and the bloodstream forms found in the mammalian host , we chose the procyclic stages , which are richer in acidocalcisomes . to isolate acidocalcisomes we used a modification of protocols described before cells were broken by abrasion with silicon carbide in a mortar and after subcellular fractionation that included 2 iodixanol gradient centrifugations , a fraction highly enriched in acidocalcisomes was isolated , as established by marker enzyme assays , marker antibody immunoblotting and electron microscopy analyses ( fig . 1 and detailed in ref . ) . after sds - page of the proteins , bands were cut , digested in - gel with trypsin and peptides analyzed by lc - ms / ms . bioinformatic analysis of the resulting peptides allowed the identification of a set of candidate proteins whose acidocalcisome localization was validated by in situ epitope - tagging and western blot analyses using specific antibodies ( fig . 1 ) . knockdown by rna interference ( rnai ) of the expression of 4 of the genes encoding these proteins ( vacuolar h - atpase subunits a and d , vacuolar iron transporter , and zinc transporter ) was done to investigate their requirement for cell growth in procyclic and bloodstream forms of the parasite . growth was greatly inhibited in both life cycle stages after knockdown of the v - h - atpase subunits or of the vacuolar iron transporter . an integrated approach for functional analyses of the newly identified acidocalcisome proteins is proposed . of the 580 proteins identified with protein probability > 0.95 , ( listed in ) over 40 were selected for further study on the basis of their presence in acidocalcisomes of other organisms , or on specific properties like presence of transmembrane domains or similarity to transporters of cations or metabolites known to be present in acidocalcisomes or lysosome - related organelles . five proteins previously identified as localized to the acidocalcisomes by immunofluorescence assays ( ifa ) using epitope tagged proteins or specific antibodies , were found in the acidocalcisome proteome : vacuolar h pyrophosphatase ( tbvp1 ) , plasma membrane - type vacuolar ca - atpase ( pmca , tbpmc1 ) , inositol 1,4,5-trisphosphate receptor ( tbip3r ) , vacuolar transporter chaperone 4 ( tbvtc4 ) , and vacuolar soluble pyrophosphatase ( tbvsp1 ) . four proteins were found in the acidocalcisome proteome and their localization validated by ifa using epitope - tagged proteins : vacuolar h - atpase subunits a and d ( tbvaa and tbvad ) , acid phosphatase ( tbap ) , and phosphate transporter ( tbpho91 ) . four proteins that localize to acidocalcisomes or acidocalcisome - like organelles of other species , such as zn transporter ( tbznt ) , vacuolar iron transporter ( tbvit1 ) , polyamine transporter ( tbpot1 ) , and vacuolar transporter chaperone 1 ( tbvtc1 ) were not detected in the acidocalcisome proteome but were validated by ifa as localized to acidocalcisomes . finally , 5 proteins were detected in the acidocalcisome proteome but were localized to different organelles such as mitochondria ( abc transporter , tbabct ) , nuclear membrane ( hypothetical protein , tbnp ) , golgi and lysosomes ( golgi /lysosome glycoprotein 1 , tbglp1 ) , lysosomes ( polyamine transporter 2 , tbpot2 ) , and flagellar tip ( cation / proton antiporter , tbftp ) . although the acidocalcisome proteome contained 580 proteins most of them corresponded to proteins known to localize in other subcellular compartments . is not unexpected because acidocalcisomes are usually observed in close contact with other organelles and intracellular structures such as the nucleus , mitochondria , subpellicular mitcrotubules , and lipid inclusions and parts of these structures and organelles could co - sediment with acidocalcisomes . in this regard , contact sites between organelles has been widely described . of the proteins validated as acidocalcisome proteins , only 2 are soluble , tbvsp1 , and tbap . several proteins were identified in both the t. brucei and the cyanidioschyzon merolae acidocalcisome proteomes such as acid phosphatase , vacuolar h - atpase subunits a and d , vacuolar pyrophosphatase , zinc transporter , vacuolar iron transporter , and vacuolar transporter chaperone 1 . there is also considerable overlap of acidocalcisome proteins with proteins localized in the yeast vacuole : v - h - atpase subunits , ca - atpase pmc1p ( ortholog of tbpmc1 ) , vtc1p and vtc4p ( orthologs of tbvtc1 and tbvtc4 ) , ca - sensitive cross - complementer 1 or ccc1p ( ortholog of tbvit1 ) , zinc transporter zrc1p ( ortholog of tbznt ) , and phosphate transporter pho91p ( ortholog of tbpho91 ) , and to the arabidopsis thaliana vacuole : v - h - atpase subunits , vacuolar h - pyrophosphatase , vacuolar iron transporter 1 ( ortholog to tbvit1 ) , and metal tolerance protein 1 ( ortholog to tbznt ) . these results suggest a close relationship between these organelles . the analysis of the acidocalcisome proteome reveals potential roles of the organelle in several metabolic and signaling pathways ( fig . a role in ca signaling is supported by the presence of mechanisms for ca uptake and release . ca uptake is driven by the plasma membrane - type ca - atpase ( tbpmc1 ) . this pump acts as a ca / h countertransporting atpase requiring an acidic intraorganellar ph for efficient ca uptake . the mechanism for ca a previous study localized this channel to the acidocalcisomes using endogenous epitope tagging , which was confirmed in this work using specific antibodies . this appears to be in contrast to the apparent endoplasmic reticulum localization of the channel in t. cruzi , although this has been disputed . figure 2.schematic representation of the acidocalcisome of trypanopsma brucei modified from ref . . the newly identified acidocalcisome proteins are marked in blue while other previously identified components are shown in black . the newly identified acidocalcisome proteins are marked in blue while other previously identified components are shown in black . in addition to enzymes involved in ppi and polyp hydrolysis , like the vacuolar soluble pyrophosphatase ( tbvsp1 ) and the exopolyphosphatase ( ppx ) activity previously reported in acidocalcisomes of t. cruzi , our work confirmed the localization of 2 components of the vacuolar transporter chaperone ( vtc ) complex , tbvtc1 and tbvtc4 , in t. brucei acidocalcisomes . the vtc complex , which was first described in the yeast vacuole , where it has 4 components ( vtc14p ) , is involved in the synthesis and translocation of polyp to acidocalcisomes and acidocalcisome - like vacuoles of yeast and chlamydomonas reinhardtii . in addition to this pi entry mechanism there is also evidence for a pi release mechanism as suggested by the identification of a na / pi symporter ( tbpho91 ) with similarity to the pho91p transporter localized in the yeast vacuole and responsible for pi efflux . furthermore , an acidocalcisome acid phosphatase ( tbap ) which could be involved in pi release from organic molecules ( r - pi ) , including perhaps polyp , is also found in acidocalcisomes . given the polyanionic nature of polyp it is not surprising that acidocalcisomes need to accumulate cations to compensate all those negative charges ( fig . previous studies revealed the presence of na / h and ca / h exchangers in acidocalcisomes of procyclic forms of t. brucei , and in the new report we describe the localization in acidocalcisomes of a putative zn transporter ( tbznt ) . tbznt is homolog to a previously described t. cruzi zinc transporter and member of the cation diffusion facilitator ( cdf ) family that includes transporters involved in zn , cd , co , and/or ni transport in exchange for protons . acidocalcisomes also possess a protein ( tbvit1 ) with similarity to the vacuolar iron transporter ( vit1p ) from yeast and plant vacuoles involved in fe and mn sequestration . polyamines , which have several positive charges , are known to be abundant in the acidocalcisome - like vacuole of yeast . interestingly , it was recently reported that during zinc limitation acidocalcisomes of c. reinardtii also accumulate copper , which was proposed as a mechanism to avoid protein mismetallation . in conclusion , acidocalcisomes are rich in polyp and cations and in addition to this storage function , most of its functions in trypanosomes could probably be attributed to their components including their role in ph and osmotic regulation , response to stress , autophagy , growth and infectivity . as several of the newly identified acidocalcisome proteins ( v - h - atpase subunits a and d , tbvit1 ) are essential for growth of procyclic and bloodstream forms of t. brucei , and acidocalcisomes possess proteins with no similarity to mammalian proteins like the vacuolar h - pyrophosphatase , and the vtc complex , the newly identified pathways could be appropriate targets for chemotherapy . this work was supported by the us . national institutes of health ( grant ai-077538 to r.d ) .

transpedicular screw fixation has been the treatment of choice for stabilizing segmental instability and in cases which wide decompression is required . however , the use of pedicle screw is not suitable for all cases of degenerative spine disease . there are some disadvantages such as postoperative back pain resulted from wide muscle dissection and long operative times . it is also associated with increased rates of cerebrospinal fluid leakage , nerve injury , deep wound infection and hardware failure5,9 ) . posterior fusion may alter the normal biomechanics of the spine , and a loss of motion at the fused levels may cause an increase in motion at adjacent segments12,18 ) . as a result , a significant amount of additional force will be placed on the facet joints at adjacent levels . one commonly observed consequence of fusing spinal motion segments is adjacent segmental degeneration ( asd ) that is attributed to the increasing forces on adjacent levels22 ) . to reduce the risks associated with the pedicle screw fixation , interspinous we report the results of patients who underwent the interspinous fusion device ( ifd ) augmented with posterior lumbar interbody fusion ( plif ) . the data acquired in these patients are compared with those obtained from patients who underwent pedicle screw fixation during the same period . this study included a total of 76 adult patients with degenerative lumbar spinal diseases , who consecutively underwent posterior ifd ( spire , medtronic sofamor danek , memphis , tn , usa ) ( fig . the ifd group included 40 patients , 17 males and 23 females , who ranged in age from 21 to 76 years ( mean 56.4 years ) at the time of surgery . lumbar spine diseases in the ifd group were as follows ; spinal stenosis in 26 patients , degenerative spondylolisthesis ( grade i ) in 12 patients , and intervertebral disc herniation with severe intervertebral disc height loss in 2 . in operated levels in the ifd group , l4 - 5 was most common ( 19 cases ) . patients with advanced degenerative spondylolisthesis ( grade ii ) , spondylolysis , infection and lesions involving more than two levels were excluded . the patients who underwent transpedicular screw fixation ( pedicle screw group ) included 36 patients . there were 15 male and 21 female patients who ranged in age from 33 to 72 years ( mean ; 55.1 years ) . the mean follow up period was 14.24 months ( range ; 12 to 22 months ) in the ifd group and 18.3 months ( range ; 12 to 28 months ) . patients ' charts were reviewed to collect data regarding estimated blood loss ( ebl ) , operative time , and hospital length of stay ( los ) from the two groups , as well as to determine whether there were surgery - related complications . dynamic and static radiographs were evaluated at 1 , 6 , and 12 months postoperatively and reviewed by the authors and neuroradiologist . lumbar computed tomography ( ct ) scan was checked 6 months after the surgery to check the fusion status quo . bone fusion was defined as a presence of trabecular bone on the sagittal view of the lumbar ct or an angle change of less than 2 degrees in comparison with flexion and extension lateral radiographs23 ) . the segmental angle was measured with the angle formed by the lines drawn on the lower endplate of the upper lumbar body of the adjacent segment and the lines drawn on the upper endplate of the lower lumbar body using simple lateral x - ray ( neutral , flexion , and extension ) radiographs , and were compared between the angle on extension and that on flexion . the total sum of intervertebral angles between flexion and extension per level meant the range of motion ( rom ) at that level . according to white and panjabi 's instability in the lumbar spine , we defined the potential instability if the segmental intervertebral angle change of l1 - 2 , l2 - 3 , l3 - 4 exceeds 15 degrees and that of l4 - 5 exceeds 20 degrees and that of l5-s1 exceeds 25 degrees between flexion and extension lateral radiographs . asd was defined as a sagittal translation of the adjacent vertebral body above the fused level greater than 3 mm on the standing lateral film , and obvious segmental instability15 ) . the clinical outcome was measured using the visual analogue scale ( vas ) score and the korean version of the oswestry disability index ( k - odi ) score . although randomly allocated , there were no significant differences in these scores preoperatively between two groups . all patients were evaluated at the outpatient clinic in regular intervals at 1 , 6 , and 12 months after surgery . chicago , il , usa ) was used to statistically analyze all of the data . the mann - whitney u test , wilcoxon signed ranks test , and fisher 's exact test were used based on the characteristics of the variables being compared . all patients in the ifd group and pedicle screw group underwent plif with a polyetheretherketone cage ( plivios cage , synthes , ontario , usa ) or a titanium alloy cage ( otis cage , bk meditech , seoul , korea ) . the ifd is composed of two titanium plates with aggressive , opposing spikes connected at their midpoint by a polyaxial stem . the ifd is inserted between the rostral and caudal portions of spinous processes , and each blades make maximal contact with their respective spinous processes21 ) . an approximately 4 cm midline skin incision was made overlying the spinous processes at the target level . after dividing the lumbodorsal fascia , electrocautery was used to perform a subperiosteal dissection to elevate the erector spinae muscles bilaterally off the rostral and caudal spinous processes down to the spinolaminar junction . the supraspinous ligament of each spinous process was left in place to facilitate anatomical closure . portions of the spinous processes and laminae may also be harvested for autograft and placed in the interspinous and laminar space for posterior arthrodesis prior to implanting the plate . care should be taken to ensure that no part of the plate protrudes above the lumbodorsal fascia . once the two plates are situated optimally with one plate on either side of the spinous processes and the stem in the interspinous space , hand - held compression instruments are used to clamp the plates towards each other , driving the spikes into the bone . while compression is maintained , a locking screw is inserted and tightened to a predetermined torque . all patients in the ifd group and pedicle screw group underwent plif with a polyetheretherketone cage ( plivios cage , synthes , ontario , usa ) or a titanium alloy cage ( otis cage , bk meditech , seoul , korea ) . the ifd is composed of two titanium plates with aggressive , opposing spikes connected at their midpoint by a polyaxial stem . the ifd is inserted between the rostral and caudal portions of spinous processes , and each blades make maximal contact with their respective spinous processes21 ) . an approximately 4 cm midline skin incision was made overlying the spinous processes at the target level . after dividing the lumbodorsal fascia , electrocautery was used to perform a subperiosteal dissection to elevate the erector spinae muscles bilaterally off the rostral and caudal spinous processes down to the spinolaminar junction . the supraspinous ligament of each spinous process was left in place to facilitate anatomical closure . portions of the spinous processes and laminae may also be harvested for autograft and placed in the interspinous and laminar space for posterior arthrodesis prior to implanting the plate . care should be taken to ensure that no part of the plate protrudes above the lumbodorsal fascia . once the two plates are situated optimally with one plate on either side of the spinous processes and the stem in the interspinous space , hand - held compression instruments are used to clamp the plates towards each other , driving the spikes into the bone . while compression is maintained , a locking screw is inserted and tightened to a predetermined torque . the mean operative time in the ifd group ( 135.8 minutes , range 110 - 170 minutes ) was significantly less than that in pedicle screw group ( 170.8 minutes , range 140 - 230 minutes ) ( p=0.03 ) . ebl was also significantly less ( mean 478.8 ml , range 120 - 1000 ml ) in the ifd group than in pedicle screw group ( mean 1130.9 ml , range 500 - 1700 ml ) ( p=0.001 ) . the mean los ( including the day of surgery ) was 9.3 days in the ifd group and 15.5 days in pedicle screw group , which was not statistically significant ( p=0.208 ) . the vas score was recorded preoperatively 7.162.1 and 8.032.3 in the ifd group and pedicle screw group , respectively , and was improved postoperatively to 1.32.9 and 1.23.2 in the ifd group and pedicle screw group , respectively in 1-year follow ups ( p<0.05 ) . at immediate postoperative follow up ( pod#1 ) , vas score was much improved to 4.63.7 in the ifd group than 7.03.5 in the pedicle screw group ( p<0.05 ) . the k - odi was reduced significantly in an equal amount in the patients of both groups in one year postoperatively ( p<0.05 ) . no statistical difference in clinical outcomes was noticed between the two groups . there was no significant difference in the preoperative segmental intervertebral angle change at each level between two groups . the postoperative radiographs in pedicle screw group indicated that the rom at the instrumented level ( 3.11.5 ) was significantly decreased compared with the preoperative rom ( 11.62.3 ) ( p<0.05 ) . in the ifd group , the postoperative rom at the instrumented level ( 4.31.9 ) was also decreased significantly compared with the preoperative rom ( 9.63.1 ) ( p<0.05 ) , although the amount of decrease was not as much as observed in pedicle screw group ( table 3 ) . the rom at the upper adjacent segment in the pedicle screw group was 5.71.6 in pre - operation and 9.12.1 in post - operation . it was increased significantly after surgery ( p<0.05 ) , while the rom in the ifd group did not significantly postoperatively change at this level ( p=0.66 ) . the rom at the lower adjacent segment was reported no statistically significant differences after surgery in both groups . the number of patients with asd was 5 ( 12.5% ) in the ifd group and 13 ( 36.1% ) in pedicle screw group . the fisher 's exact test revealed that the change in the segmental angle of the adjacent segment was much increased in pedicle screw group than the ifd group ( p=0.029 ) ( table 4 ) . in the ifd group , asd was observed at cranial segment in all cases . among 13 cases of asd in pedicle screw group , 9 cases were reported in the cranial segment and 2 cases in the caudal segment . of the 40 patients in the ifd group , bone fusion was observed in 37 patients ( 92.5% ) . among 36 patients in pedicle screw group , bone fusion was noted in 33 patients ( 91.6% ) . in the ifd group , one patient had sustained back pain , and lumbar ct revealed fusion failure and fracture on l4 inferior articular process ( fig . 4 ) . another two patients showed retropulsion of interbody cage on a follow up radiograph and required re - operation . there were no major surgery - related complications such as deep infection , nerve root injury , and csf leakage in the ifd group . however , of the 36 patients in pedicle screw group , deep infection in 3 cases , csf leakage in 2 cases , and postoperative epidural hematoma required re - operation in 1 case were reported . the mean operative time in the ifd group ( 135.8 minutes , range 110 - 170 minutes ) was significantly less than that in pedicle screw group ( 170.8 minutes , range 140 - 230 minutes ) ( p=0.03 ) . ebl was also significantly less ( mean 478.8 ml , range 120 - 1000 ml ) in the ifd group than in pedicle screw group ( mean 1130.9 ml , range 500 - 1700 ml ) ( p=0.001 ) . the mean los ( including the day of surgery ) was 9.3 days in the ifd group and 15.5 days in pedicle screw group , which was not statistically significant ( p=0.208 ) . the vas score was recorded preoperatively 7.162.1 and 8.032.3 in the ifd group and pedicle screw group , respectively , and was improved postoperatively to 1.32.9 and 1.23.2 in the ifd group and pedicle screw group , respectively in 1-year follow ups ( p<0.05 ) . at immediate postoperative follow up ( pod#1 ) , vas score was much improved to 4.63.7 in the ifd group than 7.03.5 in the pedicle screw group ( p<0.05 ) . the k - odi was reduced significantly in an equal amount in the patients of both groups in one year postoperatively ( p<0.05 ) . no statistical difference in clinical outcomes there was no significant difference in the preoperative segmental intervertebral angle change at each level between two groups . the postoperative radiographs in pedicle screw group indicated that the rom at the instrumented level ( 3.11.5 ) was significantly decreased compared with the preoperative rom ( 11.62.3 ) ( p<0.05 ) . in the ifd group , the postoperative rom at the instrumented level ( 4.31.9 ) was also decreased significantly compared with the preoperative rom ( 9.63.1 ) ( p<0.05 ) , although the amount of decrease was not as much as observed in pedicle screw group ( table 3 ) . the rom at the upper adjacent segment in the pedicle screw group was 5.71.6 in pre - operation and 9.12.1 in post - operation . it was increased significantly after surgery ( p<0.05 ) , while the rom in the ifd group did not significantly postoperatively change at this level ( p=0.66 ) . the rom at the lower adjacent segment was reported no statistically significant differences after surgery in both groups . the number of patients with asd was 5 ( 12.5% ) in the ifd group and 13 ( 36.1% ) in pedicle screw group . the fisher 's exact test revealed that the change in the segmental angle of the adjacent segment was much increased in pedicle screw group than the ifd group ( p=0.029 ) ( table 4 ) . in the ifd group , asd was observed at cranial segment in all cases . among 13 cases of asd in pedicle screw group , 9 cases were reported in the cranial segment and 2 cases in the caudal segment . of the 40 patients in the ifd group , bone fusion was observed in 37 patients ( 92.5% ) . among 36 patients in pedicle screw group , bone fusion was noted in 33 patients ( 91.6% ) . in the ifd group , one patient had sustained back pain , and lumbar ct revealed fusion failure and fracture on l4 inferior articular process ( fig . 4 ) . another two patients showed retropulsion of interbody cage on a follow up radiograph and required re - operation . there were no major surgery - related complications such as deep infection , nerve root injury , and csf leakage in the ifd group . however , of the 36 patients in pedicle screw group , deep infection in 3 cases , csf leakage in 2 cases , and postoperative epidural hematoma required re - operation in 1 case were reported . spinal fusion is commonly performed in spine surgery to improve the pain and clinical outcomes of patients with failed conservative treatment for degenerative lumbosacral disease by reducing segmental instability , which is recognized as a major cause of low back pain . currently , pedicle screw fixation or posterior interbody fusion is the gold standard for surgical management of lumbar spinal instability1,22 ) . it has proven to be effective , with reported lumbar fusion rates as high as 96%4,6,19,23 ) . transpedicular fixation is associated with significant risks as followings ; 4% cerebrospinal fluid leakage , 2% transient neurapraxia , 2% permanent nerve root injury , 4 to 5% deep tissue infection , and 3 to 12% hardware failure5,9 ) . recently , numerous complications and problems after fusion surgery have been reported , with asd being one of the most important . as a cause of asd , cunningham et al.3 ) reported a significant increase in the intervertebral disc pressures by destabilization of the adjacent lumbar spine followed by stabilization with instrumentation . still , the exact etiology is uncertain but alteration in facet loading , hypermobility , and increased intradiscal pressure at the segments adjacent to fusion mass is believed to play certain key roles7,8,12,14,16 ) . spinal fusion alters the biomechanics of the spine and the loss of motion at the fused levels is at least theoretically compensated by increased motion at other unfused segments resulting in asd11 ) . superior segment facet violation or laminectomy has recently shown in vitro to destabilize the adjacent level in pedicle screw fixation2 ) . in contrary , a recent comparative biomechanical study showed that the ifd reduced the rom and load on the disc and articular processes stresses , while it increased loads transmitted through the spinous processes13 ) . and the ifd can preserve normal anatomy and does not violate adjacent facet joints , making it less likely to cause hardware - related pain or to accelerate adjacent facet degeneration20 ) . in the present study , it tended to affect the adjacent segment less than a pedicle screw . a recent clinical study showed that the incidence of asd in the cephalad adjacent segment 10 years following 360 instrumented lumbar fusion averaged 21%17 ) . in the present study , the incidence of asd was 36.1% in the patients who received pedicle screw fixation , significantly higher incidence compared with the ifd group in which it was 12.5% ( p=0.029 ) . we showed that rom at the instrumented level was significantly decreased in both the ifd and pedicle screw group compared with the preoperative state . wang et al.21 ) have studied the biomechanical characteristics of the ifd in an in vitro test of 109 cadaveric specimens and resulted that the greatest mean limitation of flexion - extension rom was 4.14 for ifd with lumbar interbody fusion whereas it was 5.03 for pedicle screw fixation with lumbar interbody fusion , and 10.13 for the intact spine . with an interbody device , the ifd provided the greatest limitation in flexion and extension over pedicle screw constructs . in short , the ifd provides enough segmental rigidity to make it a viable alternative to pedicle screw fixation . however , the ifd has some weak point which can not effectively control the axial rotation and lateral bending . recent paper by karahalios et al.10 ) demonstrated , through cadaveric study , that the capacity controlling rom such as axial rotation and lateral bending is lack compared with transpedicular screw fixation when only interspinous device used as following ; rom of lateral bending was -3.831.47 in left and 3.921.26 in case used only interspinous device . these rom was superior to normal motion , but inferior to transpedicular screw fixation ( rom of lateral bending was -0.570.14 in left and 0.570.09 ) . however , their papers revealed that lumbar interbody fusion gave the control of rom to the ifd . via cadaveric study , there was no statistically significant difference of rom between ifd with lumbar interbody fusion and pedicle screw fixation with lumbar interbody fusion . it is less invasive and presents no risk of dural or neural injury and cerebrospinal fluid leakage because it is placed on only the spinous process . in addition to the easy maneuverability of the ifd implantation , the operative time for cases involving the ifd was shorter than that for cases involving pedicle screw fixation . similarly , intraoperative ebl was much less in cases involving the ifd ; it was 50% lower than that in patients who underwent open pedicle screw fixation . the minimal bone exposure , easy implantation , and short operative time required for implantation will contribute to decreasing ebl . because many patients undergoing lumbar fusion are elderly , a lower ebl may help prevent anemia and other postoperative complications . additionally , the ifd needs a smaller incision and requires less muscle and soft tissue retraction than the pedicle screw fixation . so , it may lead to less postoperative discomfort and we showed that immediate postoperative vas scores in the ifd group were much lower than pedicle screw group in this study . although postoperative radiographs in the ifd group showed less improvement of instability at the instrumented level compared with in pedicle screw group , patients showed a similar clinical improvement as represented by k - odi , and vas scores . if more long - term studies confirm this clinical outcome , such techniques that preserve much of normal anatomy and biomechanical function of the lumbar spine as interspinous implant , will be highly indicated in the surgical treatment of spinal stenosis with various instability . it is attractive to alter the pedicle screw fixation for selected patients requiring instrumentation augmented fixation . in particular it is important to note that the application of the ifd is contraindicated in patients with pars interarticularis defects or any incompetence in the bone between anterior and posterior spinal columns21 ) . in addition , patients with advanced degenerative spondylolisthesis ( grade ii ) are not good candidates for interspinous fixation . as a retrospective unrandomized design , it could n't exclude all the factors that affect the results . and , because the incidence of asd was investigated during short period of follow up , asd was likely to occur after follow up period . so , a prospective , randomized trial with long - term follow up period is needed to confirm the efficacy of the spire plate as ifd . although the ifd is placed only on the spinous process , it provides immediate rigid fixation of the destabilized motion segments with interbody fusion that is comparable to the pedicle screws . the plate also does not violate adjacent facet joints , making it less likely to cause asd .

triad studied a random sample of adults with diabetes enrolled in 10 health plans in eight states ( hawaii , california , texas , indiana , michigan , new york , new jersey , and pennsylvania ) that served 180,000 patients with diabetes . patients were eligible to participate if they were at least 18 years old , lived in the community , were not pregnant , had diabetes for at least 1 year , spoke either english or spanish , were continuously enrolled in the health plan for at least 18 months , used at least one service during that time , and could give informed consent . institutional review boards at each participating site approved the study . in 20002001 , we administered a survey by computer - assisted telephone interview or in writing by mail . in addition , centrally trained reviewers used standardized data collection methods to abstract medical records . each year , we obtained information on triad decedents using national death index ( ndi ) plus searches ( 8) . deaths were verified by matching name , date of birth , sex , and social security number of the decedent with data supplied by the ndi . the sensitivity of ndi has been shown to range from 87 to 98% ( 9 ) . different combinations of identifiers excluding social security number ( available for 52% of participants ) correctly identify 8392% of decedents and 9299% of living individuals , making ndi an accurate means of ascertaining vital status even without social security numbers ( 10 ) . vital status was determined for all triad participants ( n = 11,927 ) through 31 december 2007 . we included triad participants who had both survey and medical record review data ( n = 8,820 ) and excluded those with type 1 diabetes defined as age at diagnosis of diabetes < 30 years and treatment with insulin only ( n = 486 ) , leaving a final study population of 8,334 . values for variables from the patient survey that had < 15% missing , including age , sex , race / ethnicity , education , income , duration of diabetes , bmi , and smoking were imputed using single imputation with the transcan function in s - plus ( edition 6.1 ; insightful , seattle , wa ) . we used the underlying cause of death icd-10 code on the ndi file to group causes of death . the categories were as follows : diabetes ( e10e14 ) , cardiovascular disease ( i0099 ) , cancer ( c0097 ) , renal failure ( n1719 ) , infection ( a00b99 , j1018 ) , external ( injury - related ) causes ( v00y89 ) , and all other codes . we investigated all - cause , cardiovascular ( icd-10 codes i00i99 for underlying cause of death ) , and noncardiovascular mortality ( all other icd-10 codes for underlying cause of death ) . time to follow - up was calculated as interview date to date of death or date censored ( 31 december 2007 ) . we assessed demographic covariates including age , sex , race / ethnicity , education , and income . we also assessed age at diagnosis of diabetes , duration of diabetes , treatment of diabetes , and bmi . we assessed clinical variables including smoking , systolic blood pressure , ldl cholesterol , hemoglobin a1c , microalbuminuria , and charlson index measured at baseline . the charlson index weights various comorbid conditions ( including dementia , cardiovascular disease , chronic obstructive pulmonary disease , peptic ulcer disease , liver disease , connective tissue disease , and cancer ) by the strength of their associations with mortality and was used to quantify comorbidity burden ( 11,12 ) . we assessed history of hypertension , dyslipidemia , transient ischemia attack ( tia ) , stroke , carotid endarterectomy , angina , myocardial infarction , other coronary heart disease , coronary angioplasty , coronary bypass , congestive heart failure , peripheral vascular disease , peripheral vascular surgery , retinopathy , nephropathy , and diabetic peripheral neuropathy . we also assessed use of aspirin , diuretic , ace inhibitor , angiotensin receptor blocker ( arb ) , -blocker , calcium channel blocker , other antihypertensive , statin , or other anticholesterol medications at baseline . we described the percent distribution of categorical variables and the mean sd of continuous variables . unadjusted hazard rate ratios ( hrs ) were constructed using each variable singularly in three separate cox proportional hazards models predicting all - cause , cardiovascular , and noncardiovascular mortality . to simultaneously adjust for covariates predicting mortality , we constructed fully adjusted cox proportional hazards models . again , all - cause mortality , cardiovascular mortality , and noncardiovascular mortality , defined by underlying cause of death , were modeled separately , using the total alive population as the reference category . we excluded age at diagnosis of diabetes and duration of diabetes from the list of potential predictors because of their high degree of collinearity with age . continuous variables were stratified into categories to allow for interpretation of patterns and to avoid violating the multiplicative model s linearity assumption . the cox proportional hazards models also included a stratum statement for health plan / provider group cluster to account for the clustered study design and the correlation among participants within health plans and provider groups . to obtain the most parsimonious model ( defined as the best prediction model with the least number of variables ) , we used stepwise cox proportional hazards models to model all - cause , cardiovascular , and noncardiovascular mortality separately , again with each compared with the total alive population . variables that were significant at p < 0.05 were entered and remained in the model . no variables were excluded from entry into the models ; however , for some variables that we had previously categorized ( age , bmi , and charlson index ) , we now entered them into the models as continuous variables . the significance of the variables in each of the models was assessed by the wald test , and we present the estimated hrs and 95% cis . we then used logistic regression to determine the parameter estimates for the 8-year prediction equations ( the last column of tables 1 , 2 , and 3 ) . the fit of the equations was assessed by the 2 log - likelihood test and the hosmer - lemeshow goodness of fit test . we also assessed the discrimination of the equations , defined as their ability to correctly identify those who died , quantified by the concordance index ( i.e. , the c - statistic ) ( 13 ) . our final three prediction equations provide estimates of a subject s likelihood of dying over 8 years expressed as a probability between 0 and 1.0 . unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting all - cause mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting cardiovascular mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting noncardiovascular mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 we also conducted 10-fold cross - validation of the prediction models . we randomly partitioned the data into 10 equal segments and extracted one 10% segment before the prediction model was fitted . the model was fitted with the remaining 90% of the data and we calculated sensitivity , specificity , and positive predictive value ( ppv ) for the 10% subsample . all analyses were performed using sas version 9.2 ( sas institute , cary , nc ) . triad studied a random sample of adults with diabetes enrolled in 10 health plans in eight states ( hawaii , california , texas , indiana , michigan , new york , new jersey , and pennsylvania ) that served 180,000 patients with diabetes . patients were eligible to participate if they were at least 18 years old , lived in the community , were not pregnant , had diabetes for at least 1 year , spoke either english or spanish , were continuously enrolled in the health plan for at least 18 months , used at least one service during that time , and could give informed consent . institutional review boards at each participating site approved the study . in 20002001 , we administered a survey by computer - assisted telephone interview or in writing by mail . in addition , centrally trained reviewers used standardized data collection methods to abstract medical records . each year , we obtained information on triad decedents using national death index ( ndi ) plus searches ( 8) . deaths were verified by matching name , date of birth , sex , and social security number of the decedent with data supplied by the ndi . the sensitivity of ndi has been shown to range from 87 to 98% ( 9 ) . different combinations of identifiers excluding social security number ( available for 52% of participants ) correctly identify 8392% of decedents and 9299% of living individuals , making ndi an accurate means of ascertaining vital status even without social security numbers ( 10 ) . vital status was determined for all triad participants ( n = 11,927 ) through 31 december 2007 . we included triad participants who had both survey and medical record review data ( n = 8,820 ) and excluded those with type 1 diabetes defined as age at diagnosis of diabetes < 30 years and treatment with insulin only ( n = 486 ) , leaving a final study population of 8,334 . values for variables from the patient survey that had < 15% missing , including age , sex , race / ethnicity , education , income , duration of diabetes , bmi , and smoking were imputed using single imputation with the transcan function in s - plus ( edition 6.1 ; insightful , seattle , wa ) . we used the underlying cause of death icd-10 code on the ndi file to group causes of death . the categories were as follows : diabetes ( e10e14 ) , cardiovascular disease ( i0099 ) , cancer ( c0097 ) , renal failure ( n1719 ) , infection ( a00b99 , j1018 ) , external ( injury - related ) causes ( v00y89 ) , and all other codes . we investigated all - cause , cardiovascular ( icd-10 codes i00i99 for underlying cause of death ) , and noncardiovascular mortality ( all other icd-10 codes for underlying cause of death ) . time to follow - up was calculated as interview date to date of death or date censored ( 31 december 2007 ) . we assessed demographic covariates including age , sex , race / ethnicity , education , and income . we also assessed age at diagnosis of diabetes , duration of diabetes , treatment of diabetes , and bmi . we assessed clinical variables including smoking , systolic blood pressure , ldl cholesterol , hemoglobin a1c , microalbuminuria , and charlson index measured at baseline . the charlson index weights various comorbid conditions ( including dementia , cardiovascular disease , chronic obstructive pulmonary disease , peptic ulcer disease , liver disease , connective tissue disease , and cancer ) by the strength of their associations with mortality and was used to quantify comorbidity burden ( 11,12 ) . we assessed history of hypertension , dyslipidemia , transient ischemia attack ( tia ) , stroke , carotid endarterectomy , angina , myocardial infarction , other coronary heart disease , coronary angioplasty , coronary bypass , congestive heart failure , peripheral vascular disease , peripheral vascular surgery , retinopathy , nephropathy , and diabetic peripheral neuropathy . we also assessed use of aspirin , diuretic , ace inhibitor , angiotensin receptor blocker ( arb ) , -blocker , calcium channel blocker , other antihypertensive , statin , or other anticholesterol medications at baseline . we described the percent distribution of categorical variables and the mean sd of continuous variables . unadjusted hazard rate ratios ( hrs ) were constructed using each variable singularly in three separate cox proportional hazards models predicting all - cause , cardiovascular , and noncardiovascular mortality . each separate model specified the total alive population as the reference category . to simultaneously adjust for covariates predicting mortality , we constructed fully adjusted cox proportional hazards models . again , all - cause mortality , cardiovascular mortality , and noncardiovascular mortality , defined by underlying cause of death , were modeled separately , using the total alive population as the reference category . we excluded age at diagnosis of diabetes and duration of diabetes from the list of potential predictors because of their high degree of collinearity with age . continuous variables were stratified into categories to allow for interpretation of patterns and to avoid violating the multiplicative model s linearity assumption . the cox proportional hazards models also included a stratum statement for health plan / provider group cluster to account for the clustered study design and the correlation among participants within health plans and provider groups . to obtain the most parsimonious model ( defined as the best prediction model with the least number of variables ) , we used stepwise cox proportional hazards models to model all - cause , cardiovascular , and noncardiovascular mortality separately , again with each compared with the total alive population . variables that were significant at p < 0.05 were entered and remained in the model . no variables were excluded from entry into the models ; however , for some variables that we had previously categorized ( age , bmi , and charlson index ) , we now entered them into the models as continuous variables . the significance of the variables in each of the models was assessed by the wald test , and we present the estimated hrs and 95% cis . we then used logistic regression to determine the parameter estimates for the 8-year prediction equations ( the last column of tables 1 , 2 , and 3 ) . the fit of the equations was assessed by the 2 log - likelihood test and the hosmer - lemeshow goodness of fit test . we also assessed the discrimination of the equations , defined as their ability to correctly identify those who died , quantified by the concordance index ( i.e. , the c - statistic ) ( 13 ) . our final three prediction equations provide estimates of a subject s likelihood of dying over 8 years expressed as a probability between 0 and 1.0 . unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting all - cause mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting cardiovascular mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 unadjusted , fully adjusted , and stepwise cox proportional hazards regression models and final equations predicting noncardiovascular mortality in the triad population adjusted for health plan / provider group cluster ( n = 8,334 ) , 20002007 we also conducted 10-fold cross - validation of the prediction models . we randomly partitioned the data into 10 equal segments and extracted one 10% segment before the prediction model was fitted . the model was fitted with the remaining 90% of the data and we calculated sensitivity , specificity , and positive predictive value ( ppv ) for the 10% subsample . all analyses were performed using sas version 9.2 ( sas institute , cary , nc ) . of the 8,334 individuals included in our analyses , 1,616 ( 19% ) died before 1 january 2008 . one percent of decedents were 2544 years of age , 22% 4564 years of age , 65% 6584 years of age , and 12% 85 years of age . fifty percent were non - hispanic white , 15% hispanic , 18% african american , 9% asian / pacific islander , and 8% of other race / ethnicity ( supplementary appendix ) . the underlying causes of death were listed as cardiovascular for 40% of decedents and noncardiovascular for 60% of decedents . the latter causes of death included cancer ( 21% ) , other ( 16% ) , diabetes ( 13% ) , infection ( 5% ) , renal failure ( 3% ) , and external ( injury - related ) causes ( 2% ) . in all , 1,107 ( 69% of the total ) had a cardiovascular cause listed in either part i or part ii of the death certificate ( icd-1 codes i0099 ) , and 31% had no cardiovascular causes listed anywhere on the death certificate . the first column of table 1 presents the unadjusted hrs for all - cause mortality . the first columns of tables 2 and 3 present the unadjusted hrs for cardiovascular mortality and noncardiovascular mortality . in unadjusted analyses of all three outcomes they can be categorized as demographic , anthropometric , diabetes , microvascular , macrovascular , medication , and comorbidity related . the second columns present the fully adjusted multivariate cox proportional hazards models for all - cause ( table 1 ) , cardiovascular ( table 2 ) , and noncardiovascular mortality ( table 3 ) . for all - cause and cardiovascular mortality , many of the variables significant in unadjusted analyses remained statistically significant in the fully adjusted models . for noncardiovascular mortality , fewer than half of the variables remained significant in the fully adjusted model . the third column presents the most parsimonious models predicting all - cause ( table 1 ) , cardiovascular ( table 2 ) , and noncardiovascular mortality ( table 3 ) . although we allowed 31 variables to enter each model , there were only 15 variables that remained in the all - cause mortality model , 16 in the cardiovascular mortality model , and 8 in the noncardiovascular mortality model . the variables that best predict all - cause mortality are older age , male sex , non - hispanic white race , lower income , treatment with insulin with or without oral medication ( vs. oral medication only ) , current smoking , higher ldl cholesterol , history of nephropathy , history of angina , myocardial infarction , other coronary heart disease , coronary angioplasty , or coronary bypass , history of congestive heart failure , use of diuretics or -blockers , and higher charlson index ( table 1 ) . the 2 log - likelihood test was 1311 ( p < 0.0001 ) with 24 degrees of freedom . the max - rescaled r was 0.30 and the hosmer - lemeshow goodness - of - fit test was 10.29 ( p = 0.24 ) with eight degrees of freedom . there were six variables that contributed at least 1% to the explained variance of the prediction equations . these were older age , higher charlson index , male sex , lower income , smoking , and history of congestive heart failure ( partial r = 0.093 , 0.032 , 0.021 , 0.015 , 0.011 , and 0.011 , respectively ) . the c - statistic was 0.815 , and the shrinkage of the model was 0.99 . based on the classification table derived from the logistic regression and roc curve analysis for the all - cause mortality equation , the probability level that maximized sensitivity and specificity was 0.185 , yielding a sensitivity of 73% , a specificity of 73% , and a ppv of 38% . using 10-fold cross - validation , average sensitivity was 75% , specificity was 71% , and ppv was 38% . the variables that predict cardiovascular mortality are older age , male sex , non - hispanic white race , lower income , treatment of diabetes with insulin ( oral medication ) , higher bmi , current smoking , higher ldl cholesterol , history of nephropathy , history of tia , stroke , or endarterectomy , history of angina , myocardial infarction , other coronary heart disease , coronary angioplasty , or coronary bypass , history of peripheral vascular disease or peripheral vascular surgery , and use of diuretics , -blockers , or other antihypertensive or cholesterol - lowering medications ( table 2 ) . the 2 log - likelihood test was 755 ( p < 0.0001 ) with 20 degrees of freedom . the max - rescaled r was 0.29 and the hosmer - lemeshow goodness - of - fit test was 10.28 ( p = 0.25 ) with eight degrees of freedom . there were five variables that contributed at least 1% to the explained variance of the prediction equation . these were older age , history of congestive heart failure , smoking , male sex , and history of stroke ( partial r = 0.120 , 0.027 , 0.018 , 0.013 , and 0.010 , respectively ) . for the cardiovascular mortality prediction equation , the probability level that maximized sensitivity and specificity was 0.090 , yielding a sensitivity of 73% , a specificity of 78% , and a ppv of 24% . using 10-fold cross - validation , average sensitivity was 85% , specificity was 63% , and ppv was 18% . the variables that predict noncardiovascular mortality are older age , male sex , lower income , current smoking , history of nephropathy , history of congestive heart failure , use of a diuretic , and a higher charlson index ( table 3 ) . the max - rescaled r was 0.24 and the hosmer - lemeshow goodness - of - fit test was 12.63 ( p = 0.13 ) with eight degrees of freedom . there were seven variables that contributed at least 1% to the explained variance of the prediction equation . these were older age , higher charlson index , male sex , lower income , history of congestive heart failure , smoking , and use of a diuretic ( partial r = 0.137 , 0.061 , 0.026 , 0.025 , 0.021 , 0.016 , and 0.014 , respectively ) . for the noncardiovascular mortality prediction equation , the probability level that maximized sensitivity and specificity was 0.120 , yielding a sensitivity of 75% , a specificity of 70% , and a ppv of 26% . using 10-fold cross - validation , average sensitivity was 68% , specificity was 73% , and ppv was 27% . we described predictors of mortality over 8 years in an insured population with diabetes that had good access to high - quality medical care . although cardiovascular disease was the leading cause of death in this population ( 40% ) , cancer and other diabetes - related conditions , such as infections and renal failure , were also listed frequently as the underlying cause of death . in general , the predictors of 8-year mortality in fully adjusted models are similar to predictors of 4-year mortality in triad , suggesting that these models are likely generalizable enough to predict future mortality risk . socio - demographic factors including older age , male sex , white race / ethnicity , lower income , and smoking are consistently associated with mortality . diabetes - related variables including older age at diagnosis , longer duration of diabetes , and treatment with insulin with or without oral medications are associated with mortality in unadjusted models . in fully adjusted models , only treatment with insulin with our without oral medications remains a significant risk factor for all - cause and cardiovascular mortality . as one might expect , history of nephropathy predicts all - cause , cardiovascular , and noncardiovascular mortality . charlson index is not associated with cardiovascular mortality , and history of macrovascular disease is not associated with noncardiovascular mortality . to our surprise , we found that history of dyslipidemia is associated with a decreased risk of all - cause and cardiovascular mortality . we hypothesize that this might be related to our inclusion of dyslipidemia and ldl cholesterol levels and use of cholesterol - lowering medications in the models . if history of dyslipidemia is an indicator of cholesterol - lowering medication use , this would tend to attenuate the association between dyslipidemia and mortality . confounding by indication higher rates of mortality are observed for those using diuretics , -blockers , and other antihypertensive medications but those medications are more likely prescribed for those with longer duration and more severe hypertension and for those with cardiovascular disease and congestive heart failure . a previous study predicting all - cause mortality in people with diabetes was performed for 33,067 individuals included in the cleveland clinic electronic health record ( 14 ) . this study only included patients initially prescribed a single oral hypoglycemic agent , whereas our study included all people identified as having type 2 diabetes regardless of medication use . both studies found that older age , male sex , white race , smoking , insulin use , history of heart disease , history of heart failure , and not using aspirin predicted greater risk of death . however , our study did not assess glomerular filtration rate , oral diabetes medication class , diastolic blood pressure , hdl , triglycerides , new diagnosis of diabetes , or clopidogrel use . we include an indicator for history of diabetic nephropathy , which was significant in the most parsimonious model . although we included bmi , hba1c , systolic blood pressure , ldl , ace inhibitor or arb use , and use of lipid - lowering drugs as possible variables , none of these entered the most parsimonious models . our findings were also similar to the results of a study conducted in hong kong . in that study , older age , male sex , history of peripheral vascular disease , and insulin use we allowed for a continuous measurement of hba1c and bmi in our model but neither was significant in multivariate analyses . our study did not investigate albumin - creatinine ratio or glomerular filtration rate , although we did include history of microalbuminuria , nephropathy , and charlson index , two of which were significant in the most parsimonious models . the measures of area under the curve , sensitivity , and specificity for our prediction equation were very similar to those for the equation developed in the hong kong study . first , we sought to develop prediction equations to optimally discriminate the outcome of interest ( mortality ) . as a result the fact that a variable enters an equation does not mean that it is causally important . similarly , variables that do not enter an equation are not necessarily unimportant in the chain of causation . we conducted a sensitivity analysis and found that stepwise selection and backward elimination models align well . for all - cause mortality , 14 of the 17 variables were the same in the stepwise and backward elimination models . it is not surprising that the noncardiovascular disease model did not align as well as the others because many different risk factors may contribute to mortality related to cancer , infections , etc . another limitation of our study is the use of data from people enrolled in managed care plans ; our results may not be generalizable to those outside of managed care , or even those in managed care plans with different levels of access to care or quality of care . indeed , the fact that our population had good access to high - quality care may have attenuated the impact of biological risk factors on mortality . everyone had physician - diagnosed diabetes for at least 18 months before the survey , which might have resulted in higher mortality rates . in conclusion , we created three prediction equations with good discrimination and high sensitivity and specificity . although our equations need to be validated in other populations , they highlight the importance of specific demographic and biological risk factors for mortality in people with type 2 diabetes and provide a quantitative assessment of risk . after further validation , clinicians may also use such equations to inform patients about their risk for mortality and to target the most modifiable risk factors for intervention .

type 2 diabetes mellitus ( t2 dm ) is a chronic metabolic disease characterized by hyperglycemia and , resulting from insulin resistance and impaired insulin secretion . clinical management of t2 dm consists of nutrition therapy , pharmacological therapy , and exercise1 . the american college of sports medicine ( acsm ) and american diabetes association ( ada ) have published positional statements recommending the use of exercise as an intervention for t2dm2 , 3 . regular exercise and physical activity are well known as an effective strategy for the prevention and treatment of t2 dm . most studies testing the therapeutic effects of exercise on t2 dm involve continuous low- to moderate - intensity exercise , such as walking , jogging , or cycling , for 30 minutes per session2 . however , the variables inherent in these exercise training programs , such as type of exercise and intensity , have varied . two separate meta - analyses , meanwhile , have concluded that high - intensity exercise may be more effective for improving glycemic control in t2dm4 , 5 . clinical tests comparing the effects of different exercise intensities on t2 dm should be completed to better identify the precise intensity of exercise needed to achieve an optimal outcome6 . high - intensity interval exercise training ( hie ) , which involves repeated bursts of vigorous exercise interspersed with periods of recovery , may be an attractive option in implementing a high - intensity exercise training program in t2dm7 . the efficacy of hie for improving disease outcomes has been demonstrated in patients with heart failure , chronic obstructive pulmonary disease , and metabolic syndrome ( ms)8 . in low - intensity exercise training ( lie ) , energy consumption is increased , but fatigue and monotonous and generally dreary feelings make this option difficult to maintain . however , the potential benefits of hie on the disease parameters within t2 dm have yet to be fully established . recently , hie protocols have been evaluated in participants with ms and t2dm7 , 9 . these studies have demonstrated drastic improvement in body composition , glycemic control , and physical fitness factors , and although the high intensity training used may limit the feasibility of living freely under unsupervised conditions , it also highlights the potential of interval training modalities in t2 dm patients10 , 11 . recent studies have shown that hie offers either similar or superior benefits in terms of glycemic control , insulin , and -cell function outcomes compared with traditional continuous exercise11,12,13 . most of the previous studies have been centered around comparison between hie and moderate - intensity exercise ( mie ) , and research data comparing hie and lie , under the assumption that lie and t2 dm patients perform different amounts of exercise . this study looks at the progress related to diabetes management in relation to the function of the endoplasmic reticulum14,15,16 . the function of the endoplasmic reticulum and the onset of metabolic diseases are closely related . also , in the pathogenesis of genetic and metabolic disorders , such as obesity , hyperlipidemia , and diabetes , it has been reported that there is a correlation between the function of the endoplasmic reticulum15 , 16 . among the endoplasmic reticulum stress responses is a phenomenon that occurs in the regulation of synthesis and the transport of proteins that occurs through the endoplasmic reticula of eukaryotic organisms ; not only does it have a close relationship with the differentiation of cells , survival rate , immunity and physiological changes , but it also has both direct and indirect relationships with various immunology and pathology related diseases17 . unfortunately , however , research on er stress and glucagon - like peptide-1 ( glp-1 ) , in relation to diabetes has been very scarce . therefore , the aim of this study was to test the feasibility of low - intensity exercise training compared with high - intensity exercise training in adolescents with t2 dm . furthermore , we compared the efficacy of hie with energy expenditure - matched lie with regard to changes in the endoplasmic reticulum ( er ) stress level , glp-1 , and glycemic control . we hypothesized that exercise training could be successfully performed and that hie would be superior to lie with regard to improvements in er stress and glp-1 , despite expected equal improvements in body composition . adolescents with t2 dm were recruited by newspaper advertisements and by contacting local diabetes patient organizations . initially , subjects were included in the study and randomized into two groups : the hie group ( n=10 ) and lie group ( n=10 ) . their mean age was 15.3 2.2 years , mean height was 162.8 11.5 cm , mean body mass index ( bmi ) was 24.0 3.8 kgm , and mean duration of diagnosis was 4.0 2.2 years . all volunteers were selected from d university hospital outpatients with result of a 2-hour glucose tolerance test of 140 mg / dl blood sugar 200 mg / dl and no other complicating diseases . all volunteers underwent medical screening , including a health status interview , physical exam , and blood analysis . also , no subjects received insulin therapy , and no medications were altered during the exercise treatments . the study was approved by the d university hospital institutional review board ( irb ) , and the subjects were included after medical examination and diagnosis by medical specialists . before beginning the exercise program , all subjects underwent anthropometric measurements ( body weight , body mass index , percentage of fat ) . body composition was measured by the bioelectrical impedance method ( venus 5.5 , jawon medical , seoul , republic of korea ) . , gyeonggi - do , republic of korea ) exercise test and maximum oxygen uptake ( vo2max ; mlkg-1min-1 ) was determined using a quark b2 analyzer ( cosmed , rome , italy ) after a 3-minute rest . rating of perceived exertion ( rpe ) was rated every minute using the borg scale . attainment of vo2max was determined by the suspension of movement criteria of the acsm in consideration of subject safety18 . based on each subject s vo2max and maximum heart rate ( hrmax ) in a maximal graded exercise test , it was possible to calculate treadmill speeds and heart rates for superordinate and subordinate goals using the metabolic equation for running of the acsm18 . in order to achieve the target heart rate ( thr ) , workload was gradually increased during trials depending on the participant s heart rate which was monitored with a polar heart rate monitor ( polar electro , kempele , finland ) , and rpe . the lie performed supervised exercise on a treadmill six times weekly for 12 weeks at 40% heart rate reserve ( hrr ) ( 6 days / week , lie ) . lie subjects completed supervised exercise continuously during each session , which burned 200 kcal . the hie group performed supervised walking and running on treadmill three times weekly for 12 weeks at 80% hrr ( 3 days / week , hie ) . hie subjects completed supervised high - intensity interval exercise ( 30 s sprint , 30 s recovery ) continuously throughout session , which burned 400 kcal . blood samples were drawn from the antecubital vein after an 8 h overnight fast . blood samples were centrifuged for 15 minutes ( 3,000 rpm , 4 c ) , stored at 80 c , and directly analyzed . biochemical analyses of serum glucose - regulated protein 78 ( grp78 ) , glucagon - like peptide-1 ( glp-1 ) , dipeptidyl peptidase-4 ( dpp-4 ) , and leptin were performed using enzyme - linked immunosorbent assays for quantitative detection of human bip / grp78 , glp-1 , human dpp-4 ( abnova corporation , walnut , ca , usa ) and leptin ( r&d systems , minneapolis , mn , usa ) . all factors were quantified using polyclonal antibodies that recognized native human grp78 , glp-1 , dpp-4 , and leptin a series of plates containing wells coated with predetermined amounts of recombinant human grp78 , glp-1 , dpp-4 , and leptin . hba1c levels were determined by high performance liquid chromatography using commercially available kit reagents ( bio - rad , hercules , ca , usa ) . insulin resistance in the fasting state was determined by homeostasis model assessment ( homa - ir ) using the following formula : [ fasting plasma glucose ( mgdl ) fasting plasma insulin ( udl ) ] 405 . data analysis was performed ibm spss statistics for windows version 19.0 ( ibm corp . , two - way anova was used to control for differences between groups , test periods , body composition , and blood component changes caused by exercise type , and the paired and independent t - test method was used for the post hot test . a level of statistical significance of p < 0.05 was adopted for this study . the characteristics of the hie and lie groups at the beginning of the study are shown in table 1table 1.the changes in body composition after 12 weeks of exercise trainingvariablegroupprepostdiff ( % ) weight ( kg)hie62.213.360.014.42.2 ( 3.5)lie66.720.965.423.01.3 ( 1.9)bmi ( kg / m)hie25.23.324.44.00.8 ( 3.2)lie22.84.222.44.80.4 ( 1.8)fat ( % ) hie29.92.927.63.32.3 ( 7.7)lie24.24.123.94.90.4 ( 1.6)vo2max mlkgminhie2,223.5384.72,496.2577.8273 ( 12.9)lie2,382.5665.12,505.2547.2123 ( 5.2)values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise ; bmi : body mass index . * there were significant difference in body weight and percentage body fat between the lie and hie groups ( p < 0.05 , respectively ) . also , hie resulted in a greater reduction in percentage body fat than the lie ( p < 0.05 ) . values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise ; bmi : body mass index . * significant difference within group ( p<0.05 ) . significant difference compared with lie ( p<0.05 ) table 2table 2.the changes in glycemic control after 12 weeks of exercise trainingvariablegroupprepostdiff ( % ) c - peptide ( ng / ml)hie2.91.34.72.11.8 ( 62.1)lie3.92.35.42.21.5 ( 38.5)insulin ( u / ml)hie42.127.418.78.8l23.4 ( 55.6)lie58.338.846.541.411.8 ( 20.2)glucose ( mg / dl)hie174.325.5138.835.735.5 ( 20.1)lie175.318.5131.826.470.2 ( 36.6)homa - irhie17.410.56.53.410.9 ( 62.6)lie24.916.713.511.411.4 ( 45.8)hba1c ( % ) hie8.83.36.61.02.2 ( 25.0)lie7.41.26.40.41.0 ( 13.5)leptin ( ng / dl)hie1,9236701,178406745 ( 38.7)lie1,423683979618444 ( 31.2)values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise . * significant difference compared with lie ( p<0.05 ) shows the changes in glycemic control after the 12-week exercise training programs in the c - peptide level increased after the 12-week programs and was significantly different between groups ( p < 0.05 ) . the insulin level was significantly decreased after the 12-week exercise training program in the hie group . fasting glucose , homa - ir , and leptin decreased after the 12-week exercise programs and were significantly different between groups ( p < 0.05 , p < 0.05 , p < 0.05 , respectively ) . however , hba1c showed no significant difference after the 12-week exercise training programs . values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise . significant difference compared with lie ( p<0.05 ) table 3table 3.the changes in glp-1 , grp78 and dpp-4 after 12 weeks of exercise trainingvariablegroupprepostdiff ( % ) glp-1 ( ng / dl)hie0.210.020.390.040.18 ( 85.7)lie0.210.020.360.020.15 ( 71.4)grp78 ( ng / dl)hie3.884.016.336.652.45 ( 63.1)lie2.541.453.171.980.63 ( 24.8)dpp-4 ( ng / dl)hie29.2320.2322.8521.436.38 ( 21.8)lie34.1623.9829.2024.004.96 ( 14.5)values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise ; glp-1 : glucagon - like peptide-1 ; grp78 : glucose - regulated protein 78 ; dpp-4 : dipeptidyl peptidase-4 . * significant difference compared with lie ( p<0.05 ) shows the changes in er stress factors after the 12-week exercise training programs . glp-1 increased after the 12-week exercise programs and was significantly different between groups ( p < 0.05 ) . values are meanssd , hie : high - intensity exercise ; lie : low - intensity exercise ; glp-1 : glucagon - like peptide-1 ; grp78 : glucose - regulated protein 78 ; dpp-4 : dipeptidyl peptidase-4 . * the main findings of this study are that hie can be implemented as an exercise treatment method in t2 dm patients and that it is superior to energy expenditure - matched lie with regard to improvements in body composition , glycemic control , and glp-1 under the conditions used in this study . our research design included careful and successful matching of the exercise training energy expenditures and mean intensities of the training groups . the results of this study indicate statistically significant improvements in all outcomes in both groups . however , the gains in body composition ( bmi ) and er stress factors ( grp78 , dpp-4 ) were not significantly different between the groups . participants in the hie group demonstrated significant and similar gains in glycemic control and glp-1 during the 3-month trial . the acsm and ada recommend moderate- and vigorous - intensity exercise for people with t2 dm and provide evidence - based guidelines for prescribing exercise training , in terms of intensity , volume , frequency , duration , and rate of progression1 , 18 . in this study , the variable of intensity was different between the groups , whereas the variables of volume , duration , and rate of progression were identical . on the basis of the findings of this study , individuals who have type 2 diabetes and participate in exercise training at either a moderate- or high - intensity level , as defined by the acsm and ada , may experience similar gains in body composition and glycemic control . our finding indicate that the hie program resulted in more positive changes in weight , bmi , body fat percentage , and glycemic control ( c - peptide , fasting glucose , homa - ir ) in t2 dm patients than the typical lie program that is usually recommended to existing diabetic patients . hie also showed positive changes in grp78 , dpp-4 , and glp-1 , which are major factors of er stress , showing more effective improvement for t2 dm patients . as low- to moderate - intensity exercise , the acsm exercise guidelines18 for diabetes patients recommends an aerobic exercise program involving more than 150 minutes of exercise per week . however , the hie program maintains homeostasis ; increases energy consumption ; prevents metabolic diseases , such as obesity , and diabetes ; and is known to reduce stress7 , 19 , 20 . sedentary diabetic patients report many barriers to physical activity , but the most common reason given is a lack of time for exercise . however , there is a growing body of literature that compares the metabolic risk factor reduction effects of traditional endurance training programs with the reduction of risk resulting from novel exercise interventions that require minimal amounts of time for patients . for this reason , hie was recognized as an effective compromise that improves on the tediousness of lie21 , 22 . moreover , it is effective in inducing insulin sensitivity and controlling weight for t2 dm patients23 . it has been shown that the changes in physiological function , performance , and health - related markers resulting from hie are similar to those resulting from lie and that hie results in more positive change in the health of patients than lie9 . in this study , the hie group showed a significant reduction in body fat percentage after the exercise compared with before the exercise . in terms of the amount of reduction in body fat percentage , the hie group showed a higher reduction ( 7.69% ) than the lie group . both groups showed improvement in glycemic control , but the hie group was more effective . previous studies showed that exercise proved to be effective in regulating glycometabolism by bringing about changes in body weight and percentage body fat . glycemic control is medically important for t2 dm patient treatment and is an independent risk factor that can predict the possibility of diabetes patients developing complications24 . in particular , most studies demonstrated that glycemic control is improved through hie ( 80% peak power output ) after body weight loss25 , 26 . it has the effect of reducing insulin secretion , cholesterol , and triglycerides ; it also improves blood sugar levels and suppresses the direct stimulation of glucose intake in the skeletal muscle20 . the present study shows improvements in blood glucose levels according to the change in body weight , with the hie group showing a greater reduction than the lie group . it also shows that leptin was reduced in both groups , but hie was particularly more effective . t2 dm is caused by interactions between genetic and acquired factors , such as overeating and insufficient exercise , and is characterized by hyperglycemia due to relative insulin insufficiency and insulin resistacne27 . exercise has been studied regarding its role in improving insulin resistance and it has been classified by type , intensity , and activity period29 . dube et al.30 recently reported that a graded dose - response relationship exists between exercise intensity and improvements in insulin sensitivity . when matched for caloric expenditure , high - intensity exercise appears to be at least as effective as moderate - intensity exercise for improving insulin sensitivity31 . our findings indicated that the hie group showed more positive changes in c - peptide level , fasting glucose , and homa - ir than the typical lie group . the imbalance between the protein handling capability of the er and its load is referred to as er stress32 . ozcan et al.32 confirmed that reduction of er stress in the hypothalamus resulted in improvement of leptin resistance ; in addition , it was confirmed that er stress is a major cause of leptin resistance32 and that it is also a major factor that induces leptin resistance . the present study showed that er stress increased as a result of the increase in physical stress during exercise in participants in the hie group but not in those in the lie group , but the changes in weight , obesity , and insulin resistance ( characterized by positive changes in body fat ) demonstrated that exercise also had positive effects on leptin changes in adolescents with t2 dm . grp78 plays a significant role in maintaining the viability of cells exposed to various stress environments and helps in the response to immune attacks as well as in resisting the effects of various drugs33 . grp78 is increased in patients with t2 dm , and it has been reported that insulin resistance can be improved through an exercise - induced increase in grp7834 . in addition , it has been reported that exercise during hypoxia improves insulin sensitivity . treatment with hypoxia has been reported to be an effective for prevention and treatment of diabetes35 . according to previous studies , exercise increases the amount of grp-78 , which has a positive effect on improvement of insulin resistance36 . the present study showed that both groups had increased levels of expression of grp-78 , but the hie group showed a larger increase . the cause seems to be an oxygen deficiency occurring during hie , the exhaustion of glucose in the blood , and more physical stress during the exercise . recently , the role of glp-1 in the treatment of obesity and t2 dm has emerged . glp-1 , an incretin hormone that regulates blood glucose levels37 , improves insulin sensitivity by stimulating the pancreas to secrete insulin3 and is reported to increase the hydrolysis of triglyceride in adipose tissue38 . also , previous exercise - related studies have shown that healthy people have increased levels of incretin hormones such as glp-115 . people who are obese showed similar results to people with normal weight through weight loss resulting from exercise40 . since exercise is considered to be the first line of intervention for the prevention and management of t2 dm , improving glp-1 through exercise is of the utmost importance . glp-1 , secreted through food stimulation , is decomposed at a very high speed by the enzyme dipeptidyl peptidase-4 ( dpp-4 ) , and its involvement in the physiological regulation of eating causes weight loss41 . in this study , both groups were found to have significantly increased levels of glp-1 , with the hie group appearing to have a greater increase . ddp-4 also affects the amount of glp-1 , and is an important factor in predicting the onset of insulin resistance and ms ; it also has a direct association with insulin resistance42,43,44 . higher levels of it were also found in people with obesity or ms43 , and it is suggested to have a close relationship with the levels of bmi in healthy young people44 . the present study showed that dpp-4 decreased in both groups , but the hie group showed an approximately 21% greater reduction . these results are believed to be due to the reduction of bmi and body fat percentage , which resulted in suppression of dpp-4 activity and in turn creation of incretin hormones that normally control glucose levels , and this is believed to have resulted in the reduction of insulin resistance that ultimately improved the function of glycemic control in t2dm45 . therefore , high - intensity interval exercise training , as defined in this study , may cause improvements in body composition , glycemic control , and glp-1 in adolescents with t2 dm . in summary , we compared the efficacy of hie with energy expenditure - matched lie in regard to changes in body composition , glycemic control , er stress level ( grp78 , dpp-4 ) , and glp-1 . we have showed that hie can be implemented in individuals with t2 dm . furthermore , we showed that hie counteracts deteriorations in glycemic control , and that it is superior to lie with regards to improvements in body composition ( body fat percentage ) , glycemic control , and glp-1 . hie may therefore be a good option when considering which type of exercise training t2 dm patients should be recommended in primary care .

the most frequent metastasis sites of synovial sarcoma are the lungs , lymph nodes , and bone . pancreatic metastasis is quite rare ; only 3 cases have been reported worldwide to date . a 32-year - old man underwent extended excision of synovial sarcoma in the left pelvis and femur in 2009 . in 2013 , follow - up contrast - enhanced computed tomography revealed a 35-mm heterogeneously enhanced mass in the pancreas body . endoscopic ultrasound - guided fine needle aspiration of the mass revealed a diffuse proliferation of atypical spindle cells in a fascicular arrangement . because the histology was quite similar to the resected specimen of synovial sarcoma in 2009 , the mass was suspected to be a metastasis from synovial sarcoma . synovial sarcoma - specific ss18-ssx1 ( synovial sarcoma translocation , chromosome 18-synovial sarcoma x1 ) or ss18-ssx2 chimera mrna was detected in the resected specimen , confirming the diagnosis of metastasis from synovial sarcoma . this case suggests that pancreatic metastasis from synovial sarcoma can be successfully treated by metastasectomy with adjuvant chemotherapy . soft tissue sarcoma is a rare mesenchymal neoplasm that accounts for about 1% of all adult cancers . synovial sarcoma , first reported in 1893 , is one of about 50 histological types of soft tissue sarcoma but accounts for approximately 10% . the long duration of symptoms and initial slow growth sometimes give a false impression of benign characteristics . however , because of its aggressive potential , metastasis occurs in approximately 50% of patients , and the most frequent sites of metastasis are the lungs ( 74%81% ) , lymph nodes ( 3%23% ) , and bone ( 10%20% ) . pancreatic metastasis from synovial sarcoma is quite rare , and a standard management regimen has not been established . in this report , we present a case of solitary pancreatic metastasis from synovial sarcoma that was successfully treated by radical metastasectomy with adjuvant chemotherapy . a 32-year - old man without a medical history first visited osaka university hospital in 2009 for the treatment of synovial sarcoma in the left pelvis and femur ( fig . he underwent extended tumor resection and reconstruction with constrained total hip megaprosthesis with neoadjuvant adriamycin / ifosfamide ( ai ) therapy for 4 cycles , followed by adjuvant ai therapy for 1 cycle . he was regularly followed up thereafter , and no recurrence was observed on annual computed tomography ( ct ) examinations until 2012 . ( a ) t1- and ( b ) t2-weighted images of nonenhanced magnetic resonance imaging . ( c ) macroscopic and ( d ) microscopic appearance of the resected specimen ( hematoxylin eosin staining , 200 ) . in 2013 , however , follow - up contrast - enhanced ct revealed a 35-mm heterogeneously enhanced hypervascular mass in the pancreas body ( fig . enhanced magnetic resonance imaging ( mri ) , the mass was depicted as a hypo- , hyper- , and hyper - intense lesion on t1- , t2- , and diffusion - weighted images , respectively ( fig . hepatobiliary - phase images revealed no space - occupying lesions suggesting liver metastasis . on fluorodeoxyglucose positron emission tomography , the mass showed low uptake ( maximum standardized uptake value of 2.3 ) ( fig . 2 g ) , and no abnormal uptake was observed in the whole body . the patient was asymptomatic , and laboratory tests of routine biochemical parameters and tumor markers including carcinoembryonic antigen , carbohydrate antigen 199 , elastase i , and sialyl lewis x antigen were all within their respective normal ranges . ( b ) vascular - phase image of contrast - enhanced ultrasonography . ( c ) t1- , ( d ) t2- , and ( e ) diffusion - weighted images and ( f ) arterial - phase images of gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid enhanced magnetic resonance imaging . on grayscale eus , the lesion was depicted as a well - demarcated and heterogeneous mass with abundant cystic components ( fig . although the mass showed expansive growth and oppressed the splenic vein , no invasion was suspected . avoiding cystic elements , a 22-gauge puncture needle was transgastrically inserted into the mass , and specimens were collected ( fig . histological examination revealed a uniform population of small spindle - shaped cells in a fascicular arrangement ( fig . immunohistochemically , these cells were positive for bcl-2 and negative for pan - cytokeratin , epithelial membrane antigen ( ema ) , chromogranin a , synaptophysin , and vimentin . although the immunohistochemical pattern was not typical for synovial sarcoma , the mass was considered to be pancreatic metastasis from synovial sarcoma because these microscopic findings were quite similar to the resected specimen of monophasic synovial sarcoma in 2009 . endoscopic ultrasound ( eus)-guided fine needle aspiration ( fna ) of the pancreatic metastasis from synovial sarcoma ( arrowheads ) . the puncture needle is indicated by an arrow . ( a ) grayscale eus , ( b ) fna , ( c ) cytology ( papanicolaou stain , 400 ) , and ( d ) histology ( hematoxylin eosin staining , 200 ) of the tumor . we judged that the metastatic lesion could be completely resected without residual tissue and performed laparoscopic distal pancreatectomy under adequate informed consent . although a minor pancreatic fistula occurred postoperatively , it was conservatively treated and resolved in a short period of time . the resected tumor was a well - circumscribed mass measuring 45 30 mm with prominent cystic changes ( fig . microscopic examination revealed a monophasic appearance comprising only spindle cells , similar to the biopsy sample obtained by eus - fna , and glandular structures were not found ( fig . immunohistochemistry revealed a positive reaction to bcl-2 ( diffuse ) and ema ( focal ) ( fig . 4d , e ) and a negative reaction to pan - cytokeratin , -smooth muscle actin ( sma ) , cd34 , and c - kit . synovial sarcoma - specific ss18-ssx1 ( synovial sarcoma translocation , chromosome 18-synovial sarcoma x1 ) or ss18-ssx2 chimera mrna was detected by reverse transcriptase - polymerase chain reaction ( rt - pcr ) , which confirmed the diagnosis of pancreatic metastasis from synovial sarcoma ( fig . 4f ) . resected specimen of the pancreatic metastasis from synovial sarcoma ( arrowheads ) . ( f ) electrophoresis of ss18-ssx1 ( synovial sarcoma translocation , chromosome 18-synovial sarcoma x1 ) or ss18-ssx2 chimera mrna in the tumor after reverse transcriptase - polymerase chain reaction . the primers used were as follows : forward primer , 5-caacagcaagatgctacca-3 and reverse primer , 5-cacttgctatgcacctgatg-3. the patient underwent 2 cycles of adjuvant ai chemotherapy followed by 2 cycles of ifosfamide , carboplatin , and etoposide without remarkable adverse events . at the time of this writing on contrast - enhanced ct / mri , synovial sarcoma is typically depicted as a heterogeneously enhanced , well - circumscribed mass . because these imaging findings are not specific for synovial sarcoma synovial sarcoma is histologically divided into 3 subtypes : the monophasic type composed only of spindle cells ( 50%60% ) , the biphasic type composed of both epithelial and spindle cell elements ( 20%30% ) , and the poorly differentiated type ( 15%20% ) . among these histological subtypes , meanwhile , the clinical course of poorly differentiated type tends to be aggressive with early recurrence and metastasis . on immunohistochemistry , synovial sarcoma is positive for epithelial markers ( e.g. , keratins and ema ) , bcl-2 , and transducin - like enhancer of split-1 , and negative for skeletal muscle markers ( e.g. , desmin and -sma ) and cd34 . conversely , identification of the ss18-ssx1 or ss18-ssx2 fusion gene resulting from the chromosomal translocation t ( x;18)(p11;q11 ) , first published in 1994 , reportedly has a sensitivity of 94% and a specificity of 100% by rt - pcr . owing to this high diagnostic ability , demonstration of the ss18-ssx1 or ss18-ssx2 fusion gene is currently considered to be the gold standard diagnostic test for synovial sarcoma . the optimal treatment of synovial sarcoma is surgical resection , if the mass is resectable , followed by adjuvant chemotherapy using adriamycin and ifosfamide . for unresectable masses , synovial sarcoma is an intermediate- to high - grade sarcoma , and the 5- and 10-year survival rates reportedly range from 36% to 76% and 20% to 63% , respectively . pancreatic metastasis from synovial sarcoma is quite rare , and only 3 cases have been reported worldwide to date . the clinical characteristics of these 3 cases and our case are summarized in table 1 . both imaging and histological examinations were performed to achieve the correct diagnosis in all cases . however , the imaging features of pancreatic metastasis from synovial sarcoma have a broad range of differential diagnoses , including pancreatic neuroendocrine tumors , pancreatic metastases from renal cell carcinoma ( rcc ) , gastrointestinal stromal tumors , paraganglioma , solitary fibrous tumors , pancreatic hamartoma , intrapancreatic accessory spleen , and others . some of these tumors have a histological appearance similar to that of synovial sarcoma , and immunohistochemistry therefore , the presence of the ss18-ssx1 or ss18-ssx2 fusion gene should be investigated to confirm the diagnosis ; ours is the only case in which its existence was proven . because our case shows that ss18-ssx1 or ss18-ssx2 chimera mrna can also be detected in pancreatic metastasis , demonstration of the ss18-ssx1 or ss18-ssx2 fusion gene in a biopsy sample obtained by eus - fna might be the gold standard technique for the pretreatment diagnosis of pancreatic metastasis from synovial sarcoma . we performed metastasectomy to treat the tumor in this case . for unresectable synovial sarcoma , . however , sufficient evidence of drug therapy has not been established , and the effect is limited . only in cases of pulmonary metastasis can long - term survival be expected by pulmonary metastasectomy if complete resection is achieved . the question then arises regarding whether pancreatic metastasectomy enables long - term survival of patients with pancreatic metastasis from synovial sarcoma . common primary tumors are lung cancer , breast cancer , rcc , malignant melanoma , and gastrointestinal cancers . although most patients with pancreatic metastasis are not usually candidates for resection because they are considered to have systemic micrometastasis , the effectiveness of pancreatic metastasectomy has been proven in specific cancer types such as rcc . when considering surgical resection of pancreatic metastases , 4 factors are reportedly associated with a good prognosis after resection for pancreatic metastases from various malignant tumors : primary rcc , a > 3-year interval between resection of the primary tumor and development of pancreatic metastases , isolated pancreatic metastases , and no prior recurrence . with respect to synovial sarcoma , the effectiveness of pancreatectomy for metastatic lesions is completely unknown because only one case in which pancreatic metastasectomy was performed has been reported to date ( table 1 ) . in that case , however , a disease - free survival period of > 6 years was achieved after resection . similar to that case , the metastatic tumor in our case was solitary without extrapancreatic lesions . additionally , our case met 3 of the 4 criteria previously reported to be associated with a good prognosis after pancreatic metastasectomy : a > 3-year interval between resection of the primary tumor and development of pancreatic metastases , isolated pancreatic metastases , and no prior recurrence . therefore , we considered that long - term survival could be expected by pancreatic metastasectomy in our case as well . actually , the metastatic lesion was successfully resected and the patient had been disease - free for 30 months at the time of this writing . this result suggests that pancreatic metastasis from synovial sarcoma can be successfully treated by metastasectomy unless apparent extrapancreatic metastasis is present . in conclusion , we have presented a quite rare case of solitary pancreatic metastasis from synovial sarcoma that was successfully treated by radical metastasectomy with adjuvant chemotherapy . additional cases should be accumulated to investigate the characteristics of patients who are indicated for metastasectomy and to establish pancreatic metastasectomy as a treatment option for pancreatic metastasis from synovial sarcoma .

oral cancers became an important part of oro - maxilo - facial pathology because of its increased frequency , the low rate of survival , as well as the resulted functional or physiognomical defects . multiple mechanisms are acting at a molecular level and induce the neoplazic transformation of the squamous cells . at the level of the oral mucosa a continuous lesional spectrum is evidenced having the onset with preinvasive lesions , ending with invasive carcinomas and metastasis . it represents almost 95% of the head and neck cancers , predominantly developing in the oral cavity . though the therapeutic progress made in the last years is remarcable the long term rate of survival is still below 50% . the studies have shown a greater incidence among men that chronically exposed themselves to smoking and alcoholism , as well as in families with a history of head and neck cancer . there is not yet an explanation for the high frequency of oral cancer in people under 40 years of age that were not exposed to the known risk factors . the study is retrospective and was realised by selection of cases that appeared during two years ( 2007- 2008 ) . for the retrospective cases the data came from the patients charts of the clinic of oro - maxilo - facial surgery , from the emergency hospital s policlinic as well as from the anatomical pathology laboratory for all cases , there were made seriate sections which were coloured with hematoxilin - eosin . we followed a series of clinical , epidemiological and histopathological indicators like age , sex , pathological antecedents , tumor localization , macroscopical and histopathological parameters . the integration in the tnm categories was done according to the 2002 tnm classification , excepting the cases in which only biopsy was used for diagnosis . the analyzed squamous carcinomas were present at pacients with the age between the third and nineth decade of life . the incidence increse progresivelly starting with the third decade to the eighth decade of life . most of the cases ( 83,8% ) were patients with ages between 50 and 79 years old . age distribution of cases regarding the allocation by sex we observed the predominance of tumors in male patients , representing 90% of the cases . sex distribution of cases by anamnesis we pinpointed the risk factors associated with oral carcinogenesis , these being : smoking , alcoholism , family history and pre - existing lesions . risk factors distribution of cases we observed that mostly the risk factors had associated between them , especially alcoholism and smoking or alcoholism , smoking and pre - existing lesions . if smoking alone or chronic alcoholism alone found themselvesin the antecedents of the patients in a proportion of 9,40% and 4,30% , the two associated risk factors were present in a proportion of 18% of the studied cases . the association between smoking and pre - existing lesions was present in a lesser proportion of only 5,55% . the local pre - existing cancerous lesions were found in 5 cases , isolated or in association with smoking . they are represented by microtraumas , precancerous lesions ( erythroplasia , leukoplasia , lichen planus , oral fibrosis , actinic cheilitis ) , chronic inflamations of the tongue and lips . the chronic iritations of the oral mucosa ( dental malocclusion , dental cavities , defective dentures ) and chronic inflammations of the oral mucosa act as a promoter of the oral cancer . topography distribution of lesions regarding the localization of the lesions , the studied squamous carcinomas had the next locations in order of the frequency : lips , tongue , gingivae , floor of the mouth , soft palate . the study indicated the fact that tongue localization , excepting that in the lips , equals all other localizations of the oral squamous carcinomas . the tumors in this study , regarding their type , are classified in the next categories : ulcerate , vegetant , ulcero - vegetant and infiltrative . macroscopic types of squamous carcinomas lower lip vegetant carcinoma the analysis of the tumor dimensions indicated important variations about their maximum diameter . in a decreasing order of frequency we found 31 cases ( 48,8% ) of tumors with dimensions between 2 - 4 cm in the long tumoral axis and a number of 15 cases ( 23,4% ) of tumors with dimensions over 4 cm in the long tumoral axis . in 4 of the studied cases the tumors were over 4 cm in the long axis with profound invasion throgh the cortical bone to the floor of the mouth and tongue . the tumoral mass dimensions the histopathological study had in view the variety of the squamous carcinomas and the degree of differentiation , the tumoral progression stage and the presence of malignant residual cells in the surgical safety borders . corresponding to the differentiation degree , the analysis of squamous carcinomas are well differentiated in 44 cases , moderately differentiated in 32 cases and poorly differentiated in 42 cases . the stage of tumoral progression in the well differentiated squamous carcinomas group were included the keratinized forms of tumors and the verrucous carcinomas . in the moderately differentiated squamous carcinomas group were included the non - keratinized forms and the adenoid variant . in the poorly differentiated squamos carcinomas group were included some non - keratinized forms , the basal and spindle - shaped carcinomas . well differentiated squamous carcinoma in the upper lip , ob.4 , he stain moderately differentiated squamous carcinoma in the upper lip , ob.4 , he stain poorly differentiated squamous carcinoma in the upper lip , ob.10,he stain regarding the stage of tumoral progression the analyzed squamous carcinomas correspond to : microcarcinoma in 2 cases , invasive carcinomas in 57 cases and invasive carcinomas associated with metastatic adenopathy in 3 cases . the stage of tumoral progression microcarcinomas were localised in the lower lip , associated with modifications of actinic cheilitis and at the tongue associated with aspects of leukoplasia , simple dysplasia and mycelium deposits . their microscopic aspect was that of a malignant cancer proliferation with minimum invasion of the superficial chorion with the shape of a spur made up of atypical squamous cells . frank invasive carcinomas without metastatic adenopathy represent the most frequent neoplasms encountered in this study . regardless of the histopathological form and of the degree of differentiation the tumors have evolved infiltrative and destructive as islands and neoplasic lobules in the structures of origin . tongue squamos carcinoma , keratinized form , round island neoplasic invasion , ob.10,he stain frank invasive carcinomas with metastatic adenopathy were present in 3 cases . in two cases the histopathological aspect of the metastases was similar to the one of the primtive neoplasms regarding the tumors shape and degree of differentiation , specifically of non - keratinized squamous carcinomas with moderate or poor degree of differentiation . another microscopic parameter that was analysed was represented by the presence of residual malignant cells in the surgical safety borders . a number of 12 cases did not present an invasion at this level , menwhile in another 13 cases the invasion was present , taking the form of microcarcinoma in 10 cases and of invasive carcinoma in another 3 cases . in europe and australia the incidence among the annually diagnosed neoplasms is 5% compared to asia and india where it represents almost 50% . in france oral cancers are the most frequent type of cancer for the male patients and is ranked second for causes of death by cancer . research carried out in finland between 1953 and 1999 indicates a number of 17383 new cases , mostly in male patients . in the study we carried out most of the analysed cases ( 83,8% ) similar studies show that the predilection age for oral cancer is between 50 and 70 years , the mean age being 64 years . lately it is observed a rise in the incidence of lesions in women and young people , cases being decribed even in children patients , especially with tongue localization . the data from the specialized literature estimates that the male sex is affected in a proportion of 75 - 80% of cases , emphasizing that in the last years there was a rise in the incidence of these lesions among female patients . the patients in our study are in terms with these data , 89,75% being male patients . the american society of oncology estimates that in 2004 oral and pharynx neoplasms represented 3,2% and 1,7% of the new cases in male and female patients . in great britain oral cancers are ranked 15-th in all male cancers and 20-th in female cancers . world wide , oral cancer represents the fourth type of cancer in male patients and sixth type in women patients . smoking and alcoholism are dominant risk factors , being found in the past history of 75% of the patients with oropharyngeal and oral cavity cancers . it has been observed a frequency of 2 - 4 times greater of squamous carcinoma in smokers , the risk rising up to 6 - 15 times in association with chronic alcoholism . epidemiological studies showed a high risk for this disease for the families with a history of head and neck cancer . in this study we observed the fact that most often the risk factors have associated among them , especially alcoholism and smoking , or alcoholism , smoking and pre - existing lesions . approximatively 30 - 35% of the tumors have a tongue localisation , 20 - 25% a gum localisation , 5 - 7% are localised on the floor of the mouth , 4 - 6% on the soft palate and only 2 - 3% are set on cheeks . our study showed the fact that the tongue localisation , excepting the one on the lips , equals all other localisations of the squamous carcinomas that appear in the oral mucosa . in great britain the most common setting is on the floor of the mouth and tongue . in india the most affected area is the oral mucosa , being the consequence of smoking and betel chewing habit . . the found aspects , regarding the macroscopic pattern and the tongue malignant tumors are being quite similar to the data mentioned in the literature thus it is mentioned that the most commonly met form of this tumor is the ulcerate one , and rarely it can be seen in it s other aspects : vegetant , ulcero - vegetant or infiltrative . among the analysed histopathogical factors it is considered that the tumor pattern in the tumor invasion front can be one of the prediction factors of tumor evolution . string - shaped or non - cohesive islands or even isolated neoplasic cells invasion can be associated with metastasizing risk growth , though the tumors with a compact aspect in the advancement front are associated with a much less agressive evolution . also , many studies carried out on the factors of predictiction of the evolution of oral squamos carcinomas have as subject the role of the regional metastatic nodes . the presence of metastases in two or more regional limph nodes , especially associated with extracapsular tumor extension , are a major factor in the unfavourable influence of the prognosis . in the same way , the presence of malignant residual cells in the surgical safety borders has made the subject of numerous studies , being correlated with the tumor progression and with the rate of tumor recurrence . together with the presence of regional limph nodes metastases the study of the 117 cases of squamous oral carcinomas indicated the predominance of the tumour to men ( 90% ) , most of the cases being diagnosed to the age groups between 50 70 . the most frequent associated risk factors were smoking and alcohol , as they are present in the history of three fourths of the studied cases . the dimension of the tumours , the invasion pattern , the presence of metastasis and the invasion of the safety surgical limits are the most important histopathological pronostic factors .

the lh / hcg ( luteinizing hormone / human chorionic gonadotropin ) receptor ( lhr ) expressed in the gonads regulates the ovarian and testicular function123 . although gonadal tissues are the primary sites of its expression , it is now well - established that lhr is also expressed in many non - gonadal tissues4 . lhr along with follicle stimulating hormone ( fsh ) receptor and thyroid stimulating hormone ( tsh ) receptor belongs to the family of rhodopsin/2 adrenergic receptor subfamily of g protein coupled receptors5 . binding of the lh or hcg to the extracellular domain of lhr causes a conformational change leading to guanosine di - and triphosphate ( gdp - gtp ) exchange on the cognate g protein . this results in the dissociation of the inhibitory beta - gamma subunit from the alpha subunit leading to the activation of adenylatecyclase to increase the intracellular concentration of adenosine cyclic 3 , 5 cyclic monophosphate ( camp ) . cyclic amp then activates protein kinase cascade and activates multiple signaling pathways culminating in increased steroidogenesis and stimulation of other cellular functions . it has been shown that at higher concentrations , lh or hcg can also activate phospholipase c resulting in the formation of inositoltriphosphate ( ip3 ) and diacylglycerol ( dag)679 . the physiological significance of this pathway in lh - regulated ovarian function is not clearly understood . lhr is expressed in the follicles , theca cells and corpus luteum of the ovary and the expression pattern shows considerable changes throughout ovarian cycle101112 . in growing follicles , consequently , the expression of lhr increases during follicular growth as it acquires the ability to secrete estrogens13 . the receptor expression is transiently downregulated in response to pre - ovulatory lh surge , which then recovers from down - regulation after 72 h reaching maximum level by the mid luteal phase10111214 . the molecular mechanism underlying the regulation of lhr expression has been investigated in depth in our laboratory . using rodent and human ovarian models , we have determined that the regulation of lhr occurs through post - transcriptional mechanisms mediated by a specific lhr mrna binding protein1516 . this article will focus on the signaling pathways involved in the post - transcriptional regulation of lhr expression . the downregulation of lhr occurs in response to preovulatory lh surge or in response to the administration of a pharmacological dose of lh or hcg . the loss of the ligand binding activity is closely coupled to the loss of the steady state levels of lhr mrna . in human , this phenomenon occurs during ovulation induction by the administration of hcg prior to ovum retrieval for in vitro fertilization16 . granulosa cells isolated from the retrieval fluids showed complete loss of lhr mrna on the day of retrieval , when examined by northern blot analysis ( fig . the granulosa cells were then cultured for different periods of time and rna was extracted from the harvested cells for examination of the reappearance of lhr mrna . the recovery from down regulation began approximately 48 h later and reached maximum level by day 4 in culture ( fig . this phenomenon can be demonstrated in a pseudopregnant rat model by treatment with a single dose of 50 iu of hcg to down regulate lhr . the left hand panel shows the expression of lhr mrna transcripts in the saline treated control ovaries . the expression of lhr mrna transcripts in response to the administration of hcg that mimics preovulatory lh surge is shown on the right hand panel . the lhr mrna expression remains suppressed up to 48 hours and recovers from downregulation by 72 h1015 . the loss of receptor mrna could be either due to a decrease in the rate of synthesis or due to increased degradation . to examine these possibilities , nuclear run - on assays were performed to determine the transcription rate during hcg - induced lhr downregulation15 . the results showed that the transcription rate remained identical in both control and hcg treated groups suggesting that the loss of the steady state levels of lhr mrna is not due to decreased transcription , but rather resulted from increased degradation15 . furthermore , the half - life of lhr mrna was significantly reduced in the hcg treated group when compared to the mrna decay rate of the control group ( fig . there was approximately a three - fold decrease in mrna half - life in the down- regulated group . northern blot analysis of lhr mrna in human granulosa cells immediately after retrieval ( d1 ) and after 4 days of incubation in serum free media ( d4 ) . total rna was extracted from granulosa cells from day 1 and four days of incubation ( d4 ) to recover from downregulation . rna was separated on agarose - formaldehyde gel , transferred to nitrocellulose membranes , hybridized with the p - labelled hlhrcdna , and exposed to x - ray film . to monitor rna loading , the blot was stripped and rehybridized with radiolabelled cdna for 18s rrna . 16 , reproduced with permission ) . hormonal control of lhr mrna expression in the ovary . a. northern blot hybridization analysis of steady state lhr mrna levels during hcg - induced downregulation . autoradiogram of northern blot hybridization analysis of total rna isolated at the indicated times from the ovaries of saline - injected ( control ) ( lanes 1 - 6 ) or hcg - injected ( downregulated ) ( lanes 7 - 11 ) rats . blots were probed using a labelled cdna encoding the lhr carboxyl terminus and a portion of the 3-utr ( nucleotides 1936 - 2682 ) . b. lhr mrna half - life determination in control and 12 h downregulated rat ovaries . 17 , reproduced with permission ) . the steady state level of mrna , in general , is controlled by the rate of its synthesis and the rate of degradation . it is now clear that highly regulated mrnas are controlled , at least in part , by modulating their degradation . in the majority of instances of post - transcriptional regulation of mrna , the changes in the stability of mrna appear to result from changes in the binding of cytoplasmic proteins , known as trans factors , to defined sequences or structures , known as cis elements , in the target mrna , forming a ribonucleoprotein complex . the binding of proteins to mrnas can occur either on 3 end , 5 end or on the coding sequence18192021222324 . on the basis of these facts , the possible existence of an lhr mrna binding protein was examined in the cytosolic fractions from the ovaries after injection with a dose of hcg that is known to downregulate lhr mrna expression . our first attempt was to examine whether a specific lhr mrna binding protein participates in lh / hcg regulated downregulation of lhr mrna expression . cytosolic fractions from control and downregulated ovaries were prepared and the ability of these fractions to bind lhr mrna was examined by performing rna electrophoretic mobility shift assay ( remsa)17 . a 100,000 g supernatant ( s100 ) fraction of the ovarian homogenate was incubated with [ p ] labelled lhr mrna that was prepared by transcribing the full length cdna encoding the lhr in the presence of [ p ] utp . after treatment with rnases to degrade unreacted [ p ] labelled rna , the ribonucleoprotein complex was subjected to electrophoresis under non - denaturing conditions , and autoradiography . we compared the ability of the s100 fractions from the control and hcg downregulated ovaries to bind radiolabelled lhr mrna . the results showed that there were two prominent bands designated as lrbp-1 and lrbp-2 ( lhr binding protein ) , corresponding to molecular weight 50 and 45 kda , respectively , in both the control and down regulated ovaries17 . the intensity of lrbp-1 was 3-fold higher in the downregulated group compared to the saline treated control . no significant change in the intensity in lrbp-2 was noticed . since the hormonal induction of lrbp-1 was significantly higher , studies were carried out to determine the nucleotide sequence involved in the interaction of lhr mrna with proteins in the s100 fraction . truncated segments of the full length lhr mrna were incubated with ovarian s-100 fractions and electrophoretic mobility shift assays were performed to localize the region of lhr mrna that interacted with the protein . the results of these studies coupled with the results from rna hydroxyl radical foot- printing revealed that the contact site of the interaction of lrbp-1 with lhr mrna consisted of a bipartite polypyrimidine- rich sequence corresponding to the nucleotides 203 - 220 located in the region encoding the amino terminus of lhr25 . after establishing the existence of an inducible rna binding protein that recognizes a specific structure of the lhr mrna , we examined the lhr mrna binding activity under conditions that mimic follicle maturation and the lh surge26 . since the expression of the lhr is increased during follicle maturation and , conversely , the lhr expression is decreased during downregulation , these two paradigms were selected to examine whether changes in lhr expression have any bearing on the lhr mrna binding activity . in response to treatment of 23 day old rats with pregnant mare 's serum gonadotropin ( pmsg ) , as expected , there was an increase in the expression of lhr mrna by 56 h due to the induction of lhr mrna by the fsh type of activity associated with pregnant mare 's serum gonadotropin ( pmsg ) . fifty six hours after pmsg treatment , administration of 50 iu of hcg caused a transient decrease in lhr mrna expression during the ensuing 24 h period as a result of downregulation . the lhr mrna level returned to the hcg pretreatment level by 72 h. thus , the changes in lhr mrna were consistent with the expected up- and downregulation following the treatments . the lhr mrna binding activity showed a strikingly different pattern in response to these treatments . when the expression of lhr mrna was high in response to pmsg treatment , the lrbp activity showed a decrease . conversely , an increased expression of lhr mrna binding activity was seen when lhr mrna expression was low in response to lhr downregulation26 . thus , there appeared to be an inverse relationship between the expression of lhr mrna binding activity and lhr mrna expression . these results have clearly pointed out that lrbp is a regulator of lhr mrna expression . the cellular mechanism that triggers the lrbp expression in response to lh / hcg treatment that leads to lhr mrna downregulation was examined using cultured human granulosa cells . specifically , the participation of protein kinase a ( pka ) and erk signaling pathways , the two downstream signaling molecules involved in lh action was examined during hcg- induced downregulation of lhr expression . first , we examined the possible participation of pka in this process by blocking pka activity by pretreatment with 10 micromolar concentrations of a pharmacological inhibitor , h89 prior to treatment with 10 iu of hcg for 12 h to down regulate lh receptor . the effect of blockade of pka by h89 on the downregulation of lhr and induction of lrbp were determined by performing real time pcr . the results showed that , as expected , treatment with hcg , resulted in downregulation of lhr mrna ( fig . this down - regulation was abolished when cells were preincubated with h89 prior to hcg addition . these results showed that the hcg - induced activation of pka is required for inducing downregulation of lhr27 . effect of pka inhibitor h-89 on the expression of lhr mrna and lrbp protein in human granulosa cells . day 3 granulosa cells were serum - starved , treated with hcg ( 10 iu / ml ) with or without the pka inhibitor h-89 ( 10 m ; 1 h pretreatment ) for a total of 12 h , and were either processed for total rna isolation or lysed using ripa buffer . a. total rnas were reverse transcribed , and the resulting cdnas were subjected to real - time pcr quantitation using specific primers and probes for lhr . the graph represents changes in mrna levels normalized to 18s rrna , shown as fold change vs. control . error bars , mean se . * , p < 0.05 vs. control ctl ; # , p < 0.05 vs. hcg ; n = 4 . b. cell lysates were subjected to western blot analysis to detect lrbp using lrbp antibody . the lower panel represents densitometric scanning of the lrbp normalized for tubulin and expressed as fold change vs. ctl . the blot shown is representative of three independent experiments , and the results in the bar graph are average and se of three experiments . * , p < 0.05 vs. ctl ; # , p < 0.05 vs. hcg . ( source : ref 27 , reproduced with permission ) . since lh / hcg- induced lhr mrna downregulation is mediated by lrbp16252628 , the effect of preincubation with h89 on the expression of lrbp was examined27 . the results showed that while treatment with hcg alone caused an increase in lrbp expression ( fig . 3b ) , more importantly , treatment with h89 prior to hcg treatment caused a significant reduction in the expression of lrbp . from these results , we concluded that abolition of hcg - induced pka activation abrogated both lrbp expression and downregulation of lhr mrna . it has been shown that erk is downstream of pka in g protein - coupled receptor ( gpcr ) mediated signaling pathways29303132 . to demonstrate that this is also true in human granulosa cells , treatment of granulosa cells with hcg markedly increased the phosphorylation of erk1/2 in a time dependent manner , starting as early as 5 min and lasting upto 30 min , but decreased to control levels by 1 h ( fig . 4a)27 . pretreatment with the pka inhibitor , h89 blocked this increase clearly showing that erk1/2 is downstream of pka in human granulosa cells ( fig 4b ) . therefore , the involvement of erk1/2 in hcg - mediated downregulation of lhr mrna , the effect of blocking erk activation on lhr downregulation and lrbp expression were examined by depleting erk using sirna technique . first , the effectiveness of erk specific sirna to suppress erk expression was assessed by incubating human granulosa cells with erk - specific sirna for 48 h. the results showed that erk specific sirna was effective in significantly reducing erk expression while a non - specific sirna produced no change in erk level ( fig . 5a)27 . after establishing the effectiveness of erk specific sirna to block erk expression , the effect of erk depletion on lhr human granulosa cells were exposed to erk - specific sirna for 48 h to knockdown erk expression and the ability of hcg to downregulate lh receptor was tested by incubating with 10 iu of hcg for 12 h. the results showed that while hcg alone caused downregulation of lhr , this downregulation by hcg was not seen in cells pretreated with erk specific sirna ( fig . furthermore , hcg - induced lrbp expression and activity were also significantly reduced in cells where erk expression was knocked down by sirna compared to controls treated with a non - specific sirna ( fig . these results clearly demonstrated that lh - mediated erk activation is required for lhr downregulation and that this response is mediated by lrbp27 . day 4 granulosa cells were serum - starved , treated with hcg ( 10 iu / ml ) alone for different time intervals ( 5 min , 15 min , 30 min , and 1 h ; ( a ) or in the presence of h-89 ( 10 m ; 1 h pretreatment ) for 15 min ( b ) and were lysed using ripa buffer . lower panels represent densitometric scanning of the p - erk1/2 signals normalized with erk2 and expressed as fold change vs. ctl . the blots shown are representative of three independent experiments , and the results in the bar graphs are average and se of three experiments . * , erk1/2 silencing inhibits hcg - induced decrease in lhr mrna levels and increases in lrbp protein expression and binding activity . granulosa cells were transfected with either control sirna ( ctlsi ) or erk 1/2 sirna ( erksi ) and cultured for 48 h. after serum - starving for another 24 h , cells were treated with hcg ( 10 iu / ml ) for 12 h and processed for total rna isolation , for western blot analysis , or for remsa . a. erk1/2 silencing was confirmed by the western blot analysis of cell lysates using total erk2 antibody . b. total rnas were reverse transcribed , and the resulting cdnas were subjected to real - time pcr quantitation using lhr - specific primers and probes . the graph represents changes in mrna levels normalized to 18s rrna and are shown as fold change vs. control . p < 0.05 vs. ctl ; # , p < 0.05 vs. hcg ; n = 3 . c. cell lysates were subjected to western blot analysis to detect lrbp using specific antibody . the same membranes were then stripped and reprobed for erk2 and -tubulin . the blot shown is a representative of three independent experiments . d. g el mobility shift analysis was performed with [ p]-labelled rat lbs ( 1.5 105 cpm ) and s100 fractions containing equal amounts of total protein extracted from the different treatment groups . in summary , based on the present and ongoing studies , we propose a model for the mechanism for lh / hcg - mediated downregulation of lhr mrna expression , as depicted in fig . 627 . in response to preovulatory lh surge or in response to hcg administration to induce ovulation , the initial interaction of lh with its receptor results in increased cyclic amp production . this increase leads to activation of pka and erk signaling cascade to increase lrbp expression . lrbp in turn binds lhr mrna and forms an untranslatable ribonucleoprotein complex and targets lhr mrna for degradation in p bodies . this loss of lh receptor causes a temporary halt in the lh / hcg - mediated signaling during the differentiation of granulosa cells to luteal cells . thus , lh signaling through pka and erk1/2 pathway is required for lhr down regulation in response to preovulatory lh surge or in response to hcg treatment for ovulation induction . schematic model depicting the proposed signaling pathway in lh / hcg - induced lhr mrna down regulation . binding of ligand to lh receptor induces activation of erk1/2 through the camp / pka pathway . this leads to an increase in the expression of lrbp and thereby its lhr mrna binding activity , which ultimately results in lhr mrna degradation .

microbial cells may outnumber our own cells in the body , presenting an example of nature 's intricate evolutionary scheme of host microbe symbiosis ( frank and pace 2008 ; ley et al . this unique setup of human microbe interactions and its resilience is of paramount importance for health . starting from birth , various factors shape the composition and function of the human intestinal tract microbiome , defined as the entire habitat including all microbes , their genomes and surrounding environment ( see table 1 ) ( sekirov et al . 2010 ; ottman et al . the human intestinal tract microbiota consists mainly of bacteria , but also includes archaea , microeukaryotes and many viruses , mainly bacteriophages . the dominant bacterial phyla are bacteroidetes and firmicutes , while actinobacteria , proteobacteria and verrucomicrobia constitute minor phyla ( li et al . archaeal representatives consist largely of methanogens , among which methanobrevibacter and methanosphaera are the most prevalent genera ( dridi et al . the microeukaryotes commonly observed in the human intestinal tract include blastocystis spp . and fungal genera , such as candida spp . , gloetenia / paecilomyces and galactomyces . finally , various reports have detailed the presence of bacteriophages in the human intestinal tract ( minot et al . a recent deep genomic analysis of virus - like particles present in human fecal samples revealed that most bacteriophages belong to the caudovirales order ( podovridae , siphiridae and myoviridae ) , some to the microviridae and no eukaryotic viral sequences were detected ( manrique et al . the latter study revealed 64 healthy subjects to harbor a total of 44 bacteriophage groups that included a set of core bacteriophages shared among over half of the subjects , a common set that was present in 20%50% of the subjects , and a set of bacteriophages that are either rarely shared or unique to an individual . while there are indications that bacteriophages may be involved in controlling the intestinal microbiota in health and disease , additional deep studies are needed to assess how these phages are involved in the host microbe interactions ( reyes et al . interactions between the host , the prokaryotic and eukaryotic microbes and their viruses take place at various levels . while there are marked differences in microbial composition and function along the entire length of the intestinal tract , most research has focused on the fecal microbiota as fecal sampling is a cost - efficient and non - invasive method . however , the compositional and functional attributes of the microbiota vary along the intestinal tract most notably , the small intestine harbors a unique microbial ecosystem that has been characterized extensively ( zoetendal et al . moreover , the fecal microbiota may differ from that in the colonic mucosa but systematic studies are lacking because of the invasiveness of the sampling ( zoetendal et al . as bacteria are the dominant group in this ecosystem , they have received most attention in the recent studies ( lozupone et al . the infant intestinal tract becomes colonized by bacteria largely gained from the mother ( favier , de vos and akkermans 2003 ; koren et al . the programmed outgrowth , manifested as succession , and the composition of the adult intestinal microbiota depend on a variety of deterministic host factors , including age , diet , genetic - make - up , immune and health status , and gender and geographical location ( biagi et al . 2010 ; de filippo et al . 2010 ; claesson et al . 2012 ) . furthermore , stochastic factors such as colonization order or antibiotic exposure can also affect microbial colonization ( lederberg and mccray 2001 ; harris et al . 2015 ; korpela et al . overall , this process leads to the development of a dynamic and complex web of interactions between microorganisms , the host and the environment ( costello et al . 2012 ; ottman et al . microbiota homeostasis could help in defining the factors responsible for evolutionary selection and establishment of the symbiosis between human and microbes . bacteria form the dominant domain in the large intestinal microbiota and hence we focus here on the accumulated knowledge of this group . for convenience , the definition of technical terminologies used in this review is given in table 1 . we discuss the concepts of the bacterial compositional and functional core , community types and alternative stable states of the microbial ecosystem . we will further discuss how improved understanding of these features could be instrumental in microbial diagnostics , predicting changes or homeostasis and determining the efficacy of intervention strategies for microbiome modulation . a major goal in human microbiome studies is to identify and characterize the bacterial taxa and functions that are shared by the majority of individuals . viewing the bacterial community from a taxonomic perspective reveals an increasing level of complexity toward the lower taxonomic ranks from phylum level to species level . moreover , the intestinal bacteria interact with each other and the host at various levels from single cells to populations of specific bacteria to the entire community . the complexity of such multi - scale interactions presents a major challenge in understanding community functioning and its impact on host health ( greenblum , turnbaugh and borenstein 2012 ; greenblum et al . one way to approach this challenge is to focus on the most abundant bacterial species and specifically the prevalent ones that are shared by many individuals . coevolution of host and microbiome can be explained by ecological selection processes ( pianka 1970 ; ley , peterson and gordon 2006 ) . host factors such as genetics , immune regulation and age represent top down selection that plays a role in shaping the microbial community . in contrast , bottom up selection results in the growth of microbes that have specialized functions , such as those associated with degradation of specific dietary components . another concept is the r / k selection where organisms that have adapted to maximize the rate of growth are so - called r - strategists , whereas those adapted to compete and survive when resources are limited are known as k - strategists ( pianka 1970 ) . however , applying these idealized concepts to the highly complex , dense and dynamical intestinal ecosystem is challenging ( freilich et al . the large intestine is known to have spatial heterogeneity in terms of bacterial species which occupy microhabitats ( donaldson , lee and mazmanian 2016 ) . for example , the outer mucus layer is densely populated compared to the inner mucus layer ( johansson et al . the presence of microhabitats with variable population densities suggests that , rather than being present at one of the two endpoints , the bacterial community should be viewed along the r - k continuum that is governed by top down and bottom up selection processes . the aforementioned factors result in the selection of bacteria belonging to specific phylogenetic clusters with adaptive and functional features characteristic for the ecosystem . this coexisting community of microbes and their functions , which are characterized by the minimal intestinal metagenome , have co - evolved with the host for mutual benefits and are prevalent in the population , constitutes the common core ( jalanka - tuovinen et al . . the core microbiota may include specific keystone species that are important for maintaining an efficiently functioning ecosystem , and whose gain or loss may have profound influence on ecosystem structure and function through their effect on other community members ( paine 1966 ) . in the intestinal tract ecosystem , their functional capabilities include for instance breakdown of complex carbon sources to support the growth of the other core members ( ze et al . several studies focused on defining a human intestinal core microbiota in terms of phylogenetic composition ( hamady and knight 2009 ) . longitudinal surveys of fecal bacterial 16s ribosomal rna ( rrna ) sequences in individuals for extended periods of time of up to more than 10 years have revealed that a significant fraction of bacterial phylotypes is continuously present in the large intestine indicating that it comprises a stable individual core in healthy adults ( faith et al . cross - sectional comparisons of the fecal microbiota in a large population cohort have indicated the presence of a prevalent core microbiota , which comprises those bacterial taxa that can be detected in the majority of individuals ( zoetendal , rajili - stojanovi and de vos 2008 ) . in contrast , the individual core microbiota includes those taxa that are frequently detected within a particular individual ; this can overlap with the common core , but also include individual - specific features . several studies have aimed to characterize the composition of the common core but their comparison is difficult since different criteria were used ( table 2 ) . the discrepancy in categorizing the common core microbiota could be partially attributed to methodological differences in determining microbiota composition and defining the core . importantly , the depth of sequencing and analysis , number of samples and taxonomic resolution are the factors that affect the analysis . the aforementioned issues pertaining to the analytical definition of core microbiota have been addressed by using the hitchip ( human intestinal tract chip ) , a phylogenetic microarray targeting 16s rrna gene sequences of bacterial taxa that are reported to occur in the human intestinal tract ( salonen et al . this phylogenetic microarray represents a measurement platform with high reproducibility , robustness and dynamic range , allowing the incorporation of both abundant and rare taxa in microbiota profiling . however , while capturing the vast majority of intestinal microbial species , these and other phylogenetic microarrays lack the ability to detect novel taxa that can be assessed by next - generation sequencing techniques ( hazen et al . previously , the hitchip - based analysis of > 1000 rare and abundant bacterial phylotypes from 130 genus - level groups in 115 adults revealed a common core of 290 phylotypes ( jalanka - tuovinen et al . we extended this methodology to analyze the common core in a cohort of 1006 non - compromised western adults ( lahti et al . this provided a global view of the common core that revealed bacteria related to faecalibacterium prausnitzii , oscillospira guillermondii and ruminococcus obeum as the top three taxa shared by all adults ( fig . previously , we reported that the method used for dna extraction affected the microbiome analysis and that mechanical extraction is to be preferred , as further confirmed by the international human microbiome standards analysis ( salonen et al . hence , we separately analyzed the samples processed using a mechanical or enzymatic dna extraction protocol . while no major difference in the inclusion or exclusion of major core bacteria was observed , differences in their ranked cumulative fractional abundances limiting our analysis to the subset of 401 samples obtained by mechanical extraction using repeated bead - beating revealed that bacteria related to f. prausnitzii , r. obeum and subdoligranulum variabile to be the top three taxa shared by all adults . moreover , we found that 34 out of the 130 genus - like groups targeted by the hitchip are shared in 95% of the subjects at the minimum detection threshold of 0.1% relative abundance , with the highest prevalence being observed for bacteria belonging to the order clostridiales ( fig . 1 ; see supporting information for methods ) . this estimate is similar in number to the recently published data by the belgian flemish gut flora project ( fgfp ) on 1106 individuals at 95% prevalence ( falony et al . however , in the same study , additional inclusion of the subjects from the lifelines - deep study cohort ( dutch nationals ) , as well as from uk and us studies , resulted in a reduced core consisting of 17 genera that could be observed in both cohorts and there was no specific threshold on relative abundance for identifying core bacteria . in comparison , our study cohort included subjects from across mainland europe , uk / ireland and the usa ( see supporting information ) . the lower estimates of the core size in the fgfp cohort could be partially explained by technical issues , including differences in dna extraction methods , variations in sequencing depth or 16s rrna gene primer sequences . however , at genus level , the reported core taxa from these two studies are largely consistent ( table s1 , supporting information ) . furthermore , reducing the stringency in the prevalence threshold in our data set to 80% and 50% with a constant detection threshold of 0.1% resulted in an increased size of the common core from 34 to 44 and 55 genera at 80% and 50% prevalence , respectively ( fig . 1 and table s1 , supporting information ) . thus , half the population studied here shared 42% of the 130 genus - like groups investigated . this further supports previous observations that the definition of the core microbiota is sensitive to the abundance and prevalence thresholds ( salonen et al . in addition , there is a large amount of shotgun metagenomics data available in public databases which in some cases can identify species and led to the metagenome species concept defining not yet cultured and genomically sequenced taxa ( sunagawa et al . 2013 ) . this metagenomic approach is not affected by primer or pcr bias but to some extent is limited by the depth of sequencing and choice of dna extraction method . therefore , we further investigated the largest available metagenome catalog of 1267 human fecal samples to identify the core bacterial genera ( li et al . were detected to be present in > 90% of the samples ( table s1 , supporting information ) . in order to overcome the influence of lower sequencing depth , we reduced the abundance threshold by 10-fold to 0.01% ( still a conservative threshold ) and identified eight genera namely bacteroides , eubacterium , faecalibacterium , alistipes , ruminococcus , clostridium , roseburia and blautia in more than half of the studied population ( table s1 , supporting information ) . while a comparison of technologies is not the aim of the current review , we would like to highlight that the core microbiota includes a limited number of taxa that are commonly observed , irrespective of the technology used . as sequencing costs decline and new bioinformatics tools are being developed , shotgun metagenomics can be expected to become a standard in microbiome research and will lead to more uniformity between studies for comparisons . hence , it will be imperative to revisit the core microbiota analysis in the future for more conclusive evidence . studies incorporating strain - level comparison and identification of genomes without a reference genome will provide an even more detailed picture of the members , dynamics and functional aspects of the core microbiota ( qin et al . 2010 ; li et al . 2014 ; scholz et al . 2016 ) . we hypothesize that different phylogenetic groups , which share important core functions in the intestinal ecosystem , co - exist and are part of the core . analysis of the cumulative fractional abundance indicated that the common core bacteria are among the most dominant taxa in the western adult population , including bacteria related to f. prausnitzii , r. obeum , s. variabile , prevotella melaninogenica and oscillospira guillermondii ( fig . cultured representatives for the vast majority of the common core bacteria are available and have sequenced genomes with information regarding their general metabolic traits and their association with human health . to this end , an extensive survey of literature was done to identify the general metabolic traits of the common core genus - like groups , and information for a subset discussed in more detail in this review is highlighted in table 3 . for information on other common core genus - like groups , see table s2 ( supporting information ) . we observed that there is a lack of in - depth understanding of the physiology of most of the core bacteria and their potential role in improving the stability of the intestinal microbiome . we use data and analysis methodology from our previous studies ( jalanka - tuovinen et al . the data set was filtered based on dna extraction method i.e. we included only the samples processed with the repeated bead beating method ( rbb ) which has been shown to outperform other methods ( salonen et al . spearman correlation coefficient for ko profiles for 80 genomes representative of the top 50 genus - level bacterial taxa including the phylogenetic core . ( b ) the representative core bacterial genomes and their correlation to other genomes ( each point depicts an independent genome correlated to genome on y - axis ) . the genomic data were produced by the us department of energy joint genome institute http://www.jgi.doe.gov/ in collaboration with the user community and were accessed using the img system . selected core genus - like taxa in human large intestine . prevalence , general metabolic trait and health associations of few key taxa in the human intestine . the analysis is based on 130 genus - like groups as defined by ( rajilistojanovi et al . 2009 ) . for a more detailed information on all the common core genus - like taxa can be found in the supplementary table s2 . the human intestinal microbiome is a functional organ , with complex overall effects on host health . therefore , it is crucial to identify functions commonly associated with the microbiota in health and disease . initially , the metagenomic core was analyzed based on the dna isolated from fecal samples of 18 individuals with european ancestry ( turnbaugh et al . , this analysis suggested the presence of a minimal intestinal metagenome that was shared by almost all subjects in this study . in a first comprehensive metagenome study , the functional similarities of the fecal metagenome of 124 european individuals with 3.3 million unique open reading frames were reported and indicated that on average 38% of an individual 's total gene pool is shared with others ( qin et al . in a recent study , the coding capacity of the large intestinal microbiome was addressed in further detail by studying the metagenome of 1070 individuals from three continents , i.e. europe , america and asia ( li et al . this study , which included both healthy and compromised subjects , identified around 9.9 million unique microbial genes within the intestinal microbiome , and the analysis indicated that this estimate is close to the saturated coverage of core gene content and functions . in case of the intestinal ecosystem , the minimal functional core can be expected to also contain more ecosystem - specific pathways , such as those involved in resistance to bile and ability to grow in an anoxic environment using fermentation or anaerobic respiration with terminal electron acceptors other than oxygen . in the first large - scale metagenomic study , more than 1000 clusters of genes that encode vital functions necessary for survival in the large intestine were identified ( qin et al . 2010 ) . this would suggest that the genomes of core bacterial taxa would have evolved toward high functional redundancy . to assess whether the common core observed in this study also demonstrated functional redundancy , we analyzed the representative genomes of the top 50 bacterial taxa that are part of the phylogenetic core ( fig . 1 ) . since our analysis was performed at genus level , we included known intestinal genomes from different species targeted by the hitchip which resulted in selection of 80 bacterial genomes representative of 50 genus - like taxa ( table s3 , supporting information ) ( rajilistojanovi et al . we first identified the kegg orthologs ( ko ) abundances for each of the genomes using the integrated microbial genomes ( img ) system and calculated the spearman correlation coefficient ( markowitz et al . 2012 ) ( figure 2 , see supplementary data for details ) . from the inferred network , we observed that bacteria belonging to the phylum bacteroidetes have high functional redundancy , whereas the phylum firmicutes was comprised of a large number of more functionally diverse core bacteria ( fig . recently , analysis of the gyraseb encoding gene ( gyrb ) in fecal samples from humans and related primates ( wild chimpanzees , bonobos and gorillas ) indicated that bacteroides and bifidobacterium spp . have cospeciated with their mammalian hosts over thousands of generations suggesting strong symbiotic association of these taxa ( moeller et al . this would indicate that the host may also play a role in selecting functionally redundant species , which results in high competition for resources in the ecosystem but at the same time leads to higher ecosystem stability , which ultimately benefits the host ( coyte , schluter and foster 2015 ) . a striking example is ruminococcus bromii that was previously reported as a keystone species for its ability to degrade resistant starch and supporting growth of other bacteria capable of utilizing glucose , maltose , panose and isomaltose for growth ( ze et al . a similar role of providing simple carbon sources and amino acids for supporting growth of microbes in the vicinity of the mucus layer can be played by degraders of mucin , another important food source in the colon . the abundant representative of the verrucomicrobia , akkermansia muciniphila , another core member ( fig . 1 ) , is considered a keystone species in this process and is capable of using mucus as sole carbon and energy source , while signaling to the intestinal mucosa ( belzer and de vos 2012 ; everard et al . 2013 ; derrien , belzer and de vos 2016 ) . functional correlation network of 80 bacterial genomes representative of the top 50 common core bacteria . the spearman correlation matrix is represented as a network in which each genome is a node and each correlation an edge ; the width of the edges is proportional to the magnitude of the correlation ( the higher the correlation the thicker the edge line ) . the nodes with correlation coefficients below 0.5 are not connected , and genomes were placed by a graph the genomic data were produced by the us department of energy joint genome institute http://www.jgi.doe.gov/ in collaboration with the user community and were accessed using the img system . metagenomic sequencing data can provide insights into the function , taxonomic position and strain - level diversity of both known and not yet cultured species ( sunagawa et al . 2013 ; li et al . 2014 ; nielsen et al . however , the active functions within the intestinal microbiome can not be identified based on dna sequencing data alone but require analysis of transcripts and proteins . to this end , several studies have addressed the active functional capacity of the intestinal microbiota at the rna and protein level , supporting the presence of an active functional core ( verberkmoes et al . metatranscriptomics analysis of fecal samples from 10 american adults revealed alterations in gene - expression profiles after changes in dietary intake ( david et al . , metaproteomic comparison of 29 non - obese and obese adults has shown larger differences in the active functional bacteria between the two groups than those observed at the compositional level ( kolmeder et al . while these studies are a step toward functional understanding of the human microbiome , the field is still young . improved statistical techniques and clinically relevant predictions will play a key role in a wider adoption to identify changes of functional features characteristic of different disease states . several approaches have been employed to identify overarching community types , as well as alternative stable states in more specific taxonomic groups ( arumugam et al . 2011 ; holmes , harris and quince 2012 ; ding and schloss 2014 ; lahti et al . 2014 ) . moreover , longitudinal analysis of bimodal bacteria revealed temporal stability of the alternative states of low and high abundance , which are in some cases observable as ecosystem - level shifts ( lahti et al . one potential explanation for the observed bistability could be that these states are resilient to perturbations and stable over ecologically relevant timescales ( connell and sousa 1983 ) . additionally , the transition from one stable state to an alternative stable state as a result of a disturbance by e.g. dietary change , may comprise a period which can be stable for a long time relative to the extreme end state , called the alternative transient state ( fukami and nakajima 2011 ) . in alternative transient states , the communities are in a variable , non - resilient state and vary in structure and/or function as a result of processes such as priority effects ( time and order of species arrival ) , external conditions and deterministic processes ( fukami and nakajima 2011 ) . thus , the intestinal bacterial community can be viewed as a landscape representing varying global and/or local levels of stability , species richness and diversity ( arumugam et al . 2011 ; koren et al . 2013 ; thaiss et al . 2014 ; zoetendal and de vos 2014 ) . in this context , the core bacteria and their abundances can be considered as potential biomarkers that may indicate changes in the intestinal ecosystem . in an early approach to detect ecosystem - level structures in the intestinal microbiome , deep metagenome analysis suggested the presence of three clusters , termed enterotypes and characterized by co - occurring taxa named after their dominant bacterial groups i.e. bacteroides , prevotella and ruminococcus ( arumugam et al . 2011 ) . one can argue whether it is possible to infer community assembly rules for the complex and highly dynamic intestinal ecosystem based on discrete alternative community types or whether the intestinal microbiome should rather be viewed as a continuous dynamic ecosystem , which is in constant flux but existing in a microbiota host homeostatic state ( jeffery et al . recently , it was reported that the enterotype - like clustering observed in ordination analysis could be influenced by the initial abundances of prevotella and bacteroides in the samples that were analyzed ( gorvitovskaia , holmes and huse 2016 ) . moreover , it has been demonstrated that high levels of prevotella and bacteroides can co - exist in the host , suggesting that these types could be complementary rather than mutually exclusive ( lahti et al . in addition , a separate study showed that removal of these two taxa from analysis did not result in differentiation of communities into clusters , suggesting that the dominance of prevotella or bacteroides did not represent underlying differences in community structure ( gorvitovskaia , holmes and huse 2016 ) . however , that study also found that the dominance of prevotella or bacteroides correlated well with dietary habits and lifestyle . thus , it was suggested that the abundance of prevotella and bacteroides might be considered as a biomarker of diet and lifestyle instead of using the term enterotypes ( gorvitovskaia , holmes and huse 2016 ) . a different approach to clustering and classification of samples based on microbial profiling data uses a probabilistic approach , called the dirichlet multinomial mixture ( dmm ) models ( holmes , harris and quince 2012 ; ding and schloss 2014 ) . this approach found evidence for the presence of alternative community configurations in the human intestinal microbiome regardless of the extensive intra- and interpersonal variations ( holmes , harris and quince 2012 ; ding and schloss 2014 ) . ecologists have also applied graph theory - based approaches to identify species interactions networks and modularity . in most studies , the co - occurrence and co - abundance network have been inferred from correlation - based and other similarity measures ( weiss et al . one such attempt incorporated sparse regression with a number of similarity measures to predict microbial co - occurrence and co - exclusion relationships ( faust et al . it was observed that the microbial community is organized into microcommunities which would indicate a trade - off between certain combinations of microbes that may represent alternative communities ( faust et al . 2012 ) . recently , other tools have been developed to handle sparse compositional data ( friedman and alm 2012 ; gevers et al . 2014 ; kurtz et al . 2015 ) . compared to the standard correlation analyses , these new methods take better into account the sparsity , compositionality and high dimensionality of the taxonomic networks . one example is the use of ccrepe to identify crohn 's disease associated community network where it was observed that proinflammatory bacteria dominate and co - exclude other potentially beneficial taxa ( gevers et al . a similar observation of variable modularity in microbial interaction network was made in inflammatory bowel disease ( ibd ) patients and non - ibd controls ( baldassano and bassett 2016 ) . at the population level , the two states had a different modular structure and highlighted the different community memberships . however , it is vital to remember that the tools and models for identifying co - occurrences and modularity in microbiome data still need improvement ( weiss et al . these studies are observational and do not include interventions and hence inferences on causality can not be made ( de vos and de vos 2012 ) . in general , more comprehensive longitudinal analyses and intervention studies will be needed to establish causal relationships . the observed dynamics of the large intestinal ecosystem suggests the possibility for transitions between alternative and potentially stable states , with diet being one of the major driving factors ( wu et al . for instance , it has been observed that a diet rich in proteins and animal fat is associated with the bacteroides dominance , whereas carbohydrates have been linked to high abundance of prevotella ( wu et al . the extremes of dietary habits point toward a prevotella / bacteroides trade - off driven by diet and suggesting a gradient in relative abundances between the two extreme states ( high and low abundance ) ( kelsen and wu 2012 ) . the joint population frequencies ( represented as peaks and valleys ) of the bistable taxa , prevotella and bacteroides exhibit a landscape distribution in the western adult population ( fig . these two taxa exhibit high degree of functional redundancy as based on their genomic content when compared to functions shared between populations belonging to the firmicutes or bifidobacterium spp . previously , ecosystem - level models based on metagenomic and genomics data indicated that the habitat filtering ( selection pressure inflicted by the habitat ) may have greater impact than functional independence or cooperation on the community composition and thus , may result in selection of functionally redundant species ( levy and borenstein 2014 ) . it has also been proposed that the stability of intestinal microbiota is affected more by competing species than cooperating species ( coyte , schluter and foster 2015 ) . a new hypothesis emerged from the analysis of genome - based models that showed the module - derived functional redundancy to be reduced in the bacteroides spp . as compared to other genera , potentially linking this to a decreased resilience to perturbations ( vieira - silva et al . future studies aimed at mechanistic understanding of the interaction between these and other highly abundant or bistable taxa in the community and their potential role in ecosystemic transitions could provide the clues for microbiota modulation for instance based on dietary , antibiotic or other interventions . two - dimensional kernel density ( 2d - kde ) estimates . the abundance of bacteria ( log10 transformed relative abundance data ) mapped onto the x- axis and y - axis respectively , and the z - axis represents the kernel density estimate , in a 3d perspective plot showing the joint population frequencies of these two taxa demonstrating features similar to a landscape . ( a ) mapping of p. melaninogenica and b. fragilis illustrates two distinct peaks that represent low b. fragilis and high p. melaninogenica population and low b. fragilis low p. melaninogenica population . ( b ) mapping of p. melaninogenica and dialister illustrates three distinct peaks that represent low dialister and high p. melaninogenica population ; low dialister and low p. melaninogenica population ; high dialister and low p. melaninogenica population . 2014 ) . to assess the dynamics of the large intestinal microbiota , it is necessary to study microbiota composition over different time scales and investigate the influence of perturbations . compared to cross - sectional studies , analysis of time - series can provide information on intraindividual variations . longitudinal analysis of larger populations gives insights into dynamics and stability of microbiota and could be informative in identifying key species that may play a role in ecosystem shifts ( faust et al . 2015 ) . by applying a discrete time lotka - volterra model to characterize bacterial interaction networks , it was observed that a high abundance does not necessarily imply a large role in intestinal microbiota stability ( fisher and mehta 2014 ) . the identified keystone species ( bacteroides fragilis and b. stercosis ) in this study had moderate abundances but showed disproportionate influence on the interaction network . however , this study was limited by the number of individuals ( two ) included in the study . variations of the lotka - volterra model have been used widely in ecology for the understanding of two species interactions . it is well known that the use of antibiotics may have a large impact on the microbial community and makes it prone to invasion by pathobionts ( bokulich et al . a combination of lotka - volterra modeling and regression analysis in antibiotic - treated mice predicted that a microcommunity network was important in protection against clostridium difficile invasion ( stein et al . it was observed that a compromised community showed a catastrophic shift into an alternative state , which was stable and did not revert back to the initial community state after removal of the perturbations . future studies applying these and other models on larger human cohorts may give better insights and aid in understanding ecology of the large intestinal microbiome . in the intestinal tract microbiota , there exists a dynamic state and consists of species with varying fluctuations in abundances ( caporaso et al . as there are thousands of species in the ecosystem , stability may arise in multiple states within a closed domain of possible alternative states also called basins of attraction defined by a dynamic attractor ( scheffer et al . 2001 ; rosenfeld 2008 ; pepper and rosenfeld 2012 ) . the microbial community in a given individual may move from one attractor to another over time and may include transiently occurring species from external sources ( e.g. food ) ( fukami and nakajima 2011 ; lang , eisen and zivkovic 2014 ) . in many studies , single time point fecal samples are analyzed and these may represent alternative transient states and consequently explain the high individuality observed in many studies ( flores et al . 2014 ) . however , personalized patterns in the intestinal microbiota were detectable over 10 years of time , as shown by long - term monitoring of five healthy subjects ( rajili - stojanovi et al . dense sampling of the human intestinal microbiota has revealed diurnal oscillations in relative abundance of almost 10% of all bacterial taxa and revealed a repetitive pattern of fluctuations in microbiota community configurations specific for a given time of the day ( thaiss et al . these fluctuations highlight the dynamic nature of the intestinal microbiota and indicate that there exists low and high abundance states for most bacteria . when put in perspective of the alternative transient states , these observations reveal an interesting feature of the intestinal microbiota . it is possible that the daily factors such as diet result in changes in the microbiota structure and composition ( introduction of transient bacteria through food and water ) and thus result in alternative transient states . this has been experimentally verified by small studies of extreme diets ( plant - based diet or animal - based diet ) and a diet swap study of african americans and native africans ( david et al . there can be several possible paths of alternative transient states that may reach in one of multiple states within a limited set of attractors . however , if there is a major shift in dietary habit , this might push the community on an alternative transient path that ultimately results in a catastrophic shift in the community . investigation of individual bacterial groups revealed bistability ( i.e. alternative states of either high or low relative abundance ) and identified six genus - level groups that were not only bimodal in their population frequencies but also exhibited bistability , i.e. being predominantly present at either high or low relative abundance with less frequent observations and a reduced temporal stability of the intermediate abundances ( lahti et al . the intermediate abundance range ( tipping point ) exhibited reduced temporal stability within subjects ( lahti et al . these bistable groups varied relatively independently and hence were termed tipping elements. at a community level , the corresponding compositional states could be described as specific combinations of such bistable tipping elements . similar to prevotella melaninogenica and b. fragilis distribution , we were interested in observing the population frequencies of highly abundant bistable bacteria p. melaninogenica and a lower abundance bistable taxon such as dialister spp . in the cohort of western adults . we used these examples to illustrate the alternative stable states mapping of the two - dimensional kernel density estimates for bacteria related to p. melaninogenica and dialister . the less abundant dialister group can exist in its high and low abundance states with p. melaninogenica low and high abundance states . previously , the longitudinal assessment of bistability revealed that prevotella groups exist in highly bistable states and that their abundance levels are more stable in individuals over 3 months compared to other bistable taxa ( lahti et al . furthermore , it was observed that the intestinal microbiota composition of individuals with bistable taxa closer to the tipping point is more variable in time ( lahti et al . this indicates that the two extreme states are resilient , and changes in the ecosystem are more pronounced when bistable taxa have relative abundances closer to their tipping point . in the past few decades , research investigating the ecology of the intestinal microbiome has revealed various features , such as community assembly , temporal dynamics and stability and host covariates associated with intestinal microbiota ( table 4 ) . the knowledge of positive , negative or neutral effects of bacteria on the ecosystem may help in identifying targets for therapeutic modulation . additionally , features such as resilience of the ecosystem and effect of perturbations need to be understood in detail . in the modification of an ecosystem , perturbation ( random , episodic or programmed events ) plays a major role ( paine , tegner and johnson 1998 ) . the type of perturbation will affect the assembly of the community and also play a role in determining the recovery state . for example , the low and high prevotella states are highly stable and do not easily change over a short period of treatment ( lahti et al . furthermore , the initial state of a bacterial community has been reported to affect its recovery after antibiotic treatment ( raymond et al . it has also been observed that a low calorie dietary intervention could be helpful for obese individuals with lower bacterial richness ( tap et al . richness of the large intestinal microbiome is commonly associated with higher resilience ( lozupone et al . 2012 ) . resilience and stability are the key features of the intestinal microbiome that need to be addressed while attempting modulation . different alternative states may vary in both resilience ( tendency of a system to return to a position of equilibrium when disturbed ) and stability ( tendency to maintain the state when subject to disturbance ) ( walker et al . detailed understanding and identification of potential markers of these features will aid in designing intestinal microbiome modulation strategies . representative studies investigating the ecology of the intestinal microbiome employing high - throughput sequencing approach ( most recent in each category have been listed ) . in various diseased conditions , there is a shift in the composition and function of the intestinal microbiota driven by specific bacteria ( possibly pathobionts ) . these bacteria are responsible for stabilizing an alternative community state , which is unfavorable for host health by producing specific metabolites that deter growth / survival of other bacteria . for example , in an inflamed intestine , increased levels have been observed of proinflammatory bacteria , such as members of the enterobacteriaceae , associated with increased levels of the proinflammatory lipopolysaccharide produced by this group of gram - negative bacteria ( wang et al . the resulting inflammatory cascade includes an increased concentration of host - produced reactive oxygen species that hamper growth of indigenous anaerobic bacteria ( lupp et al . thus , this aberration may represent an alternative stable state , the stability of which is maintained by an inflammatory milieu created by the host and influenced by members of the enterobacteriaceae . hence , the transition from higher densities of obligate anaerobes to proinflammatory facultative anaerobes would be critical for the establishment and/or maintenance of a state of increased inflammation . in such cases , a reduction in numbers of proinflammatory bacteria will be crucial for a shift toward an alternative state ( may or may not be stable ) no longer supportive of inflammation , and thus leading to re - establishment of anoxic conditions in the intestine . along with the reduction in proinflammatory facultative anaerobes , it will be crucial to support the growth of bacteria capable of tolerating oxygen at low concentrations , such as lactobacilli and akkermansia muciniphila that have anti - inflammatory effects for re - establishment of an anoxic milieu ( tien et al . 2006 ; ouwerkerk et al . the relative abundance of diagnostic indicator taxa , such as bacteria belonging to enterobacteriaceae ( increasing relative abundance ) and a. muciniphila ( decreasing relative abundance ) , was shown to indicate particular environmental conditions such as an inflamed intestinal milieu ( png et al . in addition , indicator taxa can be sensitive to perturbations and therefore might serve as an early warning sign of unhealthy shifts in the community . such indicator taxa can be monitored as their abundance may reflect the efficacy of intervention strategies . using the knowledge of drivers and indicators , it would be possible to detect diagnostic biomarkers , identify specific therapeutic microbes ( single keystone species or mixed consortia ) for treatment and suggest lifestyle changes ( such as dietary habits ) that will result in a evidence for the predictive nature of intestinal bacteria was recently presented when investigating obese subjects undergoing dietary interventions ( korpela et al . the responsiveness of the intestinal bacterial community to a specific dietary intervention was predicted by the baseline relative abundances of clostridium clusters iv , ix and xiva , and bacilli . the members of several of the clostridium clusters also include a number of the core phylotypes in the human intestine , most notably clostridium cluster iv ( c. leptum group , a major constituent of which is the ruminococcaceae family ) and clostridium cluster xiva ( c. coccoides group , which resembles the lachnospiraceae family ) ( tap et al . 2009 , it is tempting to speculate that some core phylotypes may also serve as indicator species for responsiveness , and their relative abundance may have profound influence on the efficacy of a given intervention strategy . another example is a. muciniphila , which is a part of the intestinal core , and has been reported to be indicative of a healthy metabolic profile . furthermore , its abundance along with high gene richness was indicative of better clinical outcomes after calorie restriction diet in overweight / obese adults ( dao et al . evidence supporting the importance of bacterial community structure in determining individuals responses to dietary modulation ( in ecological terms ; ecological disturbance ) was provided by the findings of salonen et al . it was observed that the dietary intervention had little effect on the intestinal bacterial composition of non - responders who were characterized by higher initial bacterial diversity . thus , a higher baseline bacterial diversity represents a more resilient community , and increased responsiveness would be characteristic of an unstable community . this highlights that the baseline bacterial diversity could be one of the potential markers in predicting responsiveness to dietary modulation . however , it is important to acknowledge the effect of other , more specific , factors such as relative abundance of certain bacteria , e.g. bistable bacteria , in determining an individuals response to intervention . it will be interesting to investigate whether the bistable groups can serve as predictors for responsiveness or non - responsiveness to modulation strategies . the functional core and the identification of bistability of specific metabolic processes could also provide clues for predicting efficacy of interventions . for instance , also the overall bacterial gene richness has been demonstrated to exhibit bimodal population frequencies western individuals have been observed to be separated into two groups based on gene count , i.e. low gene count ( lgc ) and high gene count ( hgc ) , with low gene richness being associated with pronounced dys - metabolism and low - grade inflammation . thus , lgc and hgc could represent two alternative states in which one state ( lgc ) represents a state that is indicative of increased risk for obesity ( cotillard et al . in this study , it was observed that the lgc group after dietary intervention had improved gene richness concomitant with improvement in the clinical phenotypes but showed little change in the inflammation status of these individuals . these functional alternative states , i.e. lgc and hgc , provide us with an opportunity to predict the efficacy of any approach used for modulation . this further highlights the importance of richness of the intestinal microbiome ( both compositional and functional ) as a factor in determining resilience to ecosystem changes . it is known that species richness determines the susceptibility to invasion by non - resident species and resilience to overall ecosystem disturbances ( levine and d'antonio 1999 ) . in case of the intestinal microbiome , resilience of a community with high functional richness adds an important dimension to our understanding of its complexity . thus , there is a need for considering both species richness and functional gene richness during intervention studies aimed at microbiome modulation . in the past decade , numerous studies targeting the human intestinal microbiome have led toward remarkable advances in our understanding of the crucial role these microbes play in human health . recognizing the importance of this microbial community , the intestinal microbiome is often called a metabolic organ signifying its crucial functional role which has an effect on our health status ( bocci 1992 ) . importantly , this metabolic organ represents a functioning ecosystem , which is governed by ecological mechanisms . at present , clinically meaningful evidence for the potential application of modulating the intestinal microbiota for therapeutic gain has created considerable interest and enthusiasm ( smits et al . treatment of metabolic syndrome and clostridium difficile infection by ecosystem - level cleansing restoration via fecal microbiota transplantation ( fmt ) has demonstrated the potential for translating microbiota research to clinical practice ( li et al . while fmt is promising with proven efficacy in several cases , the underlying mechanisms that result in beneficial effects on host health are unclear and will need further refinement . one of the approaches is targeting specific group(s ) of bacteria to bring about the desired changes in the intestinal microbiome . the state of a dynamic system may be manipulated by triggering a switch to an alternative state , for instance with a strong but temporally limited , transient perturbation ( pulse , antibiotics ) , or longer impact ( press , diet ) ( costello et al . 2012 ; faust et al . these transient perturbations can also be combined with introduction of single bacteria or groups of bacteria that support or inhibit growth of specific bacteria resulting in desired changes in composition and function of the intestinal microbiome . in order to manage invasions or overgrowth of pathobionts , we first need to identify phylotypes that are common residents and their relative proportions in the intestinal ecosystem . the study of common core phylotypes and core functions is important in this aspect as this would form the baseline knowledge of comparison of this core healthy state with diseased states will assist in identifying invasive species ( potentially harmful species that are not part of the core ) and/or pathobionts ( resident microbes with pathogenic potential ) . for instance , during ibd enterobacteriacaeae are known potential pathobiont species that affect the numbers of anaerobic intestinal bacteria as mentioned above . in such cases , the use of therapeutic microbes combined with specific dietary interventions can be used to support growth of core phylotypes capable of fermenting complex polysaccharides to produce short - chain fatty acids ( scfa ) and gas ( co2 and h2 ) , which results in decreased intraluminal ph to establish an anoxic environment ( tien et al . other examples of pathobionts in the human gastrointestinal intestinal tract are helicobacter pylori , fusobacterium spp . , desulfovibrio and c. difficile ( biagi et al . 2010 ; rowan et al . the common observation in disease cases is the overgrowth of these pathobionts . in mouse models of antibiotic - mediated c. difficile infection , it was observed that a few bacteria confer protection and imbalance in their populations increases susceptibility to overgrowth by pathobionts ( stein et al . that are all part of the core and their inhibition by antibiotics was proposed to support overgrowth of c. difficile assisted by enterococcus spp . in mice ( stein et al . thus , it could be possible that the knowledge of common core phylotypes combined with the low and high abundance states of pathobionts could help in designing specific approaches for managing invasion by pathogens or increased relative abundance of pathobionts . these approaches could include changes in the diet that support higher growth of core phylotypes and functions such as butyrate production which inhibits pathogen colonization ( scott et al . . the outcome of a given intervention strategy will depend on the individual 's response to certain modulation attempts . the response of the individual to a specific intervention can be predicted and monitored by observing overall community descriptors such as species and gene richness and diversity , as well as abundance / presence / absence of key indicator taxa that may reflect the efficacy of intervention strategies . indicator taxa could include bistable bacteria , and their shift toward the tipping point could provide clues for transition from diseased to healthy alternate stable states . studying the transition between low and high abundance states or vice versa with an intermediate tipping point could provide clues for the critical time point for ecosystem restoration by modulating the microbiome toward a different , possibly healthier state . it is also possible that the shift from a diseased state may not necessarily lead to a healthy state but rather to a state different from either of the states . these alternative scenarios may be dependent on the strength of perturbation that resulted in shift to a diseased state . in any case , monitoring of microbiome composition and its dynamics over time should be incorporated in intervention strategies . human health is interlinked with the human body and its associated microbiome , and thus , maintaining a healthy microbiome is essential . the characteristics of a healthy community composition have been debated , and could be potentially identified by observing the common core and bacterial communities associated with alternate stable states . the core phylotypes have co - evolved to provide crucial ecosystem services such as production of butyrate ( energy source for colonic epithelial cells ) and protection against invasive pathogens and outgrowth of endogenous pathobionts . the process of modulating microbiome can be initiated in early life by managing certain host associated and environmental factors like diet . the mode of delivery has an effect on the pattern of development of the intestinal bacterial community ( grlund et al . caesarean delivery is associated with a reduced number of clostridium species most of which are part of the core microbiota in adult life ( fig . 1 ) ( bokulich et al . however , another recent report did not observe associations between the delivery mode and adult microbiota composition ( falony et al . maternal health status ( obesity ) and maternal stress have also been associated with an aberrant intestinal bacterial community in infants ( galley et al . however , modifying the dietary habits , breast feeding and incorporating specific oligosaccharides have shown to aid in improving the abundance of anaerobic bacteria ( haarman and knol 2006 ) . a recent study on professional athletes revealed that exercise could aid in increasing intestinal bacterial diversity ( clarke et al . 2014 ) . in elderly subjects , the intestinal microbiome is characterized by reduced bacterial diversity ( biagi et al . 2010 ; claesson et al . 2012 ) . reduced abundance of beneficial core bacteria , increased inflammation and a decrease in the concentration of scfa 's have also been observed in elderly subjects ( salazar et al . these deviations from a normal adult healthy state could be reduced by targeted modulation , which may include restoring the healthy core phylotypes ( through probiotic and/or prebiotic interventions ) , reducing proinflammatory bacteria and increasing the scfa production by incorporating dietary fibers in the daily diet . in the past few decades , we have gained significant insights into the composition , dynamics and function of our intestinal microbiome and its impact on our well - being . the western adult population shares dozens of genus - level taxonomic groups that show high functional correlation . the intestinal microbiome should be viewed as a complex landscape , which is a mixture of common and individual characteristics . in addition to continuously changing gradients across bacterial abundance and function , this intestinal microbiome landscape also includes distinct features present as alternative stable states across varied compositional , structural and functional features . it is now clear that treatment for most of the disease conditions necessities careful consideration of the intestinal microbiome . a recent example is the application of a prediction algorithm based on dietary habits , physical activity and gut microbiota that allowed predicting blood glucose levels and designing personalized diets which were effective in controlling post - prandial blood glucose levels ( zeevi et al . while the field of microbiome research rapidly moves forward , there has to be a careful consideration of the confounding variables in the studies , knowing that correlation does not imply causation , technical bias between studies , differences in statistical models , human error and most importantly reproducibility ( ioannidis 2005 ; falony et al . as the microbiome research moves into the next phase of diagnostics and therapeutics , overcoming these issues will be a major focus of future research . sas designed and coordinated the manuscript preparation , and took main responsibility on the writing . ll assisted in generation of figures . fh , ll , hs and wmdv provided critical reviews and suggestions for the content , and contributed to the text .

in 2000 , davison was the first to focus on a relatively common problem after uncomplicated cataract surgery , which was called negative dysphotopsia ( nd ) . this side effect of cataract surgery typically vanishes after a couple of weeks or months and rarely persists for longer periods of time . in most cases it does not have serious clinical consequences . since davison , these photic effects have been extensively discussed in the literature , but up to now there is no generally accepted concept which could describe causality of nd and describe the symptoms of the patients [ 28 ] . the nd phenomena refer to arc or bow shaped high contrast shadows in the temporal visual field , which appear in the early time after cataract surgery and disappear in most cases after a couple of weeks . in some cases , however , they persist for a longer time period and patients are severely disturbed and insist on surgical interventions such as intraocular lens ( iol ) explantation or implantation of an add - on iol . up to now , only f. f. marques and d. m. v. marques quantified these optical phenomena clinically by performing visual field measurements . they described a scotoma in the superior - temporal visual field at an eccentricity of about 20 , which improved upon pharmacological miosis . some authors who addressed negative dysphotopsia reported these shadows to appear mainly in the far periphery 50 to 100 away from the fovea [ 2 , 6 , 10 ] . , osher , and trattler et al . published patient drawings or sketches of these light and shadow effects without proof of their exact localization in the visual field . hong et al . investigated the causes of nd by means of optical simulations [ 2 , 7 ] . the published literature [ 1 , 5 ] reports an incidence between 0.2% and 20% . photic effects were first described with pmma lenses [ 1113 ] ; however , most authors reported nd with acrylic or silicone intraocular lenses [ 1 , 5 , 9 , 10 , 1420 ] . all patients underwent cataract surgery under local topical anesthesia with implantation of foldable acrylic single - piece monofocal hydrophobic intraocular lenses ( acrysof sa60at or sn60wf , optic diameter of 6 mm ) through a 5.5 to 6 mm capsulorhexis . the overlap of the anterior capsular remnant over the anterior optic surface was less than 0.5 mm in all cases . in all patients cataract surgery was uneventful and all iols were well centered in the capsular bag . directly after cataract surgery some patients reported on sharply bounded shadows in the temporal visual field which were majorly disturbing in every day 's life . for localization of the shadow in the visual field , we used the physiological scotoma corresponding to the blind spot . if the location of the blind spot ( approximately 15 temporal to the visual axis ) in the visual field is known , the location of the photic phenomena could be referenced in the visual field . patients were positioned at a desk in reading position and a standard preprinted form sheet , normally used for goldmann perimetry , was presented to the treated eye while the contralateral eye was occluded . at the intersection of the horizontal meridian with the 60 mark of the visual field a red spot was drawn , which referred to the blind spot . due to the short distance of the paper form and the resulting magnification , the mark at 60 the sheet was positioned on the desk so that the center was located straight in front of the eye and the red spot was located temporally . patients were requested to monocularly fixate the center of the scheme while moving the head slowly back and forth until the red spot disappeared . then , patients were asked to mark the shadow on the sheet while keeping the head position and fixation unchanged while the red spot had to remain invisible . if they had located the black shadow outside the scheme , we would have extended the form sheet laterally to enlarge the drawing canvas . photic effects were reported in one percent ( 8 out of 800 ) of our treated eyes . patient data and biometric and functional results of these eyes are summarized in table 1 . the refractive error before and after surgery did not exceed 1 d. no pathology could be found , which could explain the patients ' complaints . at the beginning of the follow - up examination the patients were asked to suspect where the black shadows were located within the visual field . all eight patients reported phenomena within the peripheral visual field between 30 and 100. we asked our patients to sketch the position of the shadow according to the above - mentioned method . most of the eight patients had difficulties in fixating the center and mark the shadow effects outside the center of the visual field scheme . finally , all of them marked the shadow directly adjacent to the blind spot ( figure 2 ) . this result was surprising for both the patient and the examiner as the shadow was expected to be located much more in the periphery . this subjective examination technique shows some limitations : we estimate the inherent accuracy to be about 5. the small red dot used for localization of the blind spot is only about 1 , whereas the blind spot has an extent of about 5. thus , the exact location of the red spot within the blind spot is not possible . this explains why scotoma between 10 and 20 temporally located from the central fixation can still be related to the blind spot , as depicted in patient # 6 . up to now , the etiology of nd is only understood if the photic effects are located in the extreme periphery [ 6 , 7 ] . we do not believe that a black shadow outside the center at 90 would disturb the patient seriously . peripheral defects in the visual field caused by glaucoma are also rarely perceived as disturbing . the shadow with the bird published by osher does more seem to be at about 15 than 90 out of the center . we interpret our results in that way that nd is multifactorial and one reason could be a change of the location and size of the blind spot due to replacement of the crystalline lens by an artificial lens . if we assume that there might be a memory effect for the blind spot , changes of the position or size due to changes in the imaging channel could be responsible for these phenomena observed by the patients . this explanation maintains that in most cases nd vanishes after a couple of weeks or months spontaneously , whereas in other cases where the location or size changes are larger or the memory effect of the blind spot which is accompanied with the image processing in the brain works differently . the reaction to a change of location or size of the corresponding image to the blind spot is individually different , and in some cases this effect disappears rapidly whereas in others it persists over a long period of time . any shift of the iol optic , different optical design , decentration , and/or tilt changes magnification or position of the image on the retina . consequently , additional implantation of a sulcus fixated lens does also change image magnification and location . in general , most patients adapt themselves to the slightly different magnification after cataract surgery and do not suffer from visual discomfort . only about 1% of the patients complain about temporary disorders such as nd . such an adaptation process is often argued to explain the spontaneous occurrence and disappearance of nd . others have described complete or partial disappearance of nd with iol exchange , nd : yag laser treatment of anterior capsule overlap , reverse optic capture , and piggyback iol implantation in some cases , whereas others report no effect of these subsequent procedures [ 2127 ] . we assume that iol exchange and piggybacking may lead to a change of magnification or repositioning of the iol image effectuating the symptoms to disappear . in contrast , anterior capsulotomy or primary reverse optic capture may be useful to treat or prevent nd caused by an anterior capsule overlap as supposed by hong et al . and masket and fram [ 7 , 27 ] . we counsel the patients with our hypothesis that a minor change in object - image magnification of the pseudophakic eye with respect to the phakic eye might change the location or size of the corresponding image to the optic disc head which might result in an arc - shaped shadow adjacent to the blind spot . our patients were generally satisfied with this explanation and did not insist on an additional surgical intervention . follow - up examinations are necessary to verify our hypothesis on nd and whether these shadows will disappear on temporary occlusion of the eye . up to now , pseudophakic negative dysphotopsia was alleged to be located in the far peripheral temporal visual field between 50 and 90. we assume that changes in the location and/or size of the blind spot in the visual field ( as a corresponding image to the optic nerve head in the object plane ) could be one reason for photic effects , which were described excessively in the literature as pseudophakic negative dysphotopsia . in that context , we present a new examination technique to localize the photic effects within the visual field in patients with negative dysphotopsia . in all of our 8 patients , the scotoma of the optic nerve head and the main arteries and veins of the phakic eye are displaced in the pseudophakic eye depending on the specific iol position , power , and shape factor . we feel that clinicians should keep in mind that beside the edge design or reflectance of iols or the direct peripheral pass of light without being refracted by the iol there might be other reasons , for example , associated with the blind spot which could cause photic effects in the pseudophakic eyes and which are typically located more centrally in the visual field .

meniscal repairs are likely to have better long - term outcomes than meniscal resections , patients report better functional outcome , and it is assumed that repair is superior to resection in preventing osteoarthritis , even if the healing is not complete . arthroscopic all - inside techniques are often used in preference to outside - in and inside - out techniques because the advantages of shorter operation time and no need for postero - medial or postero - lateral incisions . studying failure rates in a review of 19 studies , grant et al . found similar failure rates following inside - out techniques ( 17 % ) compared to all - inside techniques ( 19 % ) . the follow - up time of the studies , however , varied from 3 months to 13 years , making the comparison of the techniques hard to interpret . in a meta - analysis , comparing results following open technique , outside - in technique , inside - out technique and different all - inside techniques in 14 cohorts , nepple et al . concluded with a pooled failure rate of 23 % and no differences between the techniques ( failure rates , respectively , 23 , 24 , 22 and 24 % ) . most clinical studies on all - inside meniscal repair are retrospective , have enrolled small numbers of patients , or have included a range of different all - inside devices , and several studies have short observational time only [ 1 , 2 , 9 ] . the first - generation device biofix meniscal arrow have been reported with good results with 91 % healing within 4 months and success rates above 90 % at 23-year follow - up [ 8 , 12 , 18 ] . similarly , the second - generation all - inside devices fast - fix are reported with good functional results at 2 years and up to 90 % success of healing at 18 months . in laboratory studies , however , the biofix meniscal arrow is shown to have lower pullout strength than the fast - fix suture [ 4 , 27 ] , which have biomechanical properties comparable to inside - out vertical mattress sutures . thus , the background knowledge for what all - inside technique to choose has been limited and inconclusive . still , the use of suture devices has increased at the expense of meniscal arrows , based on the assumption that the suture device would provide higher healing rates . in our opinion , there was a need for randomized controlled trials ( rcts ) comparing different all - inside techniques . the aim of this rct therefore was to compare the survival rates and the functional results within 2 years following all - inside meniscal repair using either the biofix meniscal arrow or the fast - fix suture . our working hypothesis was that there might be differences between biodegradable meniscal arrows and suture devices regarding reoperation rates and functional outcome . during 20062010 , 46 patients enrolled to martina hansens hospital ( 39 patients ) and trondheim university hospital ( seven patients ) with vertical longitudinal meniscal tears eligible for arthroscopic all - inside meniscal repair were included in this prospective randomized double - blinded study . the patients were block randomized ( blocks of ten ) to arthroscopic meniscal repair with either biofix or fast - fix all - inside devices using the envelope method , and they were blinded for the treatment choice . blinded observers performed post - operative follow - ups after 6 weeks , 3 and 6 months , and 1 and 2 years . symptoms ( e.g. pain , stiffness , locking ) and clinical findings ( e.g. range of motion , swelling ) and potential complications were recorded . all patients that dropped out at 2-year follow - up were interviewed by telephone to verify the reoperation status . 1.fig . 1flow chart the main endpoint of the study was reoperation within 2 years as a consequence of complaints due to rerupture or impaired primary healing . reoperations were recorded when patients had recurrent symptoms of meniscal lesions ( e.g. pain , clicking , locking ) with clinical indication for reoperation , and the reoperation led to total or partial resection of the incident meniscus tear . secondary endpoints were knee function and activity level measured by knee injury and osteoarthritis outcome score ( koos ) and tegner activity scale . koos is validated for patients with meniscal tears and osteoarthritis and consists of 42 questions in five categories : pain , other symptoms ( symptoms ) , activities of daily life ( adl ) , sport and recreation ( sport ) and quality of life ( qol ) [ 21 , 22 ] . scores in each subscale are transformed to a 0100 scale , where zero represents extreme knee problems and 100 represent no knee problems . a difference of 810 points the tegner activity scale is graded from 1 through 10 , according to the patients self - esteemed level of activity . ten points are referring to pivoting sports activities ( soccer ) at international level , five points refer to heavy activities like chopping wood and one point refers to easy house cleaning . this study was approved by the regional ethical committee for south eastern norway ( registration number 1.2005.2304 ) and has been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki . inclusion criteria were patients aged 1840 years with an mri - verified vertical , longitudinal meniscal tear , 1040 mm long , located in the peripheral or the middle third of the meniscus , with a preserved central bucket handle eligible for reduction and repair with all - inside technique . the patients had no conflicting comorbidity , drug addiction or psychiatric conditions affecting surgery or post - operative rehabilitation regime . exclusion criteria were focal cartilage lesions or osteoarthritis grade 34 according to the revised international cartilage repair system ( icrs ) classification system in an area larger than 1 cm in the actual knee , ligament tears except tear of the anterior cruciate ligament ( acl ) and/or grade 1 tear of the medial collateral ligament ( mcl ) . the meniscal tears were debrided with diamond rasp , and by use of adjusted instruments for each device , the implants were inserted on both surfaces of the menisci , seeking adequate adaption and compression of the tear surfaces . post - operatively , the patients were instructed to use crutches with partial weight bearing ( within 20 kg load ) the first 6 weeks post - operatively with unlimited joint range of motion . flexion up to 90 with concomitant weight bearing was allowed from 6 to 12 weeks post - operatively . patients with acl tears had concomitant acl reconstruction or were stabilized in a brace until acl reconstruction was performed . the frequency of reoperation after meniscal repair with all - inside devices is reported ranging from 57 to 91 % [ 2 , 13 , 26 ] . in this study , a difference in reoperation rate greater than 10 % was considered as a clinical important difference . with a power of 0.90 , a level of significance of 0.05 and standard deviation ( sd ) of 15 , 100 subjects ( 50 in each group ) were needed to detect a 10 % difference . with a 20 % suggested dropout rate at 2 years , the plan was to include 120 patients ( 60 in each group ) . however , we chose to interpret our own data at the inclusion of 46 patients , since new information from other studies revealed favourable results using suture techniques compared to meniscal arrows [ 7 , 10 ] . based on the results of our own preliminary data , we found it unethical to continue the recruitment of patients . thus , the total number of patients is 46 . the value p < 0.05 was considered statistically significant and p < 0.01 was considered highly significant . comparisons between groups were performed using the chi - square test for categorical data and using the student t test or the mann whitney u test for continuous data , depending on whether normality could be assumed . due to low sample sizes , koos and tegner scores are presented with median and range values , although tests showed normality . logistic regression analysis was performed with reoperation status as the dependent variable and gender , method for meniscal repair and comorbidity in the same knee as independent variables . time to reoperation was estimated by the kaplan meier method , and differences between the treatment groups were compared using the log - rank test . both intention - to - treat and per - protocol analyses were performed inclusion criteria were patients aged 1840 years with an mri - verified vertical , longitudinal meniscal tear , 1040 mm long , located in the peripheral or the middle third of the meniscus , with a preserved central bucket handle eligible for reduction and repair with all - inside technique . the patients had no conflicting comorbidity , drug addiction or psychiatric conditions affecting surgery or post - operative rehabilitation regime . exclusion criteria were focal cartilage lesions or osteoarthritis grade 34 according to the revised international cartilage repair system ( icrs ) classification system in an area larger than 1 cm in the actual knee , ligament tears except tear of the anterior cruciate ligament ( acl ) and/or grade 1 tear of the medial collateral ligament ( mcl ) . the meniscal tears were debrided with diamond rasp , and by use of adjusted instruments for each device , the implants were inserted on both surfaces of the menisci , seeking adequate adaption and compression of the tear surfaces . post - operatively , the patients were instructed to use crutches with partial weight bearing ( within 20 kg load ) the first 6 weeks post - operatively with unlimited joint range of motion . flexion up to 90 with concomitant weight bearing was allowed from 6 to 12 weeks post - operatively . patients with acl tears had concomitant acl reconstruction or were stabilized in a brace until acl reconstruction was performed . the frequency of reoperation after meniscal repair with all - inside devices is reported ranging from 57 to 91 % [ 2 , 13 , 26 ] . in this study , a difference in reoperation rate greater than 10 % was considered as a clinical important difference . with a power of 0.90 , a level of significance of 0.05 and standard deviation ( sd ) of 15 , 100 subjects ( 50 in each group ) were needed to detect a 10 % difference . with a 20 % suggested dropout rate at 2 years , the plan was to include 120 patients ( 60 in each group ) . however , we chose to interpret our own data at the inclusion of 46 patients , since new information from other studies revealed favourable results using suture techniques compared to meniscal arrows [ 7 , 10 ] . based on the results of our own preliminary data , we found it unethical to continue the recruitment of patients . thus , the total number of patients is 46 . the value p < 0.05 was considered statistically significant and p < 0.01 was considered highly significant . comparisons between groups were performed using the chi - square test for categorical data and using the student t test or the mann whitney u test for continuous data , depending on whether normality could be assumed . due to low sample sizes , koos and tegner scores are presented with median and range values , although tests showed normality . logistic regression analysis was performed with reoperation status as the dependent variable and gender , method for meniscal repair and comorbidity in the same knee as independent variables . time to reoperation was estimated by the kaplan meier method , and differences between the treatment groups were compared using the log - rank test . both intention - to - treat and per - protocol analyses were performed . the baseline data for the two intervention groups were similar ( table 1 ) . forty - six patients ( 26 men , 20 women ) with median age 25.7 years ( range 18.740.0 ) were reviewed 2 years after meniscal repair with either biofix arrows or fast - fix sutures . twelve out of 46 patients ( 26 % ) underwent reoperation within 2 years ; nine out of 21 ( 43 % ) patients in the biofix - group and three out of 25 ( 12 % ) patients in the fast - fix - group ( p = 0.018 ) . the relative risk of reoperation was 3.6 times higher for patients in the biofix - group compared to the fast - fix - group ( 95 % confidence interval 1.111.5 ) . 2.table 1baseline databaselinebiofix - groupfast - fix - groupnumber21 patients25 patientsage median ( range)26.9 years ( 19.439.8)25.5 years ( 18.740.0)gender9 men/12 women17 men/8 womenknee9 right/12 left16 right/9 leftmeniscus20 medial/1 lateral24 medial/1 laterallength of meniscal tear median ( range)2030 mm ( 1040 mm)2030 mm ( 1040 mm)acl pathology total n 5 patients7 patients earlier acl - reconstructed n 2 patients3 patients concomitant acl - reconstructions n 2 patients3 patients remained untreated n 1 patient1 patientother concomitant injuriescartilage injuries 2 patientsosteoarthritis 2 patientscartilage injuries 4 patientstime injury - surgery median ( range)4.5 months ( 0.229.7)4.1 months ( 0.2120.0)fig . each step represents a reoperation due to rerupture or failed primary healing survival function following meniscal repair with biofix meniscal arrows and fast - fix sutures . each step represents a reoperation due to rerupture or failed primary healing the median time from operation to reoperation was 1.1 years ( range 0.41.8 ) in the biofix - group and 1.3 years ( range 0.91.5 ) in the fast - fix - group . eight of the 12 reoperated patients ( 67 % ) reported gradual recurrence of the meniscal symptoms , 3 reported minor traumas and one reported a high energetic valgus trauma . analysing comorbidity , age , gender and meniscal repair techniques in a logistic regression model with reoperation status as the dependent variable revealed that the meniscal repair technique was the only variable influencing the end point ( reoperation ) ( p = 0.019 ) . for both treatment groups , there was clinical relevant and statistical highly significant ( p < 0.01 ) increase in all koos subscales , from baseline to 2-year follow - up ( fig . 3 ) . comparing the koos profiles in the two treatment groups at 2-year follow - up with the profile from a slightly younger reference population revealed that the patients koos at 2 years were similar to the koos profiles of the reference population ( fig . , there were no major differences in any koos subscale or tegner score at any follow - ups ( table 2 ) . 3koos profiles ( expressed as median values ) for the two treatment groups preoperatively and at 2-year follow - up ( p < 0.01 for each subscale in both groups)fig . 4the koos profiles ( expressed as median values ) of the treatment groups at 2-year follow - up did not differ significantly from the koos profile of a reference population ( grey line)table 2koos and tegner activity scale scores at baseline and follow - ups at 3 and 6 months and 2 years in the two treatment groupsscoresgroupbaseline3-month follow - up p 6-month follow - up p 2-year follow - up p koos painmedian ( range)biofix 61 ( 3686)72 ( 5397)n.s94 ( 54100)0.04192 ( 69100)n.sfast - fix 63 ( 3194)83 ( 7292)82 ( 4797)94 ( 25100)koos symptomsmedian ( range)biofix 75 ( 4679)71 ( 4696)n.s.86 ( 54100)n.s89 ( 68100)n.sfast - fix 70 ( 2889)80 ( 6886)77 ( 6196)95 ( 57100)koos adlmedian ( range)biofix 75 ( 4199)94 ( 56100)n.s.100 ( 71100)n.s99 ( 78100)n.sfast - fix 68 ( 54100)91 ( 7899)88 ( 57100)98 ( 44100)koos sport - recmedian ( range)biofix 30 ( 055)40 ( 585)n.s.75 ( 595)n.s65 ( 25100)n.sfast - fix 40 ( 090)60 ( 075)68 ( 3595)85 ( 50100)koos qolmedian ( range)biofix 38 ( 663)50 ( 1975)n.s.81 ( 694)n.s75 ( 25100)n.sfast - fix 41 ( 083)47 ( 3188)63 ( 3194)78 ( 6100)tegner activity scoremedian ( range)biofix 4 ( 09)4 ( 09 ) < 0.014 ( 29)0.0465 ( 29)n.sfast - fix 4 ( 010)3 ( 09)3 ( 09)4 ( 19)results from follow - ups at 6 weeks and 1 year are not listed because there were no statistical differences between the groups koos profiles ( expressed as median values ) for the two treatment groups preoperatively and at 2-year follow - up ( p < 0.01 for each subscale in both groups ) the koos profiles ( expressed as median values ) of the treatment groups at 2-year follow - up did not differ significantly from the koos profile of a reference population ( grey line ) koos and tegner activity scale scores at baseline and follow - ups at 3 and 6 months and 2 years in the two treatment groups results from follow - ups at 6 weeks and 1 year are not listed because there were no statistical differences between the groups analysing the group of patients that was reoperated within 2 years , comparing them to the group of patients that was not reoperated , revealed that the group of reoperated patients had higher tegner activity score preoperatively ( median 5 vs. 4 ) ( range 39 vs. 19 ) ( p = 0.037 ) and at 3-month follow - up ( median 4 vs. 3 ) ( range 29 in both groups ) ( p = 0.010 ) . except for the higher tegner score , these patients did not differ from the other patients regarding baseline data or the other variables at follow - up . among the 46 patients included in this rct , one patient in the fast - fix - group suffered from a purulent arthritis caused by staphylococcus aureus diagnosed 2 weeks post - operatively . he was treated successfully with surgical lavage where the implants were left in place and with intravenous and oral antibiotics . another patient was diagnosed with deep vein thrombosis and pulmonary embolus post - operatively and was treated with thrombolytic medication for 6 months . there were no other major complications , and there were no complications related to the implants . the main finding of this prospective randomized study was a 3.6 times higher risk of reoperation within 2 years following meniscal repair with biofix meniscal arrows compared to repair with fast - fix meniscal suture . the reoperation rates are consistent with earlier studies [ 7 , 10 , 13 ] and give support to today s clinical practice where biodegradable arrows are used less frequent . logistic regression analysis demonstrated that neither comorbidity in the same knee , nor age nor gender influenced on the risk of reoperation . only the choice of meniscal repair device was found to have an impact on the outcome . the post - operative regimes and rehabilitation were identical in the two groups , and the surgical techniques were similar except for the procedures directly related to the implant . hence , the difference in risk of reoperation seems to be dependent on the implant only . biomechanical laboratory studies have shown that biofix meniscal arrows have lower pullout strength and less flexibility over the repair than fast - fix meniscal suture in cadaver knees [ 4 , 27 ] . it is possible that the combination of lower pullout strength , less flexibility and biodegradation of the implants leads to lower stability and thereby higher risk of failure in the biofix - group than in the fast - fix - group . it is also possible that the fast - fix devices tends to keep the reduced meniscal fragments more anatomically in place over a longer period of time than the biofix arrows , which possibly may reduce or delay reonset of symptoms in patients even if the meniscal tear is not healed . another finding was that patients 2 years after meniscal repair using either method had knee function assessed by koos similar to that of a reference population . the study was underpowered to explore possible between - intervention group differences in patients function assessed by koos . thus , no statistically significant differences were revealed within 2 years post - operatively , except for the koos subscale pain at 6 months where patients operated with biofix scored better than patients operated with fast - fix ( p = 0.041 ) ( table 2 ) . the mean difference was , however , only 10 points , which is recognized as being a borderline value for what is considered clinically relevant . however , we also found that biofix - patients had a significant higher tegner score at 3 and 6 months compared to the fast - fix - patients ( p < 0.01 and p = 0.046 , respectively ) ( table 2 ) . neither should this be emphasized heavily , but one explanation may be that the fast - fix sutures give rise to irritation in the joint capsule due to traction of the anchors and thereby pain and reduced activity level , whereas the biofix arrows do not since they are mainly located inside the menisci . the post - operative rehabilitation program was equal for all included patients except for the five who went through simultaneous acl reconstruction and the two who later decided not to go through reconstruction . these seven patients , however , were equally distributed in the two treatment groups . in the literature , there is no consensus concerning rehabilitation regimes following meniscal repair , but during the last decades , the trend is towards less restrictive regimes . the patients in this study were instructed to use crutches with partial weight bearing the first 6 weeks post - operatively but had no restrictions in range of motion without weight bearing . a recent study shows , however , that avoiding weight bearing after repair with fast - fix meniscal suture is unnecessary , and a laboratory study concludes that even high flexion is safe , but only when performed in closed - chain exercises . the patients in this study were not allowed to return to sports activities until 12 weeks post - operatively . in spite of this , the group of patients that later went through reoperation , had median tegner activity score 4 at 3 months , compared to the rest of the patients who had median score 3 . a tegner score of 4 corresponds to activities like moderately heavy labour , cycling , cross - country skiing and jogging . it is therefore reasonable to assume that some patients were back to knee demanding activities earlier than prescribed and that this may have contributed to failure in the healing of the meniscal repairs . however , these numbers should be interpreted with care as well , since in both groups , the scores showed a wide range ( from the value 29 ) . moreover , the subgroup of patients later being reoperated had a higher median tegner activity score at baseline compared to those who were not reoperated , again however , with wide ranges ( 39 and 19 , respectively ) . it might be possible that individuals with higher physical activity level present higher demands for knee function . they may therefore have a lower threshold for re - consulting the surgeon if they have recurrence of meniscal symptoms , which may possibly result in a reoperation . patients with lower knee demanding activity levels may accept recurrence of meniscal symptoms better and therefore avoid reoperations . nevertheless , these findings support the idea of more restrictive return to sports activities , and we suggest that these arguments should be emphasized even stronger for more active individuals the trial was prospective , randomized and double blinded ; neither the patients nor the follow - up examiners knew which device had been used . the time to final follow - up was longer than in previous studies [ 2 , 13 , 26 ] , and the main end point reoperation is absolute and assumed to be more accurate than the diagnosis of rerupture assessed by e.g. mri , which is shown to have low reliability [ 6 , 20 ] . for the main endpoint knee function and activity level , measured by koos and tegner activity scale , were the secondary outcomes in this study . the score is valid and reliable for patients with different knee injuries , including meniscal tears and acl tears . in this study , the tegner activity scale was presented as a self - assessment questionnaire as well , for which it is not validated . of course this excludes researcher bias , but on the contrary , it opens for the possibility that patients may tend to score themselves to improperly higher or lower activity levels , depending upon characteristics like personality , self - image and ambition level . the study has the following limitations : the number of patients is relatively low , the inclusion period was quite long , and there were several participating surgeons and the distribution of patients from the two hospitals was rather skew . the low number of patients was the result of stopping inclusion by ethical reasons with a considerable lower number of patients participating than actually planned . however , the difference in the failure rate between the two groups turned out much higher than estimated in the power analysis performed prior to starting the study . thus , the power for revealing a statistically significant and clinically relevant difference in the main outcome between the treatment groups turned out large enough . whether the secondary outcomes ( knee function and activity level ) would have shown some statistically significant differences mri might have given some additional information on the status of the meniscal healing of those patients not being reoperated . on the other hand , there are some pitfalls using mri as a criteria for meniscal healing , since the overall sensitivity and specificity for diagnosing a meniscal tear on conventional mri is reported to be as low as 79 and 88 % . following meniscal repair , the mri - diagnostics is even more challenging , and false positive tears may be reported still after healing of the meniscus [ 6 , 20 ] . despite the limitations in this study , it verifies that meniscal repair with an all - inside suture device is superior to meniscal arrows regarding reoperation rate during the first 2 years post - operatively . the 3.6 times higher risk of reoperation following repair with arrows will presumably have important impact on the choice of methods for meniscal repairs in clinical practice . longer term follow - up studies are necessary to determine the influence of the different methods on degenerative changes over time . saving meniscal tissue is important , and all - inside meniscal repair has benefits compared to outside - in and inside - out techniques . therefore , in clinical practice , all - inside techniques are often used and the technique and device seems to be important . the results of this study may primarily contribute to better knowledge in choosing among the wide range of devices available today and secondary in designing new devices in the future . this study showed a significant lower rate of reoperations following meniscal repair with fast - fix all - inside suture compared to biofix all - inside meniscal arrows . functional outcome was not dependent upon repair technique , but the higher tegner scores preoperatively and at 3 months post - operatively in the group of patients being reoperated at a later stage , imply that activity level may influence on the risk of reoperation . this study strongly advocates the use of a suture device instead of meniscal arrows in all - inside meniscal repair . it also suggests a restrictive activity level within the first 3 months of rehabilitation .

lack of suspicion , non - specific clinicoradiological presentation ( often mimicking tuberculosis and fungal infections ) , diagnostic intricacies , and lack of systematic reporting are the probable reasons that have hindered the true estimation of its incidence , worldwide . we present a case series of three patients of pulmonary nocardiosis , who had different clinicoradiological presentations and also responded differently to treatment . difficulties in their management have been discussed in light of the available literature that may help in improving disease management . in view of the rising incidence of cancer , organ transplant surgeries , and use of parenteral steroids , a 50-year - old female presented with complaints of breathlessness at rest , cough , and right - sided chest pain since 15 days . breathlessness was present since five years , for which she was on inhaled steroids and bronchodilators . she had completed successful anti - tubercular treatment ( att ) eight years ago and was again advised att for her current condition . on examination , she was tachypneic , with decreased intensity of breath sounds over the right hemithorax , and bilateral expiratory wheeze . hematological investigations revealed leukocytosis and neutrophilia and the values of the arterial blood gases showed type ii respiratory failure . on a chest radiograph , there was right - sided non - homogenous opacity involving all zones with air - fluid levels and left lung mid - zone infiltrates [ figure 1a ] . however , it revealed weak , acid - fast , filamentous bacilli resembling nocardia spp . the patient was started on two drugs , co - trimoxazole ( dose 40 mg / kg / day of sulfamethoxazole ) and amikacin ( sensitive to nocardia spp . ) . contrast - enhanced computed tomography ( cect ) of the thorax showed right lower lobe and left lingular necrotizing consolidation , with loculated hydropneumothorax [ figure 1c ] . , the patient developed pancytopenia , due to which , cotrimoxazole and linezolid were replaced with doxycycline . ensuring persistent improvement she successfully completed seven months of antibiotics with negative serial cultures and satisfactory clinicoradiological improvement [ figure 1d ] . ( a ) x - ray chest , pa view , showing non - homogenous opacity involving all zones on the right side and in the left para - hilar region ; ( b ) photomicrograph showing nocardia species ( black arrow ) stained with modified ziehl neelsen stain ( under 100 ) ; ( c ) axial cut section ct thorax showing a right - sided , loculated , hydropneumothorax and basilar necrotizing consolidation ; ( d ) x - ray chest pa view , showing clearing of shadows with residual fibrotic scarring over the left lung field a 72-year - old male , presented with a three - month history of progressive shortness of breath and productive cough . he had been on multiple courses of oral steroids for recurrent breathlessness , present for over five years . cect of the chest showed areas of necrotizing consolidation with a few cavitating nodules involving the upper lobe on both sides , with a background of emphysema [ figure 2 ] . on suspicion of pneumonia / tuberculosis , the sputum was subjected to microbiological tests and cytology , which was inconclusive . the patient was started on antibiotics , cotrimoxazole and linezolid ( 600 mg b.i.d . ) , along with oral steroids . however , he got readmitted after 12 days in emergency with complaints of loose stools and deranged consciousness . he had severe electrolyte imbalance , hyponatremia ( serum na : 111meq / l ) , and hypokalemia ( serum k : 1.7meq / l ) , and died due to sudden cardiac arrest . two axial cut sections of the ct thorax , with contrast showing patchy areas of necrotizing consolidation and few cavitating nodules a 42-year - old male smoker presented with cough , increased breathlessness , and high - grade fever since 20 days . the patient had been self - medicating with oral steroids for the last three years due to persistent breathlessness . the x - ray chest pa view and cect thorax showed bilateral mass - like consolidations with air bronchograms [ figure 3a and b ] . on suspicion for lung carcinoma / pneumonia , bal was performed , which showed the growth of nocardia spp . cotrimoxazole ( 40mg / kg / day of sulfamethoxazole ) and injection ceftriaxone 3 g / day were started and the steroids were continued in tapering doses . the patient completed six months of treatment with marked clinicoradiological improvement [ figure 3c ] . ( a ) x - ray chest , pa view , showing homogenous opacity involving all zones over the right hemothorax , as well as , over the mid and lower zones on the left side ; ( b ) axial section of the ct thorax below the carina showing three areas of mass - like consolidations , involving the bilateral hemothorax ; ( c ) x - ray chest , pa view , showing marked resolution of the opacities with residual fibrosis a 50-year - old female presented with complaints of breathlessness at rest , cough , and right - sided chest pain since 15 days . breathlessness was present since five years , for which she was on inhaled steroids and bronchodilators . she had completed successful anti - tubercular treatment ( att ) eight years ago and was again advised att for her current condition . on examination , she was tachypneic , with decreased intensity of breath sounds over the right hemithorax , and bilateral expiratory wheeze . hematological investigations revealed leukocytosis and neutrophilia and the values of the arterial blood gases showed type ii respiratory failure . on a chest radiograph , there was right - sided non - homogenous opacity involving all zones with air - fluid levels and left lung mid - zone infiltrates [ figure 1a ] . however , it revealed weak , acid - fast , filamentous bacilli resembling nocardia spp . the patient was started on two drugs , co - trimoxazole ( dose 40 mg / kg / day of sulfamethoxazole ) and amikacin ( sensitive to nocardia spp . ) . contrast - enhanced computed tomography ( cect ) of the thorax showed right lower lobe and left lingular necrotizing consolidation , with loculated hydropneumothorax [ figure 1c ] . , the patient developed pancytopenia , due to which , cotrimoxazole and linezolid were replaced with doxycycline . ensuring persistent improvement she successfully completed seven months of antibiotics with negative serial cultures and satisfactory clinicoradiological improvement [ figure 1d ] . ( a ) x - ray chest , pa view , showing non - homogenous opacity involving all zones on the right side and in the left para - hilar region ; ( b ) photomicrograph showing nocardia species ( black arrow ) stained with modified ziehl neelsen stain ( under 100 ) ; ( c ) axial cut section ct thorax showing a right - sided , loculated , hydropneumothorax and basilar necrotizing consolidation ; ( d ) x - ray chest pa view , showing clearing of shadows with residual fibrotic scarring over the left lung field a 72-year - old male , presented with a three - month history of progressive shortness of breath and productive cough . he had been on multiple courses of oral steroids for recurrent breathlessness , present for over five years . cect of the chest showed areas of necrotizing consolidation with a few cavitating nodules involving the upper lobe on both sides , with a background of emphysema [ figure 2 ] . on suspicion of pneumonia / tuberculosis , the patient was started on antibiotics , cotrimoxazole and linezolid ( 600 mg b.i.d . ) , along with oral steroids . however , he got readmitted after 12 days in emergency with complaints of loose stools and deranged consciousness . he had severe electrolyte imbalance , hyponatremia ( serum na : 111meq / l ) , and hypokalemia ( serum k : 1.7meq / l ) , and died due to sudden cardiac arrest . two axial cut sections of the ct thorax , with contrast showing patchy areas of necrotizing consolidation and few cavitating nodules a 42-year - old male smoker presented with cough , increased breathlessness , and high - grade fever since 20 days . the patient had been self - medicating with oral steroids for the last three years due to persistent breathlessness . the x - ray chest pa view and cect thorax showed bilateral mass - like consolidations with air bronchograms [ figure 3a and b ] . on suspicion for lung carcinoma / pneumonia , cotrimoxazole ( 40mg / kg / day of sulfamethoxazole ) and injection ceftriaxone 3 g / day were started and the steroids were continued in tapering doses . the patient completed six months of treatment with marked clinicoradiological improvement [ figure 3c ] . ( a ) x - ray chest , pa view , showing homogenous opacity involving all zones over the right hemothorax , as well as , over the mid and lower zones on the left side ; ( b ) axial section of the ct thorax below the carina showing three areas of mass - like consolidations , involving the bilateral hemothorax ; ( c ) x - ray chest , pa view , showing marked resolution of the opacities with residual fibrosis nocardiosis is caused by the aerobic , gram - positive filamentous bacteria of genus nocardia . a majority of the nocardia species responsible for respiratory and disseminated nocardiosis have been re - grouped as the nocardia asteroids complex . the existing literature from india is no better and merely consists of case series and a few case reports . lungs are the most common site of involvement and are affected in 70% of all cases of nocardiosis . the central nervous system ( cns ) , skin , and disseminated disease are other presentations , the likelihood of which depends on the individual 's immune status , time to diagnosis , and virulence of the nocardia species . all three patients in our series had isolated pulmonary involvement , as dictated by the absence of extrapulmonary symptoms . pulmonary nocardiosis usually affects patients with leukemia , human immunodeficiency virus ( hiv ) infection , organ transplantation , diabetes , or patients receiving prolonged corticosteroids . although unlikely , copd has been found as a predisposing factor in 23 - 70% of pulmonary nocardiosis . all patients in the series also had underlying airway disease consistent with chronic obstructive pulmonary disease ( copd ) and two of them had a history of prolonged steroid intake . however , it may be difficult to quantify the relative contribution of each factor in predisposing the disease . pulmonary nocardiosis may present as an acute , subacute or chronic disease , with remissions and exacerbations . symptoms include fever , cough , breathlessness , hemoptysis , and weight loss , which are often attributed to tuberculosis or community - acquired pneumonia . apart from these , nocardiosis may also mimic fungal pneumonia , anti - neutrophil cytoplasmic antibody ( anca)-associated vasculitis and lung cancer . nodules , necrotizing consolidation , cavities , and mass shadows are some of its varied but non - specific presentations . one patient in our series had a mass shadow mimicking carcinoma and the other two had non - specific consolidations . isolation and identification of the organism from the clinical specimens form the backbone for diagnosis of pulmonary nocardiosis . requirement for invasive sampling ( bal , biopsy ) , a slow growth , and varying degrees of acid fastness of the bacteria cause a hindrance to the microbiological confirmation . typically , nocardia are weak , acid - fast organisms , visualized by a modified ziehl - neelsen stain , using 1% sulfuric acid ( instead of 20% , which is used for tubercle bacilli ) . hence , the laboratory should always be notified about its suspicion so as to optimize recovery . moreover , isolation of the organism should be interpreted under the light of a relevant clinical background , as bacteria can also be isolated from colonized airways of apparently healthy individuals , like bronchiectasis . in the present series , species identification could not be done in our patients because of a lack of the requisite infrastructure . once confirmed , the extra - pulmonary spread of disease should be explored , if clinically indicated . owing to the paucity of prospective trials , there is a lack of standard treatment guidelines for pulmonary nocardiosis . in view of the difference in virulence and in vitro drug susceptibility of bacterial subtypes trimethoprim - sulfomethoxazole is the most common drug combination used , in a dose of 25 - 50 mg / kg per day of sulfamethoxazole , in divided doses . amikacin , imipenem , ceftriaxone , minocycline , levofloxacin , linezolid , and amoxicillin - clavulanic acid are the alternate drugs that have activity against nocardia . usually , the treatment is recommended for a total duration of six to twelve months , which may be prolonged in immunocompromised patients . two patients in our series were treated successfully with six and seven months of treatment , respectively , while one died during the course of treatment . the disease should always be considered in the differential diagnosis of pneumonia , not only in the immunocompromised , but also in the immunocompetent , especially when they are not responding to the standard therapy . in tuberculosis endemic countries like india ,

this was an analytical cross - sectional study of 45 treatment - nave patients with leprosy and 45 patients with other skin diseases as the control group . both groups attended the foundation of tropical dermatology and venereology alfredo da matta ( fuam ) , which is a tertiary reference centre for leprosy treatment and research located in manaus . inclusion criteria for both groups were people aged over 18 and under 60 years of either gender who agreed to participate in the study . subjects of indigenous ethnicity , pregnant women and individuals with sequelae of injury in the central nervous system and/or sequelae of head and neck cancer treatment were not included in this study . the subjects in the case and the control groups were recruited at the same institution ( fuam ) , which is a reference institution for the diagnosis of leprosy and other dermatological diseases . none of the individuals from the control group had a prior history of leprosy treatment or developed any symptoms until the end of this study . individuals in this group were diagnosed with allergies , vitiligo , acne or psoriasis . initially , all participants were subjected to an interview , followed by an evaluation of inner oral cavity sensitivity when biological samples were collected from the leprosy group . the interviews were conducted using closed questions adapted from questionnaires prepared by furkim and silva ( 1999 ) apud villar et al . the medical data for each patient were collected from the records , including the operational classification of the disease , bacteriological index ( bi ) results , degree of incapacity ( physical incapacity and neural damage ) and progression time ( time between initial symptoms and evaluation ) . in the control group , only data regarding the clinical diagnoses were obtained . during interviews , demographic data , such as the name , address , phone number , birth date , age , skin colour , education , marital status and gender , were collected . data regarding the sensitivity of the oral cavity were obtained by inquiry : difficulty in noticing the taste of food , difficulty in breathing through the nose , dry mouth , globus pharyngeus , coughing or choking during or before eating , hoarseness , throat clearing or voice disorder during or after eating , nasal reflux of food , weight loss , pneumonia and trouble swallowing . for this study , the choice of regions used to evaluate oral cavity sensitivity ( soft palate , hard palate and tongue ) was based on the anatomical regions of the oral cavity most often affected by leprosy according to the who topographic map for oral lesions in subjects with leprosy created by scheepers et al . the oral cavity thermal sensitivity evaluation followed the protocol of the brazilian ministry of health ( ms 2002 ) to estimate the sensitivity in skin lesions . tactile sensitivity was first evaluated by touching dry cotton swabs to the dorsum of the tongue and the hard and soft palates . the response was considered normal when the patient perceived and located the region touched ( hard palate , soft palate and tongue ) and the response was considered disturbed when the patient could not perceive the touch . secondly , thermal sensitivity was evaluated by tapping on the dorsum of the tongue and on the hard and soft palates ( indiscriminately ) with cotton swabs soaked in room temperature or warm ( 45c ) water . the response was considered normal when the patient felt the temperature of the swab ( hot or cold ) and the response was considered disturbed when the patient could not distinguish the temperature . finally , taste sensitivity was evaluated by offering gustatory stimuli for sweet ( 12% w / v sucrose in water ) , salty ( 2% w / v nacl in water ) , bitter ( peumus boldus tea ) and sour ( lemon ) . slit - skin smear collection and the bi procedure followed the protocols recommended by the brazilian ministry of health ( ms 2010 ) . the biological materials used for polymerase chain reaction ( pcr ) amplification were gathered directly from samples on the scalpel blade used to collect tissue from the right ear lobe ( approximately 5 - 10 l total ) and were transferred to a 1.5-ml microtube containing 200 l of 6 m guanidine isothiocyanate ( promega , madison , wi , usa ) . patients were instructed to chew a 1 cm piece of a previously sterilised latex tube ( auriflex , so roque , so paulo , bra ) to stimulate the production of saliva . all patients were instructed to swallow the saliva produced during the first 30 sec and to collect the saliva produced for the next 5 min in a sterile universal collector . a total of 500 l of saliva was collected from each universal collector and aseptically transferred to a 1.5-ml microtube with a micropipette . another 25 l of saliva was spread on a microscope slide to determine the bi . total dna from slit - skin samples was extracted using the dneasy blood & tissue kit ( qiagen , valencia , ca , usa ) , as described by the manufacturer , with some modifications . the slit - skin sample in 200 l of guanidine the same kit was used for the saliva samples ( 250 l ) , but the standard manufacturer 's instructions were followed . the extracted dna was quantified in a microvolume spectrophotometer asp-3700 ( actgene , piscataway , nj , usa ) . a quantitative pcr ( qpcr ) protocol was performed for m. leprae dna detection using a set of primers targeting the 85ac intergenic region as described by martinez et al . this protocol was originally established for conventional pcr and was adapted for real - time detection in this study using gotaq qpcr master mix ( promega , madison , wi , usa ) . the amplification reactions were performed as follows : 2.0 l of a 5.0 m mix containing the 85ac forward and reverse primers , 10.0 l of 2x gotaq qpcr master mix , 5.8 l of ultrapure water , 0.2 l of 100x carboxy - x - rhodamine and 2.0 l of the dna sample in a final reaction volume of 20.0 l . in each set of qpcr samples , three non - template controls were included as follows : a blank control containing the qpcr mix and primers only , a negative control containing human dna from a healthy person and a positive control from a multibacillary ( mb ) patient slit - skin smear confirmed by dna sequencing . the cycling program consisted of an initial denaturation at 95c for 10 min followed by eight cycles of denaturation at 95c for 15 s , annealing at a touchdown procedure starting at 72c and decreasing 1c per cycle for 30 s and an extension step at 72c for 30 s. an additional 40 cycles of denaturing at 95c for 30 s , annealing at 64c for 30 s and extension at 72c for 30 s were performed after the touchdown phase . a melt - curve analysis was included at the end of each experiment using the default parameters ( steponeplus , applied biosystems , foster city , ca , usa ) . the samples were defined as positive if they had a cycle threshold value less than 36 and a tm value of 90c ( 0.5c ) . to validate the results , the negative controls described above should test negative for the target . the data were analysed using descriptive statistics and quantitative variables were described using means and standard deviations . the categorical data measuring abnormal sensitivity in the oral cavity were analysed using a two - tailed fisher 's exact test due to small counts ( < 50 ) . all statistical analyses were conducted using graphpad prism 6 software ( graphpad software , inc , la solla , ca , usa ) . the level of agreement between the bacilloscopy of the slit - skin smear and qpcr of the slit - skin smear or saliva was calculated using cohen 's kappa test ( k ) for unweighted proportions ( cohen 1960 ) . the index was characterised as weak ( 0.00 - 0.20 ) , low ( 0.21 - 0.40 ) , medium ( 0.41 - 0.60 ) , good ( 0.61 - 0.80 ) and excellent ( 0.81 - 1.00 ) , according to the standards proposed by landis and koch ( 1977 ) . this research followed the guidelines of resolution 196/96 of the national health council and was approved by the ethical research committee ( -015/2010-erc / fuam ) . during the study period , 87 subjects with suspected leprosy were interviewed . among these , 24 subjects did not confirm the initial diagnosis , 12 subjects did not return to complete the evaluation , three subjects did not participate in the study due to meeting the exclusion criteria and three subjects refused to participate . thus , 45 subjects with leprosy were enrolled and 45 subjects diagnosed with other dermatoses were invited to participate as the control group . of the 45 subjects with leprosy , 34 were male ( 75.6% ) and 11 were female ( 24.4% ) . in the control group , the opposite trend occurred , with the majority being female ( 28 ; 62.8% ) . the mean ages of the subjects in the leprosy group and the control group were similar : 36.7 years and 36.8 years , respectively . the education level was significantly lower in the leprosy group , where only 17 subjects ( 37.8% ) had completed the elementary level . however , 29 subjects ( 64.4% ) in the control group had completed elementary school ( fisher 's exact test two - tailed p = 0.0199 ) . a brown skin colour was the most prevalent , describing 35 ( 77.8% ) and 32 ( 71.1% ) subjects in the leprosy and control groups , respectively . the majority of the patients with leprosy were operationally classified as mb ( 58% ) . the measurements of oral sensitivity revealed that 15 subjects ( 9 mb and 6 pb ) had at least one disturbed variable . a statistically significant difference was identified regarding the alteration of thermal sensitivity in subjects with leprosy compared to the control group ( fisher 's exact test two - tailed p = 0.005 ) . three ( 6.7% ) isolated cases of disturbed gustatory sensitivity were observed in the control group . there was also a significant relationship between the change in tactile sensitivity and a positive slit - skin smear sample bi ( fisher 's exact test two - tailed p = 0.015 ) ( table i ) . table irelationship between the sensitivity variable and studied groupsgroupscase ( n = 45)control ( n = 45)variablefi%fi%total ( n)p tactile sensitivitynormal4293.345100870.241disturbed36.7 - -3thermal sensitivitynormal3782.245100820.005 disturbed817.8 - -8thermal and gustatory sensitivity normal4191.145100860.111disturbed48.9 - -4a : two - tailed fisher 's exact test ; fi : simple absolute frequency . a : two - tailed fisher 's exact test ; fi : simple absolute frequency . m. leprae dna was detected in the saliva of 16 ( 35.5% ) of the 45 patients with leprosy . the operational classification and saliva qpcr results revealed that 10 ( 38.5% ) of the 26 mb patients and six ( 31.6% ) of the 19 pb patients were positive for m. leprae dna . with respect to the operational classification and slit - skin smear qpcr positivity , 10 ( 38.5% ) of the 26 mb patients and three ( 15.8% ) of the 19 pb patients were positive ( table ii ) . table iirelationship between laboratory tests results and operational classification of leprosyoperational classificationpaucibacillary ( n = 19)multibacillary ( n = 26)variablefi%fi%total ( n)p bi of slit - skin smear < 0.001positive--1246.212negative191001453.833qpcr of slit - skin0.181positive315.81038.513negative1684.21661.532bi of saliva1positive--13.81negative191002596.244qpcr of saliva0.756positive631.61038.516negative1368.41661.529a : two - tailed fisher 's exact test ; bi : bacteriological index ; fi : simple absolute frequency ; qpcr : quantitative polymerase chain reaction . a : two - tailed fisher 's exact test ; bi : bacteriological index ; fi : simple absolute frequency ; qpcr : quantitative polymerase chain reaction . when the three laboratory diagnosis protocols were analysed separately , the saliva qpcr technique was found to have the highest positivity ( 16/45 ) , followed by the slit - skin smear qpcr ( 13/45 ) and the slit - skin smear bi ( 12/45 ) techniques . a separate analysis revealed that of the 26 mb patients , 12 ( 46.2% ) were positive according to the bi test , 10 ( 38.5% ) were positive according to the saliva qpcr test and 10 ( 38.5% ) were positive according to the slit - skin smear qpcr test . of the 19 pb patients , six ( 31.6% ) were positive according to the saliva qpcr test and three ( 15.8% ) were positive according to the slit - skin smear qpcr test . when the results of the three approaches were combined , one sample was positive only in the bi test , eight samples were positive only in the saliva qpcr test and three samples were positive only in the slit - skin smear qpcr test . of the 10 positive samples in the saliva qpcr test , eight ( 3 mb and 5 pb ) were found to be positive with this technique only . the best agreement was reached between the bi test and the slit - skin smear qpcr ( k = 0.50 ) ( table iii ) . table iiirelationship between bacilloscopy of slit - skin smear and quantitative polymerase chain reaction ( qpcr ) of slit - skin smear or salivabacteriological index of slit - skin smearnegative ( n = 33)positive ( n = 12)variablefi%fi%total ( n)kqpcr of saliva0.18positive1083.365016negative2369.765029qpcr of slit - skin0.50positive515.2866.713negative2884.8433.332fi : simple absolute frequency ; k : cohen 's kappa test . fi : simple absolute frequency ; k : cohen 's kappa test . in this study , an association between m. leprae in saliva and oral sensitivity alterations was not statistically supported . among those patients with abnormalities in oral sensitivity , only three ( 18.8% ) had qpcr - detectable bacilli dna in the saliva and only one was positive in the saliva with the bi test . one of the key results of this study is that patients with leprosy were significantly more likely to have altered thermal sensitivity of the oral cavity than the control group . this finding agrees with the study of margarido and rivitti ( 2005 ) that reported a decrease in thermal sensitivity as one of the early symptoms of leprosy . it is interesting to note that eight out of the 12 cases with disturbed thermal sensitivity of the oral cavity identified in this study were in the first year of the disease progression ( data not shown ) . furthermore , half of those patients were pb , which are generally more difficult to diagnose . thus , the results indicate that evaluation of oral sensitivity could serve as a complementary test during the physical examination of suspected leprosy cases . with regard to the other sensitivity disorders in the oral cavity evaluated in this study , four leprosy patients showed concurrent alteration in thermal and gustatory sensitivity . a similar study conducted by soni and chatterji ( 1981 ) focusing on taste disorders in leprosy patients showed that 12 out of 30 ( 40% ) subjects had some alteration that was related to the disease severity . however , we were unable to observe any relationship with disease severity because two out of the four patients were pb . in this study , changes in tactile sensitivity only were observed in three mb patients who were tested subjectively , making it impossible to measure the degree of commitment ( hypoesthesia or anaesthesia ) . according to margarido and rivitti ( 2005 ) , tactile sensitivity is the last sense to be compromised ; it is observed only as hypoesthesia in the early disease phase in mb patients , but might progress to anaesthesia in the later stages of leprosy . overall , 15 subjects with altered sensitivity in the oral cavity were observed in this study ; of these , nine ( 60% ) were classified as mb . this finding is in agreement with previous studies in which most of subjects affected with lesions in the oral cavity were classified as mb patients ( scheepers et al . surprisingly , the qpcr method did not show better positivity than the bacilloscopy of the slit - skin smear ( table iii ) , which may be attributed to the difficulty of dna extraction from m. leprae . the literature indicates that variations in the positivity of pcr results may be due to the use of different primers , amplicon sizes and amplification protocols ( goulart & goulart 2008 ) , thus highlighting the need for standardisation of qpcr for leprosy detection . although the results of the slit - skin qpcr were not as sensitive as expected in mb patients , this methodology detected m. leprae dna in three out of 19 pb cases . the determination of m. leprae dna in saliva by qpcr returned a positive result in 35.5% of the 45 subjects ; moreover , in five pb cases , saliva qpcr was the only method that returned a positive result . using conventional pcr , abdalla et al . ( 2010 ) detected 10 ( 20.8% ) positive tests out of 48 subjects evaluated and martinez et al . ( 2011 ) obtained 51 ( 17.6% ) positive tests out of 290 tests performed by swabbing the oral cavity . the positive results of the saliva qpcr , especially in pb patients , suggest the possibility of a new site of sample collection . in addition to allowing the detection of dna in mb and pb subjects , it is important to mention that the collection of saliva is less invasive than the slit - skin smear and could help in situations of uncertainty in the clinical diagnosis . furthermore , the detection of bacilli in saliva is noteworthy because it suggests that the oral cavity , even in pb subjects , can be a focus of the bacillus that may correlate with the transmission of the disease , as martinez et al . altogether , the laboratory tests employed in this study indicated that 25 out of the 45 ( 55.5% ) samples from patients with leprosy had at least one positive result . although this positivity is relatively low , it is higher than that observed when the three techniques are applied separately . importantly , the use of the qpcr technique for saliva and smear , together with slit - skin smear bacilloscopy , resulted in more than twice the number of positive cases than the conventional microscopy test alone . in other words , combining different methodologies may increase the success rate of leprosy diagnosis . the successful use of qpcr to detect the presence of bacilli in the saliva was an important result because this technique was able to identify bacillus dna in subjects classified as pb patients ( i.e. , those who are not positive in the standard slit - skin smear technique ) . moreover , the possibility of including samples collected from different sites ( slit - skin smear in a skin lesion , slit - skin smear of the ear and saliva collection ) in the same tube for dna extraction , prior to molecular tests , may also serve to increase sensitivity . further research should be performed to investigate the viability of bacilli in the saliva of pb subjects . this issue has epidemiological relevance because it raises the possibility of the contribution of pb subjects to disease transmission . this study was unable to link alterations in oral cavity sensitivity to the presence of m. leprae in the saliva ; however , one - third of the enrolled patients with leprosy exhibited abnormalities in oral sensitivity . thus , further studies evaluating a larger number of patients are required to better understand the pathophysiology of the oral cavity in leprosy . in conclusion , a multidisciplinary approach , including different health professionals - physicians , nurses , laboratory technicians , dentists and speech language pathologists - may represent an important method contributing to the earlier diagnosis , assessment and rehabilitation of patients with leprosy .

although this reported prevalence is true in referral centres , it is probably much lower in the primary care outpatient setting . the importance of secondary hypertension lies in the fact that the hypertension may be remediable on treatment of the underlying cause . there are many conditions that can result in an elevation of blood pressure commonest among them being renal parenchymal disease , renovascular disease , primary hyperaldosteronism , endocrine hypertension , obstructive sleep apnea , phaeochromocytoma and various drugs , notably nsaid and cocaine . investigation for secondary hypertension follows an algorithm ultimately aimed at defining one specific cause of the hypertension . it is unusual to find three different successive causes of secondary hypertension in the same patient . we hereby present a woman who was initially diagnosed as secondary hypertension due to cushing 's disease . after undergoing a trans - sphenoidal resection of an acth secreting pituitary adenoma , she was then found to have a right renal artery stenosis for which successful percutaneous angioplasty ( ptra ) was performed . her blood pressure , however , remained extremely labile , and she was finally diagnosed with baroreflex failure . a 56-year - old caucasian woman , a past heavy smoker ( 40 pack years ) , was first evaluated in 2001 for resistant hypertension of 5-year duration . a year before , duplex sonography of the carotid arteries , performed because of visual disturbances , had demonstrated critical stenosis of the left internal carotid artery and she successfully underwent endarterectomy . during the previous years , the patient had gained 10 kg weight and had noticed the appearance of facial hirsutism and wide purple striae on her lower abdomen . her blood pressure was 180/100 mmhg , and she was moonfaced with a buffalo hump , abdominal obesity and relative wasting of limb musculature . the patient 's clinical picture and biochemical profile were consistent with a diagnosis of cushing 's syndrome either of pituitary origin or an ectopic , indolent source . on magnetic resonance imaging ( mri ) of the sella turcica , inferior petrosal sinus sampling clearly showed excessive pituitary acth excretion . in october 2002 , a 1 cm acth staining adenoma adherent to the cavernous sinus was surgically removed . a day following the surgery , , there was a gradual but complete regression of the patient 's cushingoid habitus . however , her blood pressure remained elevated and fluctuated between extremes of high to low ( table 2 ) . endocrinological workup for suspected cushing 's disease odst = oral dexamethasone suppression test ; ufc = urinary free cortisol . normal , 725 mcg / dl ; normal , 952 pg / ml ; normal , 2090 mg . twenty - four - hour urinary excretion of catecholamines as well as plasma metanephrines was all within normal limits . renal ultrasound revealed the left kidney to be 11.2 cm in length ( longitudinal axis ) versus 9.5 cm of the right kidney . duplex sonography of the renal arteries demonstrated a systolic peak ( 500 cm / s ) at the origin of the right renal artery with a parvus tardus pattern over the right intrarenal arteries . magnetic resonance angiography confirmed the presence of a significant 7 mm stenosis of the right main renal artery beginning 0.5 cm from its origin . in february 2003 , despite the success of the ptra , the patient 's blood pressure continued to fluctuate widely while being treated with maximal doses of five anti - hypertensive drugs and a pharmacological replacement dose of prednisone ( 15 mg / day ) ( table 2 ) . in view of her carotid surgery and the nature of her blood pressure profile , noninvasive measurements of beat - to - beat blood pressure and heart rate were done by using a finger pulse photoplethysmographic device ( portapres , tno institute of applied physics biomedical instrumentation ) . the patient performed a valsalva manoeuvre , maintaining an airway pressure of 30 mmhg for 12 s. during the manoeuvre , blood pressure decreased during phase 2 with an overshoot during phase 4 . baroreflex - cardiovagal gain was estimated from the relationship between interbeat interval and systolic blood pressure during phase 2 of the valsalva manoeuvre , beginning with the peak pressure and ending with the nadir blood pressure . a scatterplot was constructed relating the interbeat intervals to the systolic pressures with a 1-beat delay from the previous heartbeat . the baroreflex - cardiovagal gain ( as defined by the slope of the regression line of best fit ) was 0.07 ms / mmhg . supine and upright blood pressure and heart rate were 145/78 mmhg ; 71 bpm and 142/77 mmhg ; 79 bpm , respectively . norepinephrine levels were high normal at baseline , 487 pg / ml , and markedly increased to 927 pg / ml after 5 min of standing up . it is noteworthy that even though our patient presented with textbook features of cushing 's syndrome , she was treated for resistant hypertension for 5 years before the diagnosis was established . trans - sphenoidal resection of an acth secreting pituitary adenoma was successful in achieving complete regression of the patient 's cushingoid state . suppression of the hypothalamic - pituitary - adrenal axis , as evidenced in our patient , is an expected sequela of the operation . blood pressure , however , remained refractory to treatment . whereas previously it was persistently elevated , it was now characterized by being extremely labile swinging from excessive highs to symptomatic episodes of hypotension . as the clinical picture was highly compatible with phaeochromocytoma , investigation was focused towards this end . although renovascular hypertension is usually typified as being persistently elevated , the patient underwent ptra with stent insertion . duplex sonography a month after the procedure and computerized angiography 4 years later on showed the stent to be patent with good blood flow through the previously stenosed artery . there was , however , no demonstrable effect on blood pressure . at this stage due to the variability of the patient 's blood pressure , the baroreflex - cardiovagal gain during phase 2 of the valsalva manoeuvre was virtually zero , consistent with an inability of the heart rate to respond to a reduction in blood pressure . the blood pressure pattern during valsalva and the high plasma norepinephrine and marked increase with orthostasis rule out autonomic failure . it serves to buffer abrupt transients of blood pressure and prevents pressure from rising or falling excessively . as exemplified in our case , it is characterized by drastic changes in blood pressure due to denervation of the baroreflex . it may occur idiopathically but has mostly been reported following carotid artery endarterectomy , carotid body tumour resection and postirradiation for cervical cancer . as nearly complete loss of afferent baroreflex function is needed for baroreflex failure , it is puzzling why the condition develops following unilateral carotid endarterectomy or the unilateral resection of a chemodectoma . such pre - existing damage could be due to various causes , for example , trauma or atherosclerosis . thus , although she underwent unilateral carotid surgery , it is highly likely that the procedure contributed to her baroreflex failure . although clonidine was added to our patient 's anti - hypertensive medications , the response has been far from optimal . in summary , we have presented the unique circumstance in which three different causes of secondary hypertension were found in one patient . evolving from this is the understanding that the presence of one cause of secondary hypertension does not necessarily rule out the existence of other causes . additionally , it should be borne in mind that apart from the usually considered phaeochromocytoma , the extreme variability of blood pressure should raise suspicion of baroreflex failure .

herbal medicine continues to infuse new ideas and treatments into modern medicine for the benefit of our patients . according to the 2006 american association of poison control center ( aapcc ) data , there were a total of 972,073 exposures to pharmaceutical products resulting in adverse events , of which there were 6,809 major outcomes and 507 deaths . of these exposures , 76,364 ( 7.9% ) were due to dietary supplements and vitamins , with 42 major outcomes ( 0.6% of all major outcomes ) and only 3 deaths ( 0.6% of all deaths ) . a review of hospital admissions in hong kong found only 0.2% of the cases were due to adverse reactions from herbal products . a london unit had 5,536 enquiries on traditional medicines and food supplement exposures from 1983 to 1991 , and only 12% of the patients were symptomatic . despite the prevalent use of these products , adverse outcome and death only arise occasionally . it is safe to conclude that in most instances , dietary supplements and herbal medicines are relatively harmless when used appropriately . nevertheless , there are many reports in the literature that suggest that unnecessary or reckless use of these products can lead to problems . we see two major issues that contribute to these problems : a public perception that these products are inherently safe , and the lack of knowledge about these products in the medical profession . this article reviews the toxicities described in the literature associated with their use and when to anticipate problems with them , and describes an approach that an emergency physician can adopt when he or she encounters patients with such toxicities . the world health organization ( who ) and the united states ( us ) dietary supplements health and education act ( dshea ) of 1994 both define dietary supplements as a product ( other than tobacco ) that is meant to supplement the diet . both organizations include vitamins , minerals , herbs , botanical products , amino acids , or dietary substances in their definitions . the who also defines herbal medicines as plant - derived materials or preparations intended for human therapeutic use or for other health benefits in humans . however , most indigenous herbal traditions not only have plant matters in their material medica , but also include animal matters and mineral compounds . herbal products are usually ingested raw , as tea or as decoctions ( concentrated extracts ) . some herbal traditions , such as traditional chinese mmdicine ( tcm ) and ayurvedic medicine , have medicinal products that are packaged in the form of pills or liquids for ease of consumption and retailing . certain patterns of use or use of certain products tend to produce adverse effects and toxicities . table 1when adverse effects and toxicities may arisefactors predisposing to adverse effects and toxicitieswhen to anticipate theminappropriate usage and inherent toxicity of herbsinappropriate indications ( non - traditional indications)weight loss , athletic performance , recreational useinappropriate duration use for prolonged periods of time , usually several weeks to monthsinappropriate dosage excessive dose in order to achieve some particular resultsinadequate processing herbs that are usually consumed in a certain way in a particular herbal tradition being processed in other non - recommended waysherbs with pronounced pharmacological effects or toxic componentsadulteration with modern pharmaceuticals ( nsaids , steroids , antihistamines , sildenafil , sulfonylurea)finished products claiming fast relief of symptoms or sexual enhancementdrug interactionpatients on multiple modern pharmaceuticals , especially drugs with a narrow therapeutic index , such as warfarin , and taking dietary supplements and herbal productspatients taking multiple dietary supplements or herbal medicinesheavy metal toxicitiesfinished products from tcm , ayurvedic traditions , or mexican folk remedies when adverse effects and toxicities may arise in most instances , problems arise due to inappropriate usage of these herbs and supplements . herbs and supplements can be toxic when used for inappropriate indications , or prepared inappropriately , or used in large excessive dosages , or for a prolonged duration of time . when patients provide such a history of use , physicians should be on a look out for possible toxicities . for example , excessive doses of vitamin d due to overzealous fortification in milk resulted in hypercalemia ; similarly , excessive and prolonged intake of vitamin a can lead to osteoporosis and hepatotoxicity [ 69 ] . many serious adverse effects have arisen because of using of traditional herbs for non - traditional indications . ephedra is used in small dose in tcm for indications , such as wheezing and cough , but it was never used as a stimulant , a dieting agent , or recreational agent ; however , such uses in excessive doses and durations have resulted in serious toxicities , including death , seizures , psychosis , myocardial infarction , cardiac arrhythmia , and stroke [ 11 , 12 ] . similarly , the use of datura species for recreational purposes instead of therapeutic effects has resulted in anti - cholinergic poisoning and death . in belgium in the early 1990s aristolochia fangchi was mistakenly used instead , and this resulted in more than 100 cases of renal failure and more than 20 cases of urothelial dysplasia . the culprit is thought to be the aristolochic acid found in aristolochia fangchi . in tcm practice , stephania tetrandra and aristolochia fangchi were never meant to be used for weight loss [ 9 , 15 ] . , aristolochia fangchi requires extensive boiling into a decoction before being applied in small amounts . in the belgian sliming clinic aristolochic acid concentration is much lower in decoctions as it has low water solubility ; hence , a decoction is a safer way to administer it [ 5 , 10 ] . herbal medicines do not usually produce immediate relief of symptoms as most of them are generally concerned with homeostasis . when products claim to provide immediate relieve of symptoms , physicians should watch out for possible intentional adulteration with pharmaceuticals . these products are usually in the form of finished products meant for ingestion or occasionally for topical applications . a survey in taiwan found that of 2,609 herbal samples analyzed , 23.7% were adulterated with pharmaceuticals . a study of 243 proprietary products in california found that 7% contained undeclared pharmaceuticals . in 1999 , out of 3,320 tcm herbal products screened in singapore , 1.2% were found to contain undeclared pharmaceuticals [ 17 , 18 ] . another form of adulteration is the substitution of one herb for another that may be cheaper or more readily available , but has a less desirable safety profile . the most common adulterants are pharmaceuticals that are used to relieve uncomfortable symptoms , such as non - steroidal anti - inflammatory drugs ( nsaids ) and antihistamines [ 5 , 9 , 16 ] . adulteration in steroids and sexual - enhancing drugs , such as sildenafil , are also commonly reported [ 17 , 18 ] . serious adverse effects including death can result from such adulterations , especially since the drugs added can have serious toxic effects ; for example , sulfonylurea , phenylbutazone , phenytoin , and corticosteroids can have serious toxic effects . problems that can arise from adulterants in these products include allergic reactions , addisonian crisis , and cushing s syndrome from unsuspecting use of products with added steroids . hypotension can occur in patients on nitrates for cardiac ischemia and unsuspected use of products adulterated with sildenafil , and severe or fatal hypoglycemia can result from unsuspected use of products with sulfonylurea . for patients taking multiple medications and dietary supplements or herbal medicines , physicians should look out for herb - drug interactions . patients with chronic illnesses , who are most likely to be taking multiple medications , are also most likely to consume dietary supplements or herbal medications , putting them at risk of drug - herb interactions . patients may consume multiple dietary supplements or herbal medicines and take products that contain multiple components , putting themselves at risk of herb - herb interactions . patients most at risk of harmful drug - herbs interactions are those at extremes of age , on multiple prescriptions , with chronic illnesses or with impaired organ functions and those on prescription medications with a narrow therapeutic margin , such as warfarin . coagulative problems arising from drug - herb interactions with warfarin are commonly reported , sometimes with serious consequences , such as intracranial hematoma . garlic , gingko , ginger , and angelica sinensis ( dong quai ) are commonly implicated as they are commonly used , but any herb that may contain compounds related to salicylate or coumarin can augment anti - coagulative effects , resulting in bleeding . augmentation of sedative effects of modern pharmaceuticals is another problem , especially with popular herbs with sedative effects , such as piper methysticum ( kava ) and valeriana officinalis ( valerian ) . kava has been reported to increase the frequency and duration of off - periods in parkinson s patients , and long - term use of this herb can also lead to hepatotoxicity and dermatopathy [ 24 , 25 ] . for example , hypericum perforatum ( st john s wort ) , popularly used as an herbal anti - depressant , is a strong inducer of cyp3a , which metabolizes about 50% of all modern pharmaceuticals . this induction can lead to a sub - therapeutic effect in drugs that are inactivated or potential toxic effects in drugs that are activated by this family of enzymes . contamination of dietary supplements and herbal medicines with unwanted substances is another area of concern . problems arising from contaminations are difficult to anticipate except that they appear to be more widely reported with products from some herbal traditions , such as tcm , ayurvedic practices , and mexican folk remedies . physicians need to be aware of this problem when they encounter patients using products of these herbal traditions and presenting with non - specific symptoms . one form of contamination that is often reported is heavy metal contamination ; it can lead to heavy metal poisoning and should be suspected in patients presenting with features suggestive of heavy metal toxicities after using dietary supplements and herbal medicines [ 2628 ] . heavy metal contamination arises due to defective manufacturing processes or because the herbs were grown in polluted soil . however , in some instances , like in tcm and ayurvedic medicine , heavy metals exist as part of their formulary and are deliberately added into preparations for therapeutic effects [ 5 , 27 ] . lead poisoning is frequently reported and presents with anemia , abdominal pain , and encephalopathy . it is reported in children given mexican folk remedies for gastrointestinal symptoms [ 2931 ] and also in calcium supplements derived from animal sources [ 3234 ] . mercury , arsenic , and thallium use are reported in tcm [ 17 , 18 , 35 ] . arsenic poisoning from herbal medicine is usually chronic and presents with features , such as skin changes , leucopenia , anemia , sensory neuropathy , and malignancies . other heavy metals reported to contaminate dietary supplements and herbal products include , cadmium , copper and molybdenum [ 27 , 36 , 37 ] . other contaminants include micro - organisms , pesticides , industrial chemicals , and toxic herbs . these usually result in gastroenteritis , but more severe problems have been reported , such as eosinophilia - myalgia syndrome and death due to unknown chemical contamination of l - tryptophan supplements [ 9 , 3840 ] . chinese herbal products have been reported to be contaminated with toxic herbs such as podophyllum and datura metel . these can produce features of toxicities due to the inherent toxicities of these herbal contaminants ; for example , podophyllum poisoning can lead to agranulocytosis , and datura ingestion can lead to anti - cholinergic toxicities . dietary supplements and herbs that possess pronounced pharmacological effects or toxic constituents can be inherently poisonous , and physicians should anticipate problems with such toxicities if they encounter patients using these products . the clinical features encountered will depend on the inherent compounds present in the products . toxic herbs frequently encountered in reports are those with stimulant effects , such as ephedra species , caffeine , ginseng , and gingko ; those with cardiac effects , such as herbs containing cardioactive glycosides or aconitum species ; those with autonomic effects , such as datura species , lobelia species , and yohimbine ; those with hepatotoxic effects , such as herbs with pyrrolizidine alkaloids ; those with nephrotoxic effects , such as herbs with aristolochic acid ; and those that are used as abortifacients , such as pennyroyal oil [ 5 , 9 , 15 , 36 , 4246 ] . table 2organ toxicities and toxidromes , and common dietary supplements or herbal medicines that can cause themclinical featuresxenobioticscardiacsodium channel effects aconitum species ( widen qrs , shock)digoxin - like effects digitalis species , bufo toadscentral nervous systemseizures strychnine , thujone , essential oils ( camphor , eucalyptus)sedation valeriana species , kava kavadermatologicalblistering cantharidin ( chinese blister beetle)hematologicalcoagulopathies g - herbs ( ginger , garlic , gingko)agranulocytosis anti - mitotic agents ( colchicine , podophyllotoxin)hepatotoxichepatitis multiple agents , germander commonly reportedveno - occlusive disease pyrrolizidine alkaloids ( comfrey , senecio species , heliotropium species)nephrotoxicrenal failure aristolochia speiceshypertension , hyperkalemia licoriceanticholinergicdatura metel commonly used in tcmhexing herbs ( atropa sepcies , hyoscyamus species , mandrago officinarum ) common in western herbal practicesympathomimeticephedra species , citrus aurantium ( bitter orange)salicylate poisoningwillow bark , checkerberry organ toxicities and toxidromes , and common dietary supplements or herbal medicines that can cause them the problem of the inherent toxicity is compounded by the variation in content of the active ingredients found in these products . the chemical constituents in a plant are dependent on the soil they are grown in , rainfall and sunshine , the season of harvesting , the stage of the plant growth during harvest , diseases afflicting it , and the parts that were harvested . even in finished products , such as pills and liquids , there can be large batch - to - batch variations in content , and this can result in toxicity . for example , a survey of ginseng products found up to 200 times variation in content between different products . in another survey of ephedra products , within the same product , up to ten times variation in active ingredients among batches allergic reactions to dietary supplements and herbal medicines appear to be common and under - reported . these reactions may present as mild reactions , such as pruritus and urticaria , to more severe reactions , such as angioedema and anaphylaxis . patients may react to compounds inherent in the dietary supplements and herbal medicines , such as proteins found in animal products . patients may also develop allergic reactions to compounds that were added into these products as intentional adulterants or contaminants . it is difficult to anticipate allergic reactions to these products unless previous allergies are known . patients may not inform their physicians about herbal supplement use because they do not perceive these products as medications . when toxicities arise , patients may not be aware that the dietary supplements or herbal products are causing the problems , so they continue to use the products . the quantity of content can be different from the label given the lack of quality assurance and labeling consistency . raw herbs , dried herbs , or herbs processed into powders or liquids may prove difficult or impossible to identify . even when labeling is accurate or herbs can be identified , scientific and toxicological information regarding them may not be readily available from conventional resources . a study in the us of adverse effects from dietary supplements found that less than half of the products or ingredients were listed in the poison information software that is used in most us poison centers . nevertheless , the spectrum of toxicities from dietary supplements and herbal medicines is similar to that of toxicities from pharmaceuticals in that similar organ system effects or toxidromes can be expected . furthermore , some herbs or formulations are well known to result in certain organ toxicities or toxidromes , and their presence should be suspected when these clinical features occur ( see table 2 ) . the approach to patients with toxicities from dietary supplements and herbal medicine is similar to the approach to patients with other forms of toxicities . table 3 outlines steps generally recommended by authors for treating these patients [ 22 , 51 ] . patients who present with unstable medical conditions , such as cardiac dysrhythmias or seizures , require immediate stabilization . once they are stabilized , extended history taking , physical examination , and laboratory investigation can be done . once the problem is identified , the use of the product can be stopped or altered , and appropriate therapy can be initiated . table 3useful steps in managing patients with toxicities from dietary supplements or herbal medicines1 . laboratory studiesa . basic blood count , renal function , liver function , and electrocardiogramb . heavy metal screening if suspected or if symptoms are non - specificc . analysis methods exist for some herbal toxins only colchicines ( hplc , gcms ) , tropane alkaloids ( gcms , oxalate ( gcms ) , vinca alkaloids ( hplc ) , cardioactive steroids ( immunoassay)check with local laboratory4 . report unusual cases in the medical literature useful steps in managing patients with toxicities from dietary supplements or herbal medicines good resuscitative , symptomatic , and supportive care is paramount in these patients , as in all patients with poisoning . in such circumstances , offending agents would most likely not be identified early , and even if identified , specific antidote treatment may not exist , making resuscitative , symptomatic , and supportive care more important . patients who present early with toxic ingestion of dietary supplements or herbal products that can cause severe life - threatening effects , such as aconitium species or colchicines , should undergo gastric lavage with adequate airway protection . similarly , activated charcoal can be given in an acute overdose of toxic dietary supplements and herbal medicines if there is adequate airway protection . in patients with stimulant effects , agitation or seizures can be managed with benzodiazepines . in patients suffering from digoxin toxicity , digoxin antibody is expected to work , but a non - standard dose may be required . in patients with sodium channel effects ( wide qrs complexes , shock ) , sodium bicarbonate can be used , and class ib anti - arrhythmics such as lignocaine can be used if sodium bicarbonate fails . the local poison information centers can be good resources to assist with diagnostic or management issues . when obtaining history from patients suspected of suffering from dietary supplement or herbal medicine toxicities , it is important to remember that patients often do not volunteer information regarding the use of these products to their physicians . when suspected , physicians need to ask patients specifically if they were or currently are consuming such products . and these products include specialty teas for weight loss or calming effects . studies have shown that up to 70% of patients who use alternative therapies do not inform their physicians about it . during physical examination , features suggestive of toxidromes should be looked for , such as pupils size , mucosa moisture , skin moisture , and bowel sounds . features of organ toxicities should also be sought , especially signs of liver injuries or failure . these toxidromes and organ toxicities can often be related to certain commonly used dietary supplements and herbal medicines ( see table 2 ) . whenever possible , a sample of the actual product used by the patient should be secured ; otherwise , prescription or packaging should be secured . if this is not possible , samples from where the patient actually obtained the product may be useful . if raw herbs were involved , obtain information about the parts used and how they were processed . although immediate identification or analysis is often not possible , efforts should still be made to identify them later as some herbs and products can have long - term effects , such as hepatotoxicity or nephrotoxicity . basic diagnostic studies , such as blood count , electrolytes and renal function , liver function , and electrocardiograms , should be performed , as well as other tests based on the patient s clinical presentation . if symptoms are non - specific or suggestive of heavy metal toxicities , a heavy metal screen may be useful . analytical methods exist for herbal toxins , such as colchicines , tropane alkaloids ( anti - cholinergic ) , vinca alkaloids , and cardioactive glycosides ; however , the availability of these tests depends on local laboratories . when such analyses are indicated , it is essential to check with the local laboratory if the tests are available . the salicylate level should be available in most laboratories . for patients who can be discharged , they should be specifically instructed to stop using the dietary supplements or herbal medicines . this discussion should involve family members as well , as they may be taking similar products or be supplying them to the patients . consideration should also be given to referring patients for outpatient monitoring of liver function , renal function , and blood counts in a week or two , as toxicities in these organs may be delayed and not clinically apparent . some authors and herbal practitioners advocate that patients using tcm should have their liver function monitored regularly as many herbs can cause hepatotoxicity . the relevant regulating authorities should be informed of such events so that offending products can be investigated and if necessary taken off the market to prevent more people from being affected . the range of dietary supplements and herbal products are expanding rapidly , and medical and scientific knowledge of these products is still growing . unusual cases should be reported to the medical literature to inform the medical community of potential problems . opportunities arise at times to discuss the use of dietary supplements and herbal medicines with patients or patients family members . physicians trained in modern medicine may find themselves inadequately prepared for this task ; the simplest option practiced by most physicians would then be to denounce all such products . however , such practice may alienate patients and their family members , especially among ethnic groups where herbal traditions are important . this would also cause the patients to be less willing to approach their physicians for help when adverse effects arise . ko outlined some useful advice that can be used for patient education or discussion about dietary supplements and herbal medicines . some advice that can be easily committed to memory and applied in discussions is as follows : dietary supplements and herbal medicines should be considered as medicines . hence , dosage recommendation should be followed , and long - term therapy should be avoided . if unsure whether a product is safe or useful , or if it will interact with one s prescription , always consult a physician or pharmacist first.obtain dietary supplements and herbal medicines from reputable sources . the most reliable sources are large manufacturers , especially if they are located in countries that regulate these products like pharmaceuticals . where herbalists or traditional healers are licensed legally , licensed practitioners should be consulted.if new symptoms developed during the use of these products , stop using the product and consult a physician.vulnerable patients , such as women who are pregnant or nursing and young children , should avoid using herbal medicines or dietary supplements ( except folate and iron supplements ) if possible . hence , dosage recommendation should be followed , and long - term therapy should be avoided . if unsure whether a product is safe or useful , or if it will interact with one s prescription , always consult a physician or pharmacist first . obtain dietary supplements and herbal medicines from reputable sources . the most reliable sources are large manufacturers , especially if they are located in countries that regulate these products like pharmaceuticals . where herbalists or traditional healers are licensed legally , licensed practitioners should be consulted . if new symptoms developed during the use of these products , stop using the product and consult a physician . vulnerable patients , such as women who are pregnant or nursing and young children , should avoid using herbal medicines or dietary supplements ( except folate and iron supplements ) if possible . dietary supplements and herbal medicines play an important role in the general health - care system of many developing countries worldwide and are gaining popularity rapidly in many developed countries . most of these can be used safely if the public is given the right education and advice . physicians need to be ready to discuss or advise patients with regards to their use . adverse effects , such as allergy , drug interactions , heavy metal poisonings , reactions to adulterants or contaminants , and toxicities , can arise from the product itself . when these problems manifest , a rational approach in management emphasizing good resuscitation , symptomatic , and supportive care can be helpful .

alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) are progressive neurodegenerative disorders for which we do not have a cure . the current drug treatments are more designed to keep the symptoms at bay , but do not repair or prevent further degeneration . there is a clear need for novel treatments , and the industry has invested billions of dollars in finding new drug treatments . however , in particular in ad , the efforts have been in vain , and a string of clinical trials that did not find any improvements have discouraged some drug companies from investing into further research . the immune response strategy to remove beta - amyloid by activating the patient 's immune system or with the help of specific antibodies directed against amyloid had a string of failures , with the most recent one the antibody crenezumab developed by eli lilly ( cummings et al . , the failure to ameliorate memory impairment with this strategy has led to discussion about the sensibility of this approach ( fuller et al . , 2014 ) . the second strategy to reduce the activity of the beta- and gamma - secretases that produce amyloid also has suffered a series of setbacks : avagacestat , ( by bristol - myers squibb ) , tarenflurbil ( flurizan , myriad genetics ) , ly2886721 and semagacestat ( eli lilly and company ) ( extance , 2010 ; brooks , 2013 ; groves , 2013 ) . this led to a discussion about the validity of the ad biomarker beta - amyloid ( armstrong , 2014 ) . while these two main strategies have not been successful , another strategy has shown promising results for a fairly long time now . instead of focusing solely on amyloid levels as a biomarker , the idea of supporting cellular repair by the use of growth factors has shown promising results . growth factor signaling de - sensitization has been shown in the brains of people with alzheimer 's or parkinson 's disease ( holscher , 2014b ) . the reason for this is not entirely understood , but it may well be caused by the chronic inflammation that develops in these neurodegenerative disorders . cytokines such as tumor necrosis factor- ( tnf- ) released in the inflammation response down - regulate growth factor signaling ( chertoff et al . , 2011 ; clark et al . , 2012 ; najem et al . , a wide range of growth factors have been tested in preclinical studies and have shown a range of protective effects . one of the first growth factors that show extensive neuroprotective properties in a mouse model of ad is nerve growth factor ( ngf ) . ngf was found to protect synapses , synaptic plasticity , and learning abilities in ad mouse models or in nonprimate monkeys ( clarris et al . , 1994 ; kordower et al . , 1997 ; covaceuszach et al . , however , ngf does not cross the blood - brain barrier ( bbb ) , which severely limits the potential use of this growth factor to protect the brain ( bradbury , 2005 ; heese et al . brain derived neurotrophic factor ( bdnf ) also has been shown good effects in mouse models of ad . injecting bdnf intracerebroventricular improved memory formation , reduced the impairments of synaptic plasticity in the hippocampus , and increased synaptic numbers ( blurton - jones et al . , 2009 ) . enhancing the bdnf levels in the brain by gene delivery vectors the elevation of bdnf levels reverses synapse loss , improves synaptic plasticity and restores learning abilities of a mouse model of ad ( nagahara et al . however , bdnf does not cross the bbb and therefore it would need to be injected into the brain in order to be effective ( schulte - herbruggen et al . , 2007 ; zuccato and cattaneo , 2009 ) . other growth factors that do cross the bbb are leptin and ghrelin , which also have shown protective effects on synapses and memory formation and protect against oxidative stress ( perez - gonzalez et al . , 2011 ; tan and bloom , 2013 ; gomes et al . , 2014 ; sharma and hlscher , 2014 ) . in pd , glia - derived neurotrophic factor ( gdnf ) has shown good effects and is under intense research to develop a new treatment ( broome et al . , 1999 ; drinkut et al . does not cross the bbb either , new strategies such as nasal application are being tested ( migliore et al . , 2014 ) . the growth factors insulin and insulin - like growth factor 1 ( igf-1 ) also have shown good effects in protecting synapses and synaptic function and memory formation . in a mouse model of ad , a systemic slow - release formulation of igf-1 enhanced cognitive performance , decreased amyloid levels , protected synapses and reduced inflammation ( carro et al . , 2006 ) . in mouse models of ad , insulin and igf signaling was found to be impaired , and a reversal of this is neuroprotective ( bomfim et al . based on this observation , drugs initially developed to treat diabetes have been tested in animal models of ad and pd . the growth factors glucagon - like peptide-1 ( glp-1 ) and glucose - dependent insulinotropic polypeptide ( gip ) show good effects in animal models of diabetes and have shown good effects in several mouse models of ad ( holscher , 2014a ) . three glp-1 mimetics have been licenced and are very effective in treating type 2 diabetes ( campbell and drucker , 2013 ) . they cross the bbb ( hunter and holscher , 2012 ) and have shown good effects in animal models of ad and pd ( holscher , 2013 , 2014b ) . based on the encouraging preclinical results , several clinical trials are ongoing to test insulin of glp-1 mimetics in ad and pd patients . considering that a long list of drugs that initially showed effects in preclinical tests had shown disappointing results in subsequent clinical trials , it is noteworthy that initial data has been published that demonstrate positive clinical effects of growth factor analogues in ad and pd . biochemical tests of brain tissue demonstrate that insulin signaling in the brain is de - sensitized in ad patients , and the brain tissue shows key biochemical markers of insulin signaling failure ( moloney et al . , 2010 ; talbot et al . , 2012 ) . a study showed that in brain tissue of ad patients , igf-1 and insulin signaling was strongly de - sensitized . phosphorylation of the insulin receptor chain was reduced at positions ir py1150/1151 and ir py960 , while the insulin receptor substrate 1 ( irs-1 ) was hyperphosphorylated at positions irs-1 ps616 and irs-1 ps636 . furthermore , when incubating ad brain tissue with insulin , it revealed a reduced downstream second messenger cascade ( talbot et al . , 2012 ) . incubating the brain tissue with the glp-1 mimetic liraglutide also reversed some of the insulin signaling impairments ( talbot et al . , 2011 ) . currently , it is believed that the insulin receptor desensitization is triggered by amyloid - oligomers . in vitro cell culture experiments showed that oligomers bind to and activate receptors which are then taken up into the cell ( zhao et al . infusion of oligomers into the brains of monkeys also show these effects and trigger local inflammatory response ( lourenco et al . , 2013 ) . interestingly , glp-1 analogues that prevent the insulin desensitization can prevent this oligomer - induced effect ( bomfim et al . building on the observation that insulin signaling is impaired , nasal application of insulin has been tested in patients with mild cognitive impairment ( mci ) , which develops further to ad in the majority of cases . a pilot study testing nasal application of insulin in mci / ad patients included 26 memory - impaired subjects ( 13 diagnosed with early ad and 13 with mci ) and 35 controls randomly allocated to groups with a double - blind design . they received three intranasal treatments consisting of placebo or insulin ( 20 or 40 iu ) . insulin facilitated recall on two measures of verbal memory in memory - impaired apolipoprotein e epsilon4 ( apoee4 ) carriers . these effects were stronger for memory - impaired non - apoee4 carriers than for memory - impaired apoee4 carriers and control subjects . these findings are a proof of concept and demonstrate that insulin can indeed improve memory impairments in ad / mci patients ( reger et al . , 2006 ) . a follow - up pilot study with 24 ad / mci patients confirmed these effects of insulin ( reger et al . , 2008b ) . in a follow up study , 33 ad / mci patients and 59 control subjects received intranasal treatments of insulin or placebo . interestingly , insulin also affected plasma amyloid - beta levels in memory - impaired subjects and controls ( reger et al . , 2008a ) . following this , a randomized , double - blind , placebo - controlled clinical pilot trial was conducted . 104 patients with either mci ( n = 64 ) or mild to moderate ad ( n = 40 ) were tested . treatment with 20 iu of insulin improved memory , and both doses of insulin ( 20 and 40 iu ) preserved caregiver - rated functional ability . both insulin doses also preserved general cognition as assessed by the alzheimer 's disease assessment scale - cognitive ( adas - cog ) score for younger participants , and functional abilities as assessed by the alzheimer 's disease cooperative study activities of daily living ( adcs - adl ) scale for adults with ad . placebo - assigned participants showed decreased 18-fluoro-2-deoxyglucose with positron emission tomography ( fdg - pet ) uptake in the brain , demonstrating that neuronal metabolism was deteriorating . importantly , the improvements in episodic memory were still present two months after cessation of treatment ( craft et al . , 2012 ) . in a recent clinical trial , ad / mci patients received insulin analogue detemir ( 40 iu / d levemir ) and showed improvements in memory if they had high levels of insulin resistance at baseline ( craft , 2013 ) . this series of studies demonstrate that improving insulin signaling in the brains of ad patients has indeed positive effects on key disease parameters and biomarkers . this is a welcome change from previous experience , where encouraging effects with novel drugs in preclinical studies could not be repeated in clinical studies . the treatment of patients with insulin is unlikely to be the best strategy , as it may well enhance insulin de - sensitization if taken over longer time periods . in diabetes , the problem of progressing insulin de - sensitization by treatment with insulin is well described ( inoue et al . , the glp-1 treatment had been developed which uses a different receptor pathway ( lovshin and drucker , 2009 ) . the positive preclinical results of glp-1 in animal models of ad and pd motivated several clinical trials . this proof of concept study tested the effects of exendin-4 in a randomized open label trial . drug treated patients showed improvement at 12 months in motor tasks , compared with control patients . most interestingly , exendin-4 showed a clear improvement in the mattis drs-2 cognitive score , suggesting that exendin-4 has beneficial effects on cognition ( aviles - olmos et al . impressively , this effect was still visible 12 months after the clinical trial ( aviles - olmos et al . , 2014 ) . this result demonstrates the potential of these drugs , and confirms that the results from animal studies can be found in humans as well . the long lasting drug effect suggests that the effect is due to the normalization of growth factor signaling and the growth of new synapses and increased dopamine synthesis and release , rather than based on a short - term drug effect on synaptic release ( bertilsson et al . , a larger study has started which has a larger number of patients and a double - blind placebo controlled study design ( clinical trial identifier nct01971242 ) . to test the effects of these drugs in ad , a randomized , double - blind clinical trial of exendin-4 treatment in mci patients / early phase ad by the nih / nia furthermore , a phase ii clinical trial of the effects of the glp-1 analogue liraglutide in mci / ad patients has started ( clinical trial identifier nct01843075 ) . based on the encouraging preclinical results , several clinical trials are ongoing to test insulin of glp-1 mimetics in ad and pd patients . considering that a long list of drugs that initially showed effects in preclinical tests had shown disappointing results in subsequent clinical trials , it is noteworthy that initial data has been published that demonstrate positive clinical effects of growth factor analogues in ad and pd . biochemical tests of brain tissue demonstrate that insulin signaling in the brain is de - sensitized in ad patients , and the brain tissue shows key biochemical markers of insulin signaling failure ( moloney et al . , 2010 ; talbot et al . , 2012 ) . a study showed that in brain tissue of ad patients , igf-1 and insulin signaling was strongly de - sensitized . phosphorylation of the insulin receptor chain was reduced at positions ir py1150/1151 and ir py960 , while the insulin receptor substrate 1 ( irs-1 ) was hyperphosphorylated at positions irs-1 ps616 and irs-1 ps636 . furthermore , when incubating ad brain tissue with insulin , it revealed a reduced downstream second messenger cascade ( talbot et al . , 2012 ) . incubating the brain tissue with the glp-1 mimetic liraglutide also reversed some of the insulin signaling impairments ( talbot et al . , 2011 ) . currently , it is believed that the insulin receptor desensitization is triggered by amyloid - oligomers . in vitro cell culture experiments showed that oligomers bind to and activate receptors which are then taken up into the cell ( zhao et al . infusion of oligomers into the brains of monkeys also show these effects and trigger local inflammatory response ( lourenco et al . , 2013 ) . interestingly , glp-1 analogues that prevent the insulin desensitization can prevent this oligomer - induced effect ( bomfim et al . building on the observation that insulin signaling is impaired , nasal application of insulin has been tested in patients with mild cognitive impairment ( mci ) , which develops further to ad in the majority of cases . a pilot study testing nasal application of insulin in mci / ad patients included 26 memory - impaired subjects ( 13 diagnosed with early ad and 13 with mci ) and 35 controls randomly allocated to groups with a double - blind design . they received three intranasal treatments consisting of placebo or insulin ( 20 or 40 iu ) . insulin facilitated recall on two measures of verbal memory in memory - impaired apolipoprotein e epsilon4 ( apoee4 ) carriers . these effects were stronger for memory - impaired non - apoee4 carriers than for memory - impaired apoee4 carriers and control subjects . these findings are a proof of concept and demonstrate that insulin can indeed improve memory impairments in ad / mci patients ( reger et al . , 2006 ) . a follow - up pilot study with 24 ad / mci patients confirmed these effects of insulin ( reger et al . , 2008b ) . in a follow up study , 33 ad / mci patients and 59 control subjects received intranasal treatments of insulin or placebo . interestingly , insulin also affected plasma amyloid - beta levels in memory - impaired subjects and controls ( reger et al . , 2008a ) . following this , a randomized , double - blind , placebo - controlled clinical pilot trial was conducted . 104 patients with either mci ( n = 64 ) or mild to moderate ad ( n = 40 ) were tested . treatment with 20 iu of insulin improved memory , and both doses of insulin ( 20 and 40 iu ) preserved caregiver - rated functional ability . both insulin doses also preserved general cognition as assessed by the alzheimer 's disease assessment scale - cognitive ( adas - cog ) score for younger participants , and functional abilities as assessed by the alzheimer 's disease cooperative study activities of daily living ( adcs - adl ) scale for adults with ad . placebo - assigned participants showed decreased 18-fluoro-2-deoxyglucose with positron emission tomography ( fdg - pet ) uptake in the brain , demonstrating that neuronal metabolism was deteriorating . importantly , the improvements in episodic memory were still present two months after cessation of treatment ( craft et al . , 2012 ) . in a recent clinical trial , ad / mci patients received insulin analogue detemir ( 40 iu / d levemir ) and showed improvements in memory if they had high levels of insulin resistance at baseline ( craft , 2013 ) . this series of studies demonstrate that improving insulin signaling in the brains of ad patients has indeed positive effects on key disease parameters and biomarkers . this is a welcome change from previous experience , where encouraging effects with novel drugs in preclinical studies could not be repeated in clinical studies . the treatment of patients with insulin is unlikely to be the best strategy , as it may well enhance insulin de - sensitization if taken over longer time periods . in diabetes , the problem of progressing insulin de - sensitization by treatment with insulin is well described ( inoue et al . , 1996 ) . in order to prevent this in diabetic patients , the glp-1 treatment had been developed which uses a different receptor pathway ( lovshin and drucker , 2009 ) . the positive preclinical results of glp-1 in animal models of ad and pd motivated several clinical trials . this proof of concept study tested the effects of exendin-4 in a randomized open label trial . drug treated patients showed improvement at 12 months in motor tasks , compared with control patients . most interestingly , exendin-4 showed a clear improvement in the mattis drs-2 cognitive score , suggesting that exendin-4 has beneficial effects on cognition ( aviles - olmos et al . , 2013 ) . impressively , this effect was still visible 12 months after the clinical trial ( aviles - olmos et al . , 2014 ) . this result demonstrates the potential of these drugs , and confirms that the results from animal studies can be found in humans as well . the long lasting drug effect suggests that the effect is due to the normalization of growth factor signaling and the growth of new synapses and increased dopamine synthesis and release , rather than based on a short - term drug effect on synaptic release ( bertilsson et al . , a larger study has started which has a larger number of patients and a double - blind placebo controlled study design ( clinical trial identifier nct01971242 ) . to test the effects of these drugs in ad , a randomized , double - blind clinical trial of exendin-4 treatment in mci patients / early phase ad by the nih / nia is on the way ( clinical trial identifier nct01255163 ) . furthermore , a phase ii clinical trial of the effects of the glp-1 analogue liraglutide in mci / ad patients has started ( clinical trial identifier nct01843075 ) . in contrast to current drug treatment strategies supported by the drug industry which have not produced any protective effects in clinical trials in twenty years , the strategy of using growth factor analogues that can cross the bbb has shown first effects in humans and appears to be superior and worth investing into . currently , the drug trials of insulin and glp-1 analogues are funded by non - profit and government agencies only . the lack of funding has hampered the tests of new treatments , and there is a clear need to rethink current research strategies and a shift of research targets . the development of new growth factor analogues is currently the strategy with the greatest promise of success .

voltage - gated potassium channels of the kv1.3 type are widely expressed in many types of cells , both normal and cancer ( felipe et al . activity of kv1.3 channels plays an important role e.g. in setting the resting membrane potential , cell proliferation , apoptosis and volume regulation ( cahalan and chandy 2009 ; gulbins et al . studies performed on kv1.3 channels expressed in human t lymphocytes showed that blockage of these channels inhibited the cell proliferation in the g1 phase ( cahalan and chandy 2009 ) . specific blockers of kv1.3 channels may be applied in a selective immunosuppression ( cahalan and chandy 2009 ) . several studies demonstrated altered expression of kv1.3 channels in some cancer diseases such as breast , colon , pancreas , smooth muscle , skeletal muscle , lung , kidney and prostate cancer ( bielanska et al . inhibitors of kv1.3 channels may potentially find a clinical application in therapy of some cancer disorders characterized by an over - expression of kv1.3 channels , such as , for example , breast and lung cancer , melanoma or chronic lymphocytic leukaemia ( jang et al . for example , it was shown that inhibition of kv1.3 channels by both specific ( mgtx ) and non - specific ( tea ) inhibitors also inhibited proliferation of breast cancer ( jang et al . 2009 ) and lung cancer cells in vitro and in vivo ( jang et al . the most promising candidates for a potential clinical application may be some small molecule organic compounds , since they combine a high efficiency and specificity of cancer cell elimination with a good bioavailability and a low cytotoxity ( leanza et al . one of these compounds , clofazimine , is widely applied in medicine ( in a treatment of leprosy , for example ) since 1960s ( leanza et al . 2012 , 2013 ) . studies performed during last years in our laboratory showed that both some natural plant - derived compounds that exert antiproliferative and proapoptotic effects on cancer cells , such as an isoflavone genistein , substituted stilbene , resveratrol , and some synthetic methoxy derivatives of a substituted stilbene , piceatannol and of a flavonoid , naringenin , were inhibitors of kv1.3 channels in normal human t lymphocytes ( tl ) ( teisseyre and michalak 2005 , 2006 ; teisseyre et al . importantly , the antiproliferative effect on cancer cells and inhibition of kv1.3 channels occurred at comparable concentrations of the compounds tested ( teisseyre and michalak 2005 , 2006 ; teisseyre et al . it was concluded that the antiproliferative effect of these compounds could be , at least partially , related to the inhibition of kv1.3 channels ( teisseyre and michalak 2005 , 2006 ; teisseyre et al . our previous studies also showed that a plant - derived prenylated flavonoid 8-prenylnaringenin in contrast to its precursor naringenin was an effective inhibitor of kv1.3 channels both in normal human tl and in a human cancer t cell line jurkat ( gsiorowska et al . the estimated value of a half - blocking concentration ( ec50 ) was about 2.5 m . a complete inhibition occurred at the concentrations higher than 10 m . it was shown that 8-prenylnaringenin was the most potent inhibitor of kv1.3 channels from all plant - derived polycyclic compounds tested to date in our laboratory ( gsiorowska et al . the potency of the inhibition could be a consequence of the presence of prenyl group in the molecule of this flavonoid . if this hypothesis was correct it could be expected that other compounds from the groups of prenylated flavonoids and chalcones would also be potent inhibitors of kv1.3 channels . in this study , we tested other prenylated compounds from both groups : a chalcone : xanthohumol and a flavonoid : isoxanthohumol ( fig . 1 near here).fig . 1chemical structures of : xanthohumol ( a ) and isoxanthohumol ( b ) chemical structures of : xanthohumol ( a ) and isoxanthohumol ( b ) xanthohumol ( x ) is a structurally simple prenylated chalcone that occurs in the hop plant humulus lupulus ( 0.11 % on dry weight ) and it is used to add bitterness and flavour to beer , which is the main dietary source of xanthohumol ( stevens and page 2004 ) . isoxanthohumol , structurally related to 8-prenylnaringenin , is a product of an isomerization of xanthohumol during the brewing process and it is the main prenylflavonoid in beer ( stevens et al . both compounds have focused much attention in recent years as cancer chemopreventive agents . obtained results provide evidence that both xanthohumol and isoxanthohumol , applied at micromolar concentrations , exert antiproliferative and cytotoxic effects on human breast cancer cell line mcf-7 , colon cancer cell line ht-29 and ovarian cancer cell line a-2780 ( miranda et al . it was also shown that an incubation of xanthohumol and isoxanthohumol applied at micromolar concentrations with prostate cancer cell lines pc-3 and du145 induced a caspase - independent form of cell death ( delmulle et al . 2008 ) . moreover , both xanthohumol and isoxanthohumol exert antiangiogenic and antiinflammatory effects on human umbilical vein endothelial cells ( huvec ) and human aortic smooth muscle cells ( hasmc ) ( negrao et al . since kv1.3 channels are widely expressed in human leukemic t cell line jurkat ( attali et al . 1992 2009 ) , these cells were used in our study as a model system . obtained results provide evidence that both selected compounds effectively inhibited kv1.3 channels . the human leukemic t cell line , jurkat ( clone e6 - 1 ) , was purchased from american type culture collection ( manassas , va ) . the jurkat cells were grown in rpmi 1640 medium ( sigma - aldrich , st . louis , mo ) containing 10 % heat - inactivated fbs , 10 mm hepes and 2 mm glutamate . cells were grown on culture plates at 37 c in a 5 % co2-humidified incubator . during the experiments , cells were placed in the external solution containing , in mm : 150 nacl , 4.5 kcl , 1 cacl2 , 1 mgcl2 , 10 hepes and ph 7.35 adjusted with naoh , 300 mosm . the pipette solution contained in mm : 150 kcl , 1 cacl2 , 2 mgcl2 , 10 hepes 10 egta , and ph 7.2 was adjusted with koh , 280 mosm . the concentration of free calcium ions in the internal solution was below 100 nm , assuming the dissociation constant for egta at ph 7.2 of 10 m ( grissmer et al . 1993 ) . such a low - calcium concentration was applied to prevent the activation of calcium - activated k channels kca2.2 abundantly expressed in jurkat t cells ( grissmer et al . the chemicals were purchased from the polish chemical company ( poch , gliwice , poland ) , except of hepes and egta that were purchased from sigma . pipettes were pulled from a borosilicate glass ( hilgenberg , germany ) and fire polished before the experiment . the pipette resistance was in the range of 35 m. whole - cell potassium currents in tl were recorded applying the patch - clamp technique ( hamill et al . usa ) , low - pass filtered at 3 khz , digitised using digidata 1440 ( molecular devices corp . , usa ) analogue - to - digital converter with the sampling rate of 10 khz . the influence of selected compounds on the activity of the channels was studied by applying the voltage ramp protocol . voltage ramps depolarising cell membranes from 100 mv up to + 40 mv were applied every 20 s ; the ramp duration was 340 ms and holding potential 90 mv . upon application of the voltage ramp protocol , potassium currents in jurkat t cells were stably recorded for at least 20 min after break - in to the whole - cell configuration . during the off - line analysis , the value of kv1.3 current at the end of a voltage ramp ( + 40 mv ) was calculated . for this purpose , the leak current estimated at + 40 mv was subtracted from the total ramp current recorded at this voltage . the calculated voltage - clamp error , due to an uncompensated access resistance , was 4.0 0.5 mv ( the inhibition of the kv1.3 channels is presented in terms of a relative current recorded upon application of the studied compounds , defined as i / icontr ; where i is the kv1.3 current upon an application of an examined compound at + 40 mv , icontr is the kv1.3 current recorded on the same cell at + 40 mv under control conditions . for calculating the value of a half - blocking concentration ( ec50 ) and the hill s coefficient , the magnitude of the inhibition defined as 1 ( i / icontr ) was taken in consideration . both parameters were calculated applying the hill s equation in a form : y = a + ( b a)/(1 + ( x / c ) ) , where y is magnitude of the channels inhibition , a is the minimal value of the inhibitory effect , b is the maximal value of the inhibitory effect , x is the logarithm of the compound s concentration , c is the logarithm of the ec50 value , d is the hill s coefficient . the human leukemic t cell line , jurkat ( clone e6 - 1 ) , was purchased from american type culture collection ( manassas , va ) . the jurkat cells were grown in rpmi 1640 medium ( sigma - aldrich , st . louis , mo ) containing 10 % heat - inactivated fbs , 10 mm hepes and 2 mm glutamate . cells were grown on culture plates at 37 c in a 5 % co2-humidified incubator . during the experiments , cells were placed in the external solution containing , in mm : 150 nacl , 4.5 kcl , 1 cacl2 , 1 mgcl2 , 10 hepes and ph 7.35 adjusted with naoh , 300 mosm . the pipette solution contained in mm : 150 kcl , 1 cacl2 , 2 mgcl2 , 10 hepes 10 egta , and ph 7.2 was adjusted with koh , 280 mosm . the concentration of free calcium ions in the internal solution was below 100 nm , assuming the dissociation constant for egta at ph 7.2 of 10 m ( grissmer et al . 1993 ) . such a low - calcium concentration was applied to prevent the activation of calcium - activated k channels kca2.2 abundantly expressed in jurkat t cells ( grissmer et al . the chemicals were purchased from the polish chemical company ( poch , gliwice , poland ) , except of hepes and egta that were purchased from sigma . dishes with cells were placed under an inverted olympus imt-2 microscope . solutions containing tested compounds pipettes were pulled from a borosilicate glass ( hilgenberg , germany ) and fire polished before the experiment . the pipette resistance was in the range of 35 m. whole - cell potassium currents in tl were recorded applying the patch - clamp technique ( hamill et al . usa ) , low - pass filtered at 3 khz , digitised using digidata 1440 ( molecular devices corp . , usa ) analogue - to - digital converter with the sampling rate of 10 khz . the influence of selected compounds on the activity of the channels was studied by applying the voltage ramp protocol . voltage ramps depolarising cell membranes from 100 mv up to + 40 mv were applied every 20 s ; the ramp duration was 340 ms and holding potential 90 mv . upon application of the voltage ramp protocol , potassium currents in jurkat t cells were stably recorded for at least 20 min after break - in to the whole - cell configuration . during the off - line analysis , the value of kv1.3 current at the end of a voltage ramp ( + 40 mv ) was calculated . for this purpose , the leak current estimated at + 40 mv was subtracted from the total ramp current recorded at this voltage . the calculated voltage - clamp error , due to an uncompensated access resistance , was 4.0 0.5 mv ( n = 30 ) . the inhibition of the kv1.3 channels is presented in terms of a relative current recorded upon application of the studied compounds , defined as i / icontr ; where i is the kv1.3 current upon an application of an examined compound at + 40 mv , icontr is the kv1.3 current recorded on the same cell at + 40 mv under control conditions . for calculating the value of a half - blocking concentration ( ec50 ) and the hill s coefficient , the magnitude of the inhibition defined as 1 ( i / icontr ) was taken in consideration . both parameters were calculated applying the hill s equation in a form : y = a + ( b a)/(1 + ( x / c ) ) , where y is magnitude of the channels inhibition , a is the minimal value of the inhibitory effect , b is the maximal value of the inhibitory effect , x is the logarithm of the compound s concentration , c is the logarithm of the ec50 value , d is the hill s coefficient . examples of the whole - cell currents recorded in jurkat t cells under control conditions and in the presence of xanthohumol and isoxanthohumol at concentrations of 15 and 10 m , respectively , are presented in fig . 2 ( near here).fig . 2both examined compounds reduced the intensity of the whole - cell potassium currents recorded in jurkat t cells applying the voltage ramp protocol ( shown schematically above the current records ) : ( a ) control conditions , ( b ) application of 15 m of xanthohumol ( left panel ) and 10 m of isoxanthohumol ( right panel ) and ( c ) wash - out of the examined compounds both examined compounds reduced the intensity of the whole - cell potassium currents recorded in jurkat t cells applying the voltage ramp protocol ( shown schematically above the current records ) : ( a ) control conditions , ( b ) application of 15 m of xanthohumol ( left panel ) and 10 m of isoxanthohumol ( right panel ) and ( c ) wash - out of the examined compounds this figure depicts the raw currents ( without leak subtraction ) that were recorded applying the voltage ramp protocol . the evoked currents contained two components : small linear and much bigger non - linear . the linear current was the unspecific leak current ( reversal potential equal to 0 mv ) , whereas the non - linear component was due to activation of kv1.3 channels ( grissmer et al . 1992 ; teisseyre and mozrzymas 2002 ) . apparently , application of xanthohumol and isoxanthohumol significantly reduced the amplitude of kv1.3 current . upon application of xanthohumol at concentrations of 1 , 3 , 5 , 7 , 10 , 15 , 20 and 30 m , the relative currents ( defined in materials and methods section ) were reduced to 0.88 0.028 ( n = 5 ) , 0.57 0.076 ( n = 5 ) , 0.31 0.08 ( n = 5 ) , 0.29 0.017 ( n = 5 ) , 0.21 0.022 ( n = 5 ) , 0.20 0.05 ( n = 5 ) , 0.19 0.03 ( n = 5 ) and 0.14 0.07 ( n = 5 ) , respectively . the decrease of the relative current was statistically significant ( p < 0.05 , student s t test ) in case of all applied concentrations . in the case of isoxanthohumol applied at the same concentrations , the relative currents were reduced to 0.99 0.068 ( n = 5 ) , 0.96 0.077 ( n = 5 ) , 0.75 0.028 ( n = 5 ) , 0.55 0.047 ( n = 5 ) , 0.45 0.059 ( n = 5 ) , 0.36 0.071 ( n = 5 ) , 0.25 0.091 ( n = 5 ) and 0.13 0.046 ( n = 5 ) , respectively . the decrease of the relative current was statistically significant ( p < 0.05 , student s t test ) for concentrations equal or higher than 5 m . the inhibitory effects of xanthohumol and isoxanthohumol on kv1.3 currents in human jurkat t cells were not complete at the highest applied concentration ( 30 m ) . in order to characterize the inhibitory effects of the compounds on kv1.3 currents in more detail , the values of a half - blocking concentration ( ec50 ) and the hill s coefficient were calculated , applying the hill s equation . the estimated ec50 values were about 3.06 and 7.81 m for xanthohumol and isoxanthohumol , respectively . the difference in ec50 values between xanthohumol and isoxanthohumol was significant ( p < 0.05 , student s t test ) . this suggests that xanthohumol is much more potent inhibitor of kv1.3 currents in human jurkat t cells than its isomer isoxanthohumol . the estimated values of the hill s coefficient were 2.43 0.54 and 2.09 0.45 for xanthohumol and isoxanthohumol , respectively . these values were not significantly different from each other ( p > 0.05 , student s t test ) . figure 3 ( near here ) depicts the comparison of a magnitude of the channels inhibition by xanthohumol and isoxanthohumol as a function of a logarithm of concentrations ( in m).fig . 3comparison of a magnitude of the inhibition by xanthohumol ( filled squares ) and isoxanthohumol ( empty squares ) plotted as a function of logarithm of concentrations of both compounds ( in m ) . data points were fitted by the hill s equation comparison of a magnitude of the inhibition by xanthohumol ( filled squares ) and isoxanthohumol ( empty squares ) plotted as a function of logarithm of concentrations of both compounds ( in m ) . data points were fitted by the hill s equation our previous results showed that the inhibitory effect of 8-prenylnaringenin was accompanied by a significant increase in the inactivation rate of kv1.3 currents ( gsiorowska et al . 2012 ) . in order to prove whether the inactivation and activation rate were changed upon an application of xanthohumol and isoxantohumol , another study applying a protocol with depolarizing voltage steps described in detail in our previous article ( gsiorowska et al . in contrast to what had been observed earlier for 8-prenylnaringenin , an application of xanthohumol and isoxanthohumol did not significantly change the inactivation kinetics of the currents ( not shown ) . the activation kinetics remained unchanged upon application of the tested compounds ( not shown ) , similarly to what had been observed earlier for 8-prenylnaringenin ( gsiorowska results of our study provide evidence that both selected compounds from the groups of prenylated chalcones and flavonoids : xanthohumol and isoxanthohumol inhibit the activity of kv1.3 channels in human leukemic jurkat t cells . the inhibitory effects of xanthohumol and isoxanthohumol on kv1.3 channels were significantly different than the effect observed earlier for 8-prenylnaringenin ( gsiorowska et al . first , in contrast to what was observed for 8-prenylnaringenin , the inhibitory effects of xanthohumol and isoxanthohumol were not complete at concentrations higher than 10 m . moreover , the inhibitory effects of these compounds were not accompanied by an increase in the currents inactivation rate . these results suggest that xanthohumol and isoxanthohumol are less potent inhibitors of kv1.3 channels in human jurkat t cells than 8-prenylnarigenin . also the mechanism of the channels inhibition by these compounds is probably different . on the other hand , the inhibitory effects of xanthohumol and isoxanthohumol on kv1.3 channels were much more potent than the effects caused by other natural plant - derived compounds , which exert an antiproliferative effect on cancer cells , such as genistein or resveratrol ( teisseyre and michalak 2005 , 2006 ) . results of our previous studies provide evidence that the ec50 values were more than 10 m for genistein and about 40 m in the case of resveratrol ( teisseyre and michalak 2005 , 2006 ) . these values were significantly higher than the ec50 values estimated in this study for xanthohumol and isoxanthohumol . on the other hand , it must be taken in consideration that studies on the influence of genistein and resveratrol were not performed on jurkat t cells , but on kv1.3 channels in normal human tl ( teisseyre and michalak 2005 , 2006 ) . however , preliminary studies on kv1.3 channels in jurkat t cells demonstrated that the channels were also inhibited by resveratrol , however , the magnitude of the effect was smaller than in the case of the channels in normal human tl ( gsiorowska unpublished results ) . therefore , the observed differences in the inhibition potency of xanthohumol , isoxanthohumol , genistein and resveratrol were not due to application of different model systems . they were probably due to an involvement of different mechanisms of kv1.3 channel inhibition by compounds examined in this study , genistein and resveratrol . our earlier experiments provide evidence that a flavonoid naringenin , a precursor compound for 8-prenylnaringenin , did not inhibit kv1.3 channels in normal human tl when applied at 30 m concentration ( teisseyre et al . studies performed recently on the channels expressed in cancer cells human jurkat t cell line provide evidence that naringenin was not an inhibitor of kv1.3 channels in these cells when applied at concentrations up to 30 m ( gsiorowska unpublished results ) . the same study showed that two synthetic derivatives of naringenin : 4,7-dimethylether and 7-methylether inhibited kv1.3 channels in jurkat t cells in a concentration - dependent manner ( gsiorowska unpublished results ) . the estimated ec50 values for the inhibition were about 13 m and about 16 m for naringenin-4,7-dimethylether and naringenin-7-methylether , respectively ( gsiorowska unpublished results ) . thus , similarly to what was observed in the case of genistein and resveratrol , the ec50 values estimated for both methoxy derivatives of naringenin were significantly higher than those obtained for xanthohumol and isoxanthohumol . altogether , obtained results may indicate that prenylated flavonoids and chalcones , such as 8-prenylnaringenin , xanthohumol and isoxanthohumol , are more potent inhibitors of kv1.3 channels than non - prenylated compounds from both groups . these results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of kv1.3 channels by compounds from the groups of flavonoids and chalcones . in accordance to this hypothesis , recently performed studies with isobavachalcone a prenylated plant - derived chalcone with the chemical structure similar to xanthohumol showed that this compound also inhibited kv1.3 channels in jurkat t cells in the concentration - dependent manner . the relative current was reduced to about 0.34 at the concentration of 15 m ( gsiorowska unpublished results ) . the estimated ec50 value for isobavachalcone was about 5 m ( gsiorowska unpublished results ) . our results showed that the ec50 values for kv1.3 channel inhibition by xanthohumol and isoxanthohumol were markedly different from each other . this may be a consequence of differences both in a chemical structure and in geometry of molecules of these compounds ( fig . 1 ) . interestingly , a very significant difference in the inhibition potency occurred between isoxanthohumol and 8-prenylnaringenin ( gsiorowska et al . 2012 ) , despite a very small difference in a chemical structure replacement of a methoxy group in isoxanthohumol by a hydroxyl group in 8-prenylnaringenin . the inhibition of kv1.3 channels by prenylated chalcones and flavonoids is probably a consequence of specific interactions of these compounds with the channels proteins . it is known that inhibition of kv1.3 channels may also inhibit proliferation of some cell types , including breast , colon and prostate cancer cells ( comes et al . it is also known that xanthohumol and isoxanthohumol inhibit cell proliferation in the case of human breast cancer cell line mcf-7 , human colon cancer cell line ht-29 and ovarian cancer cell line a-2780 when applied in vitro . the ec50 values for the inhibition of proliferation of mcf-7 cells after 4 days of incubation were 3.47 and 4.69 m for xanthohumol and isoxanthohumol , respectively ( miranda et al . 1999 ) . these values are close to the ec50 values for the inhibition of kv1.3 channels in jurkat t cells . it is known that kv1.3 channels are expressed in non - lymphocyte cancer cell lines , such as human breast cancer cell line mcf-7 ( gulbins et al . a possible contribution of the inhibition of kv1.3 channels to antiproliferative effects of xanthohumol and isoxanthohumol might explain the observed phenomenon that mcf-7 cells were more sensitive to the flavonoids than ht-29 cells ( miranda et al . 1999 ) . it remains to be elucidated whether inhibition of kv1.3 channels is involved in antiproliferative effects of the examined compounds in cancer cell lines . the inhibition of kv1.3 channels by xanthohumol and isoxanthohumol might also be involved in proapoptotic activities of these compounds . studies performed in the last years showed that kv1.3 channels are expressed not only in plasma membrane but also in the inner mitochondrial membrane in normal human tl and in jurkat t cells ( gulbins et al . it is known that inhibition of mitochondrial kv1.3 channels in human t cells by a proapoptotic protein bax is the first crucial event in the process of an activation of the mitochondrial pathway of apoptosis ( szabo et al . in other studies , it was shown that inhibition of mitochondrial kv1.3 channels in jurkat t cells by membrane - permeant channel inhibitors , such as clofazimine , psora-4 and pap-1 mimicked the effects of bax and triggered the apoptotic cell death by activation of the mitochondrial pathway ( leanza et al . recently , obtained data showed that kv1.3 channels are expressed in inner mitochondrial membrane also in non - lymphocyte cancer cells , such as human prostate cancer cell line pc-3 or breast cancer cell line mcf-7 ( gulbins et al . it is possible that the examined compounds are able to diffuse across the plasma membrane and reach intracellular compartments , so they may inhibit mitochondrial kv1.3 channels . 2008 showed that an application of xanthohumol and isoxanthohumol at micromolar concentrations induced cell death in human prostate cancer pc-3 and du145 cell lines . however , it was shown that in this case , the cell death occurred in a caspase - independent manner , without typical apoptotic morphological features , probably as a result of autophagy ( delmulle et al . whether mitochondrial kv1.3 channels are as sensitive to the inhibition by the compounds as the channels in the plasma membrane and whether this blocking effect is involved in induction of the mitochondrial pathway of cancer cell apoptosis remains to be elucidated .

hepatocellular carcinoma ( hcc ) is the fifth - most prevalent malignancy worldwide and the second leading cause of cancer - related deaths . more than 700,000 newly diagnosed cases occur every year ; unfortunately , about 7580% of these cases occur in the asian pacific region , and especially in china . hcc is one of the most difficult to treat malignances . surgical resection and liver transplantation . however , fewer than 30% of all patients are eligible candidates due to poor liver function or advanced disease at the time of diagnosis . to achieve the most from available therapies , the most promising strategy is to diagnose and treat high - risk populations early on since hcc might be detected in an early stage and result in long - term survival . the most prevalent surveillance protocol in china has been ultrasonography ( us ) and serum alpha - fetoprotein ( afp ) . serum afp has been widely used as a tumor biomarker since its advent in 1950s in china . an elevated serum afp is known to be associated with hcc , but not always . according to reported data , 5060% of all patients test positive for afp . afp may also be elevated in certain patients with chronic hepatitis or cirrhosis , , thus resulting in afp s poor sensitivity and specificity at diagnosing and postoperatively surveilling hcc . an ideal alternative or complement to afp as a sensitive biomarker is crucial to detecting hcc . des--carboxyprothrombin ( dcp ) is a well - established biomarker for hcc in japan and south korea [ 1115 ] . meta analysis showed that dcp shows more diagnostic accuracy than afp , especially in diagnosing early stage hcc . during the past decade , some studies have examined the clinical usefulness of dcp in chinese patients with hcc , but they have not sufficiently demonstrated its usefulness due to limited samples and controls . theoretically , the clinical significance of dcp might vary considerably for patients with hcc due to different etiologies in different geographic regions . although hepatitis c is the underlying cause of hcc in japan and western countries , hepatitis b causes up to 85% of hcc in china . in order to further investigate the clinical utility of dcp in detecting hcc in chinese patients , the current study was conducted with a large cohort of patients with hcc and different controls . a total of 700 patients who were diagnosed with a hepatopancreatobiliary disease and who had undergone surgery were consecutively enrolled from the institute of hepatobiliary surgery , southwest hospital , third military medical university between september 2008 and september 2011 . in addition , 153 healthy volunteers were also included in the study . all subjects and volunteers subjects receiving surgery were divided into two groups : ( i ) subjects with hcc ( n=329 ) ; and ( ii ) subjects without hcc , with either benign or malignant hepatopancreatobiliary disease ( n=371 ) . hcc was clinically diagnosed based on international guidelines and pathologic findings from resected specimens were confirmed for all 329 subjects . no subjects had received any previous therapy to treat hcc such as tace , rfa , pei , or resection and they underwent surgery for the first time at this hospital . hbv infection was diagnosed based on a positive result for hepatitis b surface antigen ( hbsag ) , and hcv infection was confirmed based on a positive result for anti - hcv and hcv rna . to examine the usefulness of dcp in diagnosing hcc , a cohort with a hepatopancreatobiliary disease other than hcc based on enhanced imaging findings that were undergoing surgery at this hospital all of these subjects had been diagnosed by clinical findings as well as pathologic findings . all of the subjects with or without hcc were treated depending on their condition and stage of their disease . to ensure that healthy volunteers did not have hcc , these subjects had to have no masses according to ultrasound or ct / mri imaging and a total afp below the reference level of 10 ng / ml . . a normal liver function test and a test for hepatitis infection were also performed . a 10-ml sample of peripheral blood was obtained before surgery or at the time of enrollment and immediately centrifuged into serum and plasma and then stored in aliquots in a freezer at 80c . serum afp was examined using a commercially available immunometric assay ( st aia - pack afp , tosoh , tokyo , japan ) with enhanced chemiluminescence at the southwest hospital clinical diagnostic center . plasma dcp levels were detected using an electrochemiluminescence immunoassay ( ed036 , eisai co. , tokyo , japan ) in accordance with the manufacturer s instructions and manual . the optimal cut - off value for dcp to distinguish between normal healthy subjects and patients with hcc was previously determined to be 40 mau / ml . a p value < 0.05 in a 2-tailed test was deemed to indicate statistical significance for all calculations . data are presented as mean standard deviation and/or median ( range ) as appropriate . a wilcoxon rank - sum test and chi - square test were used for analysis of continuous and categorical data , respectively . receiver - operating characteristic ( roc ) curves were created for afp , dcp , and a combination of afp and dcp to determine the optimal cut - off value for differentiating subjects with hcc , subjects without hcc , and healthy controls . the area under the roc curves ( auroc ) with the positive ( + lr ) and negative ( lr ) likelihood ratio was examined for the aforementioned biomarkers . box plots were created and analysis of variance was used to compare descriptive statistics for the transformed tumor markers . all statistical calculations were performed using spss version 22.0 for windows ( spss , chicago , illinois , usa ) . a total of 700 patients who were diagnosed with a hepatopancreatobiliary disease and who had undergone surgery were consecutively enrolled from the institute of hepatobiliary surgery , southwest hospital , third military medical university between september 2008 and september 2011 . in addition , 153 healthy volunteers were also included in the study . all subjects and volunteers subjects receiving surgery were divided into two groups : ( i ) subjects with hcc ( n=329 ) ; and ( ii ) subjects without hcc , with either benign or malignant hepatopancreatobiliary disease ( n=371 ) . hcc was clinically diagnosed based on international guidelines and pathologic findings from resected specimens were confirmed for all 329 subjects . no subjects had received any previous therapy to treat hcc such as tace , rfa , pei , or resection and they underwent surgery for the first time at this hospital . hbv infection was diagnosed based on a positive result for hepatitis b surface antigen ( hbsag ) , and hcv infection was confirmed based on a positive result for anti - hcv and hcv rna . to examine the usefulness of dcp in diagnosing hcc , a cohort with a hepatopancreatobiliary disease other than hcc based on enhanced imaging findings that were undergoing surgery at this hospital all of these subjects had been diagnosed by clinical findings as well as pathologic findings . all of the subjects with or without hcc were treated depending on their condition and stage of their disease . to ensure that healthy volunteers did not have hcc , these subjects had to have no masses according to ultrasound or ct / mri imaging and a total afp below the reference level of 10 ng / ml . . a normal liver function test and a test for hepatitis infection were also performed . a 10-ml sample of peripheral blood was obtained before surgery or at the time of enrollment and immediately centrifuged into serum and plasma and then stored in aliquots in a freezer at 80c . serum afp was examined using a commercially available immunometric assay ( st aia - pack afp , tosoh , tokyo , japan ) with enhanced chemiluminescence at the southwest hospital clinical diagnostic center . plasma dcp levels were detected using an electrochemiluminescence immunoassay ( ed036 , eisai co. , tokyo , japan ) in accordance with the manufacturer s instructions and manual . the optimal cut - off value for dcp to distinguish between normal healthy subjects and patients with hcc was previously determined to be 40 mau / ml . a p value < 0.05 in a 2-tailed test was deemed to indicate statistical significance for all calculations . data are presented as mean standard deviation and/or median ( range ) as appropriate . a wilcoxon rank - sum test and chi - square test were used for analysis of continuous and categorical data , respectively . receiver - operating characteristic ( roc ) curves were created for afp , dcp , and a combination of afp and dcp to determine the optimal cut - off value for differentiating subjects with hcc , subjects without hcc , and healthy controls . the area under the roc curves ( auroc ) with the positive ( + lr ) and negative ( lr ) likelihood ratio was examined for the aforementioned biomarkers . box plots were created and analysis of variance was used to compare descriptive statistics for the transformed tumor markers . all statistical calculations were performed using spss version 22.0 for windows ( spss , chicago , illinois , usa ) . the 329 subjects with hcc had a mean age of 47.010.9 years and consisted of 293 males ( 89.1% ) and 36 females ( 10.9% ) . of the subjects , 258 ( 78.4% ) tested positive for hbsag and 3 ( 0.9% ) tested positive for anti - hcv while 68 ( 20.7% ) were negative for both ( table 1 ) . the median size of the tumor was 6.0 cm with a range from 1.0 to 19.0 cm . a tumor was found in the left lobe in 79 subjects ( 24.0% ) , in the right lobe in 229 ( 69.6% ) , in both lobes in 15 ( 4.6% ) , and in the caudate lobe in 6 ( 1.8% ) . all of the 329 subjects underwent surgery which was specifically described in supplementary table 1 . this study enrolled 371 subjects without hcc who were diagnosed with a benign or malignant hepatopancreatobiliary disorder . of these subjects , 90 tested positive for hbsag and 6 tested positive for anti - hcv . one subject was excluded due to missing data , and the remaining subjects were all negative for hbsag , anti - hbc and anti - hcv . the subject s diagnosis was based on pathologic findings and roughly classified into one of four categories : a common liver disease ( 33 subjects , 8.9% ) , a rare liver disease ( 13 subjects , 3.5% ) , pancreatic disease ( 79 subjects , 21.3% ) , or biliary disease ( 246 subjects , 66.3% ) ( supplementary table 2 ) . all healthy volunteers were blank controls ( mean age : 32.705.24 years ; m / f ratio=128/25 ) with a normal biochemistry and were negative for hbsag , anti - hbc , and anti - hcv . the median plasma concentration of dcp was 853.72 mau / ml in subjects with hcc , 26.43 mau / ml in subjects without hcc , and 29.91 mau / ml in healthy volunteers . there was a marked difference in the level of dcp for subjects with hcc and the other two groups ( p<0.001 ) ( table 1 , figure 1a ) . median serum levels of afp were 375.86 ng / ml in subjects with hcc , 3.00 ng / ml in subjects without hcc , and 7.00 ng / ml in healthy volunteers . a significantly higher level of afp was seen in subjects with hcc than in the other two groups ( table 1 , figure 1b ) . the correlation between the levels of dcp and afp in the 700 subjects and 153 healthy controls is shown in figure 2 . no association was seen in this cohort ( r=0.20 ) . in order to determine optimal cut - off values that could balance the false - positive and the false - negative rates with the best positive predictive value and that could best distinguish subjects with hcc from the other two groups , a roc curve was created for dcp and for afp . as shown in table 2 , when the cut - off value was set at 40 mau / ml , the sensitivity was 83.00% , the specificity was 62.30% , and the youden index ( % ) was 45.30% for subjects with hcc vs. subjects without hcc . in contrast , the sensitivity was 83.00% , the specificity was 66.00% , and the youden index ( % ) was 49.00% for subjects with hcc vs. normal subjects . interestingly , a cut - off dcp value of 87 mau / ml yielded the optimal sensitivity of 74.80% and a specificity of 83.33% with a youden index of 58.10% for differentiating subjects with hcc from subjects without hcc . the sensitivity was 74.80% , the specificity was 96.10% , and the youden index was 70.90% for differentiating subjects with hcc from normal subjects . this indicated that a cut - off dcp level of 87 mau / ml , and not 40 mau / ml , should be used to diagnose hcc in the current subjects undergoing surgery ( table 2 ) . the optimal cut - off values for afp with different serum levels were also examined . results showed that an afp level of 21.33 ng / ml should be used to diagnose hcc . this yielded a sensitivity of 70.60% , a specificity of 93.80% , and a youden index of 64.40% for differentiating subjects with hcc from subjects without hcc in contrast to a sensitivity of 70.60% , a specificity of 94.80% , and a youden index of 65.40% for differentiating subjects with hcc from normal subjects ( table 2 ) . auroc was used to evaluate the combination of the markers afp and dcp at their optimal cut - off values to gauge any potential diagnostic advantage . the combination of afp of 21.33 ng / ml and dcp of 87 mau / ml had a higher sensitivity of 88.90% and a youden index of 69.40% for subjects with hcc vs. subjects without hcc , as well as a higher sensitivity of 88.90% and a youden index of 81.10% for subjects with hcc vs. normal subjects . there was a slight decrease in the specificity of this combination , but this was acceptable given the commensurate improvement of at least 14.1% in sensitivity compared to dcp or afp alone ( table 2 ) . the roc curves comparing dcp and afp levels in subjects with hcc versus subjects without hcc indicated that the area under the roc curve was 0.846 for dcp , 0.832 for afp , and 0.890 for the combination of dcp and afp ( figure 3 ) . the 329 subjects with hcc were categorized according to tumor size and the sensitivity and specificity of dcp and afp were then estimated using roc analysis . in accordance with clinical manifestations and the population studied , tumors were classified into six sizes ranging from 2.0 cm , > 2.0 cm and 3.0 cm , > 3.0 cm and 4.0 cm , > 4.0 cm and 5.0 cm , > 5.0 cm and 10.0 cm , and > 10.0 cm . the median value of plasma dcp for 329 subjects with hcc of differing sizes is shown in table 3 . plasma dcp was 46.86 mau / ml in subjects with a tumor 2.0 cm , 98.22 mau / ml in subjects with a tumor > 2.0 cm and 3.0 cm , 332.22 mau / ml in subjects with a tumor > 3.0 cm and 4.0 cm , 359.93 mau / ml in subjects with a tumor > 4.0 cm and 5.0 cm , 1766.53 mau / ml in subjects with a tumor > 5.0 cm and 10.0 cm , and 4162.12 mau / ml in subjects with a tumor > 10.0 cm . there was an obvious incremental increase in dcp values as the tumor size increased , but the same was not true for afp ( table 3 ) . the sensitivity and specificity of dcp of 87 mau / ml , afp of 21.33 ng / ml , and a combination of both were examined in different tumor sizes . when the tumor was smaller than or equal to 3.0 cm , dcp had a relatively lower sensitivity than afp . as the tumor size increased , the sensitivity of dcp also increased markedly and was much higher than afp in subjects with hcc , as shown in table 4 . the sensitivity of the combination of the two tumor markers was higher than that of afp or dcp alone for the entire range of tumor sizes . the sensitivity of the combined markers was 82.60% for tumors smaller than or equal to 2 cm in size . larger than 5 cm , and this was significantly higher than that of dcp or afp alone ( table 4 ) . the 329 subjects with hcc had a mean age of 47.010.9 years and consisted of 293 males ( 89.1% ) and 36 females ( 10.9% ) . of the subjects , 258 ( 78.4% ) tested positive for hbsag and 3 ( 0.9% ) tested positive for anti - hcv while 68 ( 20.7% ) were negative for both ( table 1 ) . the median size of the tumor was 6.0 cm with a range from 1.0 to 19.0 cm . a tumor was found in the left lobe in 79 subjects ( 24.0% ) , in the right lobe in 229 ( 69.6% ) , in both lobes in 15 ( 4.6% ) , and in the caudate lobe in 6 ( 1.8% ) . all of the 329 subjects underwent surgery which was specifically described in supplementary table 1 . this study enrolled 371 subjects without hcc who were diagnosed with a benign or malignant hepatopancreatobiliary disorder . of these subjects , 90 tested positive for hbsag and 6 tested positive for anti - hcv . one subject was excluded due to missing data , and the remaining subjects were all negative for hbsag , anti - hbc and anti - hcv . the subject s diagnosis was based on pathologic findings and roughly classified into one of four categories : a common liver disease ( 33 subjects , 8.9% ) , a rare liver disease ( 13 subjects , 3.5% ) , pancreatic disease ( 79 subjects , 21.3% ) , or biliary disease ( 246 subjects , 66.3% ) ( supplementary table 2 ) . all healthy volunteers were blank controls ( mean age : 32.705.24 years ; m / f ratio=128/25 ) with a normal biochemistry and were negative for hbsag , anti - hbc , and anti - hcv . the median plasma concentration of dcp was 853.72 mau / ml in subjects with hcc , 26.43 mau / ml in subjects without hcc , and 29.91 mau / ml in healthy volunteers . there was a marked difference in the level of dcp for subjects with hcc and the other two groups ( p<0.001 ) ( table 1 , figure 1a ) . median serum levels of afp were 375.86 ng / ml in subjects with hcc , 3.00 ng / ml in subjects without hcc , and 7.00 ng / ml in healthy volunteers . a significantly higher level of afp was seen in subjects with hcc than in the other two groups ( table 1 , figure 1b ) . the correlation between the levels of dcp and afp in the 700 subjects and 153 healthy controls is shown in figure 2 . no association was seen in this cohort ( r=0.20 ) . in order to determine optimal cut - off values that could balance the false - positive and the false - negative rates with the best positive predictive value and that could best distinguish subjects with hcc from the other two groups , a roc curve was created for dcp and for afp . as shown in table 2 , when the cut - off value was set at 40 mau / ml , the sensitivity was 83.00% , the specificity was 62.30% , and the youden index ( % ) was 45.30% for subjects with hcc vs. subjects without hcc . in contrast , the sensitivity was 83.00% , the specificity was 66.00% , and the youden index ( % ) was 49.00% for subjects with hcc vs. normal subjects . interestingly , a cut - off dcp value of 87 mau / ml yielded the optimal sensitivity of 74.80% and a specificity of 83.33% with a youden index of 58.10% for differentiating subjects with hcc from subjects without hcc . the sensitivity was 74.80% , the specificity was 96.10% , and the youden index was 70.90% for differentiating subjects with hcc from normal subjects . this indicated that a cut - off dcp level of 87 mau / ml , and not 40 mau / ml , should be used to diagnose hcc in the current subjects undergoing surgery ( table 2 ) . the optimal cut - off results showed that an afp level of 21.33 ng / ml should be used to diagnose hcc . this yielded a sensitivity of 70.60% , a specificity of 93.80% , and a youden index of 64.40% for differentiating subjects with hcc from subjects without hcc in contrast to a sensitivity of 70.60% , a specificity of 94.80% , and a youden index of 65.40% for differentiating subjects with hcc from normal subjects ( table 2 ) . auroc was used to evaluate the combination of the markers afp and dcp at their optimal cut - off values to gauge any potential diagnostic advantage . the combination of afp of 21.33 ng / ml and dcp of 87 mau / ml had a higher sensitivity of 88.90% and a youden index of 69.40% for subjects with hcc vs. subjects without hcc , as well as a higher sensitivity of 88.90% and a youden index of 81.10% for subjects with hcc vs. normal subjects . there was a slight decrease in the specificity of this combination , but this was acceptable given the commensurate improvement of at least 14.1% in sensitivity compared to dcp or afp alone ( table 2 ) . the roc curves comparing dcp and afp levels in subjects with hcc versus subjects without hcc indicated that the area under the roc curve was 0.846 for dcp , 0.832 for afp , and 0.890 for the combination of dcp and afp ( figure 3 ) . the 329 subjects with hcc were categorized according to tumor size and the sensitivity and specificity of dcp and afp were then estimated using roc analysis . in accordance with clinical manifestations and the population studied , tumors were classified into six sizes ranging from 2.0 cm , > 2.0 cm and 3.0 cm , > 3.0 cm and 4.0 cm , > 4.0 cm and 5.0 cm , > 5.0 cm and 10.0 cm , and > 10.0 cm . the median value of plasma dcp for 329 subjects with hcc of differing sizes is shown in table 3 . plasma dcp was 46.86 mau / ml in subjects with a tumor 2.0 cm , 98.22 mau / ml in subjects with a tumor > 2.0 cm and 3.0 cm , 332.22 mau / ml in subjects with a tumor > 3.0 cm and 4.0 cm , 359.93 mau / ml in subjects with a tumor > 4.0 cm and 5.0 cm , 1766.53 mau / ml in subjects with a tumor > 5.0 cm and 10.0 cm , and 4162.12 mau / ml in subjects with a tumor > 10.0 cm . there was an obvious incremental increase in dcp values as the tumor size increased , but the same was not true for afp ( table 3 ) . the sensitivity and specificity of dcp of 87 mau / ml , afp of 21.33 ng / ml , and a combination of both were examined in different tumor sizes . when the tumor was smaller than or equal to 3.0 cm , dcp had a relatively lower sensitivity than afp . as the tumor size increased , the sensitivity of dcp also increased markedly and was much higher than afp in subjects with hcc , as shown in table 4 . the sensitivity of the combination of the two tumor markers was higher than that of afp or dcp alone for the entire range of tumor sizes . the sensitivity of the combined markers was 82.60% for tumors smaller than or equal to 2 cm in size . larger than 5 cm , and this was significantly higher than that of dcp or afp alone ( table 4 ) . this study involved a cross - sectional study of plasma dcp and serum afp in subjects undergoing surgery for hcc predominantly caused by hbv . results of this study indicated that dcp has a relatively higher sensitivity in detecting tumors larger than 3.0 cm . results also indicated that dcp is a good candidate as a compliment to afp in the diagnosis of hcc , regardless of tumor size . the combination of these two serum biomarkers could result in an optimal diagnostic accuracy for the chinese population with hepatopancreatobiliary diseases . its usefulness in diagnosing hcc at a reference level of 40 m au / ml has been established by a number of retrospective and prospective studies . however , most of these studies were completed in japan or western counties , where hcc is predominantly caused by hcv infection [ 2426 ] . dcp levels may vary widely between forms of underlying liver disease , including ald , nald , and viral hepatitis . moreover , hcc occurring in china accounts for at least half of the new cases worldwide each year . hence , there is an urgent demand for optimal serum biomarkers to help improve the early diagnosis of hcc . unfortunately , few studies have focused on the chinese population , and these studies have also involved a small sample size and limited number of controls . the current study included a consecutive cohort of 329 subjects with hcc who were undergoing curative surgery . this might be the largest sample size for a single - center study of hcc in china and the first report on all patients undergoing surgery . the subjects in the current study underwent different surgical procedures , including hepatectomy ( 317/329 ) and a liver transplant ( 12/329 ) . based on the inclusion criterion for surgery , this cohort should be classified as a cohort with early hcc , although the subjects did have a large tumor and they fell outside the milan criteria for liver transplantation . roc analysis indicated that dcp with a cut - off value of 40 m au / ml had a sensitivity of 83.00% and a specificity of 62.30% in differentiating subjects with hcc from subjects without hcc and a sensitivity of 83.00% and sensitivity of 66.00% in differentiating subjects with hcc from normal healthy subjects . however , the current results indicated that dcp of 87 m au / ml , and not the routine diagnostic level of 40 m au / ml , should be used for chinese patients . this higher level of dcp achieved a good balance between sensitivity and specificity with a higher youden index than did the level of 40 m au / ml . the current study has some advantages over previous studies of dcp . this study enrolled a relatively comprehensive set of controls that included 371 subjects without hcc and 153 normal healthy volunteers . in previous studies reported , the controls were most often subjects with viral hepatitis and/or liver cirrhosis with an underlying viral infection or ald or nald . . however , the ability of dcp to differentiate hcc from other types of hepatobiliary disease has not been fully investigated , and this might substantially limit its use as a complement to afp . controls without hcc included subjects with a wide range of hepatopancreatobiliary diseases that included the common liver diseases of chronic severe hepatitis , focal nodular hyperplasia , hepatic cavernous hemangioma , and liver cirrhosis , as well as pancreatic cancer , hepatolithiasis , and bile duct cancer . use of comprehensive controls with disease and normal controls indicated that a cut - off value of afp 21.33 ng / ml and a cut - off value of dcp 87 mau / ml had a sensitivity of 88.90% and specificity of 80.50% with a youden index 69.40% in differentiating subjects with hcc from subjects without hcc . these two markers had a much higher sensitivity and specificity than dcp or afp alone . the dcp level is known to not be associated with the serum level of afp , and the current study corroborated that finding . the logical conclusion is that dcp is an optimal biomarker that complements afp and that dcp greatly improves the sensitivity of detecting hcc predominantly caused by hbv . a previous study showed that the prognosis was poor when either afp or dcp levels were high in japanese patients with hbv . this capability is ubiquitous , as demonstrated by different controls with underlying diseases and is not limited to viral hepatitis or liver cirrhosis . there was a close correlation between plasma dcp levels and tumor size . however , the same was not true for afp . the sensitivity of dcp of 87 mau / ml and afp of 21.33 ng / ml was further examined in 329 subjects with hcc of differing sizes . results indicated that dcp had a lower sensitivity than afp for a tumor 3.0 cm in size . as the tumor size increased , the diagnostic capability of plasma dcp increased markedly in comparison to that of serum afp . this combination of two markers had a higher sensitivity than that of dcp or afp alone for tumors of all sizes . combining dcp and afp yielded an overall sensitivity of 8090% , and a sensitivity of 82.6% for tumors smaller than 2 cm in diameter . this finding has substantiated the use of dcp as a complement to afp in diagnosing early hcc . the combination of these biomarkers is superior to the current protocol , and it offers promise in terms of surveillance . there are some limitations to the current study . first , this study did involve a relatively large number of consecutive subjects with hcc , but all of those subjects underwent curative surgery . hence , subjects with hcc in a more advanced stage were not included in this study . additional information that could have been collected includes the association between dcp values and more advanced hcc , major vessel invasion and metastasis , and the prognosis for subjects with hcc . a large - scale national program to survey dcp in chinese patients with hcc should be conducted to complement this study . second , the current controls without hcc included a relatively small proportion of subjects with liver cirrhosis ( 23/371 ) in comparison to previous studies . although this selection of controls will undoubtedly yield new useful information and expand knowledge , it restricts the range of evidence supporting the usefulness of dcp in differentiating hcc from hbv - related liver cirrhosis . in conclusion , the current study involved a relatively large sample of subjects with hcc predominantly caused by hbv , and a comprehensive set of controls . this study indicated the diagnostic ability and usefulness of plasma dcp in chinese patients undergoing surgery for hcc . in the current cohort , the optimal cut - off value for dcp was 87 mau / ml . the combination of dcp with a cut - off value of 87 mau / ml and afp with a cut - off value of 21.33 ng / ml yielded great improvement in sensitivity in differentiating subjects with hcc from subjects without hcc . these two markers may be incorporated in the protocol for surveillance of hcc in high - risk subsets of the chinese population . tumor characteristics and surgical information of 329 cases of hcc . detailed information for 371 cases of non - hcc hepatopancreatobiliary disease .

isolated and unilateral granuloma annulare occurring in clusters on the finger of an adult is rare . it is frequently misdiagnosed as a rheumatoid nodule due to its clinical and histopathological resemblance . a 53-year - old female presented with multiple asymptomatic skin - colored nodules over the right ring finger since 4 years [ figure 1 ] . her past medical history was insignificant with no history of diabetes mellitus , hypertension , joint pains or connective tissue disease . clinical examination revealed multiple skin - colored , non - tender , firm to hard nodules of varying sizes located over the dorsum of the right ring finger . examination of the other fingers , toes , nails and hand joints did not reveal any abnormalities . her hematological and biochemical investigations including a complete hemogram , liver and renal function tests , blood sugar estimations were within normal limits . multiple skin - colored nodules on a finger a skin biopsy of the nodule revealed an infiltrate of histiocytes in a palisading pattern surrounding an area of increased mucin and degenerated collagen present in the lower dermis extending up to the subcutaneous tissue [ figures 2 and 3 ] . palisading granuloma with mucin deposition and degenerated collagen ( hematoxylin and eosin 10 ) palisading granuloma ( hematoxylin and eosin 40 ) based on the clinical and histopathological findings , a diagnosis of isolated unilateral deep granuloma annulare localized to one finger was made . granuloma annulare is a common , asymptomatic benign inflammatory skin disease of unknown etiology seen in children and adults . it is characterized by an annular arrangement of erythematous papules , plaques , nodules or patches . the main clinical variants of granuloma annulare include localized , generalized , papular , umblicated , perforating and subcutaneous . the presence of subcutaneous pseudorheumatoid nodules in a patient with localized ga was the keen observation of ziegler in 1941 . sga is also referred to as pseudorheumatoid nodule , deep ga ( dga ) , subcutaneous palisading granuloma and necrobiotic granuloma . the onset of sga is usually spontaneous , lesions are benign and self - limited and most patients do not report a recurrence . the factors implicated in the causation of this disease are yet to be clearly elucidated . proposed mechanisms include physical trauma , infection , immunization , insect bite , diabetes mellitus and aberrant cell - mediated reactions . the subcutaneous variety of ga is characterized by firm asymptomatic nodules in deep subcutaneous tissues which may be associated with intradermal lesions usually over the anterior pretibial area , ankles , dorsa of the feet , buttocks and scalp . however , the involvement of more superficial dermal components has been reported in 25% of subcutaneous ga lesions , which may clinically manifest as erythematous to tan discolorations in the surface skin or superficial papules overlying the subcutaneous nodules . since sga occurs almost exclusively in infants and children with maximum frequency between the ages of 3 and 6 years , the present case is unusual because it had onset in adulthood . the presence of acral , isolated , clustered deep granuloma annulare lesions confined to a single digit without classical ga lesions elsewhere is a rare finding . such a case may mimic multiple osteomas , enchondromas , rheumatoid nodules and giant cell tumor of a tendon sheath . histopathologically , ga is a collagenolytic or necrobiotic non - infectious granuloma wherein palisaded or interstitial type of granulomatous infiltrate develops around a central area of altered collagen and elastic fibers . they are called blue granulomas as the increased mucin in the interstitium imparts a bluish hue . the inflammatory infiltrate in ga comprises of histiocytes and a sparse perivascular infiltrate . occasional multinucleated giant cells and elastophagocytosis may be seen . a clue to differentiate ga from a rheumatoid nodule may be the presence of prominent mucin and a paucity of foreign body giant cells . various treatment modalities are available for the treatment of ga including topical or intralesional steroids , cryotherapy , electrocoagulation , laser destruction , puvasol , and topical imiquimod . the use of systemic agents such as systemic steroids , psoralen uv - a , isotretinoin , dapsone , pentoxifylline , hydroxychloroquine , cyclosporine , rifampicin , ofloxacin and minocycline combination , interferon gamma , chlorambucil , potassium iodide , photodynamic therapy , etarnacept , infliximab , adalimumab and efalizumab . multiple nodules of granuloma annulare occurring in clusters have been previously described on the scalp of a 4-year - old boy . isolated and unilateral involvement of a single digit with clusters of nodules of subcutaneous ga in an adult is rare . it frequently poses a diagnostic challenge and commonly mistaken for its clinical simulator rheumatoid nodule . hence , it is essential to keep in mind sga as a differential diagnosis when encountered with nodular lesions on acral areas in adults . clusters of subcutaneous granuloma annulare lesions localized and restricted to single digit in an adult is rare and may be misdiagnosed as rheumatoid nodules .

intrinsically disordered proteins ( idps ) lack a well - defined three - dimensional structure under physiological conditions of ph and salinity and in the absence of a partner or ligand . some idps however fold , partially or completely , into ordered conformations upon binding to specific interactors . the extreme structural plasticity that characterizes these proteins allows for independent tuning of affinity and specificity , recognition of multiple targets , fast association kinetics and effective regulation by post - translational modifications . according to disorder - prediction algorithms , ~30% of the eukaryotic proteins are mostly disordered and ~40% possess disordered regions longer than 50 residues . a big effort is being devoted to the investigation of idp binding to their folded interactors . the structure of numerous complexes has been solved by x - ray crystallography , describing interaction surfaces and acquired ordered structure , although complexes where idps retain their flexibility have been reported , too . to understand the mechanism of molecular recognition , it is also important to characterize the structural properties of the pre - recognition state . the flat energy landscape of idps in solution implies that they exist as highly dynamic and heterogeneous conformational ensembles , which escape structural characterization by conventional biophysical methods . nevertheless , progress has been made in conformational analysis of idps in their free state by different biophysical methods . the emerging picture is that idps in the absence of interactors populate metastable , partially folded states with preformed elements of secondary structure ( intrinsically folded structural units , ifsu ) and relatively compact tertiary structure . these partially folded conformers are thought to be functionally relevant , providing seeds for interaction surfaces and/or protecting idps from degradation and from non - specific interactions . mass spectrometry offers peculiar advantages in the analysis of complex mixtures , thanks to the possibility to detect not only distinct masses , but also different conformers endowed with variable degrees of compactness in the molecular ensemble . direct assessment of species distributions , without averaging over the molecular population , offers a valuable tool in idp analysis , complementary to other biophysical methods . this paper focuses on the contributions that charge - state distribution ( csd ) analysis by non - denaturing electrospray - ionization mass spectrometry ( esi - ms ) can give to idp identification and characterization . mild desolvation / ionization conditions allow for maintenance of native - like protein conformations during the electrospray process . experimental evidence is based on direct investigation of the structural properties of gas - phase protein ions by ion mobility , electron - capture dissociation , gas - phase hydrogen exchange and binding analysis . computational studies suggest that attractive interactions inside the protein structure can compensate to a certain extent for repulsive forces introduced by protein ionization . this effect is mainly due to hydrogen bonds and zwitterionic states , although minor contributions could also derive from dispersion forces and cation- interactions . altogether , intramolecular interactions of native protein structures provide a tremendous self - solvation potential that contribute to the stability of the gas - phase protein ions generated by electrospray . in particular , it has been shown that folded protein conformations tend to shrink upon desolvation , increasing the number of intramolecular hydrogen bonds and the exposure of hydrophobic residues on the protein surface . in order to prevent protein denaturation during electrospray , it is important to avoid the use of organic solvents and extreme ph values , and to apply mild temperature and voltage conditions to the sample source . particularly well suited to this end are nano - electrospray devices , where a micrometer - scale capillary for sample infusion leads to smaller first - generation droplets and , consequently , more effective desolvation under soft conditions of temperature and voltage . nano - esi - ms is now routinely applied to conformational studies , also offering the advantages of low sample consumption , automation , and experimental scale - up . protein csds can deliver important structural information , thanks to the fact that protein compactness in the original solution has a strong influence on the extent of protein ionization under electrospray conditions . the higher the structural compactness , the lower the average net charge that will be observed for any given protein . however , csds can also be affected by several other factors , such as instrumental parameters and solvent properties . therefore , it is important to keep the experimental conditions accurately controlled and to make sure that none of the applied instrumental settings becomes the limiting factor for csd features . it should also be taken into account that measurements in negative - ion mode could be more exposed to the risk of electrospray - induced protein unfolding . by the application of the most adequate and controlled operative conditions , good agreement between esi - ms and solution methods has been shown monitoring unfolding transitions of proteins induced by acids , organic solvents and heat . nevertheless , it should also be reminded that the signal yields of folded and unfolded conformations of the same protein can differ substantially , particularly at high flow rates and high protein concentrations , rising the need of specific control experiments for quantitative analysis . major structural heterogeneity in the molecular ensemble of a pure protein results in multimodal csds , where distinct peak envelopes can be resolved and quantified by deconvolution algorithms . the broadness of each peak envelope , in turn , is affected by structural dynamics , with narrow profiles associated to folded structures and broad profiles associated to disordered states . examples of csds obtained for idps by non - denaturing esi - ms are reported in figure 1 , in comparison to a natively folded globular protein . as it can be noticed , idps under non - denaturing conditions give rise to broad csds with high average charge , frequently present as distinct components of multimodal profiles . the different behavior of an idp and a globular standard under identical conditions can be further highlighted by control experiments where the spectrum of a mixture of the two proteins is acquired , followed by identification of the distinct components by their specific masses . the peculiar response of idps to electrospray offers the possibility to develop a signature for this class of proteins by non - denaturing esi - ms , as discussed in more detail below . examples of csds obtained by nano - esi - ms under non - denaturing conditions . the spectra were recorded on a hybrid , quadrupole time - of - flight mass spectrometer ( qstar elite , ab - sciex ) . samples were infused at room temperature , by metal - coated borosilicate capillaries with emitter tips of 1 m internal diameter ( proxeon ) . the following instrumental settings were applied : declustering potential 60/80 v , ion spray voltage 1.1/1.2 kv , curtain - gas pressure 20 psi . ( b ) 12 m -synuclein in 10 mm ammonium acetate , ph 7.4 ( negative - ion mode ) . . folded globular proteins follow a well - known , mass - to - charge relation , with the average experimental charge growing approximately as the ~0.57 power of the protein mass expressed in daltons . it has been shown that the behavior of folded and unfolded proteins can be distinguished by relating charge to protein mass , while it is unified by relating charge to solvent accessible surface area ( sasa ) . in other words , sasa seems to dictate the extent of protein ionization , regardless of the conformational state . if we relate charge to protein mass , instead , disordered protein conformations stand as clear outliers in the plot of folded globular proteins . this is true for idps under non - denaturing conditions , as well as for chemically denatured proteins , indicating that solvent effects play a marginal role in this regard . figure 2 shows the charge - to - mass plot for several idps that have been analyzed by esi - ms . these proteins were either already known as idps , or identified as such in our laboratory by independent evidence , like circular dichroism ( cd ) and nuclear magnetic resonance ( nmr ) . the data reported in figure 2 summarize the ionization behavior of these idps by nano - esi - ms under non - denaturing conditions . for those cases yielding mass spectra with bimodal distributions , the values of average charge have been calculated on each distinct component . the data are plot with reference to the curves of natively folded , globular proteins under denaturing or non - denaturing conditions . two observations are straightforward : ( 1 ) idps have at least 1 component that is a clear outlier relative to the reference curve of globular proteins measured under non - denaturing conditions and , rather , approaches the behavior of chemically denatured proteins , and ( 2 ) many idps have 1 component that is surprisingly close to the reference curve of folded , globular proteins . the relative amounts of the different forms , shown by the color code in the figure , can vary significantly , depending on the protein and the environmental conditions . these results are consistent with a remarkable conformational heterogeneity of idps in solution under non - denaturing conditions . the strong deviation from the typical ionization behavior of globular , folded proteins measured under the same conditions indicates that csd analysis by esi - ms offers a tool to identify putative idps . at the same time , these results illustrate that esi - ms can capture compact states populated by idps , even if highly dynamic and poorly represented . we discuss below the available evidence that supports the interpretation of these spectral components in terms of idp conformational states . charge - to - mass plot for distinct components of some idps analyzed by non - denaturing esi - ms . in the case of bimodal distributions , each component is represented by a circle , colored according to the relative abundance as specified in the inserted table . for unimodal distributions ( unique case of full - length sic1 ) , the global average charge is considered and the symbol is colored in black . the gray small squares and triangles represent data for globular proteins , respectively under non - denaturing and denaturing conditions . sic1 , substrate / subunit inhibitor of cyclin - dependent protein kinase ; ntail , c - terminal domain of the viral nucleoprotein n ; pnt , n - terminal domain of measles virus phosphoprotein p. as shown in figure 2 , the high - charge component is typically predominant in esi - ms spectra of idps with bimodal distributions . as generally seen for chemically denatured proteins , it is also quite broad , reflecting further heterogeneity due to structure dynamics . most importantly , when this component is analyzed as a function of mass , it follows the same power law as the denatured conformation of natively folded proteins . furthermore , its intensity varies in response to solvent composition , typically accompanied by opposite changes in the low - charge component . these observations strongly suggest that the experimental csds reflect the dynamic conformational ensemble of idps in solution . altogether , this evidence supports identification of the high - charge component as a highly disordered conformational state of idps , characterized by low structural compactness . the presence of such a component under non - denaturing conditions can be considered as a signature of idps and could be used for fast screening of putative new members of this conformational class . it is important to underscore that the anomalous ionization behavior of idps is evident also when the protein contains only a disordered tract , together with a normally folded domain , like in the case of ataxin-3 . in most of the considered idps , a sharply bimodal csd suggests that a small fraction of the molecular population exists in a highly collapsed state . it is surprising that such a component approximates the ionization behavior expected for folded , globular proteins of the same size . nevertheless , it is known by several other independent methods that idps in solution often populate partially structured states and can collapse into compact globular structures . the challenge in trying to characterize these states is that they are highly dynamic and poorly represented in the molecular ensemble , easily escaping characterization ( and even detection ) by biophysical methods . it is , therefore , extremely attractive to directly visualize minor , structured components , out from the background of the predominant conformers , thanks to the ion - sorting properties of ms measurements . at the same time , it is important to collect evidence supporting the interpretation of these spectral components in terms of protein conformation . one of the most important evidence is that these components can be progressively depleted by varying the solvent properties , and that this response is protein - specific . for instance , the low - charge component of sic1 kinase inhibitory domain ( kid ) is selectively lost by acidification whereas it accumulates in response to the same treatment in the case of -synuclein . furthermore , such a transition can be quite cooperative , as in the case of the ph - dependence of sic1-kid compact form . thus , it seems that idps compact states can be denatured by particular agents , in a way that is not so dissimilar from unfolding transitions of natively folded proteins . it is also important to point out that these transitions are characterized by changes in the relative amounts of the different components , as expected for an interconversion process , and not by progressive shift of a given peak envelope in the spectrum , as more typical for the effects of solvent composition or parameter setting on esi - ms data . furthermore , idps compact conformers corresponding to the low - charge components have been isolated and identified in gas phase by ion - mobility methods . this technique adds a second dimension to the ion sorting mechanism of ms measurements , based on drift time through a buffer gas . since ion mobility depends on collisional cross section , compact conformers will be faster and will be separated from elongated conformers inside the drift cell . thus , thanks to the survival of non - covalent interactions responsible of protein compaction , the distinct physical properties of the desolvated ions can be highlighted by a criterion that is orthogonal to the m / z measurement of conventional ms analyses . another important evidence is given by the specific ligand - binding properties of these components . such a behavior has been observed , for instance , in the case of copper binding by -synuclin . complexes with a 1:1 stoichiometry could be identified by esi - ms upon metal binding . however , csds analysis revealed that the bound form is mainly detectable in the low - charge component of the protein spectrum . this result further supports the hypothesis that such a component corresponds to a distinct conformer of the protein ensemble , displaying higher compactness and higher propensity for metal binding . the relative amount of the compact form also increases upon copper addition , indicating that the protein undergoes a process of induced folding promoted by copper binding . finally , computational studies can provide further insight on compact conformers of idps in solution . structural models have been developed by molecular - dynamics simulations for the compact states of sic1-kid . although these methods are not adequate to describe the actual equilibrium between compact and extended conformations , they effectively model the forces responsible for protein compaction and can , therefore , generate putative structures for idp compact states . the most probable structures generated by simulations on sic1-kid display ifsus and considerable degree of compaction . for instance , intramolecular interactions in the computational models were found to be prevalently of electrostatic nature , with minor contributions of hydrophobic interactions . this result is in agreement with the observed strong effect of acids and negligible effect of organic solvents on the esi - ms spectra of this protein . furthermore , the sasa estimates based on the computational models ( 5365 nm ) are in good agreement with those derived by the esi - ms data for the low - charge component of the csd ( 59.78 nm ) . these studies strongly suggest that the low - charge components frequently detected in the esi - ms spectra of idps under non - denaturing conditions correspond to metastable compact conformers with specific structural and functional properties . the ionization properties of idps under electrospray conditions reveal significant and systematic deviations from the reference behavior of folded , globular proteins . these differences emerge by measurements under non - denaturing conditions and analysis of average charge by the charge - to - mass relation . thus , csd analysis offers an effective tool for high - throughput conformational screening aimed at the identification of putative new members of this structural class . confident protein classification will then require further analysis by other techniques , such as cd , size - exclusion chromatography and nmr . non - denaturing esi - ms is also a powerful tool for the identification of partially structured states of idps , even if highly dynamic and scarcely populated . the generally applicable charge - to - surface relation , which holds independently of the conformational state , allows extracting useful structural information by sasa calculation for each detectable component . this information can be used as a constraint for computational modeling , possibly integrating other kinds of experimental measurements .

retinitis pigmentosa ( rp ) is a hereditary degenerative disorder characterized by progressive loss of photoreceptor and visual function . the visual field constricts as the disease progresses , eventually leading to severe visual loss in the advanced stage . although visual field testing is used as a standard method to assess the progression of rp , several electrophysiological , psychophysical , and imaging techniques such as full - field electroretinogram ( erg ) , multifocal erg , microperimetry , and autofluorescence imaging are being widely studied . the goldmann visual field ( gvf ) is preferred for documenting the progression of rp because it is able to probe peripheral visual field constriction , which can not be detected by automated perimeters , in the early phase . however , the large test - retest or intervisit variability of gvf , which is increased up to 50% in pathologic states like rp , makes it difficult to use this technique to evaluate disease progression or to assess the treatment outcome . on the other hand , structural assessment using optical coherence tomography ( oct ) offers high reproducibility . even in rp , macula thickness and retinal nerve fiber layer ( rnfl ) analyses show high reproducibility . the recent introduction of fourier domain oct ( fd - oct ) has improved image resolution and has enabled the analysis of various macular morphologies quantitatively . rnfl thickness measurement is widely performed by using fd - oct , and analysis of ganglion cell complex thickness has been adopted recently in glaucoma detection and progression evaluation . previous rp studies revealed that the photoreceptor inner segment / outer segment ( is / os ) length and foveal thickness , especially the outer retinal thickness , are related to retinal functions such as the visual field , visual acuity , and erg [ 35 ] . although many studies have revealed the structure - function relationship of rp by using oct , a systematic comparison to determine which structural parameter is more strongly related to visual function has not been performed . therefore , we attempted to determine the retina microstructure that reflects visual function mostly in the point of vision and the visual field . we measured the preserved outer retinal layer in 3 dimensions , including horizontal / vertical length , thickness , and area . patient data were collected from the outpatient clinic at seoul national university hospital from 2008 to 2012 . the diagnosis of rp was based on a history of night blindness , impairment in peripheral visual fields , reduced amplitudes of rod and cone electroretinograms ( erg ) , and the presence of characteristic fundus pigmentary changes . all patients underwent a comprehensive interview and ocular examination including best - corrected visual acuity ( bcva ) , slit lamp biomicroscopy , dilated fundus examination , erg , visual field testing , and oct . fd - oct was obtained in all patients by using cirrus hd - oct ( carl zeiss meditec inc . , the acquisition protocol consisted of an hd 5-line raster scan composed of 1024 a - scans at each 6 mm line and a macular cube with a 200 200 scan pattern in which a 6 mm 6 mm region of the retina was scanned [ 6 , 7 ] . further , we excluded fully advanced cases in which the elm and the pros were not discriminated as well as patients who had macular edema and vitreomacular traction . first , we determined the pros area from the en - face image by using the advanced visualization option in cirrus oct 6.0 software , as previously described by comander et al . for solar maculopathy . the en - face pros image could be visualized by using the slab image fit to the rpe layer . the width and location of the slab were finely tuned by viewing the cross - sectional image . subsequently , the margin of the pros was demarcated manually and the area was calculated by using adobe photoshop cs 3.0 ( adobe systems inc . , san jose , ca , usa ) and image j software ( national institutes of health ) . patients with a pros area exceeding the 6 mm cube scan were excluded because the actual area of the preserved pros could not be calculated . however , because an en - face pros image was available for 64% of the patients , we obtained the pros area alternatively by calculating the elliptical area derived from the horizontal and vertical pros lengths measured by using the horizontal and vertical high - definition scan across the fovea . these cases were excluded if the preserved pros was not within the 6 mm cube scan . the crt is the average thickness of the region from the ilm and rpe in a 1 mm circle , which is obtained by using macular thickness analysis . the inner retinal thickness ( irt ) was defined as the distance between ilm and elm , and the outer retinal thickness ( ort ) was defined as the distance between elm and inner border of rpe . finally , the sfct was defined as the distance between outer border of the rpe and inner border of the scleral wall . the irt , ort , and sfct were measured manually at the thinnest point of the fovea by using the internal caliper provided in cirrus oct software . the gvf test was performed by using the test target iii4e under standard conditions on the same calibrated perimeter . the extent of the centrally preserved visual field was obtained by computing the area surrounded by isopter polygons on scanned images of the perimeter charts and by calculating the solid angle subtended . the central visual field was defined as the region encompassing the central fixation , and the extents for the visual field of the scotoma were subtracted . we excluded the peripheral visual field island because an isolated preserved is / os junction in the periphery is difficult to detect by oct . the visual field area ( vfa ) this gvf extent unit , which is called the normalized solid - angle unit ( nsu ) , was log - converted for comparison with other parameters , as described in the previous literature . all values were transformed to the log scale for statistical analysis because rp is known to progress exponentially . the pearson correlation was used to examine the strength of the association between visual function ( logmar visual acuity and vfa ) and each of the oct morphology parameters ( pros area , pros length , cft , irt , ort , and sfct ) . the average visual acuity was 20/30 ( median , 20/28 ) and the average visual field was 22 ( median , 10 ) . a strong linear correlation was found between the 2 methods used for obtaining the pros area ( r = 0.953 , p < 0.001 ) in 64 patients ( figure 2 ) . we used the pros area calculated by utilizing the horizontal and vertical lengths for comparison with the other parameters . in univariable analysis using the pearson correlation , the vfa was related to the horizontal pros length ( = 0.483 , p < 0.001 ) , vertical pros length ( = 0.426 , p < 0.001 ) , and pros area ( calculated from the horizontal and vertical lengths : = 0.459 , p < 0.001 , and from the en - face image : = 0.509 , p < 0.001 ) . the correlation between the log vfa and pros length / area is presented in figure 3 . regarding the thickness of the segmented retinal layer , the crt ( = 0.276 , p < 0.005 ) and ort ( = 0.412 , p < 0.001 ) generally , the correlation coefficient was higher for the pros than for the retinal thickness . the pros area calculated by using the en - face image showed the strongest relationship to the visual field . visual acuity was correlated with the crt ( = 0.339 , p = 0.001 ) , ort ( = 0.519 , p < 0.001 ) , and irt ( = 0.297 , p = 0.003 ) . the pros horizontal length ( = 0.395 , p < 0.001 ) , pros vertical length ( = 0.376 , p < 0.001 ) , and pros area ( calculated from the horizontal and vertical lengths : = 0.389 , p < 0.001 , and from the en - face image : = 0.370 , p = 0.003 ) were also related to visual acuity . the ort was most correlated with visual acuity ( = 0.519 , p < 0.001 ) . the correlations for visual acuity , ort , and the pros area are shown in figure 4 . the correlations between visual function ( visual field test and visual acuity ) and the measured parameters ( pros values and retinal thickness parameters ) are summarized in table 2 . in multiple linear regression analysis , this study is the first to use the pros area to estimate the progression of rp . to this end , 2 methods were used : an en - face image from an advanced visualization option and a presumptive ellipsoid derived from the horizontal and vertical pros lengths . the former method is presumably more accurate and intuitive , but it could not be used in approximately 30% of the patients ( 36 patients ) . an en - face pros slab image was usually not available in cases of high myopia . because both methods showed consistent results , we used the pros area determined from the horizontal and vertical lengths to identify the relationship to visual function . these results are consistent with data suggesting that the photoreceptor is / os junction length is related to the focal erg amplitude and goldmann perimetry isopter [ 4 , 5 , 12 ] . the pros area is expected to be a better parameter than the cross - sectional pros length because it reflects the total preserved pros amount . however , the horizontal pros length showed a slightly better correlation with vfa than the pros area calculated from the pros length , and multiple linear regression analysis showed that pros horizontal length was related to the vfa . considering the intervisit variability of the gvf test , the preserved pros horizontal length can be a good progression index of rp . further research is required to confirm the test reliability and long - term follow - up of the pros length by using oct . our data regarding the relationship between each retinal layer thickness and visual functions are consistent with previous studies [ 1317 ] . in concordance with lenassi et al . and rangaswamy et al . [ 14 , 15 ] , although the total subfoveal thickness was related to visual acuity , it had a weaker correlation than the outer segment thickness . further , multivariable linear regression analysis weighted the outer retinal thickness , and only the ort was related to the vfa . instead of evaluating the is / os junction to the rpe distance , we used the elm - to - rpe distance as the ort because this value was greater than the is / os - to - rpe height and was therefore expected to provide a smaller error than the pros thickness . moreover only the elm is detectable even after the cone outer segment tip line and the is / os are disorganized in advanced rp [ 18 , 19 ] . thus , the elm - rpe thickness value is more reliable for determination of outer retinal thickness in rp . we analyzed the subfoveal choroidal thickness and found that it was not related to visual acuity or to the visual field . to our knowledge , this is the largest study to evaluate choroid thickness and to compare it with visual acuity and the visual field . reported that the sfct was not related to visual acuity , whereas ayton et al . reported that the sfct showed a significant negative correlation with visual acuity we believe that our findings are more reliable than are those of these 2 studies because our study sample was much larger . a limitation of our choroid thickness data is that we did not compare this value with a control group . many studies found that choroid thickness is reduced in rp patients compared to healthy controls [ 2022 ] . our study showed that visual acuity and the visual field are not directly related to the choroid thickness . a larger study is required to elucidate the choroid pathology in rp . in this study , we compared the correlation between macular microstructural parameters and the visual function . although the pros area from the en - face visualization showed the strongest association with the visual field , this method was not available in 1/3 of our cases . the second most correlated value , horizontal pros length , has the advantage of easy acquisition . we anticipate that measuring the pros length will be useful for evaluating preserved visual function in rp patients who retain central vision .

blue shield plan physician participation agreements serve both as a marketing device to attract subscribers and as a short - run cost - containment strategy . in most blue shield plans a participating physician agrees to accept the plan 's allowance for a procedure as payment in full . in return , the physician may be reimbursed by the plan rather than being paid directly by the patient . the advantages to the physician are smaller accounts receivable , fewer bad debts , and , of course , extra attractiveness to blue shield plan subscribers . participation does not necessarily imply a zero copayment by patients even after deductibles , if any , are met . however , since the amounts of reimbursement for procedures are predetermined in the short - run , participation makes it less risky for a plan to offer policies with low or zero copayment . moreover , even when copayment is not eliminated , subscribers benefit both in terms of the ceiling on out - of - pocket costs and by being relieved of the interest and liquidity costs of direct payment . governmental health insurance programs specifically medicare for the elderly and disabled and medicaid for the indigent use similar cost - containment strategies . physicians who treat medicaid patients must accept as full payment the amount allowed for each procedure by the state 's program . under the medicare regulations physicians may participate ( accept assignment ) on a claim - by - claim basis . administrators of both programs are vitally concerned with how sensitive physician participation ( assignment ) is to the amounts allowed for procedures . in the case of medicaid , allowances that are too low can mean insufficient suppliers of medical care for the poor . in the case of medicare , allowances that are too low can mean low physician assignment levels and higher out - of - pocket costs for the elderly and disabled . obviously , striking a desirable balance between allowance levels and participation rates is of major importance to medicare and medicaid management . unfortunately , we did not have access to medicaid or medicare assignment data for this study . however , we did have extensive data on the private market business from two blue shield plans with physician participation arrangements . all blue shield plans market one or more of three types of basic health insurance contracts : ( 1 ) usual - customary - and - reasonable ( ucr ) , ( 2 ) partial service , and ( 3 ) indemnity . although participation agreements do not apply to indemnity policies , the other two lines of private business do have certain strong parallels with medicare and medicaid . in particular , as in medicaid , a blue shield subscriber is eligible for a partial service contract only if his / her family income is below a ceiling level . also , the procedure used in setting allowances ( but not the levels ) is basically the same for medicare enrollees as it is for blue shield plan ucr subscribers . therefore , analysis of physician participation in private blue shield business should be helpful to medicare and medicaid program administrators as well as to blue shield plans themselves . claims presented to blue shield plans identify the services rendered by the physician and the amount charged for each service . the plan then sets the maximum amount allowed for each service . in partial service and indemnity business , the amount allowed is the lesser of the amount charged and a scheduled fee which is the same for all physicians . in ucr business the maximum amount allowed is called the reasonable fee and is ordinarily the minimum of the amount charged and the amounts set by one or both of the two fee screens . the first screen , called the usual fee or level 1 screen , is the physician 's mean , median , modal , or listed charge for the procedure during some prior time - period fixed by the plan . the second screen , called the customary fee or level 2 screen , is a percentile commonly but not always the 90th of the fee distribution for the procedure in the physician 's geographic area . like level 1 screens , level 2 screens are determined from past fee data and are not affected by the physician 's current charges . thus , unless the physician charges less than the screen amounts , the reasonable fee for each procedure is fixed during the current period , and , unless it happens to coincide with the level 2 screen value , it is different for each physician . finally , the plan determines the amount paid to the subscriber or physician based on the amount it allows . in partial service and indemnity business , the amount paid is equal to the allowance . in ucr business the amount paid is a percentage ( up to 100 percent ) of the allowance . in each of the two plans in this study , participating physicians nominally agree to accept plan allowances as full payment and to accept reimbursement from the plan . thus , excluding deductibles , the net prices ( average coinsurance rates ) of ucr services in the two study plans are ( small ) percentages of allowances on participating claims , and are equal to the physician 's charge minus a ( large ) percentage of allowances on nonparticipating claims . in partial service business the net prices of services are zero on participating claims , but equal to charges minus allowances on nonparticipating claims . consequently , other things being equal , the net prices to patients of participating physicians ' services are lower in the two plans than those of nonparticipating physicians ' services . given the public policy interest in physicians ' decisions to participate ( accept assignment ) , it is important to note the similarities and differences in the physician reimbursement procedures between blue shield private business and those of the medicare and medicaid programs . customary - prevailing - and - reasonable ( cpr ) , in which the level 1 and level 2 screens are labeled the customary fee and the prevailing fee respectively , and in which , as in blue shield plans , the amount allowed is called the reasonable fee . physician reimbursement under the states ' medicaid programs is either of the cpr form or , as in blue shield partial service business , based on fixed ( or de facto ) fee schedules . in medicare , the amount paid by the carrier is 80 percent of the reasonable fee . in medicaid programs , the amount paid is 100 percent of the allowance and coinsurance payments are zero ( chavkin , 1979 ) . medicare and medicaid both employ a physician payment system called accepting ( benefit ) assignment which is virtually identical to blue shield participation arrangements . a physician who accepts assignment acknowledges the amount allowed as full payment for his or her services and is reimbursed for those services except for medicare deductables and coinsurance by the carrier . the physician who does not accept assignment is free to charge and receive whatever average revenue he or she can , but must bill the patient who then files a claim for reimbursement with the carrier . unlike the case in most blue shield participation arrangements , medicare regulations permit a physician to accept assignment on a case - by - case basis . in medicaid programs , with respect to practice pricing and output policy , the most important implication of participation is that the physician 's maximum average revenue on participating claims is fixed during the current period . that is , in the current period the physician acts like a price - taker in each blue shield submarket , defined by line of business , where he elects to participate . depending on local competitive conditions , the physician may or may not be a price - taker in non - blue shield submarkets or in those blue shield submarkets where he or she does not participate . one economic model that can be applied to this institutional setting is the robinsonian model of price discrimination . the model has been used in prior research on physician participation and assignment ( sloan and steinwald , 1978 , and hadley , 1978 ) . briefly , it postulates that physicians : ( 1 ) maximize ( expected ) profit ; ( 2 ) face two or more demand functions representing the participating and nonparticipating segments of their markets ; ( 3 ) face a participating demand function that is infinitely elastic in average revenue ; ( 4 ) produce the same service in each market with one cost function ; ( 5 ) are aware of allowance levels in the participating market segments ; and ( 6 ) produce an output in each market segment and a price in each nonparticipating market segment that maximize ( expected ) profit . depending on the initial positions of the demand , marginal cost , and allowance level functions , the fraction of the physician 's output devoted to the participating market segment should : increase ( decrease ) as the allowance level is raised ( lowered ) ; increase ( decrease ) as the short - run marginal cost function shifts downward ( upward ) . for example , in a cross - section of physicians , one should observe the highest rates of participation among physicians with the shortest reimbursement lags , among those with the lowest input prices , among those of low quality ( assuming low quality is associated with low marginal costs ) , and among group rather than solo physicians if there are economies of multi - physician practice ; decrease ( increase ) as the nonparticipating demand function shifts outward ( inward ) . theoretically , factors that lead to high levels of nonparticipating demand and low physician participation rates are , for example , high physician quality , high patient income and educational levels , and a large volume of high - use patients especially medicare eligibles whose demands are financed outside of the plans ' private business . factors leading ( theoretically ) to low levels of nonparticipating demands and high participation rates are a high risk of bad debt on nonparticipating bills ( reflected , perhaps , by low per capita incomes in the physician 's market area ) , and a large volume of alternative suppliers as measured by large numbers of physicians per capita , and ample use of hospital outpatient facilities . although the model outlined here assumes profit maximization , its implications hinge only on the relative income opportunities of participation and nonparticipation . thus , the predictions can be expected to hold albeit more weakly for any type of physician optimizing behavior such as utility maximization or target net income maintenance where decision - making is sensitive to income opportunities . when the profit - maximization assumption is relaxed , however , the physician 's tastes and attitudes presumably have some impact on the participation decision . the only prior study of physician participation in blue shield plans is by sloan and steinwald ( 1978 ) . studies of medicare assignment have been carried out by huang and koropecky ( 1973 ) , muller and otelsberg ( 1978 ) , and paringer ( 1979 ) . the determinants of medicaid assignment have been explored by sloan , cromwell , and mitchell ( 1978 ) , and hadley ( 1978 ) . explicitly or implicitly , all of these studies have employed the robinsonian model , and all have used four basic groups of variables to account for variation in physician participation / assignment rates : ( 1 ) measures of reimbursement practices such as allowance levels and the stringency of claims review ; ( 2 ) proxies for the level of the short - run marginal cost function ; ( 3 ) measures of factors influencing the position of the practice 's nonparticipating / nonassignment demand function ; and ( 4 ) physician characteristics representing tastes , and possibly costs , or the position of the nonparticipating / nonassignment demand function . because of differences in analytic units , samples , definitions of variables , and estimation procedures , it is difficult to summarize the results of these studies . however , the evidence tends on balance to confirm the validity of the robinsonian model . it shows that : carrier reimbursement practices have significant effects on participation / assignment rates . the rates appear to increase significantly with allowance levels [ sloan and steinwald ( 1978 ) , sloan et al . ( 1978 ) , paringer ( 1979 ) ] , and proxies for the stringency of claims review have been found to be negatively correlated with medicare assignment tendencies [ huang and koropecky ( 1973 ) , muller and otelsberg ( 1978 ) ] . high input prices ( office wage rates ) lower assignment rates [ hadley ( 1978 ) , sloan et al . ( 1978 ) , paringer ( 1979 ) ] , but have no clearcut effect on participation tendencies [ sloan and steinwald ( 1978 ) ] . certain proxies for strong nonparticipating / nonassignment demands such as high income population , large percentages of urban , white , and elderly residents , and low volumes of hospital outpatient visits per capita are negatively correlated with assignment rates [ huang and koropecky ( 1973 ) , sloan et al . but participation rates were found to be positively correlated with population income by sloan and steinwald ( 1978 ) , and observed relationships between participation / assignment rates and the number of physicians per capita are mixed . for physician traits , the strongest results indicate that non - board - certified physicians , foreign medical graduates ( fmgs ) , young physicians , and physicians with liberal attitudes toward publicly sponsored health care have the highest participation / assignment rates [ sloan and steinwald ( 1978 ) , sloan et al . mixed results have been found with respect to relationships between participation / assignment rates and physician specialties . because of their bearing on this study , the institutional and theoretical sections of the sloan and steinwald study merit special consideration . the most prevalent ( of blue shield policies ) and full - service ( ucr ) the least prevalent . plan a had no partial service business during the sample period . in plan b , the dollar volume of ucr business exceeded that of its partial service business . the robinsonian model used by sloan and steinwald is also not completely valid for the all - or - nothing participation decision faced by physicians in our two study plans . the robinsonian model permits the physician to vary his or her proportions of participating and nonparticipating outputs continuously in effect , to participate on a claim - by - claim basis . in cases where the physician must decide whether to participate or ( as in medicaid ) to accept assignment across the board , the correct model is a discrete optimization model . if the physician maximizes profit , he or she must compare the ( expected ) profitability of the participating ( assignment ) and nonparticipating ( nonassignment ) options and choose the option with the largest anticipated profit . it is assumed here that there are two submarkets and that the physician does not discriminate in price if he or she chooses not to participate . ( the argument is substantively the same if the physician does discriminate in price . ) in submarket 1 , where the demand and marginal revenue functions are shown as the lines ab and ac respectively , the physician can not participate . in submarket 2 , the physician may participate or not . if the physician does not participate in submarket 2 , the demand and marginal revenue functions have the positions de and df , respectively ( figure 1a ) . if the physician participates in this submarket , the demand function is the line segment dl shown in figure 1b . under the nonparticipating option shown in figure 1a , the physician will choose the output oq , where the combined marginal revenue from the two submarkets ( given by a point on the line segment arij ) equals the marginal cost qs . the profit maximizing fee level in each submarket is ok , and the physician 's total profit is the area nmlk . that is , unit profit is the fee level ok minus unit cost on . under the participating option shown in figure 1b , the physician chooses the output oq , the fee level og in submarket 1 , and outputs of of and fq in submarkets 1 and 2 , respectively . total profit in this case the physician will then elect to participate only if the area hieg + ijkd equals or exceeds the area nmlk . we omit the details because of space limitations , but it can be shown that the economic implications of the discrete model are generally much more indeterminate than those of the robinsonian model . for example , in the robinsonian model an outward shift in the nonparticipating demand function ( that is , in the demand function for services on which the physician can not participate ) unambiguously lowers the profitability of participating and reduces the probability of participating . in the discrete model , the same type of shift raises the profitability of both the participating and nonparticipating options , and , on strictly logical grounds , it is not possible to tell which option becomes more profitable to the physician . hence , the effect of the shift in the participation probability can not be unambiguously predicted . as a basis for comparing the implications of the robinsonian and discrete models , the theoretically predicted impacts of shifts in allowance levels and demand and cost functions on the participation probability are summarized in table 1 . the discrete model suggests further that the physician 's participation decision will be less sensitive to shifts in any of these functions than the robinsonian model implies . this is so because in the discrete model there must be a quantum change in the relative profitabilities of participating and not participating before the physician is led to switch from one option to the other . a consequence of this consideration is that when participation or assignment ( as in medicare ) is on a claim - by - claim basis , participation / assignment decisions are more likely to be responsive to relatively small changes in plan allowances and local market conditions than when participation / assignment is on an all - or - nothing basis . the relative indeterminacy and lower sensitivity of all - or - nothing participation decisions to shifts in revenue and cost conditions do not lessen the importance of examining the decisions themselves . they merely emphasize that participation behavior must be determined empirically , and that it often can not be predicted using a priori reasoning . the data used in this study are the claims records of 942 physicians in plan a and 928 physicians in plan b covering the years 1973 - 1976 . the records apply to approximately 60 high - use medical , surgical , and other procedures , and were provided by the blue shield association . each physician in the sample practiced in the plan 's market area during the four years of the study period . the claims records contained the amounts charged by physicians and the amounts allowed and paid by the plan . they also contained the frequency with which each procedure was performed and certain additional claims data indicated in table 2 . to derive a single measure of the physician 's output , the procedure frequencies were converted into relative value units ( rvus ) using the 1974 california relative value scale . the number of rvus was then aggregated for each physician and year , and mean charges and allowances per rvu were calculated for each physician and year . finally , measures of rvus were standardized across specialties in order to generate a common output index for all physicians . the physician - specific claims data were merged with additional physician and county data taken from several sources . physician characteristics such as age , sex , specialty , board certification status , country of medical graduation , etc . , were derived from the american medical association 's masterfile of physicians . county - level data describing local market conditions were drawn from the sources listed at the end of table 2 . as already mentioned , the two study plans formally offered participation agreements on an all - or - nothing basis . in plan a the agreement applied only to ucr business . in plan b a participating physician was required to participate in both ucr and partial service business , and he or she could not elect to participate in one of the two lines alone . since partial service allowances were lower than ucr allowances in plan b , it was predicted that they represent a stronger constraint on the plan b physician 's participation decision than ucr allowance levels . in modeling the participation decision , it was assumed that , at the start of each year , the physician faces the discrete optimization problem previously described . having chosen the alternative yielding the largest anticipated profit or net income , the physician then participates or does not participate in all applicable - private business during the year . accordingly , we estimated the probability that the physician participates in year t as a function of allowance levels and other reimbursement variables , proxies for level of his or her short - run marginal cost function , proxies for the position of the demand function in the nonparticipating segment of his or her market , and a group of physician and patient - mix variables . five groups of explanatory variables were selected for this study from among those justified in the foregoing section . aprvu1 and aprvu5 denote the dollar amounts allowed per rvu in ucr and partial service business ( that is , the fee screen or fee schedule amounts set by the plans ) , respectively . both allowances were predicted to be positively correlated with the participation probability . in plan b the partial service allowance averaged about 55 percent of the ucr allowance during the four - year sample period . hence it was expected to have a somewhat stronger impact on the participation probability than the ucr allowance level . in plan a , where there were no partial service allowances , it was expected that the ucr allowance would have a stronger quantitative influence on the participation probability than the ucr allowance in plan b. lagprclm signifies the average number of days between filing a claim and receipt of reimbursement from the plan . long payment lags increase the practice 's accounts receivable , raise its interest costs , and shift its marginal cost function upward . a single measure of average amount charged was used because variation in charges across the private business lines was negligible . other things being equal , it was assumed that to the extent practice costs ( and quality ) are correlated with charges , high - priced physicians would tend to have high unit and marginal costs and to face strong demands for services produced on a nonparticipating basis . the fraction of the physician 's total number of rvus provided to hospital inpatients ( inpahosp ) was taken as proxy for the level of marginal costs and the average size of claims representing , in turn , the risk of bad debt on nonparticipating services . large values of inpahosp should imply low marginal costs and a high cost of bad debt on nonparticipating claims . it was further conjectured that large outputs of ucr and partial service rvus ( rvu1 and rvu5 ) increase the physician 's sensitivity to anticipated profit differentials between participating and not participating ( paringer , 1979 ) . the second group of explanatory variables consisted of a measure of physicians ' office wage rates ( wageindx ) and type - of - practice dummies reflecting possible economies of large scale . dummies indicating solo practice and practice in expense - sharing arrangements were deleted , so if there are important economies of scale , group practice ( group ) and partnership practice ( partner ) should denote relatively lower levels of unit costs . practice in hospitals and other institutional settings ( other em ) indicates a low level of non - physician expenses and should also denote a relatively lower level of unit production costs . the third group of explanatory variables is comprised of several county - level proxies for the position of the average revenue functions in the nonparticipating submarkets and in the participating submarket when the physician does not participate . they include per capita income ( inpercap ) , the fraction of the county population enrolled in medicare part b ( enrbprcp ) , the percentage of county residents living in urban areas ( prct - urb ) , the number of physicians per capita ( docprcap ) , and the number of hospital outpatient visits per capita ( outpprcp ) . increases in the values of each of the first three of these variables were assumed to signify outward shifts in the average revenue functions . increases in the values of the last two were assumed to denote backward shifts since they should be associated with fewer patients and/or diminished quantities demanded per physician . medical graduation in a foreign country ( fmg ) and board certification ( bordcert ) were taken as proxies for the perceived quality of the physician 's services and/or the level of production costs . in the first sense , they stand for the positions of average revenue functions with respect to all nonparticipating services , and , in the second , they stand for the position of the physician 's cost function . no hypotheses were proposed with respect to the effects of physician age ( age ) and sex ( amasex ) on the participation decision . although physician age has generally been found to be negatively correlated with assignment rates , it may capture the influence of the physician 's tastes , the perceived quality of his / her services , and the size of his / her nonparticipating and non - subscribing clienteles . thus , it was unclear on a priori grounds how age would be related to the participation probability , and similar comments apply with respect to the physician 's sex . specialty dummies denoting practice in internal medicine ( i m ) , pediatrics ( pd- ) , and the non - primary care fields ( othrspec ) were defined chiefly to reflect differences in participation propensities between the primary care and non - primary care fields . although demands in the nonparticipating markets may differ between primary care and referral practitioners , there were no obvious hypotheses concerning a systematic relationship between specialty and participation status . the final group of explanatory variables consists of three time dummies signifying the years of observation 1974 ( time74 ) , 1975 ( time75 ) , and 1976 ( time76 ) . the 1973 dummy was deleted . the variables were included as proxies for time - related events such as changes in reimbursement policies which might affect participation decisions but which could not be directly observed . with the physician designated as the analytic unit , the participation probability was specified as a regression function of the explanatory variables listed in table 2 and estimated from the combined cross - sectional and time - series sample of physician and county data . regressions were estimated separately using single - equation ordinary least squares ( ols ) , single - equation logit , and two - stage least squares ( tsls ) applied to a simultaneous system . all three sets of estimates were closely similar , and the tsls estimates , which are not shown , were nearly identical to the ols estimates indicating that simultaneity is a negligible source of bias in the single - equation regressions . for comparative purposes the ols and logit estimates of the participation probability are shown in tables 3 and 4 , respectively . the results strongly confirm the role of allowances , charges , reimbursement lags , and , in general , the relative income opportunities of participating and not participating , in the physician 's participation decision . coefficients on the allowance variables all had the expected signs and , with one exception , all were significant ( well below the 5 percent level ) . moreover , as anticipated , the ucr allowance had a much stronger influence on the participation probability in plan a ( both quantitatively and in terms of statistical significance ) than in plan b. and in plan b , the partial service allowance had a considerably stronger influence on the participation probability than the ucr allowance . the elasticities of the probability of participating with respect to allowances , estimated from the ols regressions at sample means , are : .838 for plan a 's ucr allowance ; .095 for plan b 's ucr allowance ; and .205 for plan b 's partial service allowance . by way of contrast , sloan and steinwald ( 1978 ) estimated the elasticity of the participation probability with respect to ( a proxy measure of partial service ) allowances at approximately .10 . large values of the first two signify high unit and marginal costs under the hypotheses given in the preceding section . thus , if upward shifts in the practice 's average and marginal cost functions reduce participation probabilities , wageindx and lagprclm should be negatively related to the participation probability , and the remaining four variables should be positively related to it . the signs of the coefficients on lagprclm , inpahosp , partner , and other - em were consistent with this interpretation , although the coefficients themselves were not uniformly significant . the signs of the coefficients on wageindx and group either varied between plan samples or else were not consistent with the prediction . thus , although there were some indications that participation rates fall with increasing unit or marginal costs , the results were not systematic . the effects of the county - level proxies for the positions of physicians ' average revenue functions for nonparticipating services were also somewhat mixed . per capita income and the fraction of the county population enrolled in medicare part b were negatively correlated with the participation probability . these results suggest that the relative profitability of participating is reduced by outward shifts in the average revenue functions for services on which physicians do not participate . however , the same line of reasoning would suggest that the coefficients on prct - urb should have been negative , and those on docprcap and outpprcp should have been positive . but there were no systematic patterns in the signs of the coefficients on these three variables . hence , the evidence is not conclusive that shifts in the nonparticipating average revenue functions influence participation probabilities . the surrogates for physician quality fmg , bordcert , and cprvu entered the regressions highly significantly and with the same signs for each plan sample . the findings here show unambiguously that high - quality physicians have materially lower participation rates than low - quality physicians . in terms of the theoretical model , they indicate that high - quality physicians face relatively large demands for services produced on a nonparticipating basis and have commensurately weak income incentives to enter into participation agreements where average revenues are lower . the relationships between the participation probability and physicians ' personal characteristics varied between plans . in each plan female physicians were more likely to participate than males , but the physician 's age had no systematic relationship with the participation probability . in plan a the participation probability rose significantly with the physician 's age , but in plan b the probability declined significantly with age . as a group , primary care practitioners seemed about as likely to participate as referral specialists ( othrspec ) , but there were marked differences in participation probabilities within the primary care fields . general and family practitioners had about the same participation rates as referral specialists , but pediatricians in both plans were significantly more likely to participate than general and family practitioners , and internists were significantly less likely to do so . the time dummies indicate similar patterns of autonomous shifts in the participation probabilities in the two plans over the four - year study period . in particular , the probabilities rose significantly in 1973 - 1974 , remained stable during 1974 - 1975 , and rose significantly again in 1975 - 1976 . although neither of the two plans imposed constraints on physicians ' allowances during the economic stabilization program , the common pattern of shifts in the participation probability suggests a common cause . one possibility is the restrictions on medicare allowances in effect during 1973 - 1974 and again after 1975 . restrictions on medicare allowances may have reduced the average revenue on medicare services sufficiently during 1973 - 1974 and 1975 - 1976 to make medicare business relatively less profitable during those years . such an effect would shift the average revenue function in the nonparticipating segment of the physician 's market inward and could have increased the physician 's incentives to participate in private business . unfortunately , we were unable to explore this possibility with the data available to us , but it appears to deserve further research attention . the volumes of the physician 's outputs in ucr and partial service business had no significant impacts on participation probabilities , and none was initially predicted . however , following paringer 's ( 1979 ) study of medicare assignment , where the hypothesis was first put forward , we conjectured that the responsiveness of physicians ' participation decisions to participating / nonparticipating net income differentials would increase as the volumes of participation - eligible business increase . while there are other ways of testing this conjecture , we attempted to replicate the approach used by paringer . both samples were stratified into terciles by the combined outputs of ucr and partial service business , and the single - equation version of the participation probability was re - estimated for each of the resulting subsamples . because of the close similarity of the ols and logit estimates for the full samples , the results are shown in tables 5 and 6 . if paringer 's hypothesis is correct , the absolute values and t - statistics of coefficients on all explanatory variables measuring the relative income opportunities of participation and nonparticipation should increase monotonically with output levels in private business where the physician is eligible to participate . none of the variable coefficients behaved strictly as the hypothesis predicts , but in plan a the sensitivity of the participation probability to changes in allowance and charge levels increased dramatically with the physician 's output of ucr services . indeed , the elasticity of the participation probability with respect to allowances , shown in table 7 , rose five - fold from the first to the third output terciles . the same type of pattern emerged in plan b for the first and second output terciles and for the first and third as well but not for the second and third . despite ambivalences in the evidence , it seems reasonable to conclude that there were interactions between physicians ' participation decisions and the volumes of their participation - eligible business . this , of course , is to say no more than that a physician is likely to react to relative income opportunities more strongly when the amount of business affected by his or her decision is large than when it is small . the physician participation agreements offered by the two study plans were of the all - or - nothing type . theoretically , the effects of physician , practice , and local market characteristics on participation decisions under this type of agreement have a high degree of uncertainty . except for the impacts of allowance levels , it is consequently difficult to argue that any particular group of characteristics will affect participation decisions in the same way regardless of the makeup of the physician population . this is not to say that examining all - or - nothing participation choices is irrelevant for policy purposes , but rather that the policy implications ought to be based on empirical observation . although the participation agreements offered by the two study plans have close parallels only in the present form of medicaid assignment , we believe the results have several important applications to all forms of government reimbursement policy for physicians . medical school , and high charge levels are proxies for physician quality , the evidence clearly indicates that high - quality physicians are weakly attracted into participation agreements . since the lower income portions of the population are precisely those served by or with the strongest incentives to visit participating and assignment physicians , it seems evident that the insitutions of assignment and participation tend to yield a relatively low quality of care to low income consumers . this conclusion should not be overemphasized , and , as sloan and steinwald ( 1978 ) have pointed out , it is probably an inevitable concommitant of any effort to constrain physicians ' average revenues which leaves the practitioner free to reject the program . it can also be argued that providing some type of physicians ' care to low income patients is preferable to offering little or none at all . nevertheless , the issue of controlling health care costs versus maintaining health care quality is one which policymakers must continue to confront . second , the finding that allowance levels exert a moderate to strong influence on the decision to enter into a participation agreement highlights a fundamental problem in physician reimbursement . inducing physicians to participate or to accept assignment and imposing constraints on their allowances is the cornerstone of current private and government reimbursement policy toward physicians . yet the evidence shows that raising allowance levels is arguably the only significant policy tool for increasing participation / assignment rates . and at some point , the costs to the public of increasing physicians ' allowances offset the savings due to controls on allowance levels . it is therefore reasonable to ask whether attempting to promote a 100 percent participation or assignment rate or perhaps even a rate close to 100 percent is necessarily a cost - effective method of paying for society 's medical care . third , some of our results suggest that blue shield participation rates are adversely affected when physicians ' income opportunities in the medicare program are raised . accordingly , it is reasonable to suppose that medicare and medicaid assignment is also adversely affected when physicians ' income opportunities in blue shield plans ' and other carriers ' private business are raised . if the supposition is correct , one can expect downward trends in medicare and medicaid assignment under any circumstances that lower physicians ' income opportunities from these sources ( such as new controls on medicare or medicaid allowances ) relative to those from carriers ' private business . the degree to which cost controls on government - and - privately - financed physicians ' services interact in physicians ' pricing and output decisions has not been systematically explored , and we were not able to investigate the issue here . finally , some authorities have proposed that medicare assignment be changed from its current claim - by - claim basis to an all - or - nothing system in order to strengthen the controls on medicare costs . it has also been suggested that medicaid and medicare assignment be tied together as a means of increasing the number of physicians who provide medicaid services . the essential question in the first proposal is whether changing the form of medicare assignment will , in fact , increase the rate of medicare assignment . on this point ideally , it would be necessary to compare the determinants of medicare assignment with those of all - or - nothing participation among the same group of physicians or among different physician samples with proper standardization . however , as has been explained , participating physicians in plan a whose patients submitted the claims were not effectively limited by the participation agreement to accepting amounts allowed as full payment . thus , in effect , they could participate on a case - by - case basis . however , the regressions based on proportions of rvus participating were virtually identical to those using a dummy variable for participation . this might indicate either a strong predisposition not to bend the rules or it could suggest that all - or - nothing participation is not much different from case - by - case participation in terms of the physician 's decision to participate . with respect to tying medicare and medicaid assignment together , the study 's findings do shed light on the consequences one might expect for the joint assignment rate . in plan b , physicians were allowed to participate only if they agreed to participate in high - allowance ( ucr ) and low - allowance ( partial service ) business . although the average participation rate was considerably higher in plan b than in plan a , where only high - allowance ( ucr ) participation agreements were offered , it is obviously not possible to attribute this difference to a single characteristic of the two participation agreements . indeed , in plan b the participation probability behaved as one would theoretically predict . participation probabilities varied significantly with allowance levels in both high - allowance and low - allowance business , but they were generally much more sensitive to reimbursement levels in the former than the latter . applying these results to a joint , all - or - nothing system of medicare and medicaid assignment , it is reasonable to believe that low medicaid allowances would dominate high medicare allowances in physicians ' assignment decisions . accordingly , the most likely effect of joint assignment should be an overall assignment rate between the current medicare and medicaid rates , and perhaps closer to the latter than the former . joint assignment should therefore increase access to physicians ' services by medicaid eligibles , but it should also lower the portion of medicare services subject to allowance controls on expenditures . this study does not point the way to a magic solution for controlling the costs of physicians ' care . instead , it emphasizes the tradeoffs between cost - containment on the one hand , and maintaining the quality and accessibility of physicians ' care or the market freedom of consumers and providers on the other . since it is increasingly doubtful that a magic solution exists , the time is right for reimbursement policymakers to recognize the tradeoffs and to base their calculations on them .

they alter the surrounding extracellular matrix and modify normal cell behavior to facilitate tumor cell growth , invasion , immune evasion , and angiogenesis . most of these effects are mediated by the release of small vesicles from the tumor cells into the extracellular medium . shed vesicles are known to facilitate tumor invasion [ 24 ] , mainly by proteolytic enzymes associated with their membrane [ 59 ] . indeed , the vesicle membranes are selectively enriched in some components including mmp-9 and other proteolytic enzymes [ 4 , 6 ] , together with 1 integrin and class i hla molecules . moreover , vesicles use several mechanisms to contribute to tumor escape from immune reactions [ 1116 ] . notably , vesicles carry many proangiogenic growth factors , expressed differently depending on the vesicle origin , and that act on endothelial cells to promote angiogenesis . indeed , fgf-2 was detected in vesicles shed by human hepatocarcinoma sk - hep1 cells [ 17 , 18 ] ; vegf was found to be present in vesicles shed by human ovarian carcinoma cells and in vesicles shed by neurons and astrocytes [ 20 , 21 ] ; angiogenin , il-6 , il-8 , vegf , and timps were found in vesicles shed by glioblastoma tumor cells . additionally , the sphingolipid fraction of vesicles shed by ht1080 fibrosarcoma and du-145 human prostate carcinoma cells also showed proangiogenic activity . sphingomyelin is a normal component of plasma membranes where it is largely clustered in the outer membrane leaflet . it is subjected to intense metabolism which is responsible for the formation of a number of bioactive metabolites including ceramide , ceramide-1-phosphate , sphingosine , and sphingosine-1-phosphate ( s1p ) . ceramide , generated by sphingomyelinase ( smase ) action on spingomyelin , appears to be a critical regulator of cell growth arrest , differentiation , and apoptosis [ 25 , 26 ] . sphingosine is formed by ceramide deacylation catalyzed by at least three different isoforms of ceramidase , which differ in optimal ph , primary structure , and cellular localization . the enzyme sphingosine kinase ( sphk ) catalyzes the formation of s1p from sphingosine and atp . two distinct sphk isoforms , sphk-1 and sphk-2 , have been cloned [ 29 , 30 ] . sphk-1 , the more intensely researched isoform , is primarily localized in the cytosol , but , following erk dependent phosphorylation elicited by various stimuli , it becomes translocated to the plasma membrane . sphk-1 has been shown to regulate a wide variety of cellular processes , including the promotion of cell proliferation , survival , and motility and , just as importantly , it possesses oncogenic potential . previous studies have established that sphk-1 , like fgf-2 and several other proteins , can be released in the extracellular environment although it lacks a conventional secretory signal sequence . the mechanism of sphk-1 secretion is unconventional and likely involves a nonstandard pathway independent of the endoplasmic reticulum / golgi system ; the sphk-1 secretion mechanism is only known to require functional actin dynamics . notably , the sphk product s1p , among multiple biological activities , exerts a strong proangiogenic effect which is known to act synergistically with growth factors such as fgf-2 [ 35 , 36 ] and vegf . in this study we investigated whether vesicles shed by hepatocarcinoma and carcinoma cultured cells contain s1p - generating enzymes . the data from this research demonstrates that neutral ceramidase ( ncdase ) and sphk-1 are localized in vesicles , supporting the view that s1p participates in the proangiogenic activity exerted by these particles . human sk - hep1 hepatocarcinoma cells were grown in dulbecco 's modified eagle 's medium supplemented with 10% fetal calf serum ( fcs ; euroclone , celbio ) . human breast carcinoma 8701-bc cells , kindly provided by profcessor minafra , were grown in rpmi 1640 supplemented with 10% fetal calf serum ( fcs ; euroclone , celbio ) . bovine gm7373 fetal aortic endothelial cells were grown in eagle 's minimal essential medium ( euroclone , celbio ) supplemented with 10% fcs , vitamins , and essential and nonessential amino acids . cells were removed from plate by a scraper and centrifuged at 2000 g for 5 minutes ; pelleted cells were then resuspended in 300 l of triton x100 ( 1% ) on phosphate buffer saline ( pbs ) . after 10- minute incubation at room temperature , the cell extract was centrifuged at 800 g for 10 minutes the amount of protein extracted from cells was determined using the bradford microassay method ( bio - rad , segrate , milan , italy ) employing bovine serum albumin ( sigma - aldrich ) as a standard . briefly , the medium was conditioned by culturing subconfluent healthy cells for 3 or 24 hours and were centrifuged at 2000 g for 10 minutes and at 4000 g for 15 minutes the supernatant was ultracentrifuged at 105,000 g in a ti-60 rotor ( backman ) for 90 minutes pelleted vesicles were resuspended in pbs . the amount of isolated vesicles was determined by measuring the protein concentration using the bradford microassay method ( bio - rad , segrate , milan , italy ) using bovine serum albumin ( sigma - aldrich ) as a standard . after sds - page electrophoresis were cast in 7.5% gels , proteins was blotted onto a nitrocellulose membrane ( hybond ; amersham biosciences ) that was saturated with 3% nonfat dry milk in tris buffer saline 50 mm ph 7.9/tween 0.05% ( tbs - t ) . after 5 washes in tbs - t for 5 minutes each , the nitrocellulose membranes were incubated overnight at 4c , with mouse monoclonal anti - ncdase antibody 1 : 200 ( kindly donated by professor ito , fukuoka , japan ) . the primary antibody was followed by peroxidase - conjugated anti - mouse antibodies ( 1 : 10000 ) ( amersham biosciences ) for 1 hour at room temperature . immunocomplexes were visualized with the ecl western blotting kit ( amersham biosciences ) using hyperfilm . cells , seeded at low density ( 2.000 cells / well ) onto microscope cover slips in 12-well culture plates ( nunc ) , were grown overnight in the complete medium and , when needed , for 3 more days in a serum - free medium with three medium changes . sphk-1 and sphk-2 were detected by using as primary antibodies , rabbit polyclonal anti - sphk-1 antibody ( kindly donated by prof . obeid , charleston , sc , usa ) 1 : 100 , and rabbit polyclonal anti - sphk-2 antibodies 1 : 100 ( kindly provided by dr . secondary antibodies used were antirabbit tritc - conjugated antibodies ( 1 : 200 sigma ) ; 1 integrin was detected using c27 anti-1 integrin rat primary monoclonal antibody 1 : 150 and antirat tritc conjugated secondary antibody ( 1 : 320 , sigma ) . fgf-2 was detected using mouse monoclonal anti - fgf-2 antibody ( 0.5 mg / ml 1 : 200 , upstate biotechnology type ii ) and texas redconjugated antimouse antibody ( 1 : 200 , amersham biosciences ) . in order to stain nuclei , cells were fixed in 3.7% formaldehyde and then stained for 10 minutes with propidium iodide ( sigma ) . immunostained cells were analyzed by confocal microscopy ( olympus 1x70 with melles griot laser system ) . vesicle lipids were stained with the lipophilic styryl compound fm4 - 64 ( molecular probes ) . purified vesicles ( 180 g ) were resuspended in 1 ml pbs and stained with fm4 - 64 dissolved in pbs without calcium and magnesium . fm4 - 64 was added at a final concentration of 5 g / ml ; samples were incubated at room temperature for 15 minutes . stained vesicles were collected by centrifugation at 50,000 g for 1 hour , resuspended in 50 l pbs and added to gm7373 cells to monitor vesicle targeting . sk - hep1 cells were plated in six - well culture plates at 3 10 cells / well and maintained overnight in high - glucose dmem containing 10% fetal calf serum , 2 mm l - glutamine , 100 iu / ml penicillin , and 100 g / ml streptomycin . the next day , cells were transfected with skpegfp plasmid encoding for sphk - gfp chimera ( kindly donated by professor spiegel , richmond , va , usa ) . transfection was carried out using lipofectamine reagent ( gibco life technologies ) , according to the manufacturer 's instructions . sphk activity was assayed in isolated vesicles or serum - free conditioned medium as described by olivera et al . . briefly , 50 g vesicle proteins were resuspended in 100 l of the reaction mixture which contained 20 mm tris - hcl , ph 7.4 , 20% ( v / v ) glycerol , 1 mm -mercaptoethanol , 1 mm edta , 1 mm sodium orthovanadate , 15 mm sodium fluoride , protease inhibitors ( 10 mg / ml leupeptin,10 mg / ml aprotinin , and 1 mm pmsf ) , and 0.5 mm 4-deoxypyridoxine . the serum - free conditioned medium was concentrated approximately 40-fold before being employed for enzymatic activity measurement . the enzymatic reaction was initiated by adding 50 m sphingosine and 1 mm [ p ] atp . in some cases , after 30- minutes incubation at 37c , the reaction was terminated by adding 20 l 1 n hcl and 900 l of chloroform / methanol / hcl ( 100 : 200 : 1 , v / v ) . lipids were then extracted , separated by tlc , labeled sip and quantified by liquid scintillation essentially as previously described . specific activity of sphk was expressed as pmol of s1p produced / min / mg of protein . ncdase activity was determined using c12-nbd - ceramide as a substrate as previously described . briefly , 100 pmol of c12-nbd - ceramide ( nbd - c12:0 , d18:1 ) was incubated for 2 h at 37c with an appropriate amount of proteins in 20 l of 25 mm tris - hcl buffer ph 7.5 and 0.25% ( w / v ) triton x-100 . samples were then applied to a tlc plate , which was developed with chloroform , methanol , and 25% ammonia ( 90 : 20 : 0.5 , v / v ) . spots corresponding to nbd - dodecanoic acid and c12-nbd - ceramide were scraped , incubated with methanol at 37c to extract the compounds from the silica , and their fluorescence at 470/525 nm excitation / emission wavelengths was measured using a shimadzu 9000 spectrophotofluorimeter . the compounds were quantified using a standard curve of known amounts of c12-nbd - ceramide and nbd - dodecanoic acid . in a first group of experiments , expression and localization of sphk-1 and sphk-2 were analyzed by immunofluorescence in 8701 bc carcinoma cells and in sk - hep1 hepatocarcinoma cells ( figure 1 ) . moreover , since it had been previously demonstrated that 1 integrin is clustered in shed vesicles [ 7 , 17 ] , distribution of the two proteins was compared with distribution of 1 integrin . in both cell lines , the distribution of sphk-2 was quite different from the distribution of 1 integrin ( figures 1(a ) and 1(b ) line a ) . however , it was absent in the cell membrane where instead 1 integrin was located . sphk-1 and 1 integrin appear to colocalize at the plasma membrane ( figures 1(a ) and 1(b ) line b ) . moreover , as can more clearly be seen in cells transiently transfected with gfp - sphk-1 ( figure 2 ) , both sphk-1 and 1 integrin seem to be more dense in specific areas of the plasma membrane , and clustering appears to occur in areas of the cell membrane from which vesicles are released ( figure 2(d ) ) . in a previous study , we observed that vesicles shed by sk - hep1 cells mediate fgf-2 release and that vesicle shedding and release of fgf-2 were simultaneously induced by the addition of serum to starved cells . by monitoring intracellular movements of the growth factor subsequent to serum addition , we showed that within one hour fgf-2 was targeted to the cell periphery and to the cell nucleus and nucleolus . similarly to fgf-2 , sphk-1 is a leaderless protein secreted by unconventional mechanisms [ 29 , 34 ] whose movement toward the cell periphery is mediated by actin filaments . we therefore theorized that the two proteins could share a similar export mechanism and we analyzed whether intracellular movements of sphk-1 were influenced by serum addition and whether the enzyme colocalized with fgf-2 . intracellular distribution of sphk-1 was therefore analyzed by immunolocalization in starved cells as well as at time intervals after serum addition and compared with fgf-2 distribution . as shown in figure 3 , in starved cells the two proteins did not colocalize . in sphk-1 thirty minutes after the serum was added both proteins were clustered in granules and showed a clear colocalization . one hour after the serum was added large granules containing both proteins were present near the cell membrane . these results suggest that fgf-2 and sphk-1 share a similar transport mechanism toward the cell periphery and that the two proteins are both likely to be targeted to the budding vesicles . in order to establish if an active form of sphk-1 is shed as a component of membrane vesicles , we ascertained sphk activity in vesicles or cell - conditioned media in some experiments . results reported in table 1 show that sphk-1 was clearly detectable in vesicles shed by sk - hep1 and 8701-bc cells , although the activity was found to be greater in vesicles shed by sk - hep1 cells . no enzymatic activity could be detected in serum - containing media , even when it was concentrated . instead , it was possible to detect a low sphk activity in 40-fold - concentrated serum - free medium that had been conditioned by maintaining cells in culture for 24 h. as shown in table 1 , when vesicles were collected from a medium to which 2 m nacl had been added in order to solubilize proteoglycans and other molecules unspecifically bound to the vesicle membrane , the enzymatic activity was not affected . the addition of sphingosine increased labeled s1p production ; however , the catalytic activity of the enzyme was also observed when this substrate was not added . sphingosine is likely produced by ncdase , which is known to be localized in plasma membranes and also found in the extracellular medium [ 46 , 47 ] . an enzymatically active form of ncdase was actually found to be present in vesicles shed by 8701 bc ( figure 4 and table 2 ) . moreover , in sk - hep1 cells , we observed colocalization of ncdase and 1 integrin ( data not shown ) , indicating that it is also likely that ncdase is present in vesicles shed by this cell line . since s1p can be produced in the membrane of shed vesicles , the molecule could remain , at least in part , within the vesicle . vesicles could adhere to the plasma membrane of cells surrounding the tumor and s1p could exert its effects by interacting with receptors localized at the cell surface . on the other hand , vesicles could be internalized by the host cell and consequently release s1p inside cells where it could act as intracellular messenger . in order to verify these hypotheses , we analyzed the targets of shed vesicles after adding them to gm7373 cells , an immortalized line of embryonic bovine aortic endothelial cells . for this purpose , vesicles released by sk - hep1 cells were labeled for 15 minutes with the lipid marker fm4 - 64 . labeled vesicles were then added to in vitro cultured gm7373 cells which in turn had been labeled with antibodies against 1 integrin . as shown in figure 5 , at 10 minutes after incubation vesicles were observed to be bound to the cell membrane , while after 20-minutes incubation most vesicles were internalized and visible in the cytoplasm . at 30 minutes , the signal borne by lipid marker fm4 - 64 was no longer visible , indicating that the lipids of the vesicle membranes had degraded . in principle therefore , vesicle - associated s1p could act on both membrane receptors and intracellular targets . membrane vesicles shed by tumor cells appear to exert a variety of effects on the surrounding cells . vesicles are rich in enzymatic activities able to modify extracellular medium composition , thus facilitating tumor cell migration and angiogenesis . depending on their origin , they also convey different signaling molecules which exert their effects on lymphocytes , mesenchymal cells , and endothelial cells . shed vesicles have been shown to induce angiogenesis using a variety of mechanisms including the action of proteins such as fgf-2 , vegf , angiogenin , il-6 , il-8 , and timps , and lipid molecules such as sphingomyelin . based on the present results , ncdase and sphk-1 can now be included among the signaling molecules transferred by shed membrane vesicles , suggesting that s1p formed at level of the vesicle membranes plays a role in the biological processes regulated by these particles . interestingly , ncdase , here identified as a component of shed vesicles , was previously identified in various subcellular compartments such as endosomes , mitochondria , and microdomains of the plasma membrane [ 39 , 48 ] but was also found to be involved in extracellular sphingolipid metabolism . in this regard it was demonstrated that although ncdase is localized at the plasma membrane as a type ii integral membrane protein , the enzyme is released in the extracellular medium after the proteolytic action of secretases [ 49 , 50 ] . moreover , in agreement with the present results , ncdase , together with acid smase , was identified as a component of a complex in the cell membrane domain which is subjected to budding as well as in conditioned medium associated with caveolin-1 , a key structural protein of caveole which was also detected in shed vesicles . sphk-1 is a secreted leaderless protein , and the shedding of membrane vesicles appears to represent a mechanism which accounts for its secretion . the presence of the enzymatic protein in shed vesicles does not per se exclude the face that other mechanisms may also participate in the release of sphk-1 . indeed it was reported that , in fgf-1 overexpressing nih 3t3 cells , sphk-1 is secreted together with fgf-1 as a component of a high molecular weight complex [ 53 , 54 ] . however , sphk-1 is also secreted by cells which do not express fgf-1 , and in the absence of stress signaling which induces fgf-1 secretion . here we have demonstrated that at least in some instances sphk-1 is secreted as a component of shed vesicles . since shed vesicles also contain ncdase , which provides the rate - limiting substrate for s1p production by catalyzing sphingosine generation , it is likely that these particles cause sustained s1p production . sphk-1 and s1p produced by its enzymatic activity are able to mediate a network of paracrine signaling . it is well known that acting on the two membrane receptors sip1 and sip3 , s1p induces morphogenesis in huvec cells . moreover , since vesicles carry several other molecules able to affect angiogenesis , the overall effects of vesicles on surrounding endothelial cells will be amplified and differently modulated depending on the specific composition of the vesicles . the exact mechanism by which the s1p message borne by shed vesicles is delivered to the host cell remains to be explored . indeed , an attractive hypothesis is that after interacting with the recipient cell plasma membrane , vesicles are internalized via endocytosis . sphk-1 and sip would therefore be delivered into the cytoplasm of the receiving cell , where , as already known , sip could exert its intracellular effects , regulating various processes among which cell survival is prominent [ 55 , 56 ] . indeed vesicles were shown to convey molecules , such as mrna and irna , to the cytoplasm of surrounding cells , and it was recently reported that mrna included in shed vesicles can be translated in recipient cells following endocytosis . alternatively , because sphk-1 association with cell membranes is strengthened by interaction with phosphatidylserine following its phosphorylation and since exposure of phosphatidylserine on the outer leaflet is a hallmark of shed membrane vesicles [ 58 , 59 ] , it can also be speculated that sphk-1 , localized in these particles , can generate s1p in the extracellular environment . if this is the case , the bioactive lipid generated outside the endothelial cells could determine key effects on development and proliferation of endothelial cells , acting as a ligand of s1p1 and/or s1p3 receptors [ 60 , 61 ] . independent of the action mechanism , the ability of shed vesicles to carry on key enzymes for s1p production which this study brought to light illuminates a novel aspect of their biochemical properties which is relevant to a complete understanding of their proangiogenic activity .

neuroeconomics is a general framework for studying the neural basis of economic decision making . at first glance , one may wonder how this discipline can be relevant to the sort of issues that neurologists and psychiatrists are concerned with , beyond identifying the causes of pathological gambling or compulsive spending disorders . although money is often used as a convenient proxy of value in behavioral and neuroimaging studies , economic analysis of choice behavior can be applied in various situations where individuals need to estimate the desirability of goods and services in the broadest possible sense , for example , when making choices about food items on a restaurant menu , physical exercise , career prospects , commitment in a relationship , a child 's education . the goal of economic theory is to account for choice behavior , using a restricted set of assumptions , the most fundamental one being that agents are essentially self - interested , rational maximizers , computing utility functions , weighing the expected value of an outcome by its probability or the delay in obtaining it . behavioral economics further attempts to account for observations that seem to deviate from the predictions of standard economic theory , like the fact that individuals sometimes systematically over- or underestimate probabilities or act altruistically , by taking into account how cognitive , emotional , or social factors interact to bias our choices . bringing concepts from economic theory into the realm of everyday behavior thus provides a formalization of what makes a prospect attractive or repulsive in terms of specific , tractable computations . these computations can then be traced to the activity of single neurons or populations of neurons in different brain regions , or revealed through specific selective deficits in pathological conditions . our objectives in this brief review are to introduce some of the core concepts used in behavioral economics , to make arguments based on data from animal research for the evolutionary origin of the basic neuronal mechanisms for value - based decision making , and to illustrate with clinical studies the relevance of a neuroeconomic approach in appraising different types of behavioral and cognitive disturbances . in most natural situations , our choices are rarely totally black or white , and predicting how people will choose requires an accurate model of how value is assigned to outcomes . the simplest possible computation of subjective value is a product of utility , u , and its probability of occurrence , p ( let us assume that the cost associated with 1-p is null ) . this seems like a reasonable starting point since most decisions are probabilistic : for example , expectations of a successful day at the beach hinge on rainfall statistics , and deciding to wait for a hotline operator is influenced by the probability of simultaneous calls from other customers at that particular time of the day . the prospects of winning 1 with a fixed probability of p=1 or winning 10 with a fixed probability of p=1/10 lead , in the long run , to similar average gains , but can generate different preferences depending on an individual 's attitude toward risk . furthermore , in real - world settings , probabilities are generally not fixed and fluctuate over time . experimental studies have shown that expected values are computed by taking into account the mean and variance of outcome probability distributions . decisions generally produce outcomes in the future , and outcomes that occur later are devalued relative to identical , but immediate , ones . again , experimental data show that optimal decision making in humans is based on time discounted expected values of outcomes . game - theory approaches are used to analyze scenarios in which an outcome is contingent upon mutual decisions made by two or more individuals . a typical example is the prisoner 's dilemma two players must simultaneously choose one of two possible options . payoffs are organized such that it is most advantageous for both players to choose option a. however , if only the first player picks option a and the second player opts for b , the first player will end up with a far worse outcome than had he / she chosen the less attractive option b.this type of game is a model of actual social interactions where individuals need to anticipate each other 's intentions . if the prisoner 's dilemma is played only once , game theory predicts that rational self - interested players will choose option b. if repeated multiple times , players can respond to each other 's actions and have opportunities to retaliate for or forgive noncooperative behavior , and this may engender different dominant strategies ( eg , tit for tat , win - stay / lose - shift ) . rationality assumptions posited by economic theory generate predictions that are often verified , but the literature is replete with paradoxes and violations to what is sometimes referred to as the standard model . in fact , much current work in behavioral economics is devoted to refining or proposing alternative theories in order to accommodate experimental observations . , biases such as the tendency to overestimate small probabilities can lead to an exaggerated aversion to certain types of risky choices . utilities also show nonlinearities with losses looming larger than same - sized gain , a phenomenon referred to as loss aversion . unexpected outcomes in social games occur because of individual differences in the theory of mind however , some of the most important challenges to the standard model are those that question the rationality assumption itself and highlight the impact of emotions , such as regret , envy , altruism , and inequity aversion . for example , in another widely used test , the ultimatum game , a player is given a sum of money , part of which has to be transferred to a second player ; that player can then accept the offer , and both players keep their respective share , or reject the offer , and both players leave empty - handed . strict utilitarianism predicts that the first player should transfer the smallest nonzero amount possible and the second player should accept it , because something is always better than nothing . but the common finding is that the first player transfers a sizeable share of the money , possibly because of an inherent prosocial motivation for cooperation and reciprocity or perhaps more pragmatically because , anticipating the other 's inequity aversion , he / she wishes to preempt rejection of an offer that would be considered unfair . it can thus be seen that empirical descriptions provided by models of real - world decisions can be considered as normative behavior when evaluating pathological deviations in patient populations . neuroimaging studies have contributed to the identification of the cerebral networks activated by value estimations and choice behavior in humans . many of the behavioral observations made in humans have been verified in animals , including probability distortions , long - term reward rate maximization , and inequity aversion , thus underlining the relevance of animal research . in fact , much of the human neuroimaging work has been directly inspired by findings from neuronal recording experiments conducted in rodents and nonhuman primates ( figure 1 ) . we briefly summarize some of the key results regarding the neuronal basis of reward processing and choice behavior , drawing on work conducted in nonhuman primates , the species whose cerebral organization is closest to ours . an extensive presentation of the flourishing literature based on human functional magnetic resonance imaging ( fmri ) is beyond the scope of the present paper and interested readers can find several detailed reviews in refs 7 - 9 . the biological cornerstone of valuation processes is the brain 's reward system and one of its major components , the dopamine neuronal pathway . midbrain dopamine neurons have been implicated in different functions but first and foremost in signaling the hedonic value of stimuli in the environment and are thus often considered as forming the brain 's reward retina . the response properties of macaque dopamine neurons have been found to compute variables essential for reinforcement learning , such as the difference between expected and actual reward outcomes . in simple classical conditioning experiments , dopamine neurons respond to unexpected rewards , with a sharp increase in discharge rate , and to expected but absent rewards , with a sharp decrease in discharge rate . these signals are believed to contribute to learning by signaling both positive and negative changes in the environment , corresponding to reward or utility prediction error postulated by models of economic decision . dopamine neurons also encode reward expectation through tonic discharges that , interestingly , scale with reward probability and thus convey information about risk and uncertainty . explicit reward signals are found in most midbrain dopamine neurons , as well as in subsets of neurons receiving dopaminergic projections in the orbitofrontal cortex ( ofc ) , in the ventral striatum , and in the amygdala . the ofc contains a representation of primary reinforcers , such as food or sexual stimuli . these are neurons that discriminate sensory quality within and across category , but do not merely encode the physical characteristics of stimuli . activity is elicited by a specific food item when the animal is motivated to acquire it , but stops if the animal is sated . marginal utility of that particular food is sufficiently high , ie , if it satisfies a current need or desire . rewards and reward expectation also affect the activity of more dorsal and medial regions of the prefrontal cortex involved in action selection and planning . in fact , many cortical ( prefrontal , cingulate , parietal , infero temporal cortex ) and subcortical ( striatum , amygdala , superior colliculus ) structures involved in high - level sensory and/or motor integration receive reward information . unrewarding and aversive events are also represented most commonly in distinct neuronal subpopulations in the midbrain , cortex , and amygdala . however , cells in the posterior parietal cortex have been shown to encode both rewarding and aversive stimuli , in which case activity is interpreted as reflecting motivational salience rather than value . together , these results suggest that reward information serving to select relevant stimuli and to guide goal - directed approach and avoidance behavior is broadcast widely throughout the primate brain . if reward neurons are to encode decision variables , their activity should reflect subjective value in a generic way ; that is , preferences for different goods should be encoded in a common currency . however , their preferences can be precisely estimated by offering fruits or juices in different amounts and measuring subjective equivalences ; for example , how much a grape is worth in bits of cucumber . when recorded during the performance of such a task , action potentials of dopamine neurons and reward - sensitive neurons in the ofc have been shown to be discharged in equal numbers for subjectively equivalent food rewards , irrespective of nature or amount , and to scale their firing rate in direct function of behavioral preference , thus effectively representing value in a common neural currency . further studies have focused on how the brain translates subjective value into actual choices and actions . real - world economic decisions can sometimes be straightforward but mostly involve multiple , ambiguous outcomes and require the integration of several factors , for example , delay , uncertainty , and action cost . formal models of decision generally postulate a competition between signals that accumulate in favor of each option , racing toward a threshold and culminating into a sort of winner - take - all decision . evidence for such a process has been observed in several regions , including the dorsal and ventral striatum , and the dorsolateral prefrontal , posterior parietal , and anterior cingulate cortex ( acc ) , although the models best fitting the data remain a subject of debate . identifying the elementary neural mechanisms used to compute expected values and guide rational decision making is a central objective of research in neuroeconomics , but also in the domains of reinforcement learning ( concerned with animals and humans ) and machine learning ( concerned with computer models and robots ) . all three disciplines are interested in how rewards and punishments shape responses to the environment bymeans of signals arising in midbrain dopamine neurons . studies conducted in patients in whom dopamine function is altered , like parkinson disease , tourette syndrome , or attention - deficit hyperactivity disorder , are thus directly relevant to this issue . for example , patients with tourette syndrome show early onset physical and vocal tics , believed to result from dopamine - induced basal ganglia dysfunction and often treated with neuroleptics and dopamine antagonists . in a series of elegant studies , worbe et al and palminteri they used a simple cued go / no - go task to expose participants to cue - reward associations ( monetary gain ) as well as cue - punishment associations ( monetary loss ) . patients showed learning profiles according to their treatment status that varied in a manner consistent with the hypothesis of dopamine hyperfunctioning in tourette syndrome . when off medication , patients had impaired punishment learning , whereas when on medication they had impaired reward learning . the authors interpret this pattern of findings according to the theory of dopamine 's dual action in reward and punishment learning through , respectively , positive and negative prediction error signals . in untreated patients , because baseline levels of dopamine are too high , punishments can not pull the dopamine response low enough to generate a negative prediction error signal . by contrast , in patients treated with dopamine antagonists , baseline levels of dopamine are kept too low , and rewards can not push the dopamine response high enough to generate a positive prediction error . in support of this hypothesis , a similar , but mirror - reversed dissociation in reward and punishment learning pattern has been found in patients with parkinson disease on ( high baseline dopamine ) and off ( low baseline dopamine ) l - dopa treatment . although a faulty reinforcement learning mechanism can not account for the entire spectrum of deficits observed in basal ganglia disorders , it could partly explain a number of motor and cognitive symptoms . reduced negative reinforcement due to excessive dopamine activity could be involved in the inability to suppress tics in tourette syndrome , but also in impulse control disorders , such as pathological gambling and hypersexuality , all of which have also been observed as side effects of l - dopa treatment in parkinson disease . the major drugs of addiction have a variety of pharmacological actions resulting in overstimulation of the dopaminergic reward system . reduced reward sensitivity due to low dopamine levels might be involved in early nonmotor symptoms of parkinson disease and in psychiatric conditions associated with depression and apathy . considerations about the role of dopamine imbalance must also take into account the heterogeneity of lesions and of functional anomalies in the midbrain , striatum , and associated cortical and limbic networks . understanding the specificity of behavioral disturbances in light of the known anatomy and pharmacology of the basal ganglia is a key objective for much human and nonhuman primate research in this field . the ventromedial and orbitofrontal regions are important cortical targets of dopaminergic projections , but also receive inputs from limbic structures , hippocampus , and other cortical areas . probably the first and best - known study of economic decision making in brain - lesioned patients is that of bechara and collaborators , who introduced the iowa gambling task , in which participants are presented with four different decks from which they pick cards in order to earn game money . two decks contain some cards with small gains and fewer cards with small losses , yielding , on average , positive payoffs . two other decks contain some cards with high gains and a larger number of cards with high losses , yielding , on average , negative payoffs . normal subjects , after sampling cards from each deck , develop a preference for the good decks and avoidance of the bad ones . also , as learning takes place , subjects show an increase in skin conductance before picking cards from the bad decks , even before they become aware of any difference in expected value among the four decks . this implicit stress response is considered evidence for the activation of somatic and emotional signals in guiding decisions , the so - called somatic marker hypothesis . when tested with the iowa gambling task , patients with ventromedial prefrontal cortex ( vmpfc ) lesions and patients with bilateral amygdala lesions failed to develop a preference for the safer advantageous deck and either chose randomly or had a preference for the risky gamble , leading to overall monetary losses . interestingly , while control subjects and prefrontal cortex - lesioned patients showed skin conductance responses to winning and losing , amygdala - lesioned patients failed to show such responses . furthermore , contrary to controls , neither patient group showed anticipatory skin conductance responses to the risky deck , suggesting that defective emotional signaling in the amygdala and defective routing of this information through prefrontal cortex resulted in a similar negative impact on loss ( or risk ) aversion learning . bechara et al point out that some of the vmpfc patients were aware that the decks were bad yet persevered in their risky choices ; the authors draw a parallel with other pathological conditions , such as drug addiction or psychopathy where maladaptive or pathological behavior persists despite intact awareness of the consequence of one 's actions . our group used a different type of gambling game in order to study the interactions between emotions and value - based decisions in patients with ofc lesions . subjects chose between two gambles of known probabilities and rated subjectively felt emotions after the outcome was revealed . different types of emotions could be measured depending on how much information was supplied . in the presence of feedback on the chosen gamble only , subjects experienced either satisfaction or disappointment , depending on the outcome value . when feedback was given on both gambles , they could compare what is with what could have been and hence could experience more complex forms of emotions , such as rejoicing or regret . this is an important issue , because in behavioral economics , decisions made under uncertainty generate regret aversion , leading subjects to estimate ( and sometimes overestimate ) future regret when making choices in order to minimize the risk of experiencing it later . patients with ofc lesions reported feeling elated and saddened when their chosen gamble produced wins and losses , respectively . they also showed commensurate emotional arousal , as measured by their skin conductance response , in agreement with previous findings . however , contrary to control subjects , neither their emotional responses nor their decision strategies were affected by information on the foregone option . for instance , when the chosen gamble yielded a small positive earning , their satisfaction level was not reduced by discovering that the outcome of the other gamble was even more advantageous , nor was it enhanced when discovering it was much worse . although lack of regret could also reflect blunted somatic markers , it should be emphasized that this is a cognitive - based emotion requiring a capacity to perform counterfactual comparison , a process that might be impaired in these patients . work on monkeys shows that the acc , an area closely connected to limbic and prefrontal regions , contains neurons that encode the size of rewards , obtained as well as unobtained , in a simple decision task . the acc is thus suited to provide active outcome value signals needed to compare factual and counterfactual information . neuroimaging studies in humans have shown a cerebral regret network that includes the amygdala , ofc , acc , and hippocampus . whereas simple monetary wins and losses in the two - choice gambling task modulate reward - related areas , such as the ventral striatum , experienced and anticipated regret other imaging studies have confirmed the key role of the ofc in the regret mechanism , with a possible functional distinction between lateral and medial ofc ( see ref 32 for a review ) . the tremendous success of smartphone photo exchange apps and online social networks is a patent testimony to the high value we assign to observing and learning about our conspecifics . social comparison is a basic human activity . as karl marx noted in wage labor and capital , it is a major driving force in the burning pursuit of wealth , and it is a source of no less burning frustration : a house may be large or small ; as long as the neighboring houses are likewise small , it satisfies all social requirement tor a residence . but let there arise next to the little house a palace , and the little house shrinks to a hut . the little house now makes it clear that its inmate has no social position at all to maintain , or but a very insignificant one ; and however high it may shoot up in the course of civilization , if the neighboring palace rises in equal or even in greater measure , the occupant ot the relatively little house will always find himself more uncomfortable , more dissatisfied , more cramped within his four walls . the role of inequity aversion in decision making is well documented in humans and in several nonhuman primate species . in contrast , if the other 's reward serves as reference point , ie , when an individual has to make decisions about somebody else 's money rather than about t heir 's own money , emotional engagement is reduced and risk evaluation less constrained by loss aversion . impairments in socially based cognition and communication have been reported in brain - lesioned patients . patients with vmpfc lesions engage in socially inappropriate behavior , lacking in awareness of their social norm violations , and fail to adjust their communicative behavior according to the social partner , thus raising the question of the role of prefrontal areas in social decision making . experiments in nonhuman primates have explored how neural activity is affected when decisions are made in a social context . when given the opportunity to provide food or to prevent infliction of unpleasant stimuli to conspecifics , monkeys express variable , partner - dependent social tendencies but mainly make prosocial decisions . in the ofc , we have shown that own - reward sensitive neurons respond to a fixed reward to self with different discharge rates , depending on whether monkeys are working to obtain a reward for self only or for both self and a partner monkey . activity modulations are consistent with simultaneously recorded behavior , as equity - averse animals show reduced neuronal activity to shared reward ( same amount granted to both ) and equity - seeking animals show enhanced activity . whereas neurons in the ofc and in the striatum only encode own rewards in a social context , acc neurons distinguish between rewards to self and to others , and medial prefrontal neurons track rewards and action of partner monkeys . finally , in a recent experiment using as experimental paradigm an iterated prisoner 's dilemma game , neuronal activity in the dorsal acc was shown to predict another player 's intention to cooperate or to defect , and disruption of its activity by electrical stimulation reduced the tendency to produce mutually beneficial choices . although food trading may seem quite rudimentary in comparison with complex human social exchanges , these results highlight the key role played by prefrontal cortical areas in regulating the basic mechanisms of cooperation and competition . patients with vmpfc lesions were found to reject unfair offers more often than control subjects in a single - round ultimatum game , but only when outcomes were obtained after a delay and not when outcomes were displayed and gains immediately available . thus , rather than reflecting anomalous inequity aversion , the explanation advanced by the authors is that after damage to the vmpfc , future gains are more rapidly discounted ( a so - called myopia for the future ) , thereby making the subjective value of an unfair offer appear even lower and more unacceptable than it would to a normal individual . whereas control subjects display more inequity aversion when facing a human opponent than a computer opponent , choices made by patients with lesions in the vmpfc did not distinguish between the two situations and only depended on the distribution of monetary outcomes differentiation of human versus computer partners is believed to reflect the role of social norms in economic decision making and , in the present case , the need felt to administer costly altruistic punishment to human partners who behave unfairly . by contrast , since inanimate objects can not be held responsible for their choices , the dominant strategy when playing against a computer is rational maximization ( ie , accepting a very small offer is better than nothing ) . in a later study , decisions made by vmpfc - lesioned patients were analyzed in the context of the trust game , a social exchange test evaluating trust and reciprocity . in this game , the investor has to transfer all or part of an initial endowment , knowing that the amount invested is tripled as it is transferred to the trustee , and the trustee must then decide what fraction of that amount to return to the investor . as in the previous study , in contrast to control subjects who were more risk adverse ( less trusting ) with a human than a computer partner , vmpfc - lesioned patients did not act differently in the two settings and they displayed less generosity as trustee . the human vs computer dissociation is taken as evidence of fear of betrayal , another social emotion influencing risky decisions . nevertheless , observations made in patients with ofc and vmpfc lesions suggest that this region plays a key role in regulating decision making through nonsocial emotions , such as anticipated regret and social / moral emotions , such as envy , fairness , betrayal , and guilt . identifying the elementary neural mechanisms used to compute expected values and guide rational decision making is a central objective of research in neuroeconomics , but also in the domains of reinforcement learning ( concerned with animals and humans ) and machine learning ( concerned with computer models and robots ) . all three disciplines are interested in how rewards and punishments shape responses to the environment bymeans of signals arising in midbrain dopamine neurons . studies conducted in patients in whom dopamine function is altered , like parkinson disease , tourette syndrome , or attention - deficit hyperactivity disorder , are thus directly relevant to this issue . for example , patients with tourette syndrome show early onset physical and vocal tics , believed to result from dopamine - induced basal ganglia dysfunction and often treated with neuroleptics and dopamine antagonists . in a series of elegant studies , worbe et al and palminteri they used a simple cued go / no - go task to expose participants to cue - reward associations ( monetary gain ) as well as cue - punishment associations ( monetary loss ) . patients showed learning profiles according to their treatment status that varied in a manner consistent with the hypothesis of dopamine hyperfunctioning in tourette syndrome . when off medication , patients had impaired punishment learning , whereas when on medication they had impaired reward learning . the authors interpret this pattern of findings according to the theory of dopamine 's dual action in reward and punishment learning through , respectively , positive and negative prediction error signals . in untreated patients , because baseline levels of dopamine are too high , punishments can not pull the dopamine response low enough to generate a negative prediction error signal . by contrast , in patients treated with dopamine antagonists , baseline levels of dopamine are kept too low , and rewards can not push the dopamine response high enough to generate a positive prediction error . in support of this hypothesis , a similar , but mirror - reversed dissociation in reward and punishment learning pattern has been found in patients with parkinson disease on ( high baseline dopamine ) and off ( low baseline dopamine ) l - dopa treatment . although a faulty reinforcement learning mechanism can not account for the entire spectrum of deficits observed in basal ganglia disorders , it could partly explain a number of motor and cognitive symptoms . reduced negative reinforcement due to excessive dopamine activity could be involved in the inability to suppress tics in tourette syndrome , but also in impulse control disorders , such as pathological gambling and hypersexuality , all of which have also been observed as side effects of l - dopa treatment in parkinson disease . the major drugs of addiction have a variety of pharmacological actions resulting in overstimulation of the dopaminergic reward system . reduced reward sensitivity due to low dopamine levels might be involved in early nonmotor symptoms of parkinson disease and in psychiatric conditions associated with depression and apathy . considerations about the role of dopamine imbalance must also take into account the heterogeneity of lesions and of functional anomalies in the midbrain , striatum , and associated cortical and limbic networks . understanding the specificity of behavioral disturbances in light of the known anatomy and pharmacology of the basal ganglia is a key objective for much human and nonhuman primate research in this field . the ventromedial and orbitofrontal regions are important cortical targets of dopaminergic projections , but also receive inputs from limbic structures , hippocampus , and other cortical areas . probably the first and best - known study of economic decision making in brain - lesioned patients is that of bechara and collaborators , who introduced the iowa gambling task , in which participants are presented with four different decks from which they pick cards in order to earn game money . two decks contain some cards with small gains and fewer cards with small losses , yielding , on average , positive payoffs . two other decks contain some cards with high gains and a larger number of cards with high losses , yielding , on average , negative payoffs . normal subjects , after sampling cards from each deck , develop a preference for the good decks and avoidance of the bad ones . also , as learning takes place , subjects show an increase in skin conductance before picking cards from the bad decks , even before they become aware of any difference in expected value among the four decks . this implicit stress response is considered evidence for the activation of somatic and emotional signals in guiding decisions , the so - called somatic marker hypothesis . when tested with the iowa gambling task , patients with ventromedial prefrontal cortex ( vmpfc ) lesions and patients with bilateral amygdala lesions failed to develop a preference for the safer advantageous deck and either chose randomly or had a preference for the risky gamble , leading to overall monetary losses . interestingly , while control subjects and prefrontal cortex - lesioned patients showed skin conductance responses to winning and losing , amygdala - lesioned patients failed to show such responses . furthermore , contrary to controls , neither patient group showed anticipatory skin conductance responses to the risky deck , suggesting that defective emotional signaling in the amygdala and defective routing of this information through prefrontal cortex resulted in a similar negative impact on loss ( or risk ) aversion learning . bechara et al point out that some of the vmpfc patients were aware that the decks were bad yet persevered in their risky choices ; the authors draw a parallel with other pathological conditions , such as drug addiction or psychopathy where maladaptive or pathological behavior persists despite intact awareness of the consequence of one 's actions . our group used a different type of gambling game in order to study the interactions between emotions and value - based decisions in patients with ofc lesions . subjects chose between two gambles of known probabilities and rated subjectively felt emotions after the outcome was revealed . different types of emotions could be measured depending on how much information was supplied . in the presence of feedback on the chosen gamble only , subjects experienced either satisfaction or disappointment , depending on the outcome value . when feedback was given on both gambles , they could compare what is with what could have been and hence could experience more complex forms of emotions , such as rejoicing or regret . this is an important issue , because in behavioral economics , decisions made under uncertainty generate regret aversion , leading subjects to estimate ( and sometimes overestimate ) future regret when making choices in order to minimize the risk of experiencing it later . patients with ofc lesions reported feeling elated and saddened when their chosen gamble produced wins and losses , respectively . they also showed commensurate emotional arousal , as measured by their skin conductance response , in agreement with previous findings . however , contrary to control subjects , neither their emotional responses nor their decision strategies were affected by information on the foregone option . for instance , when the chosen gamble yielded a small positive earning , their satisfaction level was not reduced by discovering that the outcome of the other gamble was even more advantageous , nor was it enhanced when discovering it was much worse . although lack of regret could also reflect blunted somatic markers , it should be emphasized that this is a cognitive - based emotion requiring a capacity to perform counterfactual comparison , a process that might be impaired in these patients . work on monkeys shows that the acc , an area closely connected to limbic and prefrontal regions , contains neurons that encode the size of rewards , obtained as well as unobtained , in a simple decision task . the acc is thus suited to provide active outcome value signals needed to compare factual and counterfactual information . neuroimaging studies in humans have shown a cerebral regret network that includes the amygdala , ofc , acc , and hippocampus . whereas simple monetary wins and losses in the two - choice gambling task modulate reward - related areas , such as the ventral striatum , experienced and anticipated regret are correlated with enhanced activity in the ofc , amygdala , and acc . other imaging studies have confirmed the key role of the ofc in the regret mechanism , with a possible functional distinction between lateral and medial ofc ( see ref 32 for a review ) . the tremendous success of smartphone photo exchange apps and online social networks is a patent testimony to the high value we assign to observing and learning about our conspecifics . social comparison is a basic human activity . as karl marx noted in wage labor and capital , it is a major driving force in the burning pursuit of wealth , and it is a source of no less burning frustration : a house may be large or small ; as long as the neighboring houses are likewise small , it satisfies all social requirement tor a residence . but let there arise next to the little house a palace , and the little house shrinks to a hut . the little house now makes it clear that its inmate has no social position at all to maintain , or but a very insignificant one ; and however high it may shoot up in the course of civilization , if the neighboring palace rises in equal or even in greater measure , the occupant ot the relatively little house will always find himself more uncomfortable , more dissatisfied , more cramped within his four walls . the role of inequity aversion in decision making is well documented in humans and in several nonhuman primate species . in contrast , if the other 's reward serves as reference point , ie , when an individual has to make decisions about somebody else 's money rather than about t heir 's own money , emotional engagement is reduced and risk evaluation less constrained by loss aversion . impairments in socially based cognition and communication have been reported in brain - lesioned patients . patients with vmpfc lesions engage in socially inappropriate behavior , lacking in awareness of their social norm violations , and fail to adjust their communicative behavior according to the social partner , thus raising the question of the role of prefrontal areas in social decision making . experiments in nonhuman primates have explored how neural activity is affected when decisions are made in a social context . when given the opportunity to provide food or to prevent infliction of unpleasant stimuli to conspecifics , monkeys express variable , partner - dependent social tendencies but mainly make prosocial decisions . in the ofc , we have shown that own - reward sensitive neurons respond to a fixed reward to self with different discharge rates , depending on whether monkeys are working to obtain a reward for self only or for both self and a partner monkey . activity modulations are consistent with simultaneously recorded behavior , as equity - averse animals show reduced neuronal activity to shared reward ( same amount granted to both ) and equity - seeking animals show enhanced activity . ofc thus appears suited to perform the neural computations involved in social comparisons . whereas neurons in the ofc and in the striatum only encode own rewards in a social context , acc neurons distinguish between rewards to self and to others , and medial prefrontal neurons track rewards and action of partner monkeys . finally , in a recent experiment using as experimental paradigm an iterated prisoner 's dilemma game , neuronal activity in the dorsal acc was shown to predict another player 's intention to cooperate or to defect , and disruption of its activity by electrical stimulation reduced the tendency to produce mutually beneficial choices . although food trading may seem quite rudimentary in comparison with complex human social exchanges , these results highlight the key role played by prefrontal cortical areas in regulating the basic mechanisms of cooperation and competition . patients with vmpfc lesions were found to reject unfair offers more often than control subjects in a single - round ultimatum game , but only when outcomes were obtained after a delay and not when outcomes were displayed and gains immediately available . thus , rather than reflecting anomalous inequity aversion , the explanation advanced by the authors is that after damage to the vmpfc , future gains are more rapidly discounted ( a so - called myopia for the future ) , thereby making the subjective value of an unfair offer appear even lower and more unacceptable than it would to a normal individual . whereas control subjects display more inequity aversion when facing a human opponent than a computer opponent , choices made by patients with lesions in the vmpfc did not distinguish between the two situations and only depended on the distribution of monetary outcomes differentiation of human versus computer partners is believed to reflect the role of social norms in economic decision making and , in the present case , the need felt to administer costly altruistic punishment to human partners who behave unfairly . by contrast , since inanimate objects can not be held responsible for their choices , the dominant strategy when playing against a computer is rational maximization ( ie , accepting a very small offer is better than nothing ) . in a later study , decisions made by vmpfc - lesioned patients were analyzed in the context of the trust game , a social exchange test evaluating trust and reciprocity . in this game , the investor has to transfer all or part of an initial endowment , knowing that the amount invested is tripled as it is transferred to the trustee , and the trustee must then decide what fraction of that amount to return to the investor . as in the previous study , in contrast to control subjects who were more risk adverse ( less trusting ) with a human than a computer partner , vmpfc - lesioned patients did not act differently in the two settings and they displayed less generosity as trustee . the human vs computer dissociation is taken as evidence of fear of betrayal , another social emotion influencing risky decisions . nevertheless , observations made in patients with ofc and vmpfc lesions suggest that this region plays a key role in regulating decision making through nonsocial emotions , such as anticipated regret and social / moral emotions , such as envy , fairness , betrayal , and guilt . the use of experimental approaches and concepts from behavioral economics is a relatively new direction in neuroscience and neuropsychology . in interaction with other domains such as reinforcement learning , computational neuroscience , and social psychology , economic analysis provides a mechanistic framework and explicit hypotheses to investigate covert reward evaluation processes and emotional regulation of individual and social decision making . further work drawing on comparative electrophysiological and neuroimaging approaches in animals and humans is needed to develop an integrated view of motivated behavior and pave the way toward novel approaches to the treatment of neuropsychiatric disorders .

su was involved in the care of the patient , performed literature search and drafted the manuscript .

occidental diet is characterized by high caloric intake , mainly saturated fatty acids and glucose consumption , contributing to the development of obesity and insulin resistance . in the past 15 years , obesity has been associated to chronic inflammation in several tissues and cells , such as liver , adipose tissue , skeletal muscle , and immune cells . in fact , chronic and subclinical low - grade inflammatory state is a hallmark of obesity , and this condition has been proposed to play a central role in the development of insulin resistance , type 2 diabetes mellitus , and steatosis . during the past decades , the prevalence of obesity has enormously increased worldwide . a possible interaction factor between metabolic disorders and inflammation is the disruption in lipid metabolism caused by high levels of saturated fatty acids from high - fat and caloric diet [ 3 , 4 ] . c57bl/6 mice develop obesity , insulin resistance ( ir ) , diabetes mellitus , advanced fatty liver , and fatty pancreatic diseases when submitted to a high - fat diet ( hfd ) , mainly enriched with saturated fatty acids . the composition of fatty acids on diet can be an important modulator of lipid metabolism and inflammation . some studies have showed that an increase in circulating levels of mono- or polyunsaturated fatty acids improves insulin sensitivity and ameliorates hepatic steatosis [ 6 , 7 ] . sunflower oil is rich in mono- ( mufa ) and polyunsaturated fatty acids ( pufa ) , mainly linoleic acid , a member of the n-6 family . diet enriched with sunflower oil decreases plasma triacylglycerol and has beneficial effects on plasma lipid profile . moreover , acute administration of sunflower oil in rats potentially prevents the gastric damages generated by indomethacin administration through increased anti - inflammatory response . however , mice fed with sunflower oil - enriched diet present increased il-6 ( a pro - inflammatory cytokine ) levels in white adipose tissue . thus , further studies are required to adequately determine the pro- or anti - inflammatory effects of n-6 fatty acids . adipose tissue , skeletal muscle , liver , and immune cells have been identified as critical targets for the disruption in metabolic and inflammatory signaling in obesity . the imbalance between metabolic alterations and inflammatory signaling pathways leads to insulin resistance , hepatic steatosis , and related diseases [ 4 , 10 ] . in this study , we evaluated the effect of sunflower oil supplementation on inflammation and insulin sensitivity in mice fed a balanced diet and a hfd ( rich in saturated fatty acids ) . the protecting or potentiating effect of sunflower oil supplementation on insulin resistance and inflammation was investigated . gtt ) and skeletal muscle ( glucose uptake and metabolism ) insulin sensitivity was examined . inflammatory markers were investigated in liver , adipose tissue , skeletal muscle , and peritoneal macrophages . the strategy used was to investigate the effect of sunflower oil supplementation in mice fed a balanced diet and a hfd . under this last condition , a clear inflammatory state is established and so a possible anti - inflammatory effect could be pronounced . all animal studies were performed according to protocols approved by the animal care and use committee from the institute of biomedical sciences , university of so paulo . c57bl/6 animals were housed in a room with light - dark cycle of 12 - 12 h and temperature of 23 2c . animals were divided in four groups : ( a ) control diet ( cd ) , ( b ) high - fat diet ( hfd ) , ( c ) control diet supplemented with n-6 ( cd + n-6 ) , and ( d ) high - fat diet supplemented with n-6 ( hfd + n-6 ) . during the first 4 weeks preceding the induction of obesity by hfd , all the four experimental groups were fed ad libitum with a control diet ( 76% carbohydrates , 9% fat , 15% proteins ) . cd + n-6 and hfd + n-6 were supplemented with sunflower oil ( n-6 pufa source ) by oral gavage at 2 g per kg of body weight , three times per week , during 12 weeks . this dosage of oil was chosen based on previous studies using different oils from our group and others [ 1113 ] . cd and hfd received water at the same dose . after the first 4 weeks , animals from the hfd and hfd + n-6 groups received high - fat diet ( 26% carbohydrates , 59% fat , 15% proteins ) during the next 8 weeks . supplementation with n-6 ( sunflower oil , 2 g / kg b.w . ) was kept until the end of the 12 weeks . tolerances to glucose ( gtt ) and to insulin ( itt ) were evaluated after 6 h fasting . for gtt , mice were intraperitoneally injected ( i.p . ) with glucose ( 2 g / kg body weight ) . blood glucose measurements were performed at 0 , 15 , 30 , 45 , 60 , and 90 min after glucose injection . glucose concentration versus time was plotted and the area under the curve ( auc ) was calculated for each animal . for itt , animals were i.p . injected with insulin ( humulin r , lilly , 0.75 ui / kg b.w . ) and glucose measurements were performed at 0 , 10 , 20 , 30 , 40 , 50 , and 60 min after injection . glucose concentration versus time was plotted and the glucose lowering rate was calculated . in both tests , serum glucose was measured by colorimetric assay commercially available ( pap liquiform glucose , labtest ) and for itt , glucose was measured by using glucometer ( one touch ultra , johnson & johnson ) . after 6 hours fasting , animals were anesthetized and blood was collected by puncturing the orbital plexus . serum glucose , triacylglycerol , total cholesterol , ldl - cholesterol , and hdl - cholesterol were determined by colorimetric assays ( labtest diagnostics , lagoa santa , mg , brazil ) . animals were euthanized by cervical dislocation and soleus muscles rapidly and carefully isolated , weighed ( 810 mg ) , attached to stainless steel clips to maintain resting tension , and preincubated for 30 min , at 37c , in krebs - ringer bicarbonate buffer ( krbb ) containing 5.6 mm glucose and 1% bovine serum albumin ( bsa ) , ph 7.4 , pregassed for 30 min with 95% o2/5% co2 , with agitation at 100 oscillations per min . after this period , muscles were transferred to other vials containing the same buffer , but added of 0.3 ci / ml d-[u - c]-glucose and 0.2 ci / ml 2-deoxy - d-[2,6-h]-glucose . phenylethylamine ( 0.2 ml ) , diluted 1 : 1 v / v in methanol , was added into a separate compartment for co2 adsorption . incubation was then performed for 1 h under similar conditions , in the absence or presence of 7 nm insulin . gasification with 95% o2/5% co2 was maintained during the pre - incubation and the first 15 min of the incubation period and then stopped . after the incubation period , muscles were briefly washed in cold krbb at 4c , dried on filter paper and frozen in liquid n2 . samples were processed for measurements of uptake of 2-deoxy - d-[2,6-h]-glucose , d-[c]-glucose incorporation , [ c]-glycogen synthesis , and decarboxylation of d-[c]-glucose , according to methods described by challiss et al . , respectively , and routinely have been used by our group [ 11 , 1722 ] . mice were euthanized on co2 chamber and visceral adipose tissue ( epididymal , retroperitoneal , and mesenteric ) , liver , gastrocnemius muscle , and peritoneal macrophages rapidly collected . about 100 mg of liver , gastrocnemius muscle and retroperitoneal adipose tissue were used for the determination of tnf- , il-6 , and il-10 content . tissues were homogenized in ripa buffer ( 0.625% nonidet p-40 , 0.625% sodium deoxycholate , 6.25 mm sodium phosphate , and 1 mm ethylenediamine tetra acetic acid at ph 7.4 ) , containing 10 g / ml of a protease inhibitor cocktail ( sigma - aldrich , st . homogenates were centrifuged at 12,000 g for 10 min at 4c , supernatant was collected , and protein concentration determined using bradford assay ( bio - rad , hercules , ca , usa ) . about 150 mg of epididymal adipose tissue were cultured in dmem sterile medium ( gibco ) , containing 10% fbs , 2 mm glutamine and antibiotics for 24 h , at 37c , 5% co2 humidified air environment . thereafter , medium culture was collected and used for the determination of leptin , adiponectin , and resistin contents . for all determinations , we used colorimetric immunoassays elisa ( duoset kits , r&d system ) . cytokine and nitric oxide ( no ) production was evaluated in macrophages obtained by washing the peritoneal cavity with 6 ml rpmi culture medium ( gibco ) , containing 10% fbs and 4 mm glutamine . macrophage - rich cultures ( more than 90% of the cells were f4/80 ) were obtained by incubating peritoneal cells in 24-well polystyrene culture plates for 2 h at 37c in a 5% co2 , humidified air environment . adherent cells were then incubated with 2.5 g / ml of lps ( e. coli , serotype 0111:b4 , sigma chemical company , usa ) for 24 h . medium was collected for determination of il-6 , il-10 , and tnf- by elisa , and nitrite content by griess method . animals fed with hfd for eight weeks showed increased ( by 3.8 fold ) body weight gain when compared to those fed with cd . despite reduced food ingestion , sunflower oil did not change body weight gain , food efficiency , or adipose tissue increased ( table 1 ) . an increase of 63% in area under the curve in glucose tolerance test ( gtt ) was also observed in animals treated with hdf , demonstrating glucose intolerance , which was associated with insulin intolerance , as observed by the reduction in glucose clearance during insulin tolerance test ( itt ) . however , the supplementation did not prevent the glucose and insulin intolerance induced by hfd . furthermore , sunflower oil supplementation induced insulin intolerance itself ( cd + n-6 group ) , reducing the clearance of glucose similar to hfd without supplementation . total cholesterol and ldl cholesterol levels were increased by hfd and sunflower oil supplementation in association with hfd showed protective effect in the prevention of the increased ldl cholesterol and cholesterol total levels found in hfd . soleus muscles from animals fed with hfd did not respond to insulin stimulus in relation to glucose uptake and metabolism ( figure 1 ) , characterizing muscle insulin resistance state . interestingly , the supplementation itself ( cd + n-6 group ) increased basal glucose uptake and glucose oxidation , but it failed to respond to the insulin stimulus when compared to the control group ( cd group ) . peritoneal macrophages stimulated with lps showed increased production of nitrite , tnf , il6 , and il10 when compared with unstimulated cells ( figure 2 ) . macrophages from animals with obesity induced by hfd for 8 weeks showed a inflammatory profile , with increased production of nitrite , tnf- , and il-6 , associated with decreased content of il-10 when stimulated with lps . for tnf- only , the association of hfd and n-6 supplementation ( hfd + n-6 group ) showed beneficial response , decreasing this cytokine production . in general , hfd induced and increase in the pro - inflammatory cytokine il6 and decrease in the anti - inflammatory cytokine il10 contents in the insulin target tissues ( liver , skeletal muscle , and adipose tissue ; figure 3 ) . interesting , sunflower oil supplementation itself ( cd + n-6 group ) for 12 weeks increased the content of the proinflammatory cytokine il6 in all tissues . however , when the sunflower oil was associated with hfd ( hfd + n-6 group ) , cytokine concentrations were similar to that found in the hfd group , in liver and adipose tissue . adipokines production by epididymal adipose tissue was determined after 24 h culture . resistin was reduced only when hfd was associated to sunflower oil supplementation ( hfd + n-6 group ) in comparison to cd + n6 group . this is the first study to investigate the effects of sunflower oil supplementation on inflammation and insulin sensitivity in mice fed a balanced diet and a hfd . we showed that hfd induces decreased glucose tolerance ( as demonstrated by gtt ) and insulin sensitivity ( as observed in itt ) in the whole body . surprisingly , this last effect was also verified in the cd + n6 group . moreover , proinflammatory response was increased in the hfd group without prevention or attenuation by sunflower oil supplementation . hfd has been associated with insulin resistance and inflammatory condition , contributing to the development of several related diseases , such as obesity , diabetes mellitus type 2 , metabolic syndrome , cancer , and cardiovascular diseases [ 2527 ] . various strategies have been investigated in order to decreasing insulin resistance and inflammation , including natural and modified compounds , cyclooxygenase inhibitors , and physical exercise [ 2835 ] . since skeletal muscle tissue represents 5060% of the body weight and it is the main insulin - responsive tissue in association to liver and adipose tissue , it has been proposed that skeletal muscle dysfunctions ( reduced oxidative capacity and impaired insulin sensitivity ) play a central role in the abnormalities of chronic diseases [ 21 , 3640 ] . in this work , we investigated the effect of sunflower oil supplementation on insulin sensitivity ( in whole animal in vivo and in soleus muscle in vivo ) and inflammatory condition ( liver , skeletal muscle , adipose tissue ) in mice fed a balanced diet and a hfd . in recent study , bjermo et al . showed that isocaloric diet with n-6 pufa for 10 weeks reduce liver fat and modestly improves metabolic conditions , without weight loss in humans . in addition , it has been proposed that n-6 pufa reduce risks of cardiovascular diseases , by improving lipid profile . this improvement in lipid profile was found in our study , whereas hfd + n6 group showed decreased plasma ldl and total cholesterol levels . some studies have shown that n-6 pufa intake positively correlate with obesity and insulin resistance [ 43 , 44 ] . treatment with subcutaneous injection of sunflower oil by 7 days in wistar rats impaired insulin sensitivity by decreasing glucose clearance during itt and glut-4 expression and translocation in white adipose tissue ( wat ) , with no effect in skeletal muscle . in the cd + n6 group , kitt was reduced , but glucose uptake and metabolism in isolated soleus muscles were not altered after insulin stimulus . these results suggest that sunflower oil supplementation per se is not able to impair insulin response in skeletal muscle , but it possibly reduces total insulin sensitivity by decreasing the response to the hormone in other tissues , such as liver and adipose tissue . in addition , both groups fed with hdf ( hfd and hfd + n6 groups ) showed skeletal muscle resistance to insulin , showing no beneficial effects of n-6 pufa on peripheral insulin sensitivity . obesity is accompanied by a chronic low grade subclinical inflammation promoted by an increase in fuel availability , leading to infiltration of macrophages into adipose tissue . this condition results in enhanced inflammatory response , which stimulates the production of cytokines by adipose tissue . moreover , in obesity , increased lipotoxicity caused by high nonesterified fatty acids ( nefa ) available , and triacylglycerol content leads to an increase in inflammatory markers and cytokine production in multiple organs [ 46 , 47 ] . hfd increased inflammatory response in macrophages , showing high content of il-6 , tnf- , and no , and low content of il-10 ( anti - inflammatory cytokine ) when compared to the control group . in peripheral tissues , il-6 content was increased in the liver and gastrocnemius muscle whereas il-10 content was decreased in liver and retroperitoneal adipose tissue . adipose tissue showed increased production of leptin in the hfd and hfd + n6 groups , compared with mice fed with control diet , and resistin was reduced in the hfd + n6 group compared with cd + n6 , in epididymal adipose tissue . whereas in retroperitoneal adipose tissue , il-6 was elevated in the hfd and cd + n6 groups compared with cd , il-10 was reduced in the hfd and cd + n6 , and adiponectin was reduced only in cd + n6 . these results showed the inflammatory characteristics the sunflower oil supplementation and the high fatty diet fed . our results showed that sunflower oil supplementation causes a clear pro - inflammatory response in mice fed on control diet and it does not have additive or prophylactic effects in mice fed on hfd . linoleic acid is an essential fatty acid that leads to inflammatory response by increasing arachidonic acid production , a fatty acid related to elevated pro - inflammatory mediator generation . high intake of linoleic acid has been associated to the development of chronic diseases in several studies [ 51 , 52 ] . however , few studies have been carried out on the direct effect of this fatty acid on inflammatory markers . in conclusion , our results showed that sunflower oil supplementation has pro - inflammatory effects in macrophages and insulin - sensitive peripheral tissues and induced insulin resistance in vivo and in vitro . these effects were demonstrated in mice fed a balanced diet and pronounced in mice fed a hfd . interestingly , sunflower oil supplementation partially prevented the dislipidemia found in mice submitted to hfd , but it did not improve or even worsened inflammatory state and insulin resistance induced by this diet .

hepatitis b virus ( hbv ) infection is a common disease that can induce a chronic carrier state and is associated with the risk of progressive disease and hepatocellular carcinoma.1 interferon ( ifn ) and several nucleoside / nucleotide analogs ( nas ) , such as lamivudine ( lam ) , adefovir dipivoxil ( adv ) , entecavir ( etv ) , and tenofovir disoproxil fumarate ( tdf ) , are currently approved for the treatment of chronic hepatitis b ( chb ) in most countries.25 because na analogs inhibit reverse transcription of the hbv polymerase but do not directly interfere with the formation of covalently closed circular dna ( cccdna ) , they require long - term administration , which is usually accompanied by the emergence and selection of drug - resistant mutations in the viral polymerase.6 tdf is widely used to treat hbv patients in the usa and europe . this agent is equally effective against multiple hbv genotypes ( a h ) as well as against lam - resistant isolates.7 no confirmed report of resistance selection during treatment with tdf in treatment - nave chb patients has yet been reported.810 in a recent study , long - term tdf monotherapy provided durable antiviral efficacy for 240 or up to 288 weeks ( 6 years ) of treatment , and comprehensive genotypic and phenotypic analyses detected no evidence of tdf resistance.11,12 additionally , longer treatment duration did not increase the incidence of virologic breakthrough.12 moreover , tdf monotherapy has demonstrated the long - term ( median 23 months ) efficacy of this agent in na - experienced patients with treatment failure , and virologic breakthrough was not observed in any patient during the entire observation period.13 here , we report for the first time a patient with chb and cirrhosis who emerged with virologic breakthrough during tdf and etv combination therapy against etv - resistant virus . a 51-year - old japanese woman with chb underwent a checkup in february 1999 and was found to be seropositive for hepatitis b surface antigen ( hbsag ) , with mild alanine aminotransferase ( alt ) elevation . hepatitis b e - antigen ( hbeag ) was positive , and serum hbv dna was > 7.6 log copies / ml ( amplicor hbv monitor assay ; f hoffman - la roche ltd , basel , switzerland ) . the hbv genotype was c , and human immunodeficiency virus ( hiv ) status was negative . she was diagnosed with cirrhosis by peritoneoscopy and liver biopsy ( moderate hepatitis [ a2 ] and severe fibrosis [ f4 ] ) in february 2000 . hbv dna levels gradually increased , and lam - resistant virus emerged ( reverse transcriptase [ rt ] m204i ) . treatment was switched from lam to etv ( 0.5 mg / day ) in october 2007 ( hbv dna 3.9 log copies / ml ) following the emergence of etv - resistant virus ( rtl180 m , rts202 g , and rtm204v ) and higher elevation in hbv dna . however , she discontinued therapy of her own volition from february 2009 to may 2010 . she returned to our hospital in may 2010 because of general fatigue and ascites , at which time serum hbv dna was > 7.6 log copies / ml , alt was 687 treatment with etv ( 0.5 mg / day ) was restarted immediately , and alt and serum hbv dna levels gradually decreased . however , because hbv dna levels remained at > 3.5 log copies / ml until september 2010 , she was started on tdf ( 300 mg / day ) and etv combination therapy ( hbv dna 3.9 log copies / ml ) . serum hbv dna then decreased to less than 2.1 log copies / ml ( cobas taqman hbv test , v2.0 ; f hoffman - la roche ltd ) at november 2011 ( month 14 of tdf and etv treatment ) and remained at this level until april 2013 ( month 31 of tdf and etv treatment ) , when it again began to increase ( hbv dna 3.9 log copies / ml ) . compliance with tdf and etv was good throughout the course of treatment . during treatment , nucleotide sequences of the polymerase gene were determined by polymerase chain reaction ( pcr ) direct sequencing , as previously described.14 the viral polymerase reverse transcriptase ( rt ) gene at the baseline of lam treatment ( september 2006 ) showed the wild type sequence ( with no lam , adv , etv , or tdf resistance substitutions ) . in october 2007 ( after switching from lam to etv ) , an amino acid substitution of the rt gene , rtm204i ( lam resistance substitution ) , was detected . moreover , amino acid substitutions of rtl180 m , rts202 g , and rtm204v ( etv resistance substitutions ) emerged during etv treatment in february 2009 . the rt gene analysis at the baseline of etv retreatment ( may 2010 ) returned the wild type sequence ( with no lam or etv resistance substitutions ) . in june 2010 ( week 3 of etv retreatment ) , amino acid substitutions of rtl180 m , rtt184i , and rtm204v ( etv resistance substitutions ) were simultaneously detected . moreover , amino acid substitutions of rtl180 m , rtt184 m , and rtm204v coexisted with the above mutants at the end of june 2010 ( week 6 of etv retreatment ) . in october 2010 ( week 4 of tdf and etv treatment ) , these amino acid substitutions were replaced by wild type virus ( no etv resistance substitutions ) . since april 2013 ( month 31 of tdf and etv treatment ) , amino acid substitutions of rtl180 m , rts202 g , and rtm204v have emerged and have been found to be associated with an increase in serum hbv dna ( figure 2 ) . in comparison with those at the start of tdf therapy , the amino acid substitutions changed from rtl180 m , rtt184 m , and rtm204v to rtl180 m , rts202 g , and rtm204v , and no other amino acid substitutions apart from these in the rt region were observed . further , there were no substitutions that could be associated with reduced tdf susceptibility ( rta181v / t , rtn236 t , or rta194 t ) in april 2013 . genotypic resistance to tdf has been detected in several patients with hiv - hbv coinfection . the substitution rta194 t ( plus rtl180 m + rtm204v ) has been associated with tdf resistance,15 albeit that a second report failed to confirm this.16 it has been shown that rta181v + rtn236 t double mutants are resistant to tdf in vitro , but clinical data suggest that patients with rta181 or rtn236 t remain susceptible to tdf.17 the substitution rtp177 g and rtf249a reduced susceptibility to tdf in an in vitro study , but no clinical findings have yet been reported.18 moreover , rescue therapy with etv and tdf in chb patients harboring viral resistance patterns ( for lam , adv , or etv ) or showing only partial antiviral responses to preceding therapies was efficient in patients both with and without advanced liver disease.19 to date , there have been no confirmed reports of resistance selection during treatment with tdf for chb.912 moreover , virologic breakthrough occurs infrequently and has been associated with nonadherence to medication in the majority of cases.12 to our knowledge , this is the first report of a patient with virologic breakthrough during tdf therapy . in our case , virologic breakthrough in compliant patients is generally related to viral resistance.6 amino acid substitutions of rtl180 m , rts202 g , and rtm204v have emerged in cases in which serum hbv dna increased during tdf and etv therapy . moreover , these amino acid substitutions changed from rtl180 m , rtt184 m , and rtm204v to rtl180 m , rts202 g , and rtm204v . this clinical course suggests that these amino acid substitutions are resistant to tdf and etv therapy , although in vitro confirmation is necessary . kim et al reported that among 18 patients who failed multiple na treatments , including lam , adv , and etv , 17 patients achieved virologic response and one patient showed a viral reduction of 3.9 log iu / ml , nearly reaching virologic response within 24 months.20 these findings indicate that genotypic resistance to etv does not affect the probability of an initial virologic response to tdf therapy.20 petersen et al reported that four patients harboring etv - resistant virus achieved a virologic response within 9 months.19 recently , seto et al reported 142 asian chb patients with at least 6 months exposure to other nas ( including etv ) who received tdf with or without lam . with a median 2.25 years of follow - up , 45 patients had detectable viremia in at least one time point.21 for these 45 patients , which included ten with virologic breakthrough , both line probe assay and direct sequencing revealed no new amino acid substitutions , including substitutions that could be associated with reduced tdf susceptibility ( rta181v / t , rtn236 t , or rta194 t ) . moreover , karatayli et al reported that hbv dna , in seven of eight patients with etv resistance mutations ( t184f / a / l / i , s202 g , and m250v ) , became undetectable with tdf and lam after 6 months of treatment.22 in vitro drug susceptibility showed that tdf displayed one- to twofold resistance to etv - resistant viral strains ( n123d + h124y + l180 m + s202 g + m204v + y257h , i163v + l164 m + l180 m + s202 g + m204v + c256s , and h124y + l180 m + s202 g + m204v + y257h ) . . in our case , virologic breakthrough occurred at month 31 of tdf and etv therapy , and the etv - resistant strain ( l180 m + s202 g + m204v ) of our case was not identical with that in the in vitro drug susceptibility study above . clarification of virologic breakthrough and resistance of tdf against patients with na - resistant virus , especially etv , will likely require further studies with a longer time frame . in conclusion , this study shows that long - term treatment of etv - resistant virus with tdf has the potential to induce virologic breakthrough and resistance , and careful follow - up should be done .

methanol is a colorless and highly toxic organic solvent and is commonly used in industry . subsequently , it is metabolized to formaldehyde by alcohol dehydrogenase , which is in turn converted into formic acid by aldehyde dehydrogenase . it has been reported that formaldehyde stays too transitory to be detected by analytical instruments , while formic acid is easily detected after methanol ingestion . consequently , formic acid has been considered as the main toxic factor in methanol poisoning . ocular toxicity is the most significant characteristic in methanol toxic effects and electroretinogram is often used to evaluate the retinal function . currently , most studies on methanol intoxication focus on a - wave and b - wave subcomponents in photopic and scotopic environment [ 3 , 4 ] . our previous research showed that a - wave and b - wave were greatly destroyed upon methanol intoxication . however , very limited researches on the characters of ops waves upon methanol poisoning were performed . garner and lee found that all ops amplitudes reduced nonspecifically and latencies delayed after acute methanol intoxication . considering that ops waves are a series of rhythmic low - amplitude potentials superimposed on the ascending phase of b - wave , systemic and deep researches on the characteristic changes of ops waves will make much sense for exploring the toxic effects of methanol poisoning . in this research , we try to explore the potential effects of methanol and its metabolite , formic acid , on rat retina function though the parameter of ops waves . this research adhered to the tenets of the declaration of helsinki or the nih statement for the use of animals in research . thirty adult ( 200250 g , national rodent laboratory animal resources , shanghai branch , china ) male sprague - dawley rats with no ophthalmopathy were supplied with food and water ad libitum and maintained at a standard temperature and humid environment . all animal experiments were approved by ethical certification and performed according to the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research . rats were maintained in a sealed chamber and given a n2o / o2 gas mixture ( 1/1 by vol ; flow rate , 2 l / min ) for 24 h before administration of methanol or saline and the gas mixture continued until the experiment 's end . initially , animals were randomly classified into control group ( a ) , 3-day group ( b ) , and 7-day group ( c ) with 10 rats per group . it has been reported that ld0 and ld50 of oral methanol for rats are 6.67 and 1214 ml / kg . groups b and c were treated with methanol by gavage ( 8 ml / kg per dose , followed by a 4 ml / kg supplemental dose 24 h later ) , and the control group a was treated with equivalent volumes of saline . erg recordings of group b were performed 72 h ( 3 days ) later after methanol treatment and those of group c were performed 168 h ( 7 days ) later . then , rats from each group were killed by decapitation and cardiac blood samples were collected into ethylenediaminetetraacetic acid anticoagulation tubes . erg recordings were performed by the electrophysiological test unit ( roland , germany ) as described by iscev . before test , then animals were anesthetized with 10% chloralhydrate ( 3.5 ml / kg , intraperitoneal injection ) and each pupil was dilated by tropicamide compound . the recording electrode was positioned on the corneal surface , the reference electrode was penetrated into the middle forehead , and the ground electrode was inserted into the skin of the ipsilateral - mastoid process . in the process of scotopic erg , the flash light intensity of 25 db was set for recording rod - response and 0 db was set for recording max - response and ops . before the photopic erg recording , the rats ' eyes needed 5 min for light adaptation . in photopic erg process , the flash light intensity of 0 db was set for recording cone - response . to diminish the external interference , the amplifier was set to be with voltage of 1 mv and narrow bandpass between 1 and 300 hz . then , the ops responses were characterized by calculating the individual amplitude of each wave , summed amplitudes of five wavelets of ops ( saop ) , elapsed time ( et ) from the appearance of the first wavelet to the end of the fifth wavelet , and interpeak interval ( ipi ) as the time interval between adjacent peaks of ops wavelets . spss 20.0 software was used to analyze data with t - tests and significant difference ( p ) set at 0.05 . thirty blood samples ( ten samples from group a , group b , and group c resp . ) were used to determine methanol concentrations by headspace gas chromatography ( gc / hs ) , as previously described . methanol concentrations were calculated from a standard curve produced using gc / hs results from a series of known concentrations of chromatographic grade methanol . the determinations of formic acid concentration were performed on thermo u3000 hplc ( thermo fisher scientific inc . , system parameters included a calesil ods-100 c18 chromatographic column ( 5 m , 4.6 250 mm ) , ambient temperature , a 1.0 ml / min flow rate , and a methanol / water mixture ( 6/4 by vol ) as the mobile phase . formic acid was measured with a diode array detector ( dad , thermo fisher scientific inc . ) at 215 nm . tissues were placed in a tube and shaken in a fastprep-24 instrument ( mp biomedicals , llc , solon , oh , usa ) for 1 - 2 min . then , 100 l blood was sufficiently mixed with the mobile phase and centrifuged at 10 000 rpm for 10 min . the 20 l supernatant was then injected into the high - performance liquid chromatography ( hplc ) and formic acid concentrations were determined from a standard curve established from hplc of standard formic acid solutions . a standard curve formula ( y = 0.621x + 0.112 ; r = 0.999 ) was established to analyze experimental methanol concentrations . the methanol concentration in blood was 1.00 0.61 mg / ml ( n = 10 ) in the 3-day group , while no effective concentration was detected in the 7-day group ( table 1 ) . similarly , a standard curve formula ( y = 15.837x 6.6471 ; r = 0.992 ) was established to analyze experimental formic acid concentrations . blood formic acid concentrations of 0.61 0.07 mg / ml and 0.60 0.05 mg / ml were found in the 3- and 7-day groups , respectively ( table 1 ) . compared with methanol , its metabolite , formic acid , persistently accumulated in blood in methanol - treated groups ( figure 2 ) . for rod - response of erg , the a and b amplitudes of group b decreased approximately to 53% and 37% , respectively , and b amplitude of group c decreased approximately to 48% compared with the control group ( p < 0.05 , the exact p value in table 2 ) . for max - response of erg , the a and b amplitudes of group b decreased approximately to 53% and 40% and b amplitude of group c decreased approximately to 44% compared with the control group ( p < 0.05 , the exact p value in table 3 ) . the latent periods of a wavelet of group b were delayed approximately to 26% in max - response , and latent period of a wavelet of group c was delayed to 28% compared with the control group ( p < 0.05 , the exact p value in table 3 ) . the summed amplitudes of oscillation potentials of groups b and c decreased to approximately 53% and 57% compared with the control ( p < 0.05 , the exact p value in table 4 ) and the elapsed time was delayed to approximately 15% and 60% ( p < 0.05 , the exact p value in table 4 ) . the amplitudes of os1 , os3 , os4 , and os5 of group b and os3 , os4 , and os5 of group c decreased obviously compared with the control group ( p < 0.05 ) . comparison of os1 value between b and c groups was statistically significant ( p < 0.05 ) . the ipi1 of group b and ipi1 - 4 of group c were broader compared with the control group ( p < 0.05 ) and the ipi1 - 4 and et of group b were broader than group c ( p < 0.05 ) ( figures 3 and 4 ) . for cone - response , the a and b amplitudes of group b decreased approximately to 53% and 40% and b amplitude of group c decreased approximately to 69% compared with the control group ( p < 0.05 , the exact p value in table 5 ) . comparison of b amplitudes between b and c groups was statistically significant ( p < 0.05 , the exact p value in table 5 ) . the latent periods for a - wave and b - wave of group c were delayed approximately to 29% and 9% compared with the control group ( p < 0.05 , the exact p value in table 5 ) . in recent years , methanol poisoning events occurred frequently because of adulterated wine and occupational exposure . methanol poisoning severely damages the retina and optic nerve resulting in the impairment of vision and visual field . in addition to methanol , its metabolites , formic acid and formaldehyde , have also been attributed as neurotoxic induced by methanol poisoning and might be more toxic than methanol itself . the severity of methanol toxicity correlates with accumulation of methanol and its metabolite , formic acid , in blood after methanol administration . chromatographic methods , including gc / hs and hplc , are powerful tools in analytical chemistry . gc / hs is widely used to detect volatile compounds , such as ethanol and methanol . although some studies have simultaneously analyzed methanol and formic acid concentrations in postmortem samples using gc / hs , unfortunately , this method was not widely recognized as an effective method to detect formic acid concentration in blood or tissues . morris successfully quantified formic acid concentrations by hplc at 210 nm absorbance , which was used in the present study to detect and analyze formic acid concentrations after methanol intoxication . in our present study , although methanol in blood was nearly completely metabolized after 7 days of intoxication , quantified and closed formic acid concentrations were detected in blood in 3- and 7-day intoxication groups . that is to say , formic acid as the continuation of methanol toxicity has a persistently cumulative phenomenon in blood comparing with methanol . it is likely that methanol and its metabolite , formic acid , commonly contributed to the toxic effects of methanol intoxication on retina structure and function and the effect of formic acid might be more persistent . erg technique is widely used to assess retinal function . international society for clinical electrophysiology of vision ( iscev ) standard for full - field clinical electroretinography ( 2008 update ) specified five responses including dark - adapted 0.01 erg ( rod - response ) , dark - adapted 3.0 erg ( max - response ) , dark - adapted 3.0 oscillatory potentials ( ops ) , light - adapted 3.0 erg ( cone - response ) , and light - adapted 3.0 flicker ( 30 hz flicker ) , the first four responses of which were recorded in this study except 30 hz flicker . the results showed that a and b amplitudes of methanol intoxication groups were decreased and some of the latent periods of a- and b - waves were delayed . methanol intoxication destroyed both of the scotopic and photopic erg recordings , which were in accordance with the previous research of our laboratory . as a scotopic response of erg recordings , ops waves separated from b - wave through fourier spectrum filtering technology can be classified into early , intermediate , and late subgroups . neurosensory retinal cells , including bipolar , amacrine , and interplexiform cells , are directly or indirectly involved in ops generation and different synaptic activities might excite different oscillatory peaks . op amplitudes measured in mercury vapor exposure patients were smaller than those of controls for o2 and o3 . in this present study the reductions of saop and delays of et were confirmed in both two methanol - treated groups indicating the damage of neurosensory retinal cell function . the et of ops in 7-day intoxication group was more delayed than in the 3-day group , which was not found in other erg responses . most of the comparisons of amplitude and latent periods of both acotopic and photopic erg recordings between 3-day and 7-day groups methanol - treated groups have no remarkable significance , the possible cause of which may be that damage of methanol poisoning was persistent on both acotopic and photopic erg recordings . although the methanol was completely metabolized , its metabolite , formic acid , was continuously accumulated in the 7-day group , which might impede the recovery of retinal dysfunction . the other possible reason is that acotopic and photopic erg recordings reflect the whole retinal function , are less sensitive than ops , and can not respond to the subtle changes of retinal function . the severe delay of et in later methanol - treated rats indicated that ops might be more sensitive to methanol poisoning . it is found that the amplitudes of later ops wavelets in methanol - treated groups , such as os3 , os4 , and os5 , were clearly decreased compared with controls . this indicated that later ops wavelets were more vulnerable to damage after methanol intoxication , which was consistent with reports by plaziac et al . but different from garner and lee . the reason for this was possibly due to a different method to measure ops wavelets by garner and lee . previous research , focused on ops chemical sensitivity , has suggested that later ops are more sensitive to ethanol than earlier ones , which could be an anastomotic explanation for our results . ipi2 , ipi3 , and ipi4 were more serious in the 7-day than the 3-day group . moreover , ipi2 , which stands for the interval between adjacent peaks of os2 and os3 , was found to be longer than the other analyzed ipis , indicating that ipi2 might have been more sensitive to retinal functional changes after methanol poisoning . combined with the methanol and formic acid concentrations in blood , deterioration of retinal function in the 7-day group was more possibly due to formic acid accumulation rather than methanol itself . this present study proposed that methanol and formic acid commonly contributed to retinal dysfunction and formic acid might play a major role in the process . the present results indicated that both of scotopic and photopic retinal functions were impaired by methanol poisoning and impairment was more serious in the 7-day than in the 3-day group , the reason for which might have been formic acid accumulation in blood after methanol ingestion . compared with other electroretinogram subcomponents , ops , especially later ops and

campylobacter concisus is a gram - negative bacterium that is commonly present in the human oral cavity [ 1 , 2 ] . in some individuals , c. concisus may colonize the intestinal tract and was found to be associated with inflammatory bowel disease ( ibd ) due to its significantly higher prevalence in the intestinal tract of patients with ibd as compared to controls [ 36 ] . ibd is a chronic inflammatory disease of the gastrointestinal tract ( git ) with unknown aetiology . crohn 's disease ( cd ) and ulcerative colitis ( uc ) are the two major clinical forms of ibd . in addition to ibd , c. concisus was also often isolated from diarrheal stool samples , suggesting a possible involvement in diarrheal disease [ 810 ] . in the literature , it was described that c. concisus requires h2-enriched microaerobic conditions for growth [ 11 , 12 ] . in laboratory cultivations of c. concisus , microaerobic conditions enriched with 510% h2 have been used [ 2 , 9 , 12 , 13 ] . the primary colonization site of c. concisus is the human oral cavity [ 1 , 2 ] . the level of h2 in the human oral cavity is extremely low . given this , it is unlikely that c. concisus is able to grow microaerobically in the human oral cavity . in previous studies , c. concisus was isolated from gingival plaque and saliva , locations where a large number of anaerobes were found , suggesting that c. concisus is more likely to use an anaerobic growth in the human oral cavity [ 1 , 2 , 15 ] . to date , there have been no studies that systematically examined the growth of c. concisus under anaerobic conditions . furthermore , there is no information available regarding the impact of h2 on c. concisus growth under anaerobic conditions . furthermore , we examined the expression of putative virulence proteins of an oral c. concisus strain grown under anaerobic conditions . a total of 63 c. concisus strains were examined in this study , including 57 oral c. concisus strains and six enteric strains . of the 57 oral c. concisus strains , 19 strains were from patients with cd , 14 strains from patients with uc , and 24 strains from healthy individuals . of the six enteric c. concisus strains , five strains were isolated from patients with ibd including two strains isolated from intestinal biopsies of patients with uc , two strains isolated from fecal samples of patients with uc , and one strain isolated from intestinal biopsies of a patient with cd . both oral and enteric c. concisus strains were isolated in our previous studies [ 1 , 3 , 6 ] . c. concisus strain 13826 , which was isolated from fecal samples of a patient with bloody diarrhea , was purchased from the american type culture collection . the growth of the above 63 c. concisus strains under the following atmospheric conditions was examined . anaero condition was generated using an25a gas - generation system as instructed by the manufacturer ( oxoid , hampshire , uk ) . anaero condition was generated using br38b gas - generation system , which was placed into a 3.5 l jar in the presence of a catalyst following the manufacturer 's instruction ( oxoid ) . a micro condition was generated using two different gas - generation systems , following the manufacturer 's instruction ( oxoid ) . the first gas - generation system was br56a ( micro ) , which was placed into a 3.5 l jar in the presence a catalyst . the second gas - generation system was cn25a ( micro ) , which was placed into a 2.5 l jar . the c. concisus strains used in this study were isolated in our previous studies [ 1 , 3 , 6 ] . the atmospheric condition used in isolation of these c. concisus strains from clinical samples in our previous studies was to place the br56a gas - generation system into a 2.5 l jar with a catalyst , which was a modification of the manufacturer 's instruction . in this study , we refer to this atmospheric condition as the original isolation condition ( ori ) . each of the 63 c. concisus strains was streaked onto three horse blood agar ( hba ) plates for each atmospheric condition . the hba plates were prepared using blood agar base number 2 supplemented with 6% ( v / v ) heat - inactivated defibrinated horse blood and 10 g / ml of vancomycin ( oxoid ) . following incubation at 37c under anaero , anaero , micro , micro , and ori conditions , respectively , for 48 hours , plates were examined for the appearance of colonies under a stereo microscope . the morphology of all grown c. concisus strains was examined using a phase - contrast microscope . to further quantitatively compare the growth of c. concisus under anaero and anaero conditions , the colony forming unit ( cfu ) of 12 c. concisus strains grown under these two conditions was determined . these 12 strains included six oral strains from patients with cd , three oral strains from uc , two oral strains from healthy controls , and one enteric strain from a patient with uc . these 12 strains were randomly selected from the 63 strains used in the experiments in section 2.2 . c. concisus strains were first cultured on hba plates under ori condition at 37c for 48 hours . the bacterial cells were collected and washed once with phosphate buffered saline ( pbs ) . the bacterial pellet of each strain was resuspended in pbs and od600 was adjusted to 0.05 , which was used as the initial inoculum for further assessment of the growth of c. concisus under anaero and anaero conditions . the initial inoculum suspension ( 50 l ) of each c. concisus strain was inoculated onto six hba plates using a sterile l - shaped glass rod . three plates were incubated under anaero condition and the remaining three plates were incubated under anaero condition for 48 hours at 37c . the bacterial cells of each c. concisus strain collected from the three plates incubated under anaero condition and the three plates incubated under anaero condition were pooled , respectively ( 1 ml of pbs was used for collection of bacterial cells from each plate ) . from each pooled c. concisus suspension , serial dilutions ( 1 : 10 to 1 : 10 ) were prepared . each of the dilutions ( 5 l ) was inoculated onto hba plates in triplicate . the plates were further incubated under ori condition for 48 hours at 37c to determine the cfu numbers . p3uco - s1 strain is an oral strain previously isolated from a patient with uc [ 6 , 16 ] . in a previous study of analysis of six housekeeping genes , we showed that the six housekeeping genes of p3uco - s1 strain were identical to the strain isolated from intestinal biopsies of the same patient ( p3ucb - s1 ) suggesting that this oral c. concisus strain was able to colonize the intestinal tract . given this , we have decided to use p3uco - s1 strain in this part of the study . p3uco - s1 strain was first cultured on a hba plate under ori condition for 48 hours at 37c . following this , bacterial cells were collected and suspended into pbs and the od600 was adjusted to 0.05 . the bacterial suspension ( 30 l ) was inoculated onto 18 hba plates . the plates were incubated under either anaerobic conditions or microaerobic conditions containing various conditions of h2 ( three plates in each condition ) . anaerobic and microaerobic conditions were generated using gas - generation system an25a and gas - generation system cn25a , respectively ( oxoid ) . hydrogen gas was supplemented by including 0.021 g , 0.042 g , or 0.083 g of sodium borohydride and 10 ml of h2o in a container placed in a 2.5 l jar , respectively , which generated 2.5% , 5.0% , and 10.0% of h2 , respectively . h2 gas was generated by the chemical reaction nabh4 + 4h2o = 4h2 + nab(oh)4 . after a period of 48 hours of incubation at 37c , c. concisus bacterial cells were collected from each plate using 1 ml of pbs . three plates cultured under each condition were pooled and eight serial dilutions ( 1 : 10 to 1 : 10 ) were prepared . each of the eight dilutions ( 5 l ) was inoculated onto hba plates in four replicates . the plates were further incubated under ori condition for 48 hours at 37c to determine the cfu numbers . proteins expressed by c. concisus cultured under anaero and anaero conditions were analysed using mass spectrometry . c. concisus strain p6cdo - s1 was used in this experiment . p6cdo - s1 is an oral strain previously isolated from saliva of a patient with cd . our previous studies showed that this oral c. concisus strain was genetically close to a c. concisus strain isolated from the intestinal biopsies of a patient with cd . therefore , we decided to investigate whether this oral c. concisus strain expresses putative virulence proteins and whether these proteins are expressed differentially when the strain is grown under anaero and anaero conditions . briefly , c. concisus p6cdo - s1 strain was grown on hba plates for 48 hours under anaero and anaero conditions , respectively . c. concisus bacteria were collected and washed with pbs and then 19 g whole cell proteins were separated on 12% sds - page as described previously . the gel lane of each sample was cut into 10 slices . in - gel the extracted peptides were separated by liquid chromatography and analysed by ms / ms as previously described [ 16 , 17 ] . mascot daemon program ( matrix science , london , uk ) was used for bacterial protein identification against the ncbi database . the spectral counts of the same proteins expressed by p6cdo - s1 under anaero and anaero conditions were compared using the scaffold-3 software ( proteome software , or , usa ) . mass spectrometry was conducted at the bioanalytical mass spectrometry facility , university of new south wales , australia . fisher 's exact test was used for analysis of the growth rate of c. concisus strains isolated from patients with ibd and controls . graphpad prism 5 software was used for statistical analysis ( san diego , ca ) . of the 57 oral strains examined , 52 strains ( 91% ) grew under anaero conditions . of the oral c. concisus strains isolated from patients with cd and uc , the positive growth rates under anaero condition were 84% ( 16/19 ) and 86% ( 12/14 ) , respectively , which were not statistically different from the positive growth rate of oral c. concisus strains ( 100% , 24/24 ) isolated from healthy controls ( p > 0.05 ) ( table 1 ) . the colonies of c. concisus strains , both the oral and enteric strains , grown under anaero conditions appeared much smaller than those grown under anaero conditions . the morphology of c. concisus grown under anaero and anaero conditions was not different under phase contrast microscopy . none of the c. concisus strains grew under microaerobic condition without h2 ; no bacterial colonies were observed on plates cultured under both micro and micro conditions . all strains grew under ori condition . to further compare the growth of c. concisus strains under anaero and anaero conditions , the cfus of 12 c. concisus strains grown under these two atmospheric conditions were determined . all strains had a greatly increased growth under anaero condition in comparison to the anaero condition . the cfu numbers of all 12 c. concisus strains grown under anaero condition were significantly higher than those of the respective strains grown under anaero condition ( p < 0.05 ) ( table 2 ) . p3uco - s1 strain was used as a representative strain to evaluate the growth of c. concisus under anaerobic and microaerobic conditions containing different concentrations of h2 . under anaerobic conditions , the cfus of p3uco - s1 strain cultured in the presence of 2.5% , 5% , and 10% h2 were ( 1.10 0.42 ) 10/ml , ( 9.15 0.82 ) 10/ml , and ( 1.90 1.33 ) 10/ml , respectively . the cfu numbers of 5% h2 were significantly higher than the cfu number of 2.5% and 10% h2 ( both p < 0.0001 ) . the cfu numbers of 10% h2 and 2.5% h2 were not significantly different ( p = 0.3 ) ( figure 1 ) . under microaerobic conditions , the cfu numbers of p3uco - s1 strain cultured in the presence of 2.5% , 5% , and 10% h2 were ( 1.0 1.15 ) 10/ml , ( 1.60 0.16 ) 10/ml , and ( 2.67 0.5 ) 10/ml , respectively . the cfu number of 2.5% h2 was significantly lower than the cfu numbers of 5% and 10% h2 ( p < 0.0001 and p < 0.005 , resp . ) . the cfu number of 5% h2 was significantly lower than the cfu number of 10% h2 ( p < 0.005 ) ( figure 1 ) . proteins expressed by strain p6cdo - s1 under anaero and anaero conditions were subjected to mass spectrometry analysis . a number of putative virulence proteins such as fibronectin - binding protein , outer membrane protein ( omp ) , protease htpx , s - layer - rtx protein , hemagglutinin / hemolysin - related protein , cjac , and evpb family type vi secretion protein were identified . the expression levels of these putative virulence proteins , indicated by the spectral counts , were not statistically different when c. concisus strain p6cdo - s1 was grown under anaero and anaero conditions ( table 3 ) . in this study , the growth of c. concisus strains under different atmospheric conditions was examined . it was previously described that c. concisus is a bacterium which requires h2-enriched microaerobic conditions for growth and some c. concisus strains may grow under anaerobic conditions if fumarate and formate are present in the culture plates [ 2 , 11 , 12 ] . in this study , we found that under anaerobic conditions the majority of oral c. concisus strains ( 91% , 52/57 ) grew on hba plates containing no formate or fumarate without the presence of h2 , suggesting that oral c. concisus is an anaerobic bacterium and that h2 gas , formate , and fumarate are not essential requirements for the anaerobic growth of oral c. concisus strains . none of the 57 oral c. concisus strains grew under microaerobic conditions without h2 , suggesting that microaerobic growth of c. concisus requires the presence of h2 , which is consistent with previous findings [ 9 , 11 ] . under anaerobic conditions , the presence of h2 greatly increased the growth of c. concisus , demonstrated by the increased colony sizes observed macroscopically and the increased cfu numbers of the same strain cultured under anaero and anaero conditions ( table 2 ) . these results suggest that under anaerobic conditions c. concisus has different metabolic pathways in generating energy for growth and oxidization of h2 is a pathway generating high energy for a rapid growth . the solubility of h2 gas in h2o is extremely low ; thus liquid culture methods are not suitable for assessing the impact of h2 gas on c. concisus growth . given this , in this study , the cfu numbers of c. concisus strains were determined using a plate culture method . in humans , h2 is produced by anaerobic bacteria predominantly in the colon [ 20 , 21 ] . h2 generated in the intestine is disposed by h2 consuming bacteria such as methanogenic bacteria , sulfate - reducing bacteria , and acetogens . some h2 is diffused into blood and this h2 can be measured by breath testing . dietary factors and the composition of an individual 's intestinal microbiota affect intestinal h2 production and consumption [ 2426 ] . the natural host of c. concisus is humans and the primary colonization site is the human oral cavity [ 1 , 2 ] . the basal level of hydrogen in healthy individuals is usually less than 10 ppm , thus having a h2 level of less than 0.001% ( 1 ppm = 0.0001% ) . in addition to anaerobic bacteria in the intestine , oral anaerobic bacteria may also produce h2 by fermentation of carbohydrate residues from food . mastropaolo and rees showed that , following a solid meal , the h2 produced by oral anaerobes was 25 ppm ( 0.0025% ) and this level was retained for only 73 minutes . given this , c. concisus colonizing the oral cavity is unlikely to have constantly available h2 for growth . the finding in this study that oral c. concisus strains were able to grow without the presence of h2 under anaerobic conditions helps to explain why c. concisus is able to colonize the human oral cavity . despite the fact that h2 dramatically increases the growth of c. concisus and the intestine is the dominant place for h2 production in humans , it is interesting to note that c. concisus has selected the oral cavity , rather than the intestinal environment , as its natural colonization site . this suggests that in healthy individuals there are some factors in the gastrointestinal tract that inhibit c. concisus intestinal colonization . it is likely that such inhibitory factors are low or lacking in patients with ibd , which contributes to the higher intestinal prevalence of c. concisus in these patients . one of such factors may be methanogenic bacteria , the dominant h2 consuming bacteria in the human intestine that produce methane . it is possible that methanogenic bacteria in the intestine compete with c. concisus for use of h2 . a study by mckay et al . examining hydrogen and methane excretion in patients with ibd and controls showed that the prevalence of methane excretion was 13% in patients with cd and 15% in patients with uc , which was significantly lower than that in healthy controls ( 54% ) . this observation was supported by a study from pimentel et al . , which showed that 97% of patients with ibd ( 75/78 ) , who had predominantly a diarrheal condition , excreted h2 only and no methane . these results suggest that there is a low level of methanogenic bacteria in patients with ibd . indeed , a study conducted by scanlan et al . detected a low prevalence of intestinal methanogenic bacteria in patients with ibd in comparison to healthy controls and other disease groups . the lack of sufficient intestinal methanogenic bacteria in patients with ibd may have generated an intestinal environment that allows c. concisus to use h2 for a rapid growth . we previously showed that some oral c. concisus strains were able to colonize the intestinal tract and have the potential to cause enteric disease [ 16 , 32 ] . in this study , we found that p6cdo - s1 strain , an oral c. concisus strain isolated from a patient with cd , expressed a number of putative virulence proteins . these proteins were previously reported to contribute to the virulence of other bacterial species [ 3239 ] . however if indeed these putative proteins play a role in c. concisus virulence , the finding in this study that the expression levels of these proteins remain similar when p6cdo - s1 strain is cultured under anaerobic conditions with and without h2 suggests that the impact of h2 on c. concisus virulence is unlikely through affecting these proteins . it is likely that h2 may affect c. concisus virulence through increasing the growth of c. concisus to a disease - causing threshold . this study also found that , under anaerobic conditions , p3uco - s1 strain , an oral strain isolated from a patient with uc , had a significantly higher cfu in the presence of 5% h2 , as compared to 2.5% h2 and 10% h2 . under microaerobic conditions , this strain had a significantly higher cfu in the presence of 10% h2 compared to 2.5% and 5% h2 . it appeared that the concentrations of h2 supplied in bacterial cultivation affect the optimal growth of c. concisus . this aspect should be further investigated by examining more c. concisus strains using systems that are able to supply fixed concentrations of co2 , n2 , and h2 , which will provide useful information to clinical laboratories in isolation of c. concisus from clinical samples . in addition to the 57 oral c. concisus strains , we have included six enteric strains , with five strains being isolated from patients with ibd , into this study . these enteric strains showed an anaerobic growth pattern that was similar to oral c. concisus strains . in summary , this study found that oral c. concisus strains were able to grow under anaerobic conditions without the presence of h2 , formate , or fumarate and that these strains did not grow in microaerobic conditions without h2 , suggesting that they are anaerobes . the presence of h2 in the anaerobic conditions greatly increased the growth of oral c. concisus strains . using mass spectrometry analysis , an oral c. concisus strain isolated from a patient with cd was found to express a number of putative virulence proteins and the expression levels of these proteins under anaerobic conditions with and without h2 remained similar . while the numbers of enteric c. concisus strains included in this study were small , these enteric strains and oral c. concisus had a similar anaerobic growth pattern . this study provides useful information in understanding the natural colonization site and pathogenicity of c. concisus .

participation in exercise and regular physical activity can provide numerous physiological , cognitive , and psychological health benefits in the aging population . impairments in fitness components such as muscle strength and balance influence the development of disability . in addition to muscle strength and balance , aerobic endurance , agility , mobility , and flexibility have also been shown to be significant determinants of physical independence . this is consistent with the notion that physical fitness factors greatly on the ability to successfully perform routine daily tasks . this includes functional tasks such as simple housework , lifting and carrying objects , negotiating steps , and walking far enough to shop and complete errands [ 3 , 4 ] . defined as the capacity of an individual to carry out the physical activities of daily living , physical function is an independent predictor of functional independence , disability , morbidity , and mortality . aging is often associated with declines in physical function affecting vital processes that are critical to independence , social engagement , and quality of life . older adults transitioning toward disability may no longer be able to appropriately utilize and execute movement patterns associated with activities of daily living ( adl ) as a result of declining physical fitness . programs of regular exercise that include cardiorespiratory , resistance , flexibility , and neuromotor exercise beyond activities of daily living to improve and maintain physical fitness and health are then essential for most adults . although mature nervous system function and motor skills are achieved in the earlier stages of life , changes in movement patterns also occur during adulthood and the latter stages of the lifespan . older adults need to practice , learn new , and relearn known motor skills as part of task training , recreational pursuits , or rehabilitation . therefore , opportunities for exercise that foster fitness , efficient functional movement skills , and self - efficacy toward performing daily tasks are needed to impede the progression of the disablement process among older adults . as such , community - based modes of exercise aimed to equip older adults with neuromotor ( balance , coordination , and agility ) , physical ( aerobic endurance , muscle strength , and flexibility ) , and functional components of fitness necessary for daily life should be explored and developed . as the population of older adults is continuing to increase and become more diverse , it will be important to recognize individual differences in level of functioning . while aging is an inevitable process older adults of the same chronological age may differ in physiological age and function due to differences in genetic makeup , lifestyle choices , cognitive ability , and several other variables . exercise programs such as the one in this study should be designed to accommodate the variable capabilities and functional levels of older adult women . additionally , they must be motivating enough to ensure active participation at an enjoyable level and increase the frequency of physical activity in a social , interactive setting . while this is true it is often the case that opportunities for exercise in community - based settings are not accommodating to individuals of all levels of functioning . exercise classes at community centers and facilities for adults will most likely contain individuals with varied levels of disability , as this is the nature of the older adult population . for example , a step aerobics class at the local senior center may have a participant who is highly functional and does not suffer from any level of disability exercising next to someone who is of the same age but has low cardiovascular endurance and poor balance . the effect and benefit of exercise on both types of individuals in this kind of environment are largely unknown . therefore , the primary purpose of this study was to observe the exercise habits of older adult women with varying levels of physical function to determine any differential effects of exercise on their physical fitness . a convenience sample of 30 women ( n = 30 , mage = 69 years ) recruited from the ywco in athens , georgia , completed participation in this study . however , seven participants were forced to withdraw for various reasons including hospitalization due to sickness , moving away , and summer travel . participants were recruited in person while they attended exercise classes at the ywco and also by posting flyers and sign - up sheets at the facility . all potential participants completed a medical history questionnaire to screen for conditions that may have inhibited safe exercise participation . exclusion criteria included history of stroke , heart attack , osteoarthritis , neurological disease , mental illness , and fracture or joint replacement within the last six months . individuals reporting one or more of such conditions were excluded from participation in this study . additionally , the participant medical history form provided demographic information related to age , sex , height , weight , marital status , employment status , and dwelling status . participants were also asked to report an average amount of time per week they routinely spent involved in planned exercise . two groups of participants were formed according to their physical ability to live independently as determined by the composite physical function ( cpf ) scale . the cpf is a self - report 12-item scale capable of assessing physical function across a wide range of activities from basic adls such as bathing and dressing to instrumental adls including gardening and shopping [ 3 , 4 ] . each item can be scored from 0 to 2 ( 0 = can not do ; 1 = can do with help ; 2 = can do without help ) based on the participants perceived ability to perform the task in question . high functioning , moderate functioning , or low functioning and at risk for loss of independence . high functioning individuals are those who indicate that they can perform all 12 items on their own without assistance , thus receiving a perfect score of 24 . both the definition of moderate functioning and its interpretation are adjusted for age . it is important that younger age groups have more stringent criteria than older age groups for being assessed as moderate functioning as age - related declines in physical capacity after the age of 60 are commonly reported to decline at least 10%15% per decade . whereas a score of 14 ( ability to perform a minimum of seven cpf activities without assistance ) is required for a rating of moderate of those aged 90 years and older , higher scores of 20 , 18 , and 16 , respectively , are needed in order for those in their 60s , 70s , and 80s to be rated as moderate functioning [ 3 , 4 ] . using this age - adjusted scoring the participants were placed in either the high functioning ( n = 13 ) or moderate / low functioning ( n = 17 ) groups . all participants were required to read and sign the university of georgia institutional review board consent form prior to commencing any screening , testing , or exercising . as part of a senior fitness initiative , each participant from both groups was encouraged to continue their normal routine of attending group exercise classes at the ywco and also to exercise on their own at home for a 10-week period . weekly exercise logs were used throughout the study as a method of self - reporting to record and compare the exercise habits of participants from each group with varying levels of functional independence . the participants were instructed to use the weekly exercise log to record each bout of planned exercise in which they participated during the 10-week study . the senior fitness test ( sft ) [ 3 , 4 ] was used to assess the overall fitness of both groups of participants at baseline , after five weeks , and again after 10 weeks of exercising . the sft is commonly used to assess physical fitness in older adults , as it represents an easy - to - use field test battery that allows for the assessment of physical fitness components vital to maintaining independent functioning . a weekly exercise log was given to each participant of them to record the type and duration of each bout of exercise . participants only received credit for planned exercise and not for other forms of physical activity such as housework , grocery shopping , or doing laundry . the weekly exercise logs were used as a method of self - reporting to record and compare the exercise habits of participants from each group with varying levels of functional independence . participants received reminders to continue tracking their exercise in - person and by email on a weekly basis . weekly exercise logs were submitted for review at the midpoint and at the end of the 10-week exercise period . the number of weekly minutes of exercise performed by the high functioning and moderate / low functioning groups was then tallied and recorded at these time points . the average number of minutes exercised was then calculated for each group after five weeks and 10 weeks of exercise . the senior fitness test consists of seven items , including one alternate test for measuring aerobic endurance . the sft items are as follows : ( 1 ) 30-second chair stand ; ( 2 ) arm curl ; ( 3 ) chair sit - and - reach ; ( 4 ) back scratch ; ( 5 ) 6-minute walk ; ( 6 ) 2-minute step ; and ( 7 ) 8-foot up - and - go . the purpose of the 30-second chair stand is to measure lower extremity strength , which is necessary for tasks such as transferring from a chair , walking , and climbing stairs . the total number of complete stands able to be completed in 30 seconds was recorded . the arm curl component of the sft measures upper extremity strength , which is needed to perform household activities such as lifting and carrying groceries , suitcases , or grandchildren . the total number of bicep curls completed with correct form with the dominant arm while holding a five - pound weight in 30 seconds was recorded . the chair sit - and - reach test will be administered to assess lower body flexibility , which is important for good posture and normal gait patterns . lower body flexibility also contributes to performing mobility tasks such as getting in and out of a car or bed . from a sitting position at the end of a chair , with one leg extended and hands reaching toward toes , the number of inches ( + or ) between extended fingers and tip of toe was measured during the chair sit - and - reach test . shoulder flexibility is vital for tasks such as combing one 's hair , putting on a coat , and reaching for a seat belt . during this test , the number of inches between middle fingers ( + and ) was measured while reaching over the shoulder with arm and up the middle of the back with the other arm . the 2-minute step test was chosen for the assessment of aerobic endurance because space and time limited the use of the 6-minute walk . aerobic endurance is important for walking distances , climbing stairs , and other activities such as shopping or sightseeing . as such , cardiovascular fitness was measured with the 2-minute step test which entails recording the number of full steps completed in two minutes while raising each knee to a point midway between the patella and iliac crest . the recorded score was equal to the number of times the right knee reaches the required height . the final component of the sft is the 8-foot up - and - go test and is intended to assess agility and dynamic balance . this fitness component is essential for quick maneuvering which is required for activities such as rushing to answer a telephone or to use the restroom . the 8-foot up - and - go test requires an individual to rise from a chair , walk forward 8 feet , change direction , and return to their seated position in the chair . the fastest time from two trials was recorded to the nearest hundredth of a second . each test item of the sft has accompanying performance standards for men and women ages 60 to 94-plus based on a national study of more than 7,000 americans [ 3 , 4 ] . additionally , the sft provides threshold values on each test item that help to identify if an older adult is at risk for mobility loss . for a 10-week period participants were monitored while performing their normal exercise routines within the athens , ga , community . the majority of this exercise took place at the athens ywco with the remaining exercise taking place at home or in other settings . while at the ywco , participants attended a variety of exercise classes designed for older adults . the classes were either 30 or 60 minutes in duration and collectively emphasized all components of physical fitness . there was no limit placed on the participants in terms of type or amount of classes they were allowed to attend in each week . it was common for the women in both groups to exercise together in the same classes and attend different classes on an individual basis . the exercise classes offered at the ywco during the time of the study focused on functional movements and included group strength training , pilates , silver sneakers , and step and sculpt . after baseline testing the participants were educated on how to execute proper posture during exercise and while performing daily activities . the researchers collaborated with the ywco instructors to ensure that proper execution of functional movements was emphasized during class time . instructors were also asked to associate the movements being taught to activities of daily living . for example , when performing arm strengthening exercises instructors were asked to relate these movements to functional tasks such as lifting and carrying items similar to laundry baskets , grocery bags , or grandchildren . the type and duration of class participation and other exercise activities were recorded on the weekly exercise log . the senior fitness test was given to each group before , at midpoint , and following 10 weeks of community - based exercise . the senior fitness test ( sft ) measures the components of fitness with a chair stand test , arm curl test , 2-minute step test , chair sit - and - reach test , back scratch test , and 8-foot up - and - go test . each test is scored separately as there is no composite score for the sft . as such , a separate mixed 3 ( time ) 2 ( group ) anova , with time as the within - subjects factor and group based on level of physical function as the between - subjects factor , analyses were conducted using spss 22 software ( spss ibm , new york , usa ) . the number of weekly minutes of exercise performed by the high functioning and moderate / low functioning groups was tallied and recorded at the midpoint and after 10 weeks of exercise . the estimation of time spent exercising reported on the health and medical history questionnaire before the study was used as the preexercise value for self - report exercise . the results of the 3 2 mixed anova statistical analysis showed no significant interaction effect for timegroup for any of the six subtests ( chair stand , arm curls , 2-minute step , chair sit - and - reach , back scratch , and 6-foot up - and - go ) of the sft . however , the main effect of time was significant for all fitness components and the main effect of group was significant for all fitness components with the exception of lower extremity flexibility ( chair sit - and - reach test ) . means and standard deviation for each of the six subtests scores for both groups are presented in figures 16 . statistical analysis showed no significant interaction effect for time group : f(2,52 ) = 0.089 , p = 0.915 , and n = 0.003 for number of chair stands at any of the three time points . the main effect of time showed a statistically significant difference in chair stands at the 3 time points : f(2,52 ) = 26.983 , p < 0.0005 , and partial n = 0.509 . the main effect of group showed a statistically significant difference in chair stands between physical function groups : f(1,26 ) = 7.387 , p = 0.012 , and partial n = 0.221 . the mean scores for the high function group for pre- , mid- , and posttests were 17.5 , 20.8 , and 22.9 , respectively . the high function group mean score for chair stands also increased from midpoint to posttest with a percent change of 10.1% . mean scores for the moderate / low function group increased from 12.5 to 15.3 yielding a percent change of 22.4% . percent change between midpoint and posttest was 14.4% with mean scores increasing from 15.3 to 17.5 . mean scores for the high function group increased over time and were 21.8 , 25.9 , and 27.15 for pre- , mid- , and posttests , respectively . percent change between baseline and midpoint was 18.8% and was 4.8% between midpoint and posttest . the moderate / low function group mean scores at pre- , mid- , and posttests were 19.4 , 21.3 , and 23.2 . baseline to midpoint percent change was calculated to be 9.8% and midpoint to posttest percent change was 8.9% . statistical analysis showed no significant interaction effect for time group : f(2,52 ) = 0.743 , p = 0.481 , and partial n = 0.028 at any of the data collection time points . the main effect of time showed a statistically significant difference in arm curls at the three time points : f(2,52 ) = 10.636 , p < 0.0005 , and partial n = 0.290 . the main effect of group showed a statistically significant difference in arm curls between physical function groups : f(1,26 ) = 6.969 , p = 0.014 , and partial n = 0.211 . the high function group averaged 113 , 131.9 , and 142.54 steps at the three data collection time points . the percent change between baseline and midpoint was 16.7% and was calculated to be 8.1% between midpoint and posttest . the moderate / low function group mean scores increased by 18.5% between baseline and midpoint and also increased between midpoint and posttest with a percent change of 8.4% . there was no significant interaction effect for time group for the 2-minute step test : f(2,50 ) = 0.032 , p = 0.853 , and partial n = 0.001 at any of the three data collection time points . the main effect of time showed a significant difference in steps at the 3 time points : f(2,50 ) = 14.08 , p < 0.0005 , and partial n = 0.360 . the main effect of group showed a significant difference in steps between physical function groups : f(1,26 ) = 10.71 , there was no significant interaction effect for time group for the chair sit - and - reach test : f(2,52 ) = 0.967 , p = 0.387 , and partial n = 0.036 . the main effect of time showed a significant difference in inches reached at the 3 time points : f(2,52 ) = 15.235 , p < 0.0005 , and partial n = 0.369 . however , the main effect of group did not show a significant difference in inches reached between physical function groups : the high function group increased their mean scores by 67.6% and 14% between baseline and midpoint and between midpoint and posttest , respectively . the moderate / low function group increased their mean scores by 47% between baseline and midpoint and also increased mean scores by 76% between midpoint and posttest . there was no significant interaction effect for time group for inches reached on the back scratch test : f(2,52 ) = 1.1 , p = 0.341 , and partial n = 0.115 at any of the three data collection time points . the main effect of time showed a significant difference in inches reached at the 3 time points : f(2,50 ) = 3.321 , p = 0.044 , and partial n = 0.117 . the main effect of group showed a significant difference in inches reached during the back scratch test between physical function groups : f(1,26 ) = 24.374 , p < 0.0005 , and partial n = 0.484 . the high function group increased their mean scores by 45.7% and 25% between baseline and midpoint and between midpoint and posttest , respectively . the moderate / low function group increased mean scores by 47.8% between baseline and midpoint and also increased mean scores by 36% between midpoint and posttest . there was a 12.6% percent change for the high function group between pre- and posttest . the percent change for the same group between midpoint and posttest was 7.6% . the moderate to low function group improved by 13.7% and 6.3% , respectively , during the two periods between data collection time points . there was no significant interaction effect between time group for the 8-foot up - and - go test : f(2,52 ) = 0.06 , the main effect of time showed a significant difference between midpoint and postexercise tests : f(2,52 ) = 4.099 , p = 0.022 , and partial n = 0.136 . the main effect of group showed a significant difference in up - and - go scores between physical function groups : f(1,26 ) = 13.071 , p = 0.001 , and partial n = 0.335 . the benefits of exercise for older adults have been established in the research literature and are well known . there is evidence that habitual exercise can minimize the physiological effects of an otherwise sedentary lifestyle and prolong active life expectancy . the heterogeneity observed among older adults and their level of physical function has important implications for research and clinical practice . this notion supports the concept that tailoring exercise programs and interventions to specific deficits and the current state of physical functioning is an important consideration . research has left little doubt that exercise interventions for older adults need to consider the individual functional needs of participants . as such , the success of aerobic and resistance training for obese men and women , balance training for individuals at risk for falls , and water exercise for those with osteoarthritis have all been shown to be effective for older adults with these specific physical function deficits . despite the plethora of data showing an overall benefit of exercise training for improving , or maintaining functional ability in older adults , attention to individual differences and identification of factors that influence efficacy of exercise as a therapy for aging - related loss of physical function have important clinical significance . moreover , the specific amount of exercise necessary to elicit maximal improvements in physical outcomes may differ between types of individuals . exercise training studies that show efficacy for improving functional ability often report main effects or mean group differences without expressing the extent of variability for these tasks . therefore , it was the primary purpose of this study to observe the exercise habits of older adult women with different levels of physical function abilities , to determine any differential effects of exercise on their physical fitness . the specific aim of the study was to determine and compare the effects of 10 weeks of community - based exercise on the cardiovascular endurance , muscular strength , flexibility , and balance fitness components of older adult women with high and moderate - low levels of physical function . the results of the statistical analysis indicate that the impact of time ( 10 weeks of exercise ) on the fitness - related outcome measures did not differ based on level of physical function at any time during the study . in other words , older adult women from this sample were able to improve fitness over time regardless of their initial level of physical function as indicated by their initial score on the composite physical function scale and group assignment . over the course of 10 weeks the women from both groups improved their sft scores related to leg strength , arm strength , cardiovascular endurance , leg flexibility , arm flexibility , and mobility and dynamic balance . this finding is consistent with the hypothesis that all participants would increase their scores on all sft subtests over time . the exercise classes offered at the ywco provided the participants with a variety of options in terms of which fitness components to train . many of the exercise classes such as pilates and silver sneakers emphasized multiple fitness components making them highly beneficial to the overall fitness of the participants . by offering programs of regular exercise that include cardiorespiratory , resistance , flexibility , and neuromotor exercise beyond activities of daily living , the athens ywco is equipped to improve and maintain the fitness and health of its members . additionally , the implementation of the senior fitness initiative at the ywco greatly influenced the participants in terms of their morale and enthusiasm toward exercise . they were highly engaged and eager to learn more about their individual level of fitness . while the high functioning group consistently had significantly better sft scores as compared to their counterparts , they did not improve at a greater rate or more significantly over time as compared to the moderate / low functioning group . however , it should be noted that the percent change during the second half of the study between midpoint and posttests was higher for the moderate / low functioning group for all six subtests of the senior fitness test . this implies that the group with lower amounts of functional ability continued to improve and were more resistant to the plateau effect shown by the high function group . the lower functioning individuals seemed to be inspired by their gains and were highly motivated to keep pace with the other women in the study . this finding suggests that there may be some benefit to integrating community - based exercise classes with people of various functional skill sets when resources or logistics are not conducive to separating them . this should only be done with qualified instructors who are able to offer modifiable exercise opportunities to the variable older adult population . as it was also an aim of this study to compare the exercise habits of older adult women functioning at different levels of independence , self - report prior to the 10-week exercise period , both groups were asked to estimate the amount of time they spent engaged in exercise on a weekly basis . an interesting finding was that the actual number of recorded minutes during the study ( at midpoint and after study ) was lower than the estimated averages reported by both groups prior to commencing the 10-week exercise period . perhaps this can be explained by social desirability , which can lead to overreporting of physical activity . this common limitation of self - report instruments appears to have held true for the weekly exercise logs and this sample of older adult women . despite this limitation , there were some benefits of utilizing the weekly exercise logs to self - report exercise activity . the participants often reported the feeling of being held accountable for their exercise activity because they were forced to write it down and track it over 10 weeks . the weekly exercise logs seemed to produce a sense of pride among the participants and may have motivated them to be more physically active . initiating exercise programs and adhering to them is often problematic for older adults . using weekly exercise logs may help to alleviate this problem among older adults . also , all participants consistently reported performing exercise activity at home and in other settings outside of the ywco . this indicates that senior fitness initiatives such as the one implemented during this study may be helpful in improving the overall exercise habits of older adults . these findings suggest that , although sometimes limited in effectiveness , self - report instruments to track the exercise habits of older adults in a community - based setting may be useful when more scientific and reliable options are not available . it is possible that these instruments may have cognitive and physical benefits for older adults . their use requires the highly complex task of recall and may motivate individuals to be more active by holding them accountable for the amount of time they choose to be engaged in exercise . additionally , study findings suggest that participation in fitness and exercise initiatives in a community - based setting can improve the perceived ability of older adults to function independently . in conclusion first , community - based exercise programs offering a variety of exercise types to people with varying levels of functional ability have both physical and psychosocial benefits . second , these programs may also be capable of improving the self - efficacy of lower functioning older adults toward performing daily tasks . finally , self - report instruments such as activity logs may be useful to track and gain an understanding of the exercise habits of older adults . this study was limited due to the nature of the community from which the participants were recruited . the ywco members were highly educated and most already had an established history of physical activity and exercise . future research should build upon using multimodal exercise regimes focusing on proper format , periodic assessment and feedback , and encouragement of home - based activities . more importantly exercise programs should teach older adults to move correctly while generalizing movements to activities commonly encountered by the population .

pets , especially domestic dogs , provide significant benefits to individuals and to society , since they can contribute to physical , social , and emotional development of children and the wellbeing of their owners ; however , hygiene and dietary habits of the animals make them particularly prone to having a range of infectious diseases during their life [ 1 , 2 ] . in this sense , especially the hookworms and the fact that they are identified as the most prevalent in these animals , as demonstrated in several parasitological surveys in brazil and in the world [ 38 ] . the hookworms may cause intestinal disorders in dogs , leading to manifestation of symptoms such as anemia , weight loss , irritability , and abdominal pain and even result in death . they are also able to trigger pathological processes in humans such as cutaneous larva migrans and eosinophilic enteritis . one of the most important steps to control hookworm is the adoption of antiparasitic treatment of infected dogs . however , despite the widespread availability of allopathic drugs for this purpose , there is a clear difficulty in decreasing the prevalence of this parasite in canine host . such issue can be attributed mainly to poor hygienic and alimentary habits of these animals and to the high environmental contamination . it is possible mainly by the existence of large amounts of stray dogs which are rarely treated and eventually spread helminth eggs and larvae . in addition , treatment with antihelminthic commercially available requires the administration of several doses and a number of repetitions throughout the life of the animal , in case of being neglected or developing resistance [ 11 , 12 ] . therefore , it is necessary to develop new strategies to control this parasite , highlighting the potential use of probiotics for this purpose . probiotics are products that consist of living and nonpathogenic microorganisms that can promote a balance in favor of the gi ( gastrointestinal ) tract microbiota , as well as an upregulation of the immune response , thereby contributing in the prevention and treatment of pathologies [ 13 , 14 ] . in this regard , species of the genus lactobacillus distinguish among microorganisms having probiotic properties , since they have the ability to tolerate acidic ph levels and , thus , to survive passage through the gi tract . these microorganisms also have the potential to inhibit or hinder the colonization of pathogenic species level in the digestive system , which can be evaluated as a potential tool for the control of various infectious processes afterwards , including the canine hookworm . thus , the present study aimed to evaluate the applicability of species of lactobacillus having probiotic properties in the control of hookworm in dogs naturally infected . pool to dogs infected with hookworm species from the zoonosis control center ( ccz ) of taubat , so paulo , from january to march 2010 . this paper was submitted to the ethics committee in animal research of fapi and approved under protocol 009/2009 . three strains of lactobacillus were evaluated whose probiotic effects were demonstrated by pereira and coutinho , namely : lactobacillus plantarum atcc 8014 ; lactobacillus acidophilus atcc 4536 ; and lactobacillus delbrueckii ufv h2b20 . l. plantarum and l. acidophilus strains were provided by the laboratory of microbiology of the university center geraldo di biasi , volta redonda , rj , brazil , and the l. delbrueckii ufv h2b20 strain was provided by the department of food technology , from the federal university of viosa , viosa , mg , brazil . all strains were maintained at 4c on mrs agar ( man , rogosa and sharp ) and were reactivated every 15 days in mrs broth , ph 6.2 , sterilized at 121 for 15 minutes , and incubated at 37c for 24 hours . the probiotic preparation was administered to animals in the form of pool , composed by mixing 1 ml of each culture containing 1 10 colony forming units ( cfu ) of each strain . in order to check the absence of contamination of the strains , gram staining and catalase test were performed during the assays . the amount of 1 10 cfu was determined based on technical regulation of bioactive substances and probiotics isolates on allegation of functional and health , according to which a preparation can only be considered as having probiotic property if it shows therapeutic property in a concentration of at least 1 10 ufc / ml . for experimental groups , animals with the following characteristics were selected : both genders , older than one - year old , without any diagnose or overt disease , and in whose feces only eggs of the ancylostomatidae family species were found by coproparasitological examinations conducted in the laboratories of parasitology from faculdade de pindamonhangaba ( fapi ) and from universidade de taubat ( unitau ) . the study excluded pregnant female dogs , one - year old or less canids , and the ones in whose fecal samples structures compatible with any intestinal parasite were found , except the ancylostomatidae family , as well as those with clinical symptoms or confirmed diagnosis of any infectious or severe disease . parasitized animals that have recently undergone treatment with antiparasitic drugs or who have undergone this type of treatment in a period shorter than 60 days were also excluded from the experiment . starting from the assumption that tests in parasitology often require the use of nonparametric methods for evaluating treatments and , therefore , each experimental group must contain at least six observations , the number of dogs in each group was established as 10 in this study and each group was set clustered in separate kennel . for the selection of the animals , fecal examinations were performed before the intervention using the willis method . the collection of fecal material was performed directly on the rectal ampulla of dogs and the samples deposited on stool specimen collectors , without added preservatives . fecal material was sent under refrigeration to the laboratory of parasitology of fapi and unitau , using the method mentioned above . after coproparasitological evaluation , the number of eggs per gram of feces ( epg ) of each animal selected for experimental groups was determined by the gordon - whitlock method . after the selection of infected animals , they were grouped as follows : group ( a ) 10 animals infected with ancylostomatidae treated with the probiotic preparation under study ; group ( b ) 10 animals infected with ancylostomatidae and not submitted to any treatment . the probiotic preparation under study , consisting of a pool of lactobacillus strains previously grown in mrs broth , was diluted in skim milk and administered on alternate days to the animals for a period of 28 days . to assess the efficacy of treatment with the probiotic preparation , a count of parasitic structures in the feces of animals was performed 1 day before the experiment and 7 , 14 , 21 , and 28 days after the start of the experiment , respectively , using the gordon - whitlock method and , particularly for group ( a ) , fecal samples were collected 14 days after completion of treatment to determine the epg number . the percentage of epg reduction was determined by using the epg mean values of the first day before the treatment started ( day zero ) and the means of the respective samples , using the following formula , were described by jacobs et al . : % of epg reduction = epg mean ( day 0 ) epg mean ( day of interest ) 100/epg mean ( day 0 ) . % of epg reduction = epg mean ( day 0 ) epg mean ( day of interest ) 100/epg mean ( day 0 ) . in order to assess the evolution of the immune and anemic status , blood samples were collected from the animals in tubes containing edta ( one day before submitting to treatment and 14 and 28 days after starting the experiment ) and blood counts were determined . the hematocrit or fused cell volume was determined , together with hemoglobin , by an automated method using a coulter act8 equipment . parametric ( anova and student 's t - test ) and nonparametric tests ( mann - whitney and kruskal - wallis ) were used , being applied according to the normality of the results , in a level of significance of 5% , using the software bio estat 5.0 as a support tool . one day before the start of the treatment ( day 0 ) , the epg values from the control of infected untreated group ( b ) and treated with probiotics group ( a ) did not differ significantly ( p > 0.05 ) from each other ( figure 1 ) . however , on the 28th day after the treatment started , the mean of group ( a ) was 455 epg , showing a reduction of 88.83% in fecal egg count ( table 1 ) , which is statistically significant ( p < 0.05 ) compared to that observed in the control group ( b ) . the animals of group ( a ) were monitored regarding the egg laying in the feces , for 14 more days after the withdrawal of the treatment with the probiotic preparation . therefore , the epg determination was performed on days 35 and 42 and the results are shown in figure 2 . an increase in epg values when compared to those determined on day 28 , which suggests that it is of critical importance the inclusion of probiotic preparations in the diet , to ensure the maintenance of low counts of epg . as far as we know , this is the first report that shows the potential applicability of lactobacillus species in the control of canine hookworm . the potential use of the strains evaluated in this study , regarding the antiparasitic activity , was also demonstrated by countinho , which evaluated the control of cryptosporidiosis in experimentally infected mice . this author evaluated a probiotic preparation containing 1 10 cfu of the same strains used in this study , which was administered for 10 consecutive days and observed 100% reduction of cryptosporidium parvum oocysts in the feces of mice . the positive effects of probiotics against parasitic infections have also been demonstrated in infections caused by species of nematodes of the family ancylostomatidae . administered a combination of anthelmintic drugs ( febantel , pyrantel pamoate , and praziquantel ) , along with a probiotic preparation of commercial use , consisting of lactobacillus acidophilus , bifidobacillus bifidum , and enterococcus faecium , and although the effects of administering microorganisms have not been evaluated in isolation , these authors observed that the use of the probiotic product had a positive influence on the clinical conditions of dogs treated . demonstrated the efficacy of administering probiotics to control another species of nematode , namely , trichinella spiralis when administering 1 10 cfu of l. casei shirota , intraperitoneally , once a week for 3 weeks to 60 mice challenged with 200 larvae of t. spiralis . the authors noticed that there was a significant decrease in the number of adult parasites in the gut of animals . in the present study , the ability of probiotic preparation in order to influence the anemia of infected animals was also evaluated . before the treatment ( t0 ) , all dogs were anemic concerning the erythrocytes blood count observed when compared with reference values ( 6000 a 8000 10 per mm of blood ) . there was no significant statistical difference regarding the number of red blood cells among the animals of group ( a ) and group ( b ) ( figure 3 ) . however , after 28 days of treatment , animals in group ( b ) had significantly lower erythrocyte counts ( p < 0.05 ) than observed in group ( a ) , indicating that the probiotic treatment was effective to stabilize the anemia that resulted from spoliation blood promoted by hookworms . this stabilization of anemia may be associated with the 88.83% reduction of the number of epg observed in group ( a ) , possibly consequent from the declining number of parasites in the gut with consequent reduction of the spoliation of blood , and may also be related to the own action of the lactobacillus species assessed . according to silva et al . the increase in the number of erythrocytes is linked with the increase of iron absorption which can be increased by the action of probiotics [ 25 , 26 ] . once absorbed , iron binds to transferrin transport iron to the bone marrow where erythroid precursors capture this metal for the production of the hemoglobin molecule . after this step , the erythroid precursors get mature and form red blood cells ( erythrocytes ) , which migrate into the circulatory system . with this result , the greater the absorption of iron is , the greater the number of erythrocytes and the concentration of serum hemoglobin produced will be [ 27 , 28 ] . the positive effects of administration of probiotic preparation may also be noticed when evaluating the concentration of hemoglobin in animals from both groups , as shown in figure 4 . it is observed that at the end of treatment ( t28 ) , there was a significant reduction in the hemoglobin concentration in group ( b ) probably related to spoiling of blood which is caused by parasites in the intestinal mucosa . moreover , the reduction of the parasitic load induced by the administration of probiotics to group ( a ) , probably induced a lower theft of blood , allowing a nonsignificant decrease in hemoglobin levels . the amount of white blood cells as well as the differential count of leukocytes are two determinations that make the cbc ( complete blood count ) , and in this study , they were used as a tool to assist in the understanding of the mechanisms involved in the healing process of the canine hookworm , induced by probiotic preparation under study and the average results of the global score are shown in figure 5 . after assessing the variations of global values of leukocytes in group ( a ) , it is possible to show that the leukocyte count observed during the last determination ( day 28 ) was significantly higher ( p < 0.05 ) than the one observed on the first day ( day 0 ) and second week ( day 14 ) of experimental monitoring . this fact may have influenced positively in the reduction of opg that was observed in this group . however , it should be emphasized that although there was an upward tendency in the number of leukocytes only in the treated group , there was no statistical difference between the two groups . concerning the reference values , there was a contrasting situation : in the beginning of the treatment ( day 0 ) , the meaning value of the leucocyte of the group not treated was above the normal value and in the end of treatment ( day 28 ) this occurred only in the treated group . according to gill , the consumption of fermented milk containing probiotic microorganisms , particularly lactobacillus species , may improve the function of the innate immune response , increasing the number of leukocytes and phagocytic activity of peripheral blood leukocytes which can remain elevated for several weeks , even after consumption ceased . that can positively influence the immune response for protection against infections as well . it is also verified ( figure 6 ) that the average values of lymphocytes observed in group ( a ) after 28 days were significantly higher than those observed on days 0 and 14 of treatment , as well as higher than the reference values ( 800 to 4160 lymphocytes per mm of blood ) , characterizing lymphocytosis . this significant increase in the number of lymphocytes was observed only in the animals of this group , thus emphasizing that the administration of probiotics positively influenced the immune response by stimulating the production of lymphocytes . this hypothesis is strengthened by the fact that the peak increase in the number of lymphocytes in group ( a ) coincideds with the period in which a significant decrease in the number of epg in the feces ( figure 6 ) was evidenced , which occurred on the day 28 of experiment . with respect to other subtypes of leukocytes , among which neutrophils and eosinophils , there were no statistically significant changes in their scores during the experiment . the administration of the probiotic preparation containing 1 10 ufc of lactobacillus plantarum atcc 8014 ; lactobacillus acidophilus atcc 4536 and lactobacillus delbrueckii ufv h2b20 to the infected dogs induced a significant increase in global leukocyte and lymphocyte counts and a decrease in the number of epg in feces after 28 days of treatment . however , after this period , the epg values tended to increase , thereby emphasizing the importance of the inclusion of a probiotic preparation in animal diets as an alternative to keep them free of parasites .

the purity and concentrations of the dna in all cases were measured . after verifying the dna quality , 219 patients were selected and were included in clonality analyses of ig and tcr gene rearrangements , as shown in table 1 . the b cell lymphomas ( 78 cases ) were comprised of 49 diffuse large b cell lymphomas ( dlbcls ) , 8 mucosa - associated lymphoid tissue lymphomas ( maltls ) , 3 nodal marginal zone b cell lymphomas ( nodal mzls ) , 6 follicular lymphomas ( fls ) , 4 mantle cell lymphomas ( mcls ) , and 8 b lymphoblastic leukemias / lymphomas ( bll / l ) . the t cell lymphomas ( 80 cases ) included of 41 peripheral t cell lymphomas , unspecified ( ptcls - u ) , 7 anaplastic large cell lymphomas ( alcls ) , 12 nasal type nk / t cell lymphomas ( n - nk / ts ) , 12 angioimmunoblastic t cell lymphomas ( aitls ) , 2 adult t cell leukemia / lymphomas ( atl / ls ) , and 6 t lymphoblastic leukemias / lymphomas ( tll / l ) . finally , 61 patients with reactive lymphoproliferative disease included 37 incidentally - detected palpable lymphadenopathies ( laps ) , 9 infection - related laps , 5 radiation - related laps , 4 autoimmune disease - related laps , 3 fevers of unknown origin laps , 2 radioactive iodine therapy - associated laps following surgery to treat thyroid papillary carcinomas , and 1 chemotherapy - related lap . sections ( 5 m thickness ) were cut from each paraffin block , and the areas of lymphoma were scraped from the unstained formalin - fixed , paraffin - embedded sections . the dna was extracted using a qiaamp dna mini kit according to the manufacturer 's protocol ( qiagen , hilden , germany ) . polymerase chain reaction ( pcr ) products were obtained using a gradual dilution of dna analyzed on metaphor agarose gel ( cambrex bio science , rockland , me , usa ) in five cases . bands of pcr products with dna over 30 ng/l were detected using the metaphor agarose gel . therefore , 219 out of the 250 samples with dna over 30 ng/l were analyzed with the full set of biomed-2 pcr assay ( invivoscribe technologies , san diego , ca , usa ) for ig and tcr gene rearrangement . thirty - one specimens with dna concentrations of less than 30 ng/l were excluded from this study . pcr amplifications were performed using a biomed-2 multiplex pcr master mix ( three tubes of complete igh [ igha : vhfr1-jh ; ighb : vhfr2-jh ; ighc : vhfr3-jh ] , two tubes of incomplete igh [ ighd : dh1 - -6-jh ; ighe : dh7-jh ] , two tubes of igk [ igka : v-j ; igkb : v-kde and jc intron - kde ] , two tubes of complete tcrb [ tcrba : v-j1/2.2/2.6/2.7 ; tcrbb : v-j2.1/2.3/2.4/2.5 ] , one tube of incomplete tcrb [ tcrbc : d-j ] , and two tubes of tcrg [ tcrga : vi/10-j ; tcrgb : v9/11-j ] ) . the biomed-2 pcr protocols19 were performed using a thermal cycler according to the manufacturer 's recommendations . each pcr was carried out in a 10-l volume ( 9 l of master mix with 1 unit of amplitaq gold dna polymerase [ applied biosystems , foster city , ca , usa ] and 1 l of template dna ) . pcr products were denatured at 95 for 10 minutes and rapidly transferred to 4 for 60 minutes to promote duplex formation . next , 480 reactions ( 40 cases ) were analyzed on both 8% non - denaturing polyacrylamide ( acrylamide : bisacrylamide=29:1 ) gel in a 1 tris - boric acid - edta buffer , and on 4% metaphor agarose gel in a 1 tris - boric acid - edta buffer at 100 v for 50 minutes simultaneously . when the pcr products appeared indistinct and one or more ill - defined bands of varying size ranges were present , the pcr products were interpreted as polyclonal . the rearrangements were interpreted as clonal when one or more discrete bands appeared within the expected size range , and they were often accompanied by one or more bands in ig or tcr.20 co - existing clonality was defined when one or more discrete bands within the expected size range appeared in ig and tcr . co - existing clonality was regarded as a subtype of clonality in the current study . in cases with weak pcr bands or equivocal results , the amplification was repeated and the amplicons were analyzed on both 8% non - denaturing polyacrylamide gel and 4% metaphor agarose gel . for this study , we selected 250 cases diagnosed with b cell lymphomas , t cell lymphomas and reactive lymphadenitis from 2004 - 2012 at chonnam national university hospital ( gwangju , korea ) and chonnam national university hwasun hospital ( hwasun , korea ) . the purity and concentrations of the dna in all cases were measured . after verifying the dna quality , 219 patients were selected and were included in clonality analyses of ig and tcr gene rearrangements , as shown in table 1 . the b cell lymphomas ( 78 cases ) were comprised of 49 diffuse large b cell lymphomas ( dlbcls ) , 8 mucosa - associated lymphoid tissue lymphomas ( maltls ) , 3 nodal marginal zone b cell lymphomas ( nodal mzls ) , 6 follicular lymphomas ( fls ) , 4 mantle cell lymphomas ( mcls ) , and 8 b lymphoblastic leukemias / lymphomas ( bll / l ) . the t cell lymphomas ( 80 cases ) included of 41 peripheral t cell lymphomas , unspecified ( ptcls - u ) , 7 anaplastic large cell lymphomas ( alcls ) , 12 nasal type nk / t cell lymphomas ( n - nk / ts ) , 12 angioimmunoblastic t cell lymphomas ( aitls ) , 2 adult t cell leukemia / lymphomas ( atl / ls ) , and 6 t lymphoblastic leukemias / lymphomas ( tll / l ) . finally , 61 patients with reactive lymphoproliferative disease included 37 incidentally - detected palpable lymphadenopathies ( laps ) , 9 infection - related laps , 5 radiation - related laps , 4 autoimmune disease - related laps , 3 fevers of unknown origin laps , 2 radioactive iodine therapy - associated laps following surgery to treat thyroid papillary carcinomas , and 1 chemotherapy - related lap . sections ( 5 m thickness ) were cut from each paraffin block , and the areas of lymphoma were scraped from the unstained formalin - fixed , paraffin - embedded sections . the dna was extracted using a qiaamp dna mini kit according to the manufacturer 's protocol ( qiagen , hilden , germany ) . polymerase chain reaction ( pcr ) products were obtained using a gradual dilution of dna analyzed on metaphor agarose gel ( cambrex bio science , rockland , me , usa ) in five cases . bands of pcr products with dna over 30 ng/l were detected using the metaphor agarose gel . therefore , 219 out of the 250 samples with dna over 30 ng/l were analyzed with the full set of biomed-2 pcr assay ( invivoscribe technologies , san diego , ca , usa ) for ig and tcr gene rearrangement . thirty - one specimens with dna concentrations of less than 30 ng/l were excluded from this study . pcr amplifications were performed using a biomed-2 multiplex pcr master mix ( three tubes of complete igh [ igha : vhfr1-jh ; ighb : vhfr2-jh ; ighc : vhfr3-jh ] , two tubes of incomplete igh [ ighd : dh1 - -6-jh ; ighe : dh7-jh ] , two tubes of igk [ igka : v-j ; igkb : v-kde and jc intron - kde ] , two tubes of complete tcrb [ tcrba : v-j1/2.2/2.6/2.7 ; tcrbb : v-j2.1/2.3/2.4/2.5 ] , one tube of incomplete tcrb [ tcrbc : d-j ] , and two tubes of tcrg [ tcrga : vi/10-j ; tcrgb : v9/11-j ] ) . the biomed-2 pcr protocols19 were performed using a thermal cycler according to the manufacturer 's recommendations . each pcr was carried out in a 10-l volume ( 9 l of master mix with 1 unit of amplitaq gold dna polymerase [ applied biosystems , foster city , ca , usa ] and 1 l of template dna ) . pcr products were denatured at 95 for 10 minutes and rapidly transferred to 4 for 60 minutes to promote duplex formation . next , 480 reactions ( 40 cases ) were analyzed on both 8% non - denaturing polyacrylamide ( acrylamide : bisacrylamide=29:1 ) gel in a 1 tris - boric acid - edta buffer , and on 4% metaphor agarose gel in a 1 tris - boric acid - edta buffer at 100 v for 50 minutes simultaneously . when the pcr products appeared indistinct and one or more ill - defined bands of varying size ranges were present , the pcr products were interpreted as polyclonal . the rearrangements were interpreted as clonal when one or more discrete bands appeared within the expected size range , and they were often accompanied by one or more bands in ig or tcr.20 co - existing clonality was defined when one or more discrete bands within the expected size range appeared in ig and tcr . co - existing clonality was regarded as a subtype of clonality in the current study . in cases with weak pcr bands or equivocal results , the amplification was repeated and the amplicons were analyzed on both 8% non - denaturing polyacrylamide gel and 4% metaphor agarose gel . among 70 cases of mature b cell lymphomas , 67 ( 95.7% ) showed clonality for ig , 2 ( 2.9% ) exhibited co - existing clonality for ig and tcr , and 3 ( 4.3% ) showed polyclonality . four out of eight ( 50% ) bll / l cases revealed clonality for ig , five cases ( 62.5% ) had clonality for tcr , two cases ( 25% ) exhibited co - existing clonality , and one ( 12.5% ) had polyclonality . sixty - two out of 74 cases ( 83.8% ) diagnosed with mature t cell lymphoma revealed clonality for tcr , 12 cases ( 16.2% ) showed polyclonality , and 6 ( 8.1% ) cases revealed co - existing clonality . four out of six tll / l cases showed clonality for tcr , and two cases showed polyclonality . among 61 cases of reactive lymphadenitis , 57 cases ( 93.4% ) were polyclonal for ig and tcr ; however , 4 cases ( 6.6% ) had clonality for igk or tcrg . two cases ( 3.3% ) were clonal for each igka and tcrgb ( table 2 ) . mature b cell lymphoma revealed clonality for igh in 60 out of total 70 cases . among 70 mature b cell lymphoma cases , 56 cases ( 64.0% ) had clonality for the vdj region of igh and 35 ( 50.0% ) revealed clonality for the dh - jh region of igh sixty - seven out of 70 ( 95.7% ) cases had clonality for igh or igk . the dlbcls revealed clonality for igh in 41 out of 49 ( 83.7% ) cases , including 38 ( 77.6% ) that showed clonality for the vdj region and 29 ( 59.2% ) for the dh - jh region . two cases of dlbcl showed combined tcr gene rearrangement , and three cases revealed polyclonality . other mature b cell lymphomas showed 100% clonality for ig . of the cases with maltls , eight ( 100% ) had clonality for igh , and four ( 50.0% ) had clonality for igk . of those cases with nodal mzls , three ( 100% ) had clonality for igh and two ( 66.7% ) showed clonality for igk . three of the four cases with mcls ( 75.0% ) had clonality for igh and all 4 ( 100% ) had clonality for igk . all fl cases showed clonality for igh or igk , but no clonality for the dh - jh region of igh was found . bll / l revealed four cases ( 50% ) with clonality for ig , including three ( 37.5% ) that showed clonality for igh and one ( 12.5% ) with clonality for igk . five out of eight bll / l showed tcr gene rearrangements including two tcrb clonalities and/or five tcrg clonalities . co - existing clonal cases were two and polyclonal case was one in bll / l ( table 3 ) . among 74 mature t cell lymphomas , 62 cases ( 83.8% ) had tcr clonality . there were 38 clonal cases ( 51.4% ) each for tcrb and tcrg ( table 4 ) . clonal bands were observed in 23 ( 31.1% ) and 26 cases ( 35.1% ) for the v-j and d-j regions of tcrb , respectively . thirty - seven of 41 ( 90.2% ) ptcl - u cases had clonality for tcr , including 19 cases ( 46.3% ) with clonality for tcrb and 21 cases ( 51.2% ) with clonality for tcrg . n - nk / ts had clonality for tcr in 8 cases ( 66.7% ) . aitl had clonality in 10 ( 83.3% ) , and atl / ls had clonality for tcr in 2 ( 100% ) . n - nk / ts had clonality for tcrb in seven cases ( 58.3% ) and for tcrg in four cases ( 33.3% ) . tcrb and tcrg gene rearrangements of aitl were revealed in six cases ( 50% ) and eight cases ( 66.7% ) , respectively . alcls had clonality for total tcr in five cases ( 71.4% ) including four cases ( 57.1% ) of clonality for tcrb and three cases ( 42.9% ) for tcrg . all tll / l cases had no clonality for igh or igk , and four out of six ( 66.7% ) tll / l showed tcr clonality , including two tcrb and two tcrg clonal cases ( table 4 ) . in all , 480 reactions spanning 40 cases were analyzed on non - denaturing polyacrylamide gels and metaphor agarose gels ( cambrex bio science ) simultaneously . the clonality detection rate of lpds of koreans is lower than that of europeans but higher than that of the taiwanese . all mature b cell lymphomas in this study from a korean population showed 67 out of 70 cases ( 95.7% ) of clonality for the ig gene compared to 367 out of 369 ( 99.0% ) in a european study.21 twenty - two cases ( 95.7% ) in b cell lymphomas of southern taiwan had clonality for ig , which was the same finding as our data.17 sung et al . 's study22 in koreans using biomed-2 multiplex pcr reported 87.5% ( 14/16 ) clonality for ig . our cases showed clonality for igh in 60 out of 70 cases ( 85.7% ) , whereas for igk , clonality was shown in 33 out of 70 cases ( 47.1% ) . igk clonality was significantly lower compared to that in a european patient sample , which revealed 323 out of 369 cases ( 88.0% ) ( p<.05 ) of clonality , and it was also lower compared to that in a taiwanese patient sample , which found 16 of 23 cases ( 69.6% ) of clonality.17,21 however , in a korean patient sample , sung et al.22 reported that 7 of 16 cases ( 43.8% ) showed clonality for igk , which was similar to our findings . of the dlbcl cases , 93.8% showed clonality for ig , which was similar to a european study that revealed 98.0% clonality.15 the other types of b cell lymphomas showed 100% clonality , similar to what was observed in a european study.14,15 the fl cases showed 100% ( 6/6 ) clonality for ig including igh and igk ; however , no clonality was observed for dh - jh rearrangements of igh in this study , as compared to a 19.0% rearrangement rate in a european study.15 bll / l showed quite different clonality results from other types of b cell lymphomas . bll / l had clonality for ig in four of eight cases ( 50% ) , which was much lower than other types of b cell lymphomas , and clonality for tcr in five of eight cases ( 62.5% ) , which was much higher than other types of b cell lymphomas . in addition , co - existing clonality was presented in two cases ( 25% ) in bll / l , which was higher than other b cell lymphomas except dlbcl . this finding might be because bll / l are arrested at various stages of pre - b cell development and cross lineage ig / tcr gene rearrangement occurs particularly in immature lymphoid malignancy . therefore , in the case of lpd of unknown origin , the ig / tcr gene rearrangement result should not be used directly for determination of b or t cell lineage.15 there were three polyclonal cases out of 70 ( 4.3% ) b cell lymphomas , all of which were dlbcls . the ratio of polyclonal b cell lymphomas was higher among the korean sample than the european sample , which revealed polyclonality in two dlbcls out of 369 b cell lymphomas . there was one polyclonal case out of 23 b cell lymphomas in taiwan , which was also dlbcl . polyclonality may occur in lymphoid malignancies when the number of clonal b cells is too low to be detected or the cases have a significant somatic hypermutation without gene rearrangement.21 moreover , there were two co - existing clonal cases in dlbcls and bll / l , respectively . one case out of two co - existing dlbcls showed bands in igka and tcrbc , while the other case revealed discrete bands in ighc , ighd , igka , and tcrga . one case of bll / l was positive for igkb and tcrgb , and the other case showed bands in all tubes of igh , tcrba and tcrbb , and tcrgb . the study of europeans reported that co - existing clonality was 27% in mature b cell lymphomas . additionally , the incidence of cross lineage ig / tcr gene rearrangement has been reported to be up to 90% in bll / l.21 although the causes are currently not well understood , the rearrangements might arise from somatic hypermutations or point mutations.21 the clonality of t cell lymphomas in this study was lower than that of europeans but higher than that of the taiwanese . t and tll / l in this study was 87.1% ( 54/62 ) , as compared to 94.0% ( 177/188 ) in a european study and 76.0% ( 19/25 ) in a taiwanese study.12,17 among t cell lymphomas , including n - nk / t and tll / l , tcr gene rearrangement was revealed in 66 out of 80 cases ( 82.5% ) in this study . there was clonality for tcrb in 38 out of 74 cases ( 51.4% ) mature t cell lymphomas in koreans compared with 171 out of 188 cases ( 91.0% ) in a european study and 11 out of 28 cases ( 39.3% ) in a taiwanese study . additionally , clonality for tcrg was found in 38 cases ( 51.4% ) in this study , which was significantly lower than the data reported in the european - based study ( 168/188 , 89.0% ; p<.05 ) and similar to that of the taiwanese study ( 17/29 , 58.6%).12,14,15,17 in ptcl - u cases , 21 out of 41 cases ( 51.2% ) showed clonality for tcrg compared with 177 out of 188 ( 94.0% ) in a european study and 10 out of 14 ( 71.4% ) in a taiwanese study . europeans have minor allele frequencies of 0.33 , whereas african americans have frequencies of 0.50 , and asians 0.42 for the allele of a single nucleotide polymorphism ( rs 2392542 ) for tcrg.23 n - nk / t showed clonality for tcr in eight out of 12 ( 66.7% ) , which was higher than those of a thai group ( 20/67 , 30% ) and a taiwan study ( 0/8 , 0%).17,18 of seven alcl cases , five ( 71.4% ) had clonality for tcr , which was similar to data from a european study that reported clonality in 34 out of 43 ( 79.0%).12 of 12 aitl cases , 10 ( 83.3% ) had clonality for tcr compared with 35 out of 37 ( 95.0% ) in the european data.12 twelve polyclonal cases were observed in 74 t cell lymphomas . they were four ptcls - u , four n - nk / ts , two aitls , and two anaplastic lymphoma kinase ( alk)-positive alcls . the european study revealed 11 polyclonal cases in 188 ( 5.9% ) t cell lymphomas , which consisted of eight alk - positive alcls , one alk - negative alcl , and two aitls . there were no polyclonal cases for tcr among ptcls - u in a european study . european data reported that 24.0% of alk - positive alcls did not demonstrate tcr clonality.12 four ptcl - u with co - existing clonality showed various ig and tcr gene rearrangements , which were ighc and ighd , igkb , tcrba and tcrbb , and tcrgb . two aitls revealed co - existing clonality for ighb , ighc , ighd , igkb , and tcrba . in this study , six cases ( 8.1% ) with co - existing clonality were identified in 74 mature t cell lymphomas . the rate of 0 - 9% for co - existing clonality has been reported in t cell lymphomas of europeans.12 four out of six ( 66.7% ) tll / l showed clonality for tcr , which is similar to that of other t cell lymphomas . in contrast to bll / l , all of the tll / l cases showed no co - existing clonality . . however , four out of 61 cases ( 6.6% ) with reactive lymphadenitis had clonality as well . a case with clonality for igk involved a 53-year - old female with inguinal lap who had a history of radiation therapy due to uterine cervical cancer . the clonality may have resulted from the radiation therapy , as previously reported.24 the second case with clonality for igk was a 28-year - old male who was infected with human immunodeficiency virus ( hiv ) . it has been reported that ig rearrangements are associated with hiv - related lap syndrome.20 the third case with clonality for tcrg was a 53-year - old male who had received radioactive iodine therapy after a total thyroidectomy due to papillary thyroid carcinoma . radioactive iodine therapy is reported to cause clonality of the lymph nodes.25 the fourth case was a 37-year - old female with a history of virus - induced cervical lap . the clonality for tcrg may have been induced by a viral infection in this case.20 there was no occurrence of lymphoma in these cases during the three - year follow - up period . cho et al.8 reported on ig and tcr gene rearrangements in lpds based on a conventional nested pcr - based clonality assay using a friiia - ljh / vljh consensus primer for igh and a v-j consensus primer for tcrg in a korean population . the clonality for igh in b cell lymphomas was 97.1% ( 34/35 ) , which was higher than the 85.7% clonality in the current study . however , the specificity of igh gene rearrangements was 97% , which was lower than the 100% specificity in biomed-2 found in this study . in addition , the clonality for tcrg in t cell lymphoma was 79.3%,8 which was higher than the 51.2% ( 21/41 ) reported herein . the specificity of tcrg gene rearrangements using the previous method was 99.4% , which was higher than the 96.7% found by the current study . the higher clonality ratio for ig and tcr might result from the semi - nested pcr technique , which runs the pcr twice . electrophoresis of 480 pcr products was performed using 4% metaphor agarose gels and 8% non - denaturing polyacrylamide gels , which revealed the same results on both gels . although the manufacturer of the biomed-2 multiplex pcr kit recommends electrophoresis of the pcr products using a non - denaturing polyacrylamide gel or gene scan , the metaphor agarose gel is as sensitive as a non - denaturing polyacrylamide gel . metaphor agarose gel has several advantages over non - denaturing polyacrylamide gel , such as ease of handling , safe and quick preparation . therefore , all of the pcr products in this study were analyzed using 4% metaphor agarose gels . in cases with weak pcr bands or equivocal results , the analysis of both 8% non - denaturing polyacrylamide gel and 4% metaphor agarose gel will help confirm the diagnosis we performed igh , igk , tcrb , and tcrg tests , but the pcr amplification for igl and tcrd was not carried out . it has been reported that the effect of igl or tcrd testing for clonality detection is negligible , ranging from1 to 2%.12,21 sung et al.22 also found a similar result . they observed the clonality detection rate using igh and igk was not changed irrespective of the igl test . additionally , the number of cases in this study was less than that of the european study . more cases of koreans would be of help in comparing among races and establishing a definitive diagnosis . although there were some differences in clonalities for igk and tcrg of b and t cell lymphomas between korean and european populations , the overall clonality ratio in b or t cell lymphomas of koreans was similar to those of europeans . we conclude that the biomed-2 multiplex pcr reaction can be a useful adjuvant test for diagnosing lpds when the histologic and immunohistochemical evidence is insufficient .

in the pancreas , the cholecystokonin-2 receptor ( cck-2r ) , formerly the cck - b receptor , has been located exclusively on the somatostatin - delta cells of six different species , including man . to date , nothing is known about the role of this cck receptor subtype on the physiology of these delta cells . it has been reported however that chronic injections of caerulein , a cck analog , and secretin caused significant increases in rat pancreatic somatostatin ( ss ) content without any effect on total delta cell numbers . similar changes in ss contents were also observed with the same hormones combination in islets cultured for 10 days with no difference in islets ' total dna contents . it was initially demonstrated that gastrin and cck-8 , at relatively high doses , caused a quick and transient twofold increase in ss release by the perfused canine pancreas . when cck-8 was infused at a dose that stimulates the secretion of protein in the pancreatic juice of dogs to the levels normally observed with a meal , ss in the pancreatic vein rose immediately about 2-fold above basal and remained elevated for the 15 minutes of the hormone infusion . these data indicate that gastrin and cck can induce ss secretion from the pancreatic gland which was later directly confirmed from rat islet cell in culture . in order to have an exclusive and direct access to the pancreatic somatostatin cells and to avoid mixtures of cell types from islet cell cultures , investigators established and cloned a transplantable rat islet cell tumor which secretes insulin and somatostatin . clones of the original somatostatin cell line were used to study somatostatin secretion ( rint3 ) , intracellular signalling pathways activated in response to cck receptor subtypes occupation ( rin1027-b2 ) and cck receptor subtypes characterization and ss release ( rin-14b ) [ 911 ] . this study was undertaken to establish the presence of the two cck receptor subtypes on the rin-14b cells and to determine if one or both of these types can affect growth of these cells . to our knowledge , no study has yet been performed to explore if the gastrointestinal hormones cck and gastrin , two specific cck-1 and cck-2 receptors agonists , respectively , have any effects on growth of the pancreatic somatostatin cells . productssomatostatin-14 ( ss-14 ) was purchased from bachem bioscience ( king of prussia , pa , usa ) . pentagastrin ( pg ) was purchased from sigma - aldrich ( oakville , on , canada ) . v. j. lotti , merck sharp and dohme research laboratories ( west point , pa ) . cell culture media reagents were obtained from life technologies ( burlington , on , canada ) . the somatostatin ( barbar ) antibody , purified by affinity chromatography , was a gift from dr . p. brazeau , universit de montral , montreal , canada . the goat polyclonal anti - cck-1 receptor antibody ( 7509 ) was raised against the rat internal peptide ( kfdasqkksakekr ) and the goat polyclonal anti - cck-2 receptor antibody ( 6767 ) was raised against the human internal peptides cck-2 ( fdgdsdsdsqsrvrnq ) . these two antibodies and corresponding peptides were gifts from dr . somatostatin-14 ( ss-14 ) was purchased from bachem bioscience ( king of prussia , pa , usa ) . pentagastrin ( pg ) was purchased from sigma - aldrich ( oakville , on , canada ) . v. j. lotti , merck sharp and dohme research laboratories ( west point , pa ) . cell culture media reagents were obtained from life technologies ( burlington , on , canada ) . the somatostatin ( barbar ) antibody , purified by affinity chromatography , was a gift from dr . p. brazeau , universit de montral , montreal , canada . the goat polyclonal anti - cck-1 receptor antibody ( 7509 ) was raised against the rat internal peptide ( kfdasqkksakekr ) and the goat polyclonal anti - cck-2 receptor antibody ( 6767 ) was raised against the human internal peptides cck-2 ( fdgdsdsdsqsrvrnq ) . these two antibodies and corresponding peptides were gifts from dr . cell culturethe rat rin-14b cell line is a secondary clone derived from the rin - m rat islet cell line ; these cells synthesize and secrete somatostatin . routinely , the cells were grown on petri dishes in rpmi 1640 ( for rin-14b ) and dmem ( for cos cells ) media containing 25 mm glucose , 10% fetal bovine serum ( fbs ) , penicillin ( 100 uml ) , streptomycin ( 100 g ml ) , 2 mm l - glutamine , 10 mm hepes , 1 mm sodium pyruvate , and 1.5 g l sodium bicarbonate . cells were kept in culture at 37c in a 5% co2 humidified atmosphere . the culture media were changed every other day ; they were passed weekly by trypsin ( 0.25%)-edta ( 2.2 mm ) detachment and subcultured every week at a ratio of 1 : 7 . the rat rin-14b cell line is a secondary clone derived from the rin - m rat islet cell line ; these cells synthesize and secrete somatostatin . routinely , the cells were grown on petri dishes in rpmi 1640 ( for rin-14b ) and dmem ( for cos cells ) media containing 25 mm glucose , 10% fetal bovine serum ( fbs ) , penicillin ( 100 uml ) , streptomycin ( 100 g ml ) , 2 mm l - glutamine , 10 mm hepes , 1 mm sodium pyruvate , and 1.5 g l sodium bicarbonate . the culture media were changed every other day ; they were passed weekly by trypsin ( 0.25%)-edta ( 2.2 mm ) detachment and subcultured every week at a ratio of 1 : 7 . growth assaycells were plated in 60 mm diameter petri dishes at a density of 0.2 10 ml ( 4 ml / dish ) . the next day , cells were transferred to rpmi 1640 - 0.5% heat inactivated fbs and adapted overnight to grow in this medium before addition of peptides , antiserum , and cck - receptor antagonist . the medium was changed daily and cells were supplemented also daily with cae ( 10 m ) , pg ( 10 m ) , and ss-14 ( 10 m ) alone or in combination with l-364,718 ( 10 m ) or barbar ( 0.25 g ml ) . cell growth was measured after 1 , 2 , 3 , 4 , and 5 days with a cell counter ( coulter counter ) . cells were plated in 60 mm diameter petri dishes at a density of 0.2 10 ml ( 4 ml / dish ) . the next day , cells were transferred to rpmi 1640 - 0.5% heat inactivated fbs and adapted overnight to grow in this medium before addition of peptides , antiserum , and cck - receptor antagonist . the medium was changed daily and cells were supplemented also daily with cae ( 10 m ) , pg ( 10 m ) , and ss-14 ( 10 m ) alone or in combination with l-364,718 ( 10 m ) or barbar ( 0.25 g ml ) . cell growth was measured after 1 , 2 , 3 , 4 , and 5 days with a cell counter ( coulter counter ) . sds - page and western blotthe membranes of the rin-14b and cos cells were prepared as described in . briefly , cells were resuspended in a homogenization buffer ( 10 mm hepes , ph 7.5 , 250 mm sucrose , 1 mm egta , 1 mm edta , 0,5 mm pmsf , 20 m leupeptin , 1 m aprotinin , and 2 m pepstatin ) , homogenized using a potter homogenizer and sonicated for 10 seconds ( 40% power ) . the membranes were then collected by centrifugation at 30 000 g for 30 minutes at 4c using a beckman tls-55 rotor ( palo alto , calif , usa ) and resuspended at 17 mg ml in the homogenization buffer . proteins were determined with the bca assay of pierce using bovine serum albumin as standard.membrane proteins ( 100 g ) were separated by electrophoresis on a 10% sds - polyacrylamide gel according to and transferred to nitrocellulose membranes ( sigma - aldrich ) . the membranes were blocked for 2 hours with nap - blocker ( g biosciences , st - louis , mo , usa ) in tbst ( 1 : 2 v / v ) and incubated overnight at 4c in nap - blocker : tbst ( 1 : 2 v / v ) with either the anti - cck-1r 7509 ( 0.1 g ml ) or the anti - cck-2r 6767 ( 0.1 g ml ) antibodies . the blots were then washed in tbst and incubated for 1 hour at rt in nap - blocker : tbst ( 1 : 2 v / v ) with horseradish peroxidase - conjugated antigoat ( 1 : 10 000 ) . blots were washed with tbst and proteins were detected by chemiluminescence using ecl immunodetection system ( amersham biosciences , baie durf , qc ) . specificity of each cck receptor antibody was established by preincubation of each antibody for 2 hours at rt with its specific peptide antigen : 1 g ml of 7509 and 0.1 g ml of 6767 peptides . the membranes of the rin-14b and cos cells were prepared as described in . briefly , cells were resuspended in a homogenization buffer ( 10 mm hepes , ph 7.5 , 250 mm sucrose , 1 mm egta , 1 mm edta , 0,5 mm pmsf , 20 m leupeptin , 1 m aprotinin , and 2 m pepstatin ) , homogenized using a potter homogenizer and sonicated for 10 seconds ( 40% power ) . the membranes were then collected by centrifugation at 30 000 g for 30 minutes at 4c using a beckman tls-55 rotor ( palo alto , calif , usa ) and resuspended at 17 mg ml in the homogenization buffer . proteins were determined with the bca assay of pierce using bovine serum albumin as standard . membrane proteins ( 100 g ) were separated by electrophoresis on a 10% sds - polyacrylamide gel according to and transferred to nitrocellulose membranes ( sigma - aldrich ) . the membranes were blocked for 2 hours with nap - blocker ( g biosciences , st - louis , mo , usa ) in tbst ( 1 : 2 v / v ) and incubated overnight at 4c in nap - blocker : tbst ( 1 : 2 v / v ) with either the anti - cck-1r 7509 ( 0.1 g ml ) or the anti - cck-2r 6767 ( 0.1 g ml ) antibodies . the blots were then washed in tbst and incubated for 1 hour at rt in nap - blocker : tbst ( 1 : 2 v / v ) with horseradish peroxidase - conjugated antigoat ( 1 : 10 000 ) . blots were washed with tbst and proteins were detected by chemiluminescence using ecl immunodetection system ( amersham biosciences , baie durf , qc ) . specificity of each cck receptor antibody was established by preincubation of each antibody for 2 hours at rt with its specific peptide antigen : 1 g ml of 7509 and 0.1 g ml of 6767 peptides . statistical analysisresults were analyzed by the student 's t - test for comparison of independent samples with a probability value of < .05 which is considered significant . results were analyzed by the student 's t - test for comparison of independent samples with a probability value of < .05 which is considered significant . to establish the presence of both cck receptor subtypes on the rin-14b cells , we used specific antibodies raised against both receptors . with the specific cck-1 receptor antibody ( 7509 ) , we can see that the rin-14b cells express the cck-1 receptor as a 75 kda protein . this recognition by the antibody is specific because the band totally disappeared with preincubation of the antibody with the peptide used to develop this antibody ( see figure 1(a ) ) . similarly , these cells also express the cck-2 receptor , also as a 75 kda protein identified with the antibody 6767 ; specificity of the receptor - antibody binding was also revealed with preincubation of the antibody with the peptide with disappearance of the band ( see figure 1(b ) ) . to further strengthen the specificity of both cck-1 and cck-2 receptor antibodies , we established the absence of these two cck receptors in cos cells known to be free of these two receptors . as shown in figures 1(a ) and 1(b ) , both antibodies 7509 ( anti - cck-1 ) and 6767 ( anti - cck-2 ) did not recognize the 75 kda band observed in the rin-14b cells . this observation further supports the specificity of the two cck receptor antibodies we used in this study to demonstrate their presence in the rin cells . to determine if the cck-1 analog caerulein can affect the growth of the rin-14b cells , growth assays were performed . as shown in figure 2 , caerulein , used at a concentration which totally occupies the high affinity cck-1 receptors and causing maximal amylase release from rat dispersed pancreatic acini , caused significant reductions in rin-14b cell growth from day 3 to day 5 of culture . the observation that this inhibition by caerulein was totally reversed by the specific cck-1 receptor antagonist l-364718 indicates that the cck-1 receptors are involved in this process of growth inhibition . to eliminate the possibility that somatostatin released in response to caerulein could be responsible for growth inhibition by the cck analog , cells were also incubated with an ss antibody along with caerulein ; in no time did we observe a difference between growth in the caerulein group alone and caerulein plus the ss antibody ( data not shown ) . as shown in figure 4 , the ss antibody prevented growth inhibition by ss alone . to establish if the cck-2 agonist pentagastrin could affect growth of these rin-14b cells , growth assays were also performed . the results indicate that contrary to what was observed with occupation of the cck-1 receptors , occupation of the cck-2 receptors by 10 m pentagastrin , a gastrin analog , had no effect on rin-14b cell growth over the 5-day period examined ( see figure 3 ) . this strongly indicates that occupation of the cck-2 receptors did not alter the growth of the rin-14b cells . to verify if somatostatin could have an inhibitory effect on growth of the cells responsible for its synthesis , the rin-14b cells were incubated for up to five days in the presence of 1 nm somatostatin-14 . as shown in figure 4 , somatostatin significantly reduced growth of the rin-14b cells by 11.3% after 5 days of culture ; this inhibitory effect was already significant after 2 days of culture . addition of a somatostatin antibody to the culture medium to neutralize endogenous ss released by the cells had no effect on their growth ; however its addition to exogenous somatostatin reversed its growth inhibition . these results indicate that endogenous somatostatin released from these cells did not reach the concentration needed to inhibit their growth but when applied at the needed concentration , exogenous somatostatin can inhibit their growth . this study reports for the first time that ( 1 ) the ss - secreting rin-14b cells express the two known and characterized cck receptor subtypes ; ( 2 ) occupation of the cck-1 receptor subtype leads to inhibition of these cells ' growth , while occupation of the cck-2 receptor subtype fails to stimulate or inhibit such growth ; ( 3 ) somatostatin can also inhibit growth of these rin-14b cells probably through an autocrine mechanism . in contrast to substances which can control somatostatin secretion , nothing is currently known about those responsible for delta cells ' growth . the observation that the rin-14b cells express the two cck receptor subtypes is not unique to these cells . indeed , expression of both receptor subtypes has been documented in normal rat pancreatic acini with a predominance of the cck-1 subtype over that of the cck-2 . these two receptors were also reported in normal human and rat islets but on different cell types , the cck-1 receptors on alpha and beta cells and the cck-2 receptors on the delta cells using two different antibodies . studies performed in rats demonstrated that in pancreatic malignancies , the cck-1 receptor is overexpressed while the cck-2 receptor is newly expressed . in human pancreatic tumors indeed , by using the pcr technique , one study reported the presence of the cck-2 receptors in all samples of normal pancreas and pancreatic adenocarcinoma ; the cck-1 receptor expression could not be detected in normal pancreatic samples but it appeared in all samples of pancreatic adenocarcinomas . by receptor autoradiography , the cck-2 receptor was found occasionally in pancreatic tumors while the cck-1 receptor was mostly expressed in these tumors . these data emphasize that the expression of these two cck receptor subtypes in many cancer cell types may be an important indicator of the influence of cck and gastrin of local or systemic origin on the growth of these cancers . indeed , in elas cck-2 receptor transgenic mice , the growth rate of their pancreas was increased by 40% after birth between 40 and 110 days of age ; this expression had a key role in the development of pre- and neoplastic lesions in their pancreas . while everyone agrees that occupation of the cck-1 receptors in the pancreas of rats and other rodents led to growth of the organ , its presence and stimulation in miapaca-2 and panc-1 cells led to growth inhibitory responses . occupation of the cck-2 receptor also resulted in surprising opposite effects when transfected in cho and swiss 3t3 cells . it inhibited proliferation and dna synthesis in the cho - cck-2 cells while stimulation occurred in the swiss 3t3-cck-2 cells ; these opposite effects on growth happened while cck-8 stimulated the same common second messenger pathways . growth inhibition was also observed with occupation of the transfected cck-2 receptors in the human pancreatic miapaca-2 and panc-1 cells . interestingly , coexpression of gastrin and cck-2 receptors were observed in 5/5 and 7/8 human gastric and colorectal cell lines and these cells maintain an autocrine growth pathway ; the rin-14b cells also express gastrin although in small quantities ( data not shown ) . when comparing all the above results with those obtained with the rin-14b cells , it seems that growth inhibition observed in response to caerulein in this study renders these rin cells comparable to the transfected cck-1 miapaca-2 and panc-1 cells . endogenous cck also resulted in growth inhibition of human cholangiocarcinoma ; however , no specific antagonists of either cck receptor subtypes were used to confirm which cck receptor was involved . this study points out that cck can inhibit growth not only in cells transfected with both cck receptor subtypes . moreover , the inhibition observed in these rin cells really resulted from occupation of the cck-1 receptors because it has been reversed by l-364718 , a specific cck-1 receptor antagonist . somatostatin can inhibit endogenous ss secretion from the delta cells through an auto - feedback mechanism . although we have not investigated which somatostatin receptors are present on the rin-14b cells , it has been reported that about 70% of the rat pancreatic delta cells express the ss receptor subtypes 14 . since it is accepted that the antiproliferative effect of somatostatin results from its action via the endocrine pathway , evidence also exists that somatostatin can also act via an autocrine / paracrine pathway which has been recently described in pc-3 and lncap cells , two human prostate adenocarcinoma cell lines . we do not believe that growth of these rin-14b cells is autoregulated by somatostatin they release into the medium . this conclusion is based on the following observations : ( a ) these cells grew in a 0.5% inactivated fbs medium when they release into the medium approximately 200 pg ml of somatostatin per 4 hours , and ( b ) they also grew at their control rates even in the presence of 0.25 g ml of a specific somatostatin antibody ; an autocrine regulated pathway would have shown these cells grow at a rate above the controls in the presence of the antibody . this was previously observed in vivo when the trophic effect of caerulein on the rat pancreas was significantly enhanced by a simultaneous administration of a somatostatin antiserum , the same used in this study . although endogenous somatostatin released by the rin cells does not seem sufficient to sustain growth inhibition , these cells are however sensitive to somatostatin as they exhibited growth inhibition in its presence at a higher concentration . the magnitude of inhibition is comparable to what was previously described in ar4 - 2j cells where at 10 nm , the hormone caused a 25% growth inhibition over a 96-hour period . this inhibitory effect of somatostatin on the rin cells proliferation is specific since prevented by the presence of the somatostatin antibody . in conclusion , our data indicate for the first time that the somatostatin rin-14b cells possess the two cck receptor subtypes , and that their proliferation can be negatively affected by occupation of the cck-1 receptor subtype and exogenous somatostatin .

parkinson s disease ( pd ) is the second most common neurodegenerative disease behind alzheimer s disease ( ad ) . an essay on the shaking palsy . the cardinal motor signs of pd ( bradykinesia , resting tremor , cogwheel rigidity , postural instability ) are emphasized in making the diagnosis and in tracking progression of the disease ( gibb and lees 1988 ) . most recently , significant attention has been given to the non - motor symptoms of pd , including constipation , depression , olfactory dysfunction and dementia ( chaudhuri et al 2006 ) . parkinson ( 1817 ) was keenly aware of many non - motor aspects of pd including constipation and disturbed sleep . he did not recognize , however , impaired olfaction and dementia as a part of the disease describing the senses and intellects as being uninjured ( parkinson 1817 ) . his lack of recognition of pd as a dementing illness is understandable given life expectancy in great britain in the early 19th century was under 40-years , and we now know that the risk of parkinson s disease dementia ( pdd ) increases with age ( levy et al 2002 ) . pdd has perhaps been more studied and emphasized of late for several reasons : ( 1 ) greater emphasis on the non - motor symptoms of pd ( chaudhuri et al 2006 ) , ( 2 ) the emergence of cholinesterase inhibitors as effective treatments in ad and pdd , and ( 3 ) ruling - out dementia is important in pd patients being considered for deep brain stimulation surgery ( given it is exclusionary ) . we do know that pdd is under recognized and under treated in routine clinical practice . recent studies have demonstrated that cognitive decline and/or pdd afflicts the majority of patients with pd over time ( aarsland et al 2003 ; hely et al 2005 ) and that this significantly contributes to increased morbidity and mortality ( levy et al 2002 ; hughes et al 2004 ; de lau et al 2005 ) . according to dsm - iv criteria ( apa 2000 ) , dementia is characterized by the development of multiple cognitive deficits that include memory impairment and at least one of the following cognitive disturbances : aphasia , apraxia , agnosia , or disturbance in executive dysfunction . the cognitive deficits must be sufficiently severe to cause impairment in occupational or social functioning and must represent a decline from a previously higher level of functioning . major cognitive domains that can be affected in dementia include : ( 1 ) executive function , ( 2 ) recent memory , ( 3 ) language , and ( 4 ) visuospatial function . pdd is more of a subcortical dementia with prominent deficits in executive and visuospatial function typically more so than language and recent memory ( cummings 1988 ; rippon and marder 2005 ) . pdd is also characterized by generalized cognitive slowing ( bradyphrenia ) and impaired attention ( cummings 1988 ; rippon and marder 2005 ) . mild cognitive impairment ( mci ) is differentiated from dementia in that it represents a borderland between normal cognition and dementia . mci remains controversial with some clinicians viewing this entity as a disease along a pathway leading to fulminant ad , while others view mci as a heterogeneous syndrome representing an early stage of different forms of dementia ( fernandez et al 2005 ) . cognitive decline is perhaps the rule as pd progresses ( hely et al 2005 ) ; however , the definition of mci in pd is even less well - defined than the definition of pdd ( fernandez et al 2005 ) . given clinical and neuropathological overlap , the most important distinction in the diagnosis of pdd is attempting to distinguish it from dementia with lewy bodies ( dlb ) ( aarsland et al 2005 ; mckeith et al 2005 ) . clinically , patients with dlb are typically characterized as having early dementia and parkinsonism with less robust response to levodopa , fluctuations in level of alertness , visual hallucinations in the absence of dopaminergic treatment , rapid progression of dementia , and severe sensitivity to typical and some atypical antipsychotics ( fuchs et al 2004 ; mckeith et al 2005 ) . unfortunately , neuropsychological testing may not be able to distinguish between patients with these two entities ( noe et al 2004 ) , and the timecourse of the development of the dementia is perhaps most important . for an operational method of distinguishing pdd from dlb , pdd has recently been defined as the onset of dementia at least one year after the appearance of parkinsonian motor symptoms ( mckeith et al 2005 ) . this definition is artificial and not typically representative of clinical experience ( where patients become demented after many years of pd ( aarsland et al 2005 ) ) and maybe difficult to apply in practice . the largest clinical trial in pdd ( emre et al 2004 ) included patients meeting established clinical diagnostic criteria for pd ( gibb and lees 1988 ) and fulfilling dsm - iv criteria for pdd with onset at least two years after the diagnosis of pd ( emre et al 2004 ) . neuropathologically pdd overlaps with dlb and ad in numerous studies ( mahler and cummings 1990 ; perl et al 1998 ; aarsland et al 2004 ; galvin et al 2006 ) . dementia with lewy bodies ( dlb ) and pdd are typically categorized as two separate disease states with distinct pathologies , but debate continues as to whether dlb and pdd are , in fact , along the same disease spectrum ( levy et al 2006 ; galvin et al 2006 ; padovani et al 2006 ) . ad pathology has been noted in many demented and non - demented patients with pd and estimates of ad pathology among pd patients at autopsy are as high as 42%91% ( galvin et al 2006 ; padovani et al 2006 ) . although these changes are present in a large proportion of patients with pd , it is difficult to define the role these changes play in the cognitive decline in pd . it is also suggested that the cognitive dysfunction developing later in parkinson s is due to the loss of cholinergic neurons in the nucleus basalis of meynert ( whitehouse et al 1987 ) . supporting this hypothesis , there is evidence that cortical cholinergic deficits are more pronounced in pd than in ad and well - correlated with cognitive decline and neuropsychiatric disturbances in pd ( bohnen et al 2003 ; hilker et al 2005 ) . most recently , aarsland et al ( 2005 ) reported the neuropathology of 22 pd patients identified in the community and followed prospectively until death . eighteen of the 22 patients ( 82% ! ! ) were diagnosed with dementia with none of the 18 meeting established neuropathological criteria for ad ( aarsland et al 2005 ) . all 18 demented patients had limbic and neocortical lewy bodies ( aarsland et al 2005 ) however . in accord with clinical practice , the average disease duration for these patients was approximately 16 years with a diagnosis of dementia occurring a mean of three years before death ( aarsland et al 2005 ) . it thus appears that limbic and neocortical lewy bodies associated with significant cholinergic deficits are perhaps the main substrate for the development of pdd . the apo 2 allele , older age at onset , the presence of depression , development of visual hallucinations , severity of disease , and an akinetic - rigid phenotype are established risk factors for the development of pdd ( marder et al 1995 ; levy et al 2002 ; de lau et al 2005 ; burn et al 2006 ) . the incidence and prevalence of pdd depends upon the neuropsychological tests and the criteria used to define pdd and neuropsychological testing batteries can vary considerably from center to center ( defer et al 1999 ; saint - cyr and trepanier 2000 ; pillon 2002 ; burn et al 2006 ; voon et al 2006 ) . the incidence of pdd varies widely across studies with the greatest incidence in elderly pd patients . the risk of developing dementia in pd is estimated to be 6 times that of age - matched controls ( padovani et al 2006 ) . early estimates of the prevalence of dementia in pd were in the range of 20% ( brown and marsden 1984 ) . cummings ( 1988 ) estimated that as many as 40 percent of pd patients cross - sectionally have pdd . more recently , aarsland et al ( 2003 ) found that 78% of pd patients followed for 8 years develop pdd ( aarsland et al 2003 ) . . cognitive decline , perhaps consistent with mci , was present in 84% of survivors from a cohort of pd patients followed 15-years after diagnosis ( hely et al 2005 ) . forty - eight percent of these survivors fulfilled criteria for dementia ( hely et al 2005 ) . consistent with these findings , most pd patients undergoing neuropsychological testing to exclude dementia before deep brain stimulation surgery display some level of cognitive dysfunction ( typically executive dysfunction ) ( saint - cyr et al 2000 ; pillon 2002 ) . acetylcholinesterase ( ache ) inhibitors have been the mainstay of treatment in ad for many years . in ad , these drugs have been shown to slow symptomatic decline in cognition over time , delay nursing home placement , and result in lower long - term healthcare costs for patients / families ( geldmacher 2005 ) . ache inhibitors theoretically improve cognitive function by increasing acetylcholine in the brain through preventing its breakdown . there are numerous ache inhibitors commercially available including donepezil , galantamine , rivastigmine , and tacrine . all of these drugs are effective in ad , however there is relatively little data on their use in pdd . rivastigmine is a second generation , carbamate - type , reversible , brain selective cholinesterase inhibitor ( enz et al 1991 ) . it appears to be more selective for the monomeric form of ache that is predominantly in the cortex and hippocampus relative to peripheral forms of ache ( enz et al 1993 ) . it is dosed twice daily titrating from 1.5 mg twice per day up to 6 mg twice per day as tolerated . rivastigmine is slightly different than donepezil and galantamine given it has both ache inhibitory activity and butyrylcholinesterase inhibitory activity . inhibition of both forms of cholinesterase has theoretical advantages in allowing greater endogenous levels of acetylcholine , but it is unknown if this drug provides benefits above and beyond other ache inhibitors given head - to - head studies have not been done . donepezil and galantamine have only been studied in open - label studies or small scale clinical trials in pdd ( table 1 ) . rivastigmine has been studied in large randomized , placebo - controlled clinical trials in dlb ( mckeith et al 2000 ) and pdd ( emre et al 2004 ) ( table 1 ) . the data available for rivastigmine has led to the approval of this medication in the treatment of pdd in europe , the united states and elsewhere . mckeith et al ( 2000 ) studied the efficacy of rivastigmine in dlb . in this randomized , double - blind , placebo - controlled study , participants were given placebo or titrated up to 12 mg / day of rivastigmine for 20-weeks followed by a 3-week washout period . significant clinical and cognitive performance improvements were noted in those receiving rivastigmine as compared with placebo . thirty - seven subjects ( 63% ) in the rivastigmine group showed at least a 30 percent improvement from baseline as compared to only 18 subjects ( 30% ) of the placebo group . patients improved most in the areas of apathy and indifference and had fewer hallucinations than those receiving placebo . as previously discussed , it is becoming more evident that dlb and pdd are on a continuum of disease , with significant overlap in terms of clinical and cognitive signs and symptoms ( aarsland et al 2005 ; galvin et al 2006 ; padovani et al 2006 ) . given the obvious clinical benefits of rivastigmine in dlb ( mckeith et al 2000 ) , this led to further study in patients defined as having pdd . in an open label trial by reading et al ( 2001 ) , twelve patients with pd - related cognitive impairment and psychosis were given rivastigmine at an initial dose of 1.5 mg twice daily and then titrated to 6 mg twice daily or the highest tolerated dose . patients were assessed at 8-weeks after the maximum titration level was reached and once again 6-weeks after that period . the drug was discontinued at that point and participants were assessed once again 3-weeks after withdrawal of rivastigmine . at each session , the folstein mini - mental status exam ( mmse ) was utilized as a measure of cognition and the unified parkinson s disease rating scale ( updrs ) was administered to evaluate the motor symptoms of pd . neuropsychiatric symptoms were evaluated using the neuropsychiatric inventory ( npi ) , which evaluates behaviors over the preceding four weeks . cognitive assessment utilizing the mmse revealed significant improvement of 5 points in patients on rivastigmine relative to their baseline ( z = 2.81 , p < 0.005 ) . motor symptoms and signs were unchanged as measured by udprs ( z = 1.18 , p > 0.2 ) . npi scores were also significantly lowered ( improved ) from baseline on treatment ( z = 2.85 , p < 0.004 ) . patients worsened significantly three weeks after withdrawal of the rivastigmine ( reading et al 2001 ) . giladi et al ( 2003 ) also studied the efficacy of rivastigmine in the treatment of pdd . in this open label study , 20 of 28 patients completed 26-weeks of treatment with rivastigmine therapy ( as tolerated up to 12 mg / day ) . the mmse and alzheimer s disease assessment scale ( adas - cog ) were used as cognitive measures and the updrs for documenting parkinsonian features . patients were assessed prior to starting therapy , at 12 and 26-weeks while on rivastigmine , and 8-weeks after withdrawal of therapy . an increase in udprs score was observed from baseline to week 26 ( p > 0.06 ) and a non - significant improvement was noted from week 26 to 8-weeks after treatment washout . significant improvement was noted in the area of the attentional components of the mmse at week 26 ( p < 0.002 ) . in terms of the adas - cog , a significant increase in total score ( worsening ) was noted throughout the study period ( p = 0.002 ) . the authors indicated that the objective cognitive measures ( mmse and adas - cog ) did not at all reflect the clinical impressions of the caregivers for the patients in the study . this , again , suggests the need for a cognition assessment tool that is validated and best utilized in pd . giladi et al ( 2003 ) also noted increased tremor in eleven of the original 28 participants in the study and dose reduction was required . in this study , it would be expected for patients to worsen on the updrs over 6-months whether they were on an ache inhibitor or not . a non - significant trend for improvement in the updrs scores following washout , however would make one consider that rivastigmine negatively influenced motor performance . an increase in tremor and parkinsonism was a legitimate concern in this study given we still use anticholinergics for some patients with pd ( certainly not demented ones , though ! ) ( morgan and sethi 2005 ) . there were prior case reports of worsening of tremor and parkinsonism in pd patients treated with ache ( richard et al 2002 ) . the results of the reading et al ( 2001 ) and giladi et al ( 2003 ) studies indicated further need to study the effects of this drug on cognition and parkinsonism in pdd in a prospective , double - blind , placebo - controlled fashion . in 2003 , fogelson et al ( 2003 ) performed another open label study examining the effects of rivastigmine on quantitative eeg ( qeeg ) in pdd patients ( n = 19 ) , given they frequently have a slowing of alpha activity on eeg . patients were treated with rivastigmine at an initial dose of 3 mg / day and titrated to a dose of 12 mg / day or highest tolerated dosage . quantitative eeg recordings were performed prior to introduction of rivastigmine and repeated when the patients had been on treatment for 12-weeks . a significant increase in the relative alpha activity was noted after treatment with rivastigmine ( p = 0.019 ) , however , no correlation between qeeg changes and cognitive improvement was identified ( fogelson et al 2003 ) . it is difficult , therefore , to determine whether these qeeg changes were due to improvement in cognitive state rather than just an increase in arousal . a sound neuropathological and pharmacological basis and promising open label studies were followed by the publication of a multi - center , placebo - controlled , double - blind study in 541 patients with pdd ( emre et al 2004 ) . pd was diagnosed using the uk pd brain bank criteria and dementia was diagnosed using dsm - iv criteria . participants were randomly assigned to receive placebo or 312 mg ( titrated to the maximum tolerated dose over a 16-week dose escalation period ) of rivastigmine divided twice daily for 24-weeks . the primary outcome measures were the adas - cog scores as in previous open label studies ( fogelson et al 2003 ; giladi et al 2003 ) and the alzheimer s disease cooperative study - clinician s global impression of change ( adcs - cgic ) . activities of daily living ( adcs - adl ) , npi , mmse , cognitive drug research power of attention tests ( cdr ) , delis - kaplan executive function system ( d - kefs ) verbal fluency test , and the ten point clock - drawing test . safety monitoring included recording of adverse events , monitoring ecgs and laboratory data , vital signs and body weight . patients were also assessed for changes in parkinsonism from baseline scores at weeks 16 and 24 using the updrs part iii ( motor section ) score . patients were included in the efficacy analysis if they underwent a baseline evaluation and if they took one dose of study medication followed by an assessment of the one of the efficacy variables after baseline ( regardless if they were taking study medication at the time ) . if no follow - up information was available emre et al ( 2004 ) used the last - observation - carried - forward method to impute missing values for missing follow - up information . approximately 30%35% of patients enrolled in the study suffered with co - morbid psychiatric disorders ( including depression , anxiety , and psychosis ) ( emre et al 2004 ) . the average age of patients was approximately 72 with pd diagnosis 9-years earlier on average . the mean time since the diagnosis of pdd for patients entering the study was approximately 1315 months . as compared to patients in the placebo group , patients who received rivastigmine demonstrated significant improvements in adas - cog and adcs - cgic scores ( primary efficacy variables ) ( emre et al 2004 ) . patients that were treated with rivastigmine had a mean improvement of 2.1 points in the adas - cog , while patients in the placebo group had a 0.7 point worsening ( p , 0.001 ) . clinically meaningful improvement was observed in the investigator rated ads - cgic in 19.8% or rivastigmine patients and 14.5% in placebo - treated patients , while clinically meaningful worsening was evident in 13.0% and 23.1 percent , respectively ( p = 0.007 ) . at week 24 , rivastigmine provided improvement in all six secondary efficacy variables relative to the baseline evaluation , while placebo - treated patients remained the same ( npi ) or worsened ( adcs - adl , mmse , cdr , d - kefs , ten point clock - drawing test ) ( emre et al 2004 ) . these benefits should be considered moderate and in - line with other clinical trials of ache inhibitors in ad . ninety - nine patients dropped out of the study in the rivastigmine arm with 32 dropping - out in the placebo arm . approximately two - thirds of the drop - outs in the rivastigmine arm were due to adverse events and less than half of the drop - outs in the placebo arm were due to the same . cholinergic symptoms typical of ache inhibitors were the most common adverse events , with nausea reported by 29% of rivastigmine vs 11.2% of placebo - treated patients ( p < in general , parkinsonian symptoms as a whole were more often reported by patients in the rivastigmine group relative to placebo ( 27.3% vs 15.6% , p = 0.002 ) . tremor ( 10.2% vs 3.9% ) and dizziness ( 5.8% vs 1.1% ) were also reported more often as an adverse event in rivastigmine - treated patients . tremor only caused withdrawal of 1.7% of patients in the rivastigmine group and no one in the placebo group ( p = 0.19 ) . there was no significant difference in updrs motor scores and tremor related items between the groups , however . interestingly , hallucinations ( 4.7% vs 9.5% ) and orthostatic hypotension ( 1.7% vs 5.0% ) were reported more often in patients treated with placebo . in a letter to the editor , harada et al ( 2005 ) argued that the number needed to treat in order to reach what emre et al ( 2004 ) defined as clinically meaningful improvement does not outweigh the side effects experienced by the participants in the study . harada et al ( 2005 ) only considered those who had clinically meaningful benefit in their analysis , however , and an important factor in any progressive neurodegenerative dementia is how many patients were prevented from clinically meaningful worsening as well . the results of the emre et al ( 2004 ) study are encouraging for the use of rivastigmine in pdd , however the efficacy is modest . adverse events are common with rivastigmine and typically cholinergic in nature , however they usually do not result in discontinuation of the drug . the adas - cog and the adcs - cgic as primary efficacy variables are appropriate , however there needs to be further validation of this scale in pdd . fortunately , six additional tests of cognitive domains that would be impaired in pdd were also used ( cdr , etc . ) . the finding of fewer reported hallucinations in the rivastigmine - treated arm is interesting given this drug may not only provide stabilization and slower decline of cognitive function in pdd , it may also help reduce hallucinations , which can be quite troubling for patients and caregivers alike . is the modest benefit obtained enough to delay nursing home placement and is it cost - effective over time ? should nmda - receptor antagonists such as memantine be added to pdd as it advances ? are the other ache inhibitors equally efficacious in pdd ? there was an open - label extension to the emre et al ( 2004 ) study published recently by poewe et al ( 2006 ) . of 433 patients that completed the double - blind trial , 334 entered and 273 completed the active treatment extension study ( 312 mg rivastigmine / day ) . at 48-weeks the adas - cog score had improved by 2 points above baseline for the entire group of patients . patients in the placebo treatment arm in the original emre et al ( 2004 ) trial also had a 2-point improvement in their adas - cog score . the safety profile of rivastigmine in the open label extension was similar to the double - blind phase ( poewe et al 2006 ) . wesnes et al ( 2005 ) looked specifically at the effects of rivastigmine on attention in pdd in patient enrolled in the emre et al ( 2004 ) trial . in this sub - study , 487 patients with pdd were given rivastigmine or placebo and assessed at baseline , 16 and 24-weeks . assessment of attention on the cognitive drug research ( cdr ) computerized assessment was performed at each visit in order to assess attention during various tasks . as compared with placebo , significant benefits on attention difficulty maintaining attention is a common finding in pdd and further asserts that rivastigmine is helpful for this impairment in these patients ( wesnes et al 2005 ) . willan et al ( 2006 ) prospectively examined the cost effectiveness of rivastigmine in the emre et al ( 2004 ) treated patients . quality adjusted survival time ( qast ) score was transformed from the mmse score and utilized as a measure of cost effectiveness . although an increase in qast in the rivastigmine arm of 2.81 quality - adjusted life - days was noted ( two - sided p - value 0.13 [ 90% ci 0.243 , 5.86 ] ) , no between - treatment differences in cost were seen . the high variability in cost of medications ( the study looked at canadian and uk prices ) as well as the short duration of the study ( six months ) could have interfered with the examination of cost effectiveness and further studies need to be performed . patients with pdd and dlb can often have autonomic nervous system dysfunction , and ache can potentially increase acetylcholine and contribute to cardiac dysfunction . ballard et al ( 2006 ) reviewed cardiac safety of rivastigmine in dlb and pdd . reviewing the emre et al ( 2004 ) pdd trial ( n = 541 ) and the mckeith et al ( 2000 ) dlb trial ( n = 120 ) , no clinically meaningful treatment differences in bradycardia or abnormalities on ecg were noted . patients treated with rivastigmine did have a mean reduction of 1.52 beats per minute in heart rate , however . in fact , compared with placebo , it appears that rivastigmine was associated with fewer adverse events ( p = 0.002 ) and fewer syncopal episodes ( p = 0.018 ) among pdd patients ( ballard et al 2006 ) . a recent practice parameter from the american academy of neurology indicated that there was level b evidence ( moderately strong ) for the treatment of pdd with either donepezil or rivastigmine ( miyasaki et al 2006 ) . given the lack of published large , randomized , placebo - controlled trials of donepezil in pdd , we feel that rivastigmine currently has the best data for use in pdd . the benefits are modest and further studies are needed , but the emre et al ( 2004 ) data are the best that we have in the use of ache inhibitors in pdd and the clinically and pathologically similar condition dlb ( mckeith et al 2002 ) . the dropout rate in the placebo - controlled phase was higher in the rivastigmine - treated pdd patients vs placebo - treated patients ( 27.3% vs 17.9% ) ( emre et al 2004 ) and using locf analytical techniques in a progressive condition like pdd would artifactually show less worsening in the rivastigmine treated group . the number needed to treat to provide a clinically meaningful significant outcome in the emre et al ( 2004 ) trial is perhaps six if you consider both meaningful improvement in the rivastigmine arm combined with the group with clinically meaningful worsening in the placebo - treated arm ( aarsland et al 2006 ) . given the relatively high drop - out rate in rivastigmine - treated patients , the safety profile of rivastigmine should be further examined . future studies need to specifically explore the gastrointestinal side effects and patient perceived worsening of motor symptoms ( ie , especially tremor ) . to this end , a recent study of 26 patients with pdd on rivastigmine revealed only mild worsening of tremor by accelerometry and a global tremor rating scale based upon the updrs ( gurevich et al 2006 ) . further study of the possible positive influence of rivastigmine on the neuropsychiatric symptoms of pdd ( hallucinations and delusions ) is warranted given these problems were reported significantly less as adverse events in the rivastigmine arm of the emre et al ( 2004 ) placebo - controlled study . it will be interesting to see if the other ache inhibitors like donepezil ( a drug with fewer reported adverse events in ad trials ( birks 2006 ) ) provide similar efficacy in pdd with fewer side effects ( aarsland et al 2002 ; leroi et al 2004 ; ravina et al 2005 ) . it is also necessary to look at the cost of rivastigmine versus the benefits gained by patients and caretakers . while there is good pharmacoeconomic data in ad for various ache inhibitors ( geldmacher 2005 ) , it is lacking for these drugs in pdd . a pharmacoeconomic assessment performed by willan et al ( 2006 ) was possibly not significant because of the variability of drug costs and the short , six - month study period . a confounding factor in pdd is that progression and severity of motor symptoms can lead to long - term care in patients with pd , which is somewhat different than in ad where patients are typically put in a nursing home due to severe dementia . a well - defined , clinically and neuropathologically relevant definition of pdd must be developed . the movement disorders society has assembled a task force to develop this definition and the outline of this new definition was presented at the 10th international conference of parkinson s disease and movement disorders in kyoto , japan in 2006 . while a decline in premorbid function will be necessary for the diagnosis of pdd with impairment in at least two cognitive domains , it appears that memory impairment on the mmse ( score < 25 ) will not be a required feature to make the diagnosis of pdd by these criteria . the current definition of pdd is largely based on the definition of dementia as cited in the dsm - iv with a decline in functional level , memory decline and at least one additional form of cognitive impairment ( executive dysfunction , apraxia , agnosia , aphasia ) ( fuchs et al 2004 ) and by exclusion of pdd from those with dlb ( mckeith et al 2005 ) . screening and assessment tools for mci in pd patients and in pdd must be identified , developed and validated . if you examine various neuropsychological testing batteries recommended for evaluation of cognitive function in pd , you will find a hodge - podge of cognitive scales and tests with many lacking widespread use and validation in pd ( defer et al 1999 ; saint - cyr et al 2000 ; pillon et al 2002 ; burn et al 2006 ; voon et al 2006 ) . emre et al ( 2004 ) , giladi et al ( 2003 ) , fogelson et al ( 2003 ) , and reading et al ( 2002 ) used cognitive scales that were assessed and validated in ad ( adcs - gcis , adcs - adl , npi ) without significant validation in pdd . miyasaki et al ( 2006 ) identified the most accurate screening tools for pdd as the mmse and the cambridge cognitive examination ( camcog ) . both tools had similar sensitivities for the diagnosis of pdd ; however , the camcog ( 94% ) was more specific than the mmse ( 77% ) ( miyasaki et al 2006 ) . a more precise definition and characterization of mild cognitive impairment ( mci ) in pd is also warranted ( fernandez et al 2005 ) . we know that pd is a progressive illness and patients with mci and without pd typically go on to develop ad over time . in patients with pd , how many patients have mci and how many of these go on to develop pdd over time ? there is much additional work that needs to be done in these areas to characterize and understand the course our patients will take as they progress . the montreal cognitive assessment ( moca ) has recently entered into the literature as an assessment tool for mci . unlike the mmse , the moca incorporates 8 additional cognitive domains including more complex visuospatial evaluation , naming , attention , memory , language and abstraction tasks . it appears sensitive to the kind of deficits that are evident in pdd ( executive and visuospatial dysfunction , etc . ) . in a validation study performed by nasreddine et al ( 2005 ) , the moca was found to have high sensitivity and specificity for detecting mci in patients who performed in normal ranges on the mmse . using a normal cut - off score of 26/30 , the mmse had a sensitivity of 18% to detect mci whereas an abnormal moca score detected 90% . while the moca seems to have much promise in detection of mci , further validation in pd and pdd is necessary . the publication of the first large - scale , randomized , double - blind controlled trial of rivastigmine in pdd has generated much interest in the definition , recognition , and treatment of pdd . while the clinical benefits of rivastigmine for pdd patients and their families appear moderate , and drop - out rates due to adverse events is a concern , this drug shows promise in an under recognized and under treated condition . rivastigmine provides clinically meaningful benefits in cognition and activities of daily living for some pdd patients in the short - term ( emre et al 2004 ; poewe et al 2006 ) ; however , future studies are necessary to validate its use in the long - term treatment of pdd .

the year 2011 was a banner year for oral health reports ; the institute of medicine published two key reports entitled advancing oral health in america which outlined the seriousness of poor oral health and lack of access in the oral health care system in america . the second report was entitled improving access to oral health care for vulnerable and underserved populations . the first report was written to describe the aims of oral health as integral to the overall health of the population beyond just dental conditions . it was also a call to arms not only for those in the professional dental community but also for all health care professionals who have interactions with patients . in any given health care encounter , clinicians and in particular nurses have the opportunity to screen for oral cancer , tooth decay , and gum disease and consider any necessary health care referrals as well as provide health education on the role of fluorides , sealants , nutrition , and oral health . there is clear evidence related to the oral - systemic connection and how oral disease can lead to poor health outcomes such as premature birth and cardiac valve disease . . the secretary of hhs should give the leaders of the new oral health initiative ( nohi ) the authority and resources needed to successfully integrate oral health into the planning , programming , policies , and research that occurs across all hhs programs and agencies . all relevant hhs agencies should promote and monitor the use of evidence - based preventative services in oral health , ( both the clinical and community based ) and counseling across the lifespan . all relevant hhs agencies should undertake oral health literacy and education efforts aimed at individuals and communities and health care professionals . cms should explore new delivery and payment models for medicare and medicaid and chip to improve access , quality , and coverage of oral health care across the lifespan . hhs should place a high priority on efforts to improve open , actionable , and timely information to advance science and improve oral health through research . to evaluate the nohi the leaders of the nohi should convene an annual public meeting of the agency heads to report on the progress of the nohi . this report , commissioned in 2009 by the united states department of health and human services , asked the institute of medicine to convene a panel to think about strategies for oral health . we have long known that our oral health system is challenged when it comes to access and that many people who encounter a dental visit may , unfortunately , receive less than a comprehensive health assessment while there . the important distinction in the advancing oral health in america this change of term reminds all of us that oral health can be addressed by nurses , physicians , social workers , pharmacists , and other allied health professionals who come in contact with patients . by simply asking questions about the patient 's oral health , an important difference can be made in the screening and triage of those in need of better oral health care . there are numerous reports and ample data to document the need for better access and higher - quality oral health care in america . the department of health and human services ( dhhs ) has conducted a number of surveys through the cdc and its national health and nutrition examination survey ( nhanes ) , in which data are gathered from a sample of the civilian us population for the purpose of better understanding how health and nutrition impact health status outcomes . in 2007 , the national center for health statistics ( nchs ) compared two reports from nhanes ( nhanes iii , 19881994 , and nhanes , 19902004 ) , on the dental health of the public . dye and colleagues have reported optimistic data because in older adults , dental health , specifically edentulism , and periodontitis have improved and the number of dental caries in adolescents decreased from 68% to 59% . according to nhanes data , the dental health of the general adolescent and adult population has improved with a decreased prevalence of dental caries and periodontal disease , likely due to the impact of fluoridation . only a subset of the adult population , consisting of mexican - american and non - hispanic black men , showed a decrease in oral and dental health in their self - reports , which could be associated with socioeconomic factors . further , treatment of dental decay increased in most ethnic and racial groups , except for non - hispanic black persons and youths living at or over 200% of the federal poverty level . the iom report , however , documents a discouragingly high percentage of adults , including older adults who present with untreated dental caries at the time of examination . finally and alarmingly , there has been an 1820% reported increase in dental caries in preschool children . oral health includes dental health and conditions such as cleft palate ( 1 in 1,000 live births ) , neoplasms , and neuromuscular or joint disorders of the oral region . oral cancer has decreased over the past decade , presumably because of a reduction in the use of alcohol and tobacco ; however , there is a high mortality because most oral cancers are diagnosed in advanced stages , especially in african americans , who have a 5-year survival rate of 42% compared to a 63% in whites . in the latest seer cancer statistics review , oral cancer is among the top 15 cancers . chevarley has estimated that approximately 1 in 10 persons in the us noninstitutionalized population ( approximately 30 million persons ) was not able to get or had a delay in accessing needed dental care . specifically , approximately 5.5 percent of the population was unable to access dental care when needed . the alarming death of deamonte driver , a 12-year - old who died in 2007 due to a lack of access to dental care , was a shocking wakeup call for all health care professionals . every primary care visit is an opportunity to provide oral health assessment and care . in the united states , primary care is provided by several categories of health care practitioners including nurse practitioners , family physicians , physician assistants , general internists , pediatricians , and geriatricians . further , registered nurses , health educators , and medical assistants are an important part of the healthcare workforce who can bring to bear their own knowledge and skills to oral health assessment and care . private practices , hospital outpatient departments , community health centers , and integrated care systems are all setting for oral health intervention and the mandate to do so is growing . for example , in 2007 there were 88.9 million visits to hospital outpatient departments ( opds ) in the united states and each encounter , an opportunity for an oral health examination , with 54.7% of those visits to primary care providers . black or african - american persons ( 58.4 visits per 100 persons ) had higher opd visit rates . if you want to provide dental care , go to dental school . this sentiment is a major barrier to oral health screening for those who are committed to the specialty model of health care . all providers are increasingly under pressure to show productivity in the workplace and argue that the oral examination is not in their scope of practice . further , there is a concern that the requisite knowledge and skills for the oral health examination are not well understood . nursing and medical licensing exams require knowledge of oral health , but few medical or nursing schools include oral health in their curriculum . there is an opportunity to fundamentally reconceptualize healthcare delivery practice patterns and interprofessional collaboration as a bidirectional relationship between oral health care and physical health care to improve patient outcomes and access [ 11 , 12 ] . in 2008 , the american association of medical colleges added oral health in their learning objectives but much is to be done before oral health examinations are a standard practice . only 50% of pediatricians receive oral health training during residency and report a lack of training as a barrier to incorporating oral health in their practices . there are 3.1 million nurses and over 140,000 nurse practitioners in the united states and few have received adequate education related to oral health assessment . while the majority received some instruction related to oral hygiene , few nurses place a high priority on mouth care in the practice setting [ 11 , 15 ] . in 2006 , family medicine residencies included oral health as a requirement ; however , only three - quarters of residency directors were aware of this requirement , and in 2006 , only two - thirds of the programs were including oral health in the curriculum . these deficiencies in physician training on oral health lead not only to a lack of diagnosis of oral conditions but also to a low rate of referrals to dentists , as 23% of internal medicine residents educational competencies for nurse practitioners , physicians , and dentists have a great deal in common with opportunities to capitalize on practice expectations . this can be used to promote synergies between educators in the different programs , enriching students ' education with knowledge of another discipline which can be incorporated in their future professional practice in benefit of patients . in 2007 , the national hospital ambulatory medical care survey reported that 55.3% of opd visits were to a provider other than the patient 's primary care provider . it also reported that established patients ( those with previous visits to the opd clinic ) made 82.9% of opd visits and that only 43.1% of visits by these patients were to their primary care provider . the lack in continuity of patients to a constant primary care provider ( pcp ) may add to the minimization of oral health during examination . it has been described that when used at least twice a year , fluoride varnish reduces dental decay by 38% . in some states , medicaid reimburses physicians and nurse practitioners $ 18.18 per visit to apply fluoride varnish 3 times a year to children under 19 years of age . furthermore , quionez et al . reported that the application of fluoride varnish improves clinical outcomes by 1.52 cavity - free months at a cost of us$7.18 for each cavity - free month gained per child and us$203 for each treatment . these authors have concluded that the use of fluoride varnish in the medical setting is effective in reducing early childhood caries in low - income populations . this is an extremely important data point and adds power to the argument that all of us need to practice oral health assessment and intervention . despite the evidence that only four percent of pediatricians regularly apply fluoride varnish , there are no data for nurse practitioners . lack of practitioner confidence , minimal education during formal training , time constraints during the visit , and an absence of referral strategies after examination except in locations where there are dental schools all lead to lack of access and underserved populations . the two recent iom reports provide specific recommendations that must be addressed and nurse practitioners and physicians are a powerful voice in addressing our current shortfalls in practice [ 1 , 2 ] . with the looming retirement of the dental workforce there is an urgency to implement some of the creative strategies that are now in pilot phase ( dolce , in press ) . our professional practice associations ( ama , ana ) can do much to move the agenda forward by giving voice to the inadequacies of our current educational and payment systems as they relate to oral health .

the word autophagy is derived from the greek language auto ( self ) and phagy ( eating ) ( self - eating ) and generally refers to the cell catabolic and recycling processes in which cytoplasm non - essential and old or damaged components are transported to lysosomes for lysosome - mediated degradation and turnover process . it plays a significant homeostatic function in cells and keeps the balance between formation , synthesis , elimination and subsequent recycling of cell elements . de duve first used the word autophagy to describe an observable fact associated with single and/or double - membrane vesicles that had cytoplasm cargo inside at different stages of digestion / degradation . autophagic cargo consists of cytoplasm constituents including misfolded or aggregated proteins , damaged organelles such as endoplasmic reticulum , mitochondria and peroxisomes as well as intracellular pathogens . moreover , autophagy can be differentiated from heterophagy , in which the cell degrades extracellular substances . autophagy machinery involves induction and presentation of target components such as organelles and proteins to the lysosomes via three major distinct autophagy processes : macroautophagy , microautophagy and chaperone mediated autophagy , which we will describe as autophagy which is the aim of this review . autophagy is a degradation system / process involved in the clearance / elimination of aging / damaged organelles and pathologically changed proteins . during the process of autophagy , pre - autophagosomal structures , also called phagophores , fuse and elongate with engulfing the cytoplasm material within double - membrane vesicles , named autophagosomes . the autophagosomes initially fuse with endosomes to shape amphisomes , which later fuse with lysosomes where cytoplasm cargo is degraded . thus , a normal activity of autophagy guarantees the physiological turnover of damaged and aging organelles as a garbage recycling process . however , a considerable accumulation of autophagosomes is considered as an alternative route of cell death or an eventual cell survival pathway in response to a pathogen . autophagy today is recognized as an arbiter of neuron survival and death decisions in neurodegenerative disorders . for example , in alzheimer s disease and after brain ischemia the presence of hyperphosphorylated tau protein and aggregated -synuclein has been observed . given that the autophagy machinery is an important route by which intracellular proteins aggregates are cleared , it is of no surprise that autophagy is postulated to remove these neurotoxic aggregates . it is evident that autophagy can play a role in cell death according to the type and degree of a pathogen . neuronal cell reaction might shift step by step from degradation of toxic proteins and damaged organelles via autophagy , which leads to its recovery and the initiation of apoptotic routes determining neuronal death . on the other hand , there is a suggestion that along with neurodegenerative diseases induction of autophagy is a protective reaction , and that the autophagy anomaly , rather than excessive autophagy , induces neuron death . autophagy in animals like in humans occurs physiologically and can be stimulated by starvation and different pathologies including alzheimer s disease . besides its activity in maintaining cell homeostasis by producing substrates for both energy metabolism and vital protein synthesis as well as by supporting the clearance of damaged organelles and misfolded proteins , autophagy is important in various pathological episodes , including the degradation of aggregating proteins in neurodegenerative disorders . dysfunction of the autophagy system has been presented to accompany a range of pathological situations including neurodegenerative disorders such as debilitating alzheimer s disease . in this review in a paper by yu et al . a novel -amyloid peptide - generating pathway activated in alzheimer s disease has been presented , which occurs in autophagic vacuoles during their maturation defect in alzheimer s disease brain . a great number of autophagic vacuoles were noted in dystrophic axons and neurites , earlier than the appearance of extracellular -amyloid peptide as plaques in diseased brains indicating that autophagic vacuoles deposition / aggregation constitute an early hallmark of the disease in neurons . the above mentioned authors presented that -amyloid peptide is generated within the autophagic vacuoles . to support their point of view they found -amyloid peptides and -secretase on the membranes of autophagic vacuoles in alzheimer s disease brains . additionally , in experiments in vitro they presented a correlation between autophagic activity and -amyloid peptide production after starvation . on the other hand , -amyloid peptide secretion was clearly suppressed when autophagy was inhibited e.g. by an excess of amino acids . the hypothesis that chronic and incomplete autophagic elimination involves itself in the overload formation of -amyloid peptide today moreover , the new data presented by different laboratories fully support and provide clear insights into the role of autophagy in the processing of amyloid precursor protein and production -amyloid peptide in alzheimer s brains [ 911 ] . abnormal processing and altered clearance by autophagic - lysosomal machinery of -protein is implicated in the formation of neurofibrillary tangles in brains of alzheimer s disease . the observations that have been presented are supported by a study of tau protein transgenic animals brains , which demonstrated altered autophagic - lysosomal system [ 12 , 13 ] . aforementioned models provoked the development of axonal spheroids , which were well - equipped with tau protein filaments and autophagosomes . furthermore , this pathology appeared similar to abnormalities observed in the dystrophic neurites in alzheimer s disease brains [ 12 , 13 ] . it was demonstrated that -protein aggregation and accumulation were induced not due to abnormalities in -protein phosphorylation . on the contrary , other data showed that autophagy could eliminate soluble -protein , and its related aggregates and autophagy activity inhibition led to intensification in -protein aggregation and neurotoxicity . apart from adaption role of autophagy , it plays a role in cell death , as well . cell death is associated with continuous dysfunction of autophagy and may not be caused by the triggering of autophagy . other studies have demonstrated that autophagy might promote cell death [ 18 , 19 ] . autophagy - lysosome pathway dysfunction can cause neuron death potentially associated with dysregulation of ubiquitin proteasome pathway substrates . a former and current study presenting ubiquitination as a significant regulatory element for autophagy [ 2224 ] , which is important as a key mechanism for clearing pathological protein aggregates from neuronal cells [ 25 , 26 ] , support the above mechanism . regardless of the key role of above pathological proteins aggregates , -amyloid peptide aggregates or neurofibrillary tangles their existence can be a sign of dysregulation or a problem of neuronal degradation mechanisms maturation . it is becoming evident that autophagy can be a part of the cause in induction of cell death associating with different pathogens . neuronal response might shift step by step from clearance of old proteins and damaged organelles via autophagy , which finally leads to its recovery to a normal condition , to the initiation of apoptotic mechanisms determining neuronal cell death . finally , in the situation of neurodegenerative diseases the hypothesis is that generation of autophagy is a neuroprotective reaction and that defective or inadequate autophagy supports neuron death in the majority of these diseases , including alzheimer s disease [ 2 , 20 , 27 , 28 ] . active and efficient transport from synapses and neurites along microtubules to the neuronal body where lysosomes are accumulated is important for autophagic clearance in neuronal cells . in alzheimer s disease there occurs a massive concentration of autophagosomes in swelling and degenerating neurites due to problems with maturation of autophagosomes and their retrograde trafficking along defected microtubules towards the neuronal soma . a common hypothesis is proposed that autophagy is developing in alzheimer s disease because of ineffective degradation of autophagosomes which hold amyloid precursor protein and secretases important for generation of -amyloid peptides from amyloid precursor . as a result -amyloid peptides accumulate within autophagosomes in dystrophic neurites and become the most important intracellular pool of neurotoxic peptides in alzheimer s disease patients brains . it is documented that -amyloid peptides can damage autophagosomes membranes and trigger the release of different toxic substances into cytoplasm compartment . it has been presented that accumulation of autophagosomes in neuronal cells can increase -amyloid peptides plaques generation and that abnormal activity of autophagy especially in damaged neuropil could be an important source of toxic -amyloid peptides [ 10 , 11 , 29 ] . in addition to this , the autophagy - related protein beclin 1 shows reduced expression in early alzheimer s disease , what induces -amyloid peptides accumulation in transgenic mice overexpressing amyloid precursor protein due to reduced autophagy . thus , this observation is consistent with an observation that autophagosomes maturation is impaired in alzheimer s disease . on the other hand , it is important to notice that -amyloid peptides accumulation is affected by both generation and clearance [ 3133 ] . moreover , problems with autophagosomes maturation triggering -amyloid peptides production , but decreased autophagy connected with beclin 1 deficiency , probably disturb -amyloid peptides clearance by altered amyloid precursor protein metabolism . despite ongoing interest in alzheimer s disease , the basis of this entity is not yet clear . at present , the best - established and accepted culprit in alzheimer s disease pathology by most scientists is the amyloid , as the main molecular factor responsible for neurodegeneration in this disease . abnormal upregulation of amyloid production or a disturbed clearance mechanism may lead to pathological accumulation of amyloid in brain according to the amyloid hypothesis . we and others critically reviewed these observations and highlighted inconsistencies between the predictions of the amyloid hypothesis and the published data [ 5 , 32 , 33 ] . a question arises whether amyloid is responsible for the neurodegeneration or if it accumulates because of the neurodegeneration . recent evidence suggests that the pathophysiology and neuropathology of alzheimer s disease comprises more than amyloid accumulation , -protein pathology and finally brain atrophy with dementia . nowadays , a handful of researchers share a newly emerged view that the ischemic episodes of brain best describe the pathogenic cascade , which eventually leads to neuronal loss , especially in hippocampus , with amyloid accumulation , -protein pathology and irreversible dementia of alzheimer type [ 5 , 32 , 33 ] . the most persuasive evidences come from investigations of ischemically damaged brains of patients and from experimental ischemic brain studies that mimic alzheimer - type dementia . the resulting brain ischemia dysregulated expression of amyloid precursor protein and amyloid - processing enzyme genes [ 32 , 33 ] that , in addition , ultimately compromise brain functions , leading over time to the complex alterations that characterize advanced sporadic alzheimer s disease . data suggest that ischemia promotes overproduction and aggregation of -amyloid peptide in brain , which is toxic for ischemic neuronal cells as byproduct [ 5 , 33 ] . the identification of the genes involved in alzheimer s disease induced by ischemia will enable to further define the events leading to sporadic alzheimer s disease - related abnormalities . additionally , knowledge gained from the above investigations should facilitate the elaboration of the effective treatment and/or prevention of alzheimer s disease . autophagy can occur in neurons following brain ischemia giving mixed features of neuronal cell death . the interaction after brain ischemia among autophagy , apoptosis and necrosis are complex with much crosstalk , and their roles in ischemia and alzheimer s disease need further studies . aggregate - prone age - dependent and disease - related proteins like -amyloid peptide and -protein can be a substrate of the autophagy activity . still , the processes underlying the elimination / recycling of particular neurodegeneration - related proteins are now completely unclear . new data suggested possible influence of -protein dysfunction on autophagy clearance / elimination of cytoplasm garbage , which probably contributes to alzheimer s disease development / maturation . -protein has been found to control the stability of microtubules but phosphorylation of -protein or an increase in the total level of -protein can cause dysfunction of neuronal cells microtubules . this put neurons in enormous danger due to abnormal function and placement of cellular organelles such as mitochondria and lysosomes . in particular autophagosome degradation by lysosomes is a microtubule dependent route . as a result , pathological activity of -protein may affect neuron function like neurites and axonal transport , which as well involves transportation of autophagic rubbish and completely develops autophagosomes with all consequences in dystrophic axons and neurites in alzheimer s disease brains . as a consequence of the above , autophagosomes dynamically form along neurites , axons and in synaptic area . however , effective clearance of these parts requires their retrograde transportation towards the neuronal body , where lysosomes are mainly packed . in alzheimer s disease brains the maturation of autophago - lysosomes in above parts of neurons and their retrograde transportation are obstructed , what leads to an enormous aggregation and accumulation of autophagy products ( e.g. -amyloid peptides ) within dystrophic and degenerating axons and neurites . the combination of massive induced autophagy with faulty clearance of -amyloid peptides producing autophagic vacuoles establishes circumstances helpful for -amyloid peptides aggregation and accumulation in alzheimer s disease brains . autophagy may contribute to alzheimer s disease amplification through secondary mechanisms such as dysregulated metabolism of amyloid precursor protein and increased production of -amyloid peptides [ 5 , 32 , 33 ] . autophagy is now recognized as an arbiter of neuronal survival and death decisions in alzheimer s disease . in conclusion , in full - blown alzheimer disease , investigation of the regulation of autophagosome maturation and movement especially to neuronal cell body / soma is a promising area [ 24 , 39 ] . thus , autophagy regulation and stimulation might serve as a future therapy target for preventing and/or stopping alzheimer s disease neuropathogenesis .

, extensions of the key functions are replaced by updating the level set function based on an artificial image that is composed of the diffused image and the original image . the low convergence problem of the classical algorithm is efficiently solved in the proposed approach .

methicillin - resistant staphylococcus aureus ( mrsa ) is frequently resistant to the majority of commonly used antimicrobial agents , including beta - lactam antibiotics , aminoglycosides , macrolides , chloramphenicol , tetracycline , and fluoroquinolones . the prevalence of methicillin resistance is known to be more than ~6070% among s. aureus isolates from hospitals in korea [ 2 , 3 ] . mrsa has become a one of the most important causes of nosocomial pathogenic infections , and the use of vancomycin for the treatment of mrsa infection has increased . unfortunately , vancomycin - resistant enterococcus ( vre ) and vancomycin - resistant coagulase - negative staphylococcus ( vrcns ) have been reported , as well as vancomycin - resistant s. aureus ( vrsa ) [ 57 ] . to prevent the spread of vre , vrcns , and vrsa , the use of vancomycin has to be reduced . this will require the introduction of a new class of antibiotics that can replace vancomycin . pharmacokinetic advantages such as concentration - dependant bactericidal activity and prolonged post - antibiotic effect are more appreciable than vancomycin . however , only a few reports of clinical data describing this new kind of antibiotic exist outside of japan , which was the first country that approved its use against mrsa infections . therefore , presently , mrsa infection is treated mainly with vancomycin and teicoplanin , which are glycopeptide antibiotics . we studied the clinical and bacteriological efficacy and safety of arbekacin compared to vancomycin in the treatment of infections caused by mrsa . this was a retrospective case control study of patients who were admitted to chonbuk national university hospital , a 1,100-bed tertiary care university hospital in jeonju , korea , from january 1st , 2009 to may 31st , 2010 , and received the antibiotics arbekacin or vancomycin . all of the mrsa - infected patients who received arbekacin were enrolled during the study period . the vancomycin group infected by mrsa was selected by age and sex that matched the arbekacin group . the study protocol was approved by the institutional review board of chonbuk national university hospital . in this study , nephrotoxicity was defined as when at least 50% reduction was seen in the glomerular filtration rate ( gfr ) using the abbreviated modified diet in the renal disease ( mdrd ) equation , which was gfr ( ml / min/1.73 m ) = 186 pcr1.154 age ( 1.212 if black ) ( 0.742 if female ) . hepatotoxicity was defined as when the aspartate aminotransferase / alanine aminotransferase ( alt / ast ) levels were raised over two times the baseline values during treatment . leukocytopenia was defined as a continuous decrease lower than 4.8 10/l in the number of white blood cells found in the complete blood cell count during treatment . drug fever was defined as a disorder characterized by fever coinciding with the administration of a drug and disappearing after the discontinuation of the drug . the bacteriological efficacy response ( ber ) the improved ber was defined as no growth of mrsa , whereas failure was defined as the growth of mrsa culture at the end of therapy or during treatment . improved cer was defined as resolution or reduction of the majority of signs and symptoms related to the original infection . failure was defined as no resolution and no reduction of the majority of the signs and symptoms , or the worsening of one or more signs and symptoms , or new symptoms or signs associated with the original infection or a new infection . categorical variables were compared by the chi - squared test and continuous variables were compared by the unpaired t - test . spss software ( version 15.0 ) was used throughout and p - values of less than 0.05 were considered to be statistically significant . seventy - three patients receiving arbekacin were compared with the same number of patients receiving vancomycin ( table 1 ) . the mean age of the arbekacin group was 54.1 16.4 years , and that of the vancomycin group was 56.3 14.7 years . the arbekacin group was more common in surgical section than the vancomycin group ( p < 0.001 ) . the clinical status ( p = 0.063 ) or the medication duration ( 19.4 15.7 days vs. 18.2 11.3 days ) was not different between the two groups.table 1general characteristics of the study populationarbekacin ( n = 73)vancomycin ( n = 73)p - valueage ( years)54.1 16.456.3 14.70.397sex male43 ( 58.9%)43 ( 58.9%)1.000 female30 ( 41.1%)30 ( 41.1%)department medical15 ( 20.5%)38 ( 52.1%)<0.001 surgical58 ( 79.5%)35 ( 47.9%)clinical status sepsis5 ( 6.8%)6 ( 8.2%)0.063 wound- and catheter - related45 ( 61.6%)31 ( 42.5% ) others23 ( 31.5%)36 ( 49.3%)medication duration ( days)19.4 15.718.2 11.30.608otitis media ( 13 ) , meningitis ( 6 ) , pneumonia ( 29 ) , peritonitis ( 6 ) , urinary tract infection ( uti ) , cellulitis , neutropenic fever ( 1)analyzed by the t - test and the chi - squared test general characteristics of the study population otitis media ( 13 ) , meningitis ( 6 ) , pneumonia ( 29 ) , peritonitis ( 6 ) , urinary tract infection ( uti ) , cellulitis , neutropenic fever ( 1 ) analyzed by the t - test and the chi - squared test the complications of the antibiotics between the two groups were different ( table 2 ) . the complication rate was significantly higher in the vancomycin group ( 32.9% ) than in the arbekacin group ( 15.1% ) ( p = 0.019 ) . however , individual complications such as nephrotoxicity , leukopenia , and hepatotoxicity were not significantly different between the two groups . skin rash and drug fever occurred only among patients in the vancomycin group . in the outcome section , the ber ( improved , arbekacin vs. vancomycin ; 71.2% vs. 79.5% ) and the cer ( improved , arbekacin vs. vancomycin ; 65.3% vs. 76.1% ) were not statistically different between the two groups ( table 2).table 2safety and outcomes in patients receiving arbekacin or vancomycinarbekacin ( n = 73)vancomycin ( n = 73)p - valuecomplicationsno62 ( 84.9%)49 ( 67.1%)0.019yes11 ( 15.1%)24 ( 32.9%)nephrotoxicity5 ( 6.8%)6 ( 8.2%)0.754leukopenia4 ( 5.5%)5 ( 6.8%)0.731hepatotoxicity3 ( 4.1%)3 ( 4.1%)1.000skin rash0 ( 0.0%)5 ( 6.8%)n / adrug fever0 ( 0.0%)6 ( 8.2%)n / aoutcomesberimproved52 ( 71.2%)58 ( 79.5%)0.249failure21 ( 28.8%)15 ( 20.5%)cerimproved47 ( 65.3%)54 ( 76.1%)0.157failure25 ( 34.7%)17 ( 23.9%)n / a , not applicable ; ber , bacteriological efficacy response ; cer , clinical efficacy responseanalyzed by the chi - squared test safety and outcomes in patients receiving arbekacin or vancomycin n / a , not applicable ; ber , bacteriological efficacy response ; cer , clinical efficacy response analyzed by the chi - squared test in this study , arbekacin had a similar efficacy to vancomycin in patients with mrsa . in addition , the side effects in the arbekacin group were significantly lower than in the vancomycin group . this showed that arbekacin may be a good alternative drug for the treatment of infectious disease with mrsa . furthermore , this could lead to the decrease of vancomycin usage in hospitals and decrease antibiotic - resistant microorganisms , particularly vre and vancomycin - intermediate s. aureus ( visa ) , in clinical settings . approximately 80% of s. aureus strains isolated in intensive care units ( icus ) are resistant to methicillin in asia . more than 60% of mrsa were found in central line - associated bloodstream infections in icus from the united states . glycopeptides such as vancomycin and teicoplanin are still the most frequently chosen antibiotics for the treatment of mrsa infections , but the susceptibility to vancomycin diminished in mrsa strains . in addition , vrsa strains have emerged in clinical settings . although vrsa infections continue to be rare and no transmission has been identified , it remains a serious public health concern all over the world . it was shown that , with more vancomycin exposure , there was a higher risk for vrsa or vre . prudent use of vancomycin as well as the development of alternative therapeutic options against mrsa is , therefore , required . arbekacin , a derivative of the aminoglycoside dibekacin , has been reported to have good in vitro activity against mrsa [ 18 , 19 ] . previous reports showed that the majority of mrsa isolates in europe and japan were susceptible to arbekacin . arbekacin has been used for the treatment of mrsa infections is shown to be as useful as vancomycin based on clinical data [ 21 , 22 ] . showed that arbekacin - based combination regimens could be an alternative option for glycopeptides in the treatment of mrsa or hetero - visa infection . in this study , the major side effects of vancomycin include local phlebitis , fever , neutropenia , skin rash , and renal toxicity . the major side effects of arbekacin were renal and ear toxicity , like other aminoglycosides . in this study , however , the clinical status was not different between the two groups . in conclusion , we suggest that arbekacin could be a good alternative drug for vancomycin in mrsa treatment in the hospital . more well - designed studies are required in order to evaluate the exact clinical response between arbekacin and vancomycin .

the workshop leaders will present a panoramic view of the community mental health care in holland : some figures of psychiatric disorders , organizational features of mental health care , treatment of depression and anxiety disorders in primary health care , treatment and care of the severe mentally ill and public mental health solutions . they will show examples of community mental health care and indicate innovations and future trends . advantages and disadvantages of the system will be discussed and compared with arrangements of community mental healthcare in other countries . in an interactive way participants will be invited to indicate their community mental health care so that the workshop gives the opportunity to compare and weigh current community - based care for psychiatric patients and new directions .

the zebrafish has become an attractive model for human disease modeling as there are a large number of orthologous genes in the zebrafish genome , and the encoded proteins exhibit similar functions to those expressed in humans . the methods that are available to manipulate zebrafish gene expression are limited , but principal among these methods is morpholino - based antisense technology that enables transient downregulation in order to study the effects of reduced gene expression during early vertebrate development . to create stable mutant fish lines , chemical mutagenesis ( enu ) is the method of choice ; however , this forward genetics approach is driven by phenotypic outcomes and is not able to target and knock down specific genes . in contrast , zinc finger nucleases ( zfns ) are an emerging technology that has proved successful in targeted gene knockdown in zebrafish [ 24 ] . unfortunately , the design and production process is arduous and requires specialised knowledge , thus restricting its use in the wider research community . zfns also do not offer the option of causing gene knockdown in a tissue - specific manner or of providing conditional gene knockdown ( the ability to turn on or off gene knockdown ) . this latter outcome would provide a more subtle disease model that is suited for studying late onset disorders , and it is in this context that rna interference may offer a way forward . the use of rna interference ( rnai ) technology has proven to be a very useful tool in disease modelling in mammalian systems . the use of different promoters has allowed for tissue - specific expression of mirnas , therefore restricting gene knockdown to targeted areas / organs , and also conditional knockdown with the use of tet - on promoters ( the cre / loxp system is used in mice ) . in brief , small noncoding rnas enable fine - tuning gene expression at the posttranscriptional level during development , apoptosis , and metabolism [ 7 , 8 ] . these noncoding rnas cause cleavage / degradation , or the stalling of translation , of a target mrna ( refer to for a detailed review ) and can be synthetically created to cause gene knockdown . mirnas can be expressed by a dna - based vector system in which mirna transcripts , consisting of a double - stranded hairpin structure , are processed by several endonucleases to form 22 nucleotide mature mirnas , which then form a complex with the rna - induced silencing complex ( risc ) to carry out targeted gene silencing . the use of rnai in zebrafish has been controversial as there are varying opinions regarding its effectiveness , and specificity , in causing gene knockdown . it has been suggested that the expression of mirnas in zebrafish embryos leads to off - target / toxic phenotypes due to flooding the endogenous mirna pathway [ 9 , 10 ] . however , transgenic zebrafish lines have been constructed that express mirnas designed to target desired genes of interest with no apparent toxic effects . dong et al . and ho et al . successfully used mirnas to mediate gene knockdown in zebrafish and have created a conditional knockdown model and a heritable gene knockdown fish line . given the above background , we have considered that rnai - based technology might allow the development of zebrafish models of heritable cardiac disorders such as long qt syndrome ( lqts ) . this syndrome is a congenital cardiac disorder , which is characterized by a prolonged qt - interval that can lead to fatal arrhythmias . of the many genes ( encoding for cardiac ion channels / scaffolding proteins ) implicated in lqts , we have focussed on the kcnh2 gene , which encodes for the -subunit of the rapid delayed inward rectifier potassium channel and is vital for the repolarisation phase in the cardiac cycle . the dominant mutations that have been detected in lqts type 2 patients encompass nonsense mutations , as well as exon deletions and duplications , which suggest that haploinsufficiency of kcnh2 plays a role in the manifestation of disease [ 13 , 14 ] . two kcnh2-type genes have been identified in zebrafish , termed zerg-2 and zerg-3 [ 1 , 2 ] . the former appears to be an orthologue of the human kcnh2 gene , while the latter appears to encode a protein with kcnh2 function but is an orthologue of the human kcnh6 gene , which is another potassium channel that belongs to the same family as kcnh2 . critically , the ( unregulated ) knockdown of both zebrafish genes leads to a cardiac phenotype ( bradycardia , atrioventricular block , and prolonged qt interval ) that mimics the phenotype seen in patients with lqts [ 1 , 3 ] . here , we show that a commercially available mirna vector system is capable of mediating gene knockdown in zebrafish . we evaluate this approach in a transient in vivo method that targets various regions of the zebrafish zerg-2 and zerg-3 gene transcripts as a prelude to mimicking the haploinsufficiency of zerg-2 and zerg-3 in zebrafish in a stable transgenic fish line and to ensure that the custom - designed mirnas are able to cause the knockdown of their intended targets . wild - type zebrafish ( danio rerio ) were bred and maintained as described by tang et al . . linearised pcdna 6.2 gw / emgfp - mir was recircularised by the insertion of synthesised double - stranded oligonucleotides ( dsoligo ) containing the same cohesive ends as the mirna cloning site . single - stranded dnas ( ssoligos ) were synthesised ( invitrogen ) and annealed and cloned into the pcdna 6.2 gw / emgfp - mir vector as described below . the dsred express coding region was amplified by pcr from the ptre - tight - bi - dsred express vector ( clontech ) . the emgfp coding region was removed from the pcdna 6.2 gw / emgfp - mir vector by drai restriction enzyme digestion , treated with calf intestinal phosphatise , and purified . the amplified dsred express coding region was cloned into the pcdna 6.2 gw vector and bidirectionally sequenced to confirm the correct orientation of dsred express . mirna target sites were designed using the block - it rnai designer ( invitrogen ) under the mir rnai design option . the entire zerg-2 ( genbank i d : hm209079 ) and zerg3 ( ensembl i d : ensdarp00000085242 ) gene transcript sequences were used for the design process , and targets were chosen based on location ( close to or within the 3 utr ) and the score the designer programme gave to each target site . the top and bottom ssoligos ( table 1 ) of the mature mirna were synthesised ( invitrogen ) and cloned into the pcdna 6.2-gw / dsred - mir expression vector according to the manufacturer 's instructions . as the pcs2 + vector does not contain an egfp reporter gene , one had to be inserted into the vector before the egfp sensor vector could be used . the kozak sequence and the egfp coding region was excised from pt2al200r150 g ( a gift from dr koichi kawakami , national institute of genetics ) using bamhi and clai restriction enzymes . the pcs2 + vector was restriction enzyme digested with bamhi and clai and the egfp coding region was cloned into the linearised vector ( figure 1 ) . the 22 nucleotides that form the mature mirna ( table 1 ) were used as the target site and the top and bottom ssoligos were synthesized ( invitrogen ) . the ssoligos were annealed according to the manufacturer 's instructions for the construction of the mirna vectors , and this was ligated downstream of the egfp reporter gene in the pcs2 + egfp vector . the pcs2 + egfp vector was linearized with xhoi and xbai to create sites for ligation . both mir rnai and the target vectors were linearised using eagi and xhoi ( mirna vector ) and bsshii ( egfp sensor vector ) . the digested products were gel - purified using minelute gel extraction kit ( qiagen ) . in vitro transcription was carried out using the mmessage mmachine kit ( ambion , inc ) , and transcribed mrnas were purified using the licl precipitation method ( according to manufacturer 's instructions ) . rna quality was determined by gel electrophoresis and quantified using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , inc ) . 1 nl of solution containing 300 pg of mirna and 50 pg of egfp sensor mrna was microinjected into 1 - 2-cell stage embryos ( or 300 pg of scrambled sequence mirna , which was designed not to target any known zebrafish transcript , with 50 pg of egpf sensor mrna ) . 24 hours post - fertilization ( hpf ) embryos were observed and imaged using the zeiss axiovert s100 microscope . all images were taken from nikon eclipse ti inverted microscope . to quantify the raw fluorescence output , each group of embryos were loaded into a 96-well optical bottom plates in triplicate and the fluorescence was quantified using an envision multilabel microplate reader ( perkinelmer , inc ) . the settings were as follows : 485 nm excitation filter and 535 nm emission filter with a constant incubation temperature of 28c . the raw fluorescence was averaged and the difference in fluorescence was compared to determine the efficiency of each mirna . the readings were then normalized against the negative control embryos and expressed as a percentage . the mechanism behind mirna - mediated gene knockdown is still widely discussed ( mechanisms ranging from translation stalling to mrna cleavage ) , so it was considered prudent to design several mirnas that targeted different regions in the two genes of interest . to mediate gene - specific knockdown in zebrafish , four different mirnas were designed against each of the two zebrafish kcnh2 genes ( zerg-2 and zerg-3 ) . each target site had a five - star ranking based on the ranking system using the invitrogen block - it rnai designer , which ensured that the designed mirnas would form the most effective rnai molecules . at least one of the mirna targets was located within the 3 untranslated region ( 3 utr ) , while the others were located at various locations in the open reading frame ( orf ; figure 1(a ) ) . dong et al . have shown that mirna target sites located at the 3 utr are more effective at causing gene knockdown compared to mirna sites in the middle of an orf , and our own laboratory has shown that it is also the case with the block - it system ( data not shown ) . it has been suggested that this bias in efficiency is due to the translational machinery displacing bound mirnas in the orf regions as the machinery moves from the cap - binding complex to the orf [ 7 , 16 ] . despite this , it has also been found that many mirna target sites are found within orfs . therefore , it was thought that gene expression knockdown could be maximized by targeting four different locations within each transcript . the use of mirna in mediating gene knockdown in zebrafish has been successfully achieved by dong et al . here we have used a commercially available mirna backbone that is based on the endogenous mouse mir-155 , which has previously been shown to be effective in causing gene knockdown in mammalian cell lines . despite the fact that dong et al . found the efficiency of the zebrafish orthologue of mir-155 to be less than mir-30e , the data did not suggest that the mouse mir-155 backbone would not be effective in causing gene knockdown in zebrafish . it was decided to validate the block - it mirna system in the zebrafish and also to test the effectiveness of the custom - designed mirnas by using a transient in vivo sensor assay . if the custom - designed mirnas effectively caused knockdown in the transient in vivo sensor assay , then the mirnas could be stably incorporated into the zebrafish genome to create transgenic fish lines . the transient in vivo sensor assay involved cloning the putative mirna target sites into the 3 utr of a reporter / sensor gene [ 9 , 18 , 19 ] ( figure 1(a ) ) . each mirna target was inserted downstream of an egfp reporter gene ( egfp sensor ) , and both the mirna and their respective sensor were coinjected into one - cell stage embryos . the duration of this assay was restricted by the half - life of the capped mirna transcripts , and the embryos were screened for egfp expression at 24 hours post - fertilization ( hpf ) . at this stage , egfp and dsred expression could be detected and the effect of the mirnas assessed . in the event of targeted knockdown , egfp fluorescence as the mirna target site was linked to egfp , the emgfp reporter gene in the block - it expression vector was replaced with the dsred express reporter gene . the assessment of the assay was based solely on the level of egfp expression produced by the injected embryos , and any phenotypic outcomes ( i.e. , heart rate , electrocardiogram ) were not scored . all eight mirnas were able to decrease egfp expression ( figures 2 and 3 ) . zerg-2 mirna 4 caused the greatest level of egfp expression knockdown ( approximately 88% ) with zerg-2 mirna 1 and 2 causing approximately 6973% egfp knockdown ( figure 2 ) . zerg3 mirna 3 caused the greatest level of egfp knockdown ( approximately 98% ) with zerg3 mirna 1 and 4 causing approximately 7790% knockdown ( figure 3 ) ; the extent of knockdown caused by zerg-3 mirna 2 was not statistically significant . the results from the in vivo sensor assay demonstrate that the block - it mirna backbone is functional in zebrafish and the custom - designed mirna is capable of causing gene knockdown as assessed by the in vivo sensor system . one of the main issues in using rnai knockdown technology is the possibility of causing off - target gene knockdown . in zebrafish , this has proven to be one of the main downfalls of using rnai as it has been found that the mirna is vital for early development and the introduction of exogenous mirna might cause competition for mirna processing machinery [ 9 , 10 ] . this in turn leads to a decrease in endogenous mirna which is needed to clear maternal mrna during development . zhao et al . have reported phenotypes that are associated with toxic effects from the use of rnai in zebrafish , which included the loss of the midbrain - hindbrain boundary in sirna - injected embryos . in the current study there has been no evidence of the loss of the midbrain - hindbrain boundary ; however , it can not be ruled out that there is tissue - specific toxicity that is not readily apparent ( figure 1(c ) ) . the transient in vivo egfp sensor assay has shown that the block - it is functional in the zebrafish system and also that the designed mirnas are capable of causing knockdown at the protein level . these pilot data lay the foundation for constructing transgenic zebrafish expressing the mirnas used here in order to model lqts .

many enzymes are activated by specific binding of one or more monovalent cations ( mvcs ) ; these either contribute directly to the catalysis at the active site or indirectly modulate the activity by binding to an allosteric site . potassium and sodium ions are the most common activating mvcs , with a strong correlation between mvc preference and intracellular ( k ) or extracellular ( na ) location of the enzyme . examples of intracellular , potassium - activated enzymes are the molecular chaperones hsp70 and groel ( k binding at the active sites is crucial for the atp - dependent reaction cycles ) , ribokinase ( k binding near but not at the active site activates the enzyme ) , and the hydrolase histone deacetylase 8 ( hdac8 ; binding to two k binding sites affects activity ) . it is generally found that enzymes that are activated by the binding of k ( ionic radius 1.33 ) are either not activated or are activated to a lesser extent by na ( ionic radius 0.97 ) , and vice versa . however , enzymes that are activated by k can often be activated by an mvc of similar ionic radius , such as rb ( 1.48 ) or nh4 ( 1.44 ) . in some cases it has even been possible to engineer a potassium - independent enzyme by introducing a lysine residue in such a way that its terminal r - nh3 occupies the binding site , thereby taking the place of the regulatory potassium ion . using nh4 as a proxy for k offers the opportunity to characterise potassium binding sites by nmr spectroscopy , as isotopically labelled nh4 is readily available and the use of nh4 as a k mimic has been reported previously for the characterisation of potassium binding sites of dna quadruplexes by nmr spectroscopy . however , the previously investigated dna quadruplexes are relatively small compared to many enzymes , and quadruplexes also exhibit highly symmetric mvc binding sites . it is therefore not immediately obvious that the approach of using nh4 to probe potassium binding sites will also be applicable to larger , potassium - dependent enzymes . herein we show that nh4 can be used to probe k binding sites in medium - sized enzymes ( > 40 kda ) by nmr spectroscopy . the technique is facilitated by local reorientation and cross - correlated relaxations of the symmetric nh4 molecule within its binding site ; this results in line - narrowing of the n and h resonances , thereby improving the sensitivity of heteronuclear nmr experiments . in contrast to other experimental approaches used to probe mvc binding sites in proteins , nmr measurements of nh4 have distinct advantages : 1 ) these probe the binding of the mvc directly , not indirectly ( e.g. , by enzyme activity or stability ) ; 2 ) they have a high spatial resolution ( in contrast to , for example , fluorescence spectroscopy ) ; 3 ) they cause relatively little perturbation ( unlike site - directed mutagenesis - based experiments ) ; and 4 ) they are suitable for monitoring weak binding events ( up to the high mm range ) , where other methods to study mvc binding ( e.g. , isothermal titration calorimetry ) have limitations . the critical point is that the binding of nh4 to an mvc binding site can decrease the exchange of the ammonium protons with the bulk solvent to such an extent that these become observable even under conditions where free nh4 in solution is undetectable in proton nitrogen - correlated experiments because of rapid exchange with the solvent . for the hdac8 enzyme , we first show that ammonium and potassium regulate the activity similarly , thus supporting the use of nh4 as a proxy for k. using n - edited nmr methods , we probed the binding of nh4 to one of the potassium binding sites of hdac8 and to two potassium binding sites of the bacterial hsp70 homologue dnak . furthermore , we developed a strategy for mapping potassium binding sites within protein structures that does not require a protein chemical shift assignment . the strategy relies on selective isotope labelling , and utilises through - space noe measurements between nh4 protons and residue - specific side - chain protons . in addition to the catalytically important zn ion at the active site , crystal structures of hdac8 reveal two mvc binding sites ( figure 1 a c ) , as has been observed in most structurally characterised hdacs and hdac - related deacetylases . biochemical characterisation and computational approaches have highlighted the importance of potassium binding for activity and potentially even for regulation of this enzyme . specifically , the effect of kcl concentration on hdac8 activity was investigated by fierke and co - workers using hdac8 with two different divalent metal ions ( co and zn ) bound at the active site . these experiments revealed a bell - shaped activity profile , with an activating effect at lower concentrations of k and an inactivating effect as the concentration was increased . the resulting activation ( k1/2,act ) and inhibition ( k1/2,inhib ) constants , which describe the apparent dissociation constants for an activating and an inhibiting binding site , respectively , were k1/2,act=14 mm and k1/2,inhib=130 mm for zn - bound hdac8 . a ) crystal structure of hdac8 ( pdb i d : 2v5w ) with the active - site zn ( black sphere ) and the two k ions ( purple spheres ) . b ) residues within approximately 10 of the two potassium binding sites in hdac8 . c ) two - dimensional representations of the two binding sites ( generated with ligplot ) . concentrations of k ( red ) , nh4 ( green ) and choline ( blue ) were in 25 mm trishcl at ph 8.0 . the two - site binding model described previously was used to fit the activity profile for kcl and nh4cl according to the equation v = v1/(1+k1/2,act/[mvc]+[mvc]/k1/2,inh ) , where k1/2,act and k1/2,inh are the apparent binding affinities ( dissociation constants ) for activation and inhibition , v is the relative kcat / km value , v1 is the kcat / km value of the fully activated state and [ mvc ] is the concentration of the respective monovalent cation . kcat / km of the fully inhibited state was set as zero ; data are normalised such that v1 ( k)=1.0 . k1/2,act ( k ) : 4526 mm , k1/2,inh ( k ) : 610530 mm , k1/2,act ( nh4 ) : 258 mm , k1/2,inh ( nh4 ) : 26001700 mm . although the errors for the affinities of the inhibitory site are large , these data agree with a bell - shaped activity profile for k and nh4 . in order to test whether nh4 is a good mimic of k for enzyme activation , we measured the deacetylase activity of hdac8 as a function of nh4 , k and choline concentration . choline was used as a negative control to detect any possible nonspecific influence of ionic strength on hdac8 activity . for k and nh4 the activity profiles were bell - shaped ( figure 1 d ) , similar to those reported previously , whereas addition of choline had only a very small effect on hdac8 activity . nh4 activation of hdac8 was 60 % of that of k , with comparable k1/2,act ( compare the curves in figure 1 d ) , thus suggesting that nh4 effectively mimics k in modulating the activity of hdac8 . next , we tested whether it was possible to observe binding of nh4 to hdac8 by nmr . as expected , bulk nh4 was not observed in n - edited , proton - detected 1d nmr spectra in buffer containing 200 mm nh4 at ph 8.0 ( figure 2 a ) because of rapid exchange of the ammonium protons with the bulk solvent . the n chemical shift of the free ammonium was therefore determined by direct n - detection to be 20.5 ppm ( figure 2 a , inset ) . addition of [ n]hdac8 , however , resulted in a distinct peak in the n - edited experiment , indicative of hdac8-bound nh4 with a proton chemical shift of 7.1 ppm ( figure 2 b ) . a n chemical shift of 25.5 ppm was subsequently determined by recording a 2d h , n hsqc spectrum ( figure 2 c ) . a ) n - edited 1d proton spectrum of the buffer ( 25 mm trishcl ph 8.0 , 200 mm nh4cl , 0.5 mm tcep ) and direct n - detected 1d spectrum ( inset ) . b ) as above but after addition of 100 m [ n]hdac8 to the sample . d ) titration of nh4 ( ) with the observed binding site in hdac8 ( using n - edited 1d proton spectra ) . the intensities were obtained by integration of the ammonium peak , and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration . at 100 mm nh4 , addition of 100 mm kcl ( ) led to approximately 50 % reduction of the signal , thus indicating that k and nh4 bind to the same site with similar affinities . a hyperbolic binding equation y = ax/(kd+x ) ( ) was fitted to the normalised peak intensities to yield kd=(138 ) mm . the n line - width of nh4 at 298 k bound to hdac8 ( 42 kda ) would be expected to be 45 hz , with the n nucleus relaxed principally by dipolar interactions with its four protons , with a rotational correlation time of approximately 25 ns . the observation of a peak in the n - edited spectra , shifted from the position for free ammonium , shows that binding of the nh4 ion to hdac8 is sufficiently strong to significantly limit exchange of the ammonium protons with the bulk solvent . although cross - correlated relaxations within the nh4 molecule cause line - narrowing , the experimental line - width ( 25 hz ) , which also includes contributions from the residual proton exchange and proton r1 , indicates that the ammonium ion is undergoing local reorientation within the binding site . the spectral parameters obtained from the peak in the n - edited spectrum report directly on nh4 in the mvc binding site , with the intensity of this peak directly related to the amount of nh4 bound to hdac8 , thus allowing an estimation of the dissociation constant . proton - detected , n - edited 1d spectra were recorded for a titration of hdac8 with nh4 , and the resulting nh4 peak integrals were used to determine the dissociation constant for binding of nh4 to hdac8 ( kd=(138 ) mm ; figure 2 d ) . addition of 100 mm kcl to a sample containing 100 mm nh4 led to a reduction ( 50 % ) in the nh4 integral , thus indicating both that nh4 and k compete for the same binding site and that they bind to this site with similar affinities ( figure 2 d ) . we therefore conclude that nh4 binds to at least one of the two k binding sites in hdac8 . binding of nh4 to this site decreased proton - exchange with the solvent to such an extent that it was observable in h , n - correlated experiments , under conditions where bulk nh4 can not be detected . the change in n chemical shift upon binding ( =5 ppm ) indicates a significant change in the chemical environment , and given the different natures of the two potassium binding sites in hdac8 ( see below and figure 1 c ) , it is unlikely that both the proton and n chemical shifts of the two hdac8-bound ammonium ions would be so similar as to lead to overlap of the corresponding peaks in the h , n - correlated spectra . we propose instead that hdac8 binds a second nh4as it binds a second k but that this second site does not protect the bound nh4 from solvent proton exchange sufficiently to be detectable in our experiments . two potassium ions in the atp - binding domain ( figure 3 a ) have been identified to be crucial for the atpase cycle,[6b ] which in turn regulates the binding and release of substrate proteins . in a similar way to that for hdac8 , these potassium ions can be replaced by ammonium , thereby resulting in approximately half of the atpase activity observed with potassium.[6b ] based on our success in characterising the potassium binding sites of hdac8 with nh4 , we tested the general applicability of our approach by probing the potassium binding sites of the 41 kda atp - binding domain of dnak , a bacterial hsp70 homologue from thermus thermophilus . a ) crystal structure of the atpase domain of hsp70 ( pdb i d : 1hpm ) : purple spheres : k , green sphere : mg , yellow sticks : adp and pi . a ligplot analysis showing two - dimensional representations of the binding sites is shown in figure s2 . b ) h , n hsqc spectra and 1d projections ( above ) with the abd of bacterial hsp70 homologue dnak . red spectra : dnak - abd ( 100 m ) in 30 mm trishcl , 150 mm nh4cl , 5 mm mgcl2 and 1 mm dtt , ph 7.5 . blue spectra : the same sample after addition of 0.5 mm adp and 50 mm nh4h2po4 . note : because of the low ph of the nh4h2po4 stock solution , tris base was also added to achieve ph 7.5 , thus increasing the total tris concentration to 75 mm . unlike for the experiments with hdac8 , addition of nucleotide - free dnak atp - binding domain ( dnak - abd ) to a nh4cl - containing buffer gave only a very weak signal in n - edited proton - detected and h , n correlation spectra . the signal was barely above the noise level ( figure 3 b , figure s1b in the supporting information ) , thus suggesting either that nh4 binds only weakly to nucleotide - free dnak - abd , or that the binding sites are solvent - exposed and do not shield the ammonium protons from exchange with bulk solvent ( figure s1 ) . interestingly , addition of adp and pi resulted in the appearance of two clear peaks ( figure 3 b ) . the n chemical shifts of both these peaks ( 19.3 and 21.9 ppm ) are different from the n chemical shift that we observed for free ammonium in this buffer when using a direct - detect n experiment ( 20.6 ppm ) . our interpretation of these results is that binding of adp and pi protects the ammonium protons from solvent exchange , by themselves blocking solvent molecules and/or by inducing a conformational change that decreases solvent accessibility . both effects agree well with molecular models of nucleotide binding to hsp70 , and it is likely that a combination of both leads to the increased protection of ammonium ions.[2 , 14 ] to verify that the two resonances observed in figure 3 b report on two different sites , we recorded n - edited noesy experiments ( figure s3 ) . in the resulting n - edited noesy spectrum , the two resonances gave rise to very different sets of cross - peaks , thus confirming that the resonances report on two different sites in the protein , in agreement with the observation of two different potassium binding sites in the crystal structures of hsp70 chaperones . the ammonium protons bound in a protein environment are close to protons of nearby amino acid side chains ; this proximity serves as a potential means for mapping the mvc binding site within the protein structure . the key restraints used for protein structure determinations are nuclear overhauser enhancements ( noes ) between protons , where the intensity of the observed noe is approximately proportional to r ( r is the distance between the two interacting protons ) . similarly , noes between bound ammonium protons and protein side - chain protons report on the location of the mvc binding site . a n - edited noesy experiment was performed to identify side chains in the vicinity of the observed mvc binding site of hdac8 . the resulting spectrum shows a number of noe crosspeaks between the hdac8-bound ammonium and the aliphatic side chains , as well as between the ammonium and the amide protons ( figure 4 ) . for proteins where a full chemical shift assignment is available , the binding site is readily mapped from the chemical shifts of the noe cross - peaks . however , for hdac8 , as well as many other enzymes of interest , a full chemical shift assignment is not available , so alternative approaches must be used . site - directed mutagenesis and mutation of k - binding side chains might be an initial and intuitive approach . however , such an approach fails for hdac8 , as the potassium is mostly coordinated by the protein backbone , and also because mutation of side chains that do participate in the coordination of k ( s199 and d176 , see figure 1 c ) resulted in insufficient purified protein for further nmr characterisation . generally , identification of mvc binding sites by sitedirected mutagenesis could prove difficult , as such sites are either close to the active site or in allosteric regions of the enzyme . we therefore prefer a non - invasive approach ( described below ) that is based on residue - specific isotope labelling . n - edited noesy experiments with different isotope labelling schemes used to map the k / nh4 binding site of hdac8 ( pdb i d : 2v5w ) . shaped n pulses at 25 ppm ( see the experimental section ) ensured selection of ammonium - specific cross - peaks . the fully protonated hdac8 sample ( left , black ) gives rise to many cross - peaks that arise from the side chain and backbone protons of hdac8 in the vicinity of the ammonium binding site . expressing hdac8 in a medium supplemented with protonated histidine in an otherwise deuterated background ( middle , blue ) reduces the number of crosspeaks significantly . the only observed cross - peaks in the aliphatic region are those from non - exchangeable histidine side - chain protons . a control experiment shows hdac8 expressed in deuterated medium without labelled histidine supplement ( right , red ) . only solvent - exchangeable protons give rise to cross - peaks . the graph on the right shows the distributions of expected chemical shifts for the exchangeable protons in hdac8 ( red ) and all histidine protons ( blue ) based on bmrb statistics . the hdac8 structures above the spectra ( modelled from pdb i d : 2v5w ) highlight protons introduced by the corresponding labelling scheme in blue ( histidine ) , red ( exchangeable ) and grey ( all ) . this set of experiments , in combination with the distribution of histidine residues , localises the observed ammonium binding site in the hdac8 structure . inspection of the two potassium binding sites in the hdac8 crystal structure revealed different distributions of amino acids in the immediate vicinities of the two binding sites . whereas the binding site close to the active site ( here termed site 1 ) is surrounded by histidines ( figure 1 b and figure 4 ) and leucines , these amino acids are not found in the vicinity of site 2 . we therefore produced hdac8 that was selectively protonated at histidine side chains , by adding h , n - labelled histidine in an otherwise fully h , n background . histidine is at the end of a biosynthetic pathway and therefore little scrambling with other amino acids is expected . this is confirmed by the fact that we observed a maximum of 11 cross - peaks in the amide region of a h , n hsqc spectrum of this sample , compared to 13 histidines in hdac8 ( figure s4 ) . although a specifically histidine - labelled sample has aliphatic protons only in the histidine side chains , additional protons are present at exchangeable sites , such as unprotected backbone amides . to account for exchangeable protons introduced by the h2o buffers , we expressed and purified a sample ( u - n , h ) in the same deuterated background and without the addition of protonated histidine . by comparing the results of the subsequent noesy experiments ( figure 4 ) this demonstrates that the observed nh4 binds to site 1 , and thus we were able to assign the observed k / nh4 binding site in hdac8 even in the absence of a full chemical shift assignment . in general , identification and assignment of mvc binding sites is possible provided that 1 ) the corresponding nh4 is sufficiently protected from solvent exchange that a signal is observed in n - edited nmr spectra , and 2 ) the mvc binding sites of interest have different local distributions of amino acids . in addition to the catalytically important zn ion at the active site , crystal structures of hdac8 reveal two mvc binding sites ( figure 1 a c ) , as has been observed in most structurally characterised hdacs and hdac - related deacetylases . biochemical characterisation and computational approaches have highlighted the importance of potassium binding for activity and potentially even for regulation of this enzyme . specifically , the effect of kcl concentration on hdac8 activity was investigated by fierke and co - workers using hdac8 with two different divalent metal ions ( co and zn ) bound at the active site . these experiments revealed a bell - shaped activity profile , with an activating effect at lower concentrations of k and an inactivating effect as the concentration was increased . the resulting activation ( k1/2,act ) and inhibition ( k1/2,inhib ) constants , which describe the apparent dissociation constants for an activating and an inhibiting binding site , respectively , were k1/2,act=14 mm and k1/2,inhib=130 mm for zn - bound hdac8 . a ) crystal structure of hdac8 ( pdb i d : 2v5w ) with the active - site zn ( black sphere ) and the two k ions ( purple spheres ) . b ) residues within approximately 10 of the two potassium binding sites in hdac8 . c ) two - dimensional representations of the two binding sites ( generated with ligplot ) . concentrations of k ( red ) , nh4 ( green ) and choline ( blue ) were in 25 mm trishcl at ph 8.0 . the two - site binding model described previously was used to fit the activity profile for kcl and nh4cl according to the equation v = v1/(1+k1/2,act/[mvc]+[mvc]/k1/2,inh ) , where k1/2,act and k1/2,inh are the apparent binding affinities ( dissociation constants ) for activation and inhibition , v is the relative kcat / km value , v1 is the kcat / km value of the fully activated state and [ mvc ] is the concentration of the respective monovalent cation . kcat / km of the fully inhibited state was set as zero ; data are normalised such that v1 ( k)=1.0 . k1/2,act ( k ) : 4526 mm , k1/2,inh ( k ) : 610530 mm , k1/2,act ( nh4 ) : 258 mm , k1/2,inh ( nh4 ) : 26001700 mm . although the errors for the affinities of the inhibitory site are large , these data agree with a bell - shaped activity profile for k and nh4 . in order to test whether nh4 is a good mimic of k for enzyme activation , we measured the deacetylase activity of hdac8 as a function of nh4 , k and choline concentration . choline was used as a negative control to detect any possible nonspecific influence of ionic strength on hdac8 activity . for k and nh4 the activity profiles were bell - shaped ( figure 1 d ) , similar to those reported previously , whereas addition of choline had only a very small effect on hdac8 activity . nh4 activation of hdac8 was 60 % of that of k , with comparable k1/2,act ( compare the curves in figure 1 d ) , thus suggesting that nh4 effectively mimics k in modulating the activity of hdac8 . next , we tested whether it was possible to observe binding of nh4 to hdac8 by nmr . as expected , bulk nh4 was not observed in n - edited , proton - detected 1d nmr spectra in buffer containing 200 mm nh4 at ph 8.0 ( figure 2 a ) because of rapid exchange of the ammonium protons with the bulk solvent . the n chemical shift of the free ammonium was therefore determined by direct n - detection to be 20.5 ppm ( figure 2 a , inset ) . addition of [ n]hdac8 , however , resulted in a distinct peak in the n - edited experiment , indicative of hdac8-bound nh4 with a proton chemical shift of 7.1 ppm ( figure 2 b ) . a n chemical shift of 25.5 ppm was subsequently determined by recording a 2d h , n hsqc spectrum ( figure 2 c ) . a ) n - edited 1d proton spectrum of the buffer ( 25 mm trishcl ph 8.0 , 200 mm nh4cl , 0.5 mm tcep ) and direct n - detected 1d spectrum ( inset ) . b ) as above but after addition of 100 m [ n]hdac8 to the sample . d ) titration of nh4 ( ) with the observed binding site in hdac8 ( using n - edited 1d proton spectra ) . the intensities were obtained by integration of the ammonium peak , and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration . at 100 mm nh4 , addition of 100 mm kcl ( ) led to approximately 50 % reduction of the signal , thus indicating that k and nh4 bind to the same site with similar affinities . a hyperbolic binding equation y = ax/(kd+x ) ( ) the n line - width of nh4 at 298 k bound to hdac8 ( 42 kda ) would be expected to be 45 hz , with the n nucleus relaxed principally by dipolar interactions with its four protons , with a rotational correlation time of approximately 25 ns . the observation of a peak in the n - edited spectra , shifted from the position for free ammonium , shows that binding of the nh4 ion to hdac8 is sufficiently strong to significantly limit exchange of the ammonium protons with the bulk solvent . although cross - correlated relaxations within the nh4 molecule cause line - narrowing , the experimental line - width ( 25 hz ) , which also includes contributions from the residual proton exchange and proton r1 , indicates that the ammonium ion is undergoing local reorientation within the binding site . the spectral parameters obtained from the peak in the n - edited spectrum report directly on nh4 in the mvc binding site , with the intensity of this peak directly related to the amount of nh4 bound to hdac8 , thus allowing an estimation of the dissociation constant . proton - detected , n - edited 1d spectra were recorded for a titration of hdac8 with nh4 , and the resulting nh4 peak integrals were used to determine the dissociation constant for binding of nh4 to hdac8 ( kd=(138 ) mm ; figure 2 d ) . addition of 100 mm kcl to a sample containing 100 mm nh4 led to a reduction ( 50 % ) in the nh4 integral , thus indicating both that nh4 and k compete for the same binding site and that they bind to this site with similar affinities ( figure 2 d ) . we therefore conclude that nh4 binds to at least one of the two k binding sites in hdac8 . binding of nh4 to this site decreased proton - exchange with the solvent to such an extent that it was observable in h , n - correlated experiments , under conditions where bulk nh4 can not be detected . the change in n chemical shift upon binding ( =5 ppm ) indicates a significant change in the chemical environment , and given the different natures of the two potassium binding sites in hdac8 ( see below and figure 1 c ) , it is unlikely that both the proton and n chemical shifts of the two hdac8-bound ammonium ions would be so similar as to lead to overlap of the corresponding peaks in the h , n - correlated spectra . we propose instead that hdac8 binds a second nh4as it binds a second k but that this second site does not protect the bound nh4 from solvent proton exchange sufficiently to be detectable in our experiments . two potassium ions in the atp - binding domain ( figure 3 a ) have been identified to be crucial for the atpase cycle,[6b ] which in turn regulates the binding and release of substrate proteins . in a similar way to that for hdac8 , these potassium ions can be replaced by ammonium , thereby resulting in approximately half of the atpase activity observed with potassium.[6b ] based on our success in characterising the potassium binding sites of hdac8 with nh4 , we tested the general applicability of our approach by probing the potassium binding sites of the 41 kda atp - binding domain of dnak , a bacterial hsp70 homologue from thermus thermophilus . a ) crystal structure of the atpase domain of hsp70 ( pdb i d : 1hpm ) : purple spheres : k , green sphere : mg , yellow sticks : adp and pi . a ligplot analysis showing two - dimensional representations of the binding sites is shown in figure s2 . b ) h , n hsqc spectra and 1d projections ( above ) with the abd of bacterial hsp70 homologue dnak . red spectra : dnak - abd ( 100 m ) in 30 mm trishcl , 150 mm nh4cl , 5 mm mgcl2 and 1 mm dtt , ph 7.5 . blue spectra : the same sample after addition of 0.5 mm adp and 50 mm nh4h2po4 . note : because of the low ph of the nh4h2po4 stock solution , tris base was also added to achieve ph 7.5 , thus increasing the total tris concentration to 75 mm . unlike for the experiments with hdac8 , addition of nucleotide - free dnak atp - binding domain ( dnak - abd ) to a nh4cl - containing buffer gave only a very weak signal in n - edited proton - detected and h , n correlation spectra . the signal was barely above the noise level ( figure 3 b , figure s1b in the supporting information ) , thus suggesting either that nh4 binds only weakly to nucleotide - free dnak - abd , or that the binding sites are solvent - exposed and do not shield the ammonium protons from exchange with bulk solvent ( figure s1 ) . interestingly , addition of adp and pi resulted in the appearance of two clear peaks ( figure 3 b ) . the n chemical shifts of both these peaks ( 19.3 and 21.9 ppm ) are different from the n chemical shift that we observed for free ammonium in this buffer when using a direct - detect n experiment ( 20.6 ppm ) . our interpretation of these results is that binding of adp and pi protects the ammonium protons from solvent exchange , by themselves blocking solvent molecules and/or by inducing a conformational change that decreases solvent accessibility . both effects agree well with molecular models of nucleotide binding to hsp70 , and it is likely that a combination of both leads to the increased protection of ammonium ions.[2 , 14 ] to verify that the two resonances observed in figure 3 b report on two different sites , we recorded n - edited noesy experiments ( figure s3 ) . in the resulting n - edited noesy spectrum , the two resonances gave rise to very different sets of cross - peaks , thus confirming that the resonances report on two different sites in the protein , in agreement with the observation of two different potassium binding sites in the crystal structures of hsp70 chaperones . the ammonium protons bound in a protein environment are close to protons of nearby amino acid side chains ; this proximity serves as a potential means for mapping the mvc binding site within the protein structure . the key restraints used for protein structure determinations are nuclear overhauser enhancements ( noes ) between protons , where the intensity of the observed noe is approximately proportional to r ( r is the distance between the two interacting protons ) . similarly , noes between bound ammonium protons and protein side - chain protons report on the location of the mvc binding site . a n - edited noesy experiment was performed to identify side chains in the vicinity of the observed mvc binding site of hdac8 . the resulting spectrum shows a number of noe crosspeaks between the hdac8-bound ammonium and the aliphatic side chains , as well as between the ammonium and the amide protons ( figure 4 ) . for proteins where a full chemical shift assignment is available , the binding site is readily mapped from the chemical shifts of the noe cross - peaks . however , for hdac8 , as well as many other enzymes of interest , a full chemical shift assignment is not available , so alternative approaches must be used . site - directed mutagenesis and mutation of k - binding side chains might be an initial and intuitive approach . however , such an approach fails for hdac8 , as the potassium is mostly coordinated by the protein backbone , and also because mutation of side chains that do participate in the coordination of k ( s199 and d176 , see figure 1 c ) resulted in insufficient purified protein for further nmr characterisation . generally , identification of mvc binding sites by sitedirected mutagenesis could prove difficult , as such sites are either close to the active site or in allosteric regions of the enzyme . we therefore prefer a non - invasive approach ( described below ) that is based on residue - specific isotope labelling . n - edited noesy experiments with different isotope labelling schemes used to map the k / nh4 binding site of hdac8 ( pdb i d : 2v5w ) . shaped n pulses at 25 ppm ( see the experimental section ) ensured selection of ammonium - specific cross - peaks . the fully protonated hdac8 sample ( left , black ) gives rise to many cross - peaks that arise from the side chain and backbone protons of hdac8 in the vicinity of the ammonium binding site . expressing hdac8 in a medium supplemented with protonated histidine in an otherwise deuterated background ( middle , blue ) reduces the number of crosspeaks significantly . the only observed cross - peaks in the aliphatic region are those from non - exchangeable histidine side - chain protons . a control experiment shows hdac8 expressed in deuterated medium without labelled histidine supplement ( right , red ) . the graph on the right shows the distributions of expected chemical shifts for the exchangeable protons in hdac8 ( red ) and all histidine protons ( blue ) based on bmrb statistics . the hdac8 structures above the spectra ( modelled from pdb i d : 2v5w ) highlight protons introduced by the corresponding labelling scheme in blue ( histidine ) , red ( exchangeable ) and grey ( all ) . this set of experiments , in combination with the distribution of histidine residues , localises the observed ammonium binding site in the hdac8 structure . inspection of the two potassium binding sites in the hdac8 crystal structure revealed different distributions of amino acids in the immediate vicinities of the two binding sites . whereas the binding site close to the active site ( here termed site 1 ) is surrounded by histidines ( figure 1 b and figure 4 ) and leucines , these amino acids are not found in the vicinity of site 2 . we therefore produced hdac8 that was selectively protonated at histidine side chains , by adding h , n - labelled histidine in an otherwise fully h , n background . histidine is at the end of a biosynthetic pathway and therefore little scrambling with other amino acids is expected . this is confirmed by the fact that we observed a maximum of 11 cross - peaks in the amide region of a h , n hsqc spectrum of this sample , compared to 13 histidines in hdac8 ( figure s4 ) . although a specifically histidine - labelled sample has aliphatic protons only in the histidine side chains , additional protons are present at exchangeable sites , such as unprotected backbone amides . to account for exchangeable protons introduced by the h2o buffers , we expressed and purified a sample ( u - n , h ) in the same deuterated background and without the addition of protonated histidine . by comparing the results of the subsequent noesy experiments ( figure 4 ) this demonstrates that the observed nh4 binds to site 1 , and thus we were able to assign the observed k / nh4 binding site in hdac8 even in the absence of a full chemical shift assignment . in general , identification and assignment of mvc binding sites is possible provided that 1 ) the corresponding nh4 is sufficiently protected from solvent exchange that a signal is observed in n - edited nmr spectra , and 2 ) the mvc binding sites of interest have different local distributions of amino acids . we successfully used nh4 to characterise a k binding site in the 42 kda enzyme hdac8 by nmr spectroscopy . by using nh4 instead of k , we could employ noesy experiments in combination with selective amino acid labelling of the protein to probe the immediate vicinity of the binding site and map it onto the hdac8 structure . as hdac8 has two binding sites for potassium and these are connected by one -strand , one would expect perturbations of these sites to have mutual effects on each other . chemical shift perturbations upon titrating with k or assays based on site - directed mutagenesis could therefore lead to ambiguous results . the assignment of the sites by nh4-detected noesy spectra does not suffer from such ambiguity . secondly , this strategy does not rely on an available chemical shift assignment for the protein , and is generally applicable provided a crystal structure is known and the amino acid compositions of the binding sites in question are different . and thirdly , most of the experiments presented here do not require isotopically labelled protein samples and are therefore relatively cost - effective compared to many other experiments involving nmr spectroscopy . the bell - shaped activity profile and the presence of two mvc binding sites in crystal structures of hdac8 led to a model of one activating and one deactivating k binding site . combination of this model with site - directed mutagenesis experiments has led to the proposition that occupation of the k binding site close to the active site ( site 1 ) is deactivating , whereas occupation of the second site ( site 2 ) is activating . our results , however , suggest that site 1 is occupied even at relatively low concentrations of k ( nh4 kd 13 mm ) , that is , in the concentration range of enzyme activation . recently , we were able to show that binding of potassium has an effect on the structural integrity of hdac8 in a similar concentration range ( k kd42 mm ) as probed by an arginine side chain close to site 2 ( 12 from site 2 , 21 from site 1 ) . based on these findings , we therefore propose that both potassium binding sites are important for the stabilisation and activation of the enzyme , and we speculate that the deactivating effect at higher potassium concentrations could result from secondary effects , such as that of high ionic strength on km or inhibition by high concentrations of chloride.[6a ] in a second example , we monitored ammonium binding to the 41 kda atp binding domain of the hsp70 chaperone dnak . after addition of adp and pi , we observed two crosspeaks in the h , n hsqc spectrum , in agreement with the two potassium ions observed in the crystal structure of hsp70 with adp and pi . these results are in agreement with previous findings of a closed and less solvent - accessible active site in response to nucleotide binding.[14a ] overall , we detected three potassium binding sites in two proteins by using nh4cl and n - edited nmr spectra . the observed chemical shifts range from 6.8 to 7.5 ppm in the h dimension and from 19 to 25.5 ppm in the n dimension . the level of dispersion suggests that this approach can be used to observe and identify multiple potassium binding sites in even large proteins , where the relaxation properties might be improved by cross - correlated relaxations and local reorientation of the nh4 ion within the binding site . the methodology presented above could be further exploited by combination with the powerful array of existing nmr techniques to describe the kinetic and dynamic aspects of mvc binding . for example , employing longitudinal exchange - type experiments would allow measurement of the exchange on the millisecond - to - second timescale between two or more bound mvcs , or the exchange of bound mvcs with those from the bulk solvent , as has been shown in the context of dna quadruplex structures.[8b ] the latter experiment depends on detection of bulk ammonium and thus requires a low ph ( e.g. , ph 5.0),[8b ] which might prove a limitation for many proteins . it would also be intriguing to shed light on the relative mobility of ammonium ions in the binding sites of enzymes , and n , h - relaxation - based experiments could give access to such information in the future . given this potential , correlating characteristics of nh4 binding with functional enzymatic states , as exemplified here , will ultimately lead to a better understanding of the role of monovalent cations in proteins and enzymes . expression and purification of hdac8 : recombinant hdac8 was expressed and purified as described previously . isotope - labelled samples were prepared from culture grown in m9 minimal medium . for the u - n , h sample , the m9 medium was made with nh4cl ( 1 g l ) . for the u - n , h sample , the m9 medium was made up in 99 % d2o with nh4cl ( 1 g l ) and h7-glucose ( 2.5 g l ) . for the his - n , h / u - n , h sample , the m9 medium was made up in 99 % d2o with nh4cl ( 1 g l ) and h7-glucose ( 2.5 g l ) ; [ n , h]histidine ( 60 mg l ) was added to the culture 1 h prior to induction . expression was induced with zncl2 ( 0.2 mm ) and isopropyl--d - thiogalactopyranoside ( 1 mm ) , and growth continued overnight at 21 c before harvesting . recombinant dnak - abd ( residues 1381 of thermus thermophilus dnak ) was expressed in 2yt medium from bl21(de3 ) cells and purified essentially as described previously for dnak . the nucleotide content ( 2 % ) of the preparation was determined by hplc analysis . activity assay : hdac8 activity was measured by a modified fluorescence - based assay . the substrate analogue boc - lys(ac)-7-amino-4-methylcoumarin ( mal ) was synthesised according to hoffmann et al . and kindly provided by christopher matthews and charles marson ( ucl , london ) . the deacetylation reaction was performed in trishcl ( 25 mm , ph 8.0 ) with different concentrations of kcl , nh4cl and choline chloride . hdac8 ( 0.4 m ) was added to these buffers and incubated for at least 30 min at room temperature . it should be noted that this addition introduced kcl ( final concentration 1.5 mm ) from the enzyme stock . the reaction was started by adding substrate ( 200 m ) , and quenched after 30 min at 25 c by addition of an equal volume of developer solution ( trypsin ( 10 mg ml ) and trichostatin a ( 4 m ) in trishcl ( 50 mm ph 8.0 ) , nacl ( 137 mm ) , kcl ( 2.7 mm ) , mgcl2 ( 1 mm ) , peg ( 10 % , avg . 8000 da ) and bsa ( 1 mg ml ) ) . after incubation at room temperature for 30 min , the degree of relative deacetylation was quantified in a fluostar optima plate reader ( excitation : 380 nm , detection : 460 nm ; bmg labtech , aylesbury , uk ) . nmr experiments : unless indicated otherwise , samples for nmr experiments were prepared in trishcl ( 25 mm , ph 8.0 ) , with tcep ( 0.5 mm ) , nan3 ( 1 mm ) and nh4cl ( 200 mm ) . the spectra presented in figure 2 and 3 were measured with an external d2o reference insert ( wilmad coaxial insert z278513 ( sigma aldrich ) ) , in which case no d2o was added to the sample buffer . all n - edited proton 1d spectra and the direct - observed n 1d spectra were recorded at 298 k on a 500 mhz avance iii spectrometer ( bruker ) equipped with a room temperature , inverse , triple - resonance probe . the pulse sequence for the n - edited proton 1d spectra was based on the first increment of the fast n hsqc sequence . n - edited noesy spectra were recorded at 298 k on a 700 mhz avance iii spectrometer ( bruker ) equipped with a cryogenic inverse triple - resonance probe . the pulse sequence for the noesy spectra was based on a standard noesy / n hsqc sequence , with ammonium selectivity achieved by a shaped iburp1 nitrogen pulse ( 1714 s , centred at 25 ppm ) in the first inept of the n hsqc element . broadband n decoupling during the indirect proton chemical shift evolution period was achieved by using an adiabatic smoothed chirp pulse ( 1000 s , 30 khz sweep - width , centred at 100 ppm ) . data were processed and visualised by using topspin ( bruker ) , nmrdraw and sparky . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

dyadic contests for limited resources are common in animals ( huntingford and turner 1987 ; hardy and briffa 2013 ) . in these contests , asymmetrical fighting ability and/or resource holding potential ( rhp ; parker 1974 ) are among the most important factors that determine outcomes . since rhp is often determined by body or weapon size , contestants with a larger body or weapon size show a higher , whereas smaller or weaponless contestants show a lower , probability of winning ( maginnis 2006 ; fleming et al . 2007 ; arnott and elwood 2009 ) . such weaker contestants often pay a greater cost associated with aggression ( parker 1974 ) than stronger contestants ( searcy and nowicki 2005 ) , especially when contests escalate to more intense or costly phases . ( 1998 ) suggests that , in the cichlid fish tilapia zilli , male contestants incur injury and energy costs during escalated fights . although the contest escalation is costly for both stronger and weaker individuals , weaker contestants face a more severe condition . potentially weaker contestants therefore avoid contests by avoiding escalation or adopting a lower level of aggression ( e.g. , okada and miyatake 2010 ; yasuda et al . decapod crustaceans are one of the most studied taxa in terms of the functions of their morphological weapons in contest competition ( searcy and nowicki 2005 ) . most of these animals possess an enlarged ( i.e. , major ) cheliped , and this is a critically important predictor of the winner in dyadic contests ( emlen 2008 ; briffa 2013 ) . contestants often use their major cheliped as both a visual advertisement of their fighting ability and a substantial weapon ( e.g. , crane 1975 ; hughes 1996 ; sneddon et al . 1997 ; moore 2007 ) , and , in some species , having a larger major cheliped improves fighting success more than having a large body ( barki et al . if a contestant has lost its major cheliped , it has a lower probability of winning contests than an intact contestant ( smith 1992 ; juanes and smith 1995 ; daleo et al . 2009 ) , especially when the weaponless contestant initiates a contest with a resource - holding opponent ( abello et al . intruders without a major cheliped also engage in fewer contests in the hermit crab pagurus bernhardus ( neil 1985 ) and the fiddler crab uca annulipes ( booksmythe et al . such intruders might therefore avoid fruitless contests and minimize their potential fighting costs by abandoning contests at an early stage , instead of escalating . males of pagurus hermit crabs use an enlarged right ( i.e. , major ) cheliped as a weapon in contests for females during the reproductive season ( yasuda et al . males grasp the aperture of the gastropod shell occupied by a sexually mature female for several days ( imafuku 1986 ; elwood and neil 1992 ; goshima et al . 1998 ) , and male male contests over the female often occur between a solitary intruder and a guarding male . in contests between intact males , the outcomes are determined by major cheliped size rather than body size in p. middendorffii ( yasuda et al . males without a major cheliped , therefore , are expected to be less willing to escalate in male male contests . however , 2011 ) have reported that in p. nigrofascia , intruders without a major cheliped actively escalate the fight against intact guarding males even though the loss of the major cheliped clearly decreases the probability of winning in male contestants . since p. nigrofascia intruders typically escalate the contest whenever they encounter a guarding opponent ( e.g. , 100 % of the time ; suzuki et al . 2012 ; yasuda et al . 2015 ) , this species might not be a suitable choice to examine the factors affecting an intruder s decision about whether to give up or escalate a contest . further study using other pagurus species will be needed to clarify the relationship between an individual without a major cheliped and the decision to give up . in the present study , we examined whether loss of the major cheliped by males of p. minutus affected an intruder s decision to give up at an early stage ( i.e. , before escalation ) in male male contests . in this species , some intact intruders give up contests before escalation if their major cheliped is smaller than those of their opponents ( yasuda and koga , in submission ) . the decision of a p. minutus intruder would therefore be affected by the status of their own major cheliped , and intruders without a major cheliped might show different contest behavior from intact intruders . when the contests were escalated by intruders , we also compared the success of takeover in intact intruders and intruders without a major cheliped to examine the magnitude of the disadvantage caused by loss of the major cheliped in this species . we collected 174 precopulatory guarding pairs of p. minutus from a sandy mud flat at nunohiki , in the waka river estuary , wakayama , japan ( 341023n , 1351049e ) , from 19 december 2014 to 9 january 2015 ; the mating season of this species at this site occurs from november to april ( koga unpublished data ) . in the laboratory , if a male was still guarding the female , we placed the pair in a container ( 8 12.5 8 cm ) containing natural seawater to a depth of 2.5 cm . all the pairs were acclimated to laboratory conditions for at least an hour before the experimental tests of male male contest , and all tests were conducted within 6 h of collection . for each contest , we placed one male ( the owner ) and his partner in an arena ( 11 19.5 8.5 cm ) . to prevent any appendages from protruding from the water surface during the interaction , the arena contained seawater to a depth of 3 cm . after confirming guarding by the owner , we introduced another male ( the intruder ) randomly chosen from the other pairs into the arena after separating him from his partner . all owners ( guarding males ) had a major cheliped ; that is , they were intact . using a digital camera ( wg-10 , pentax ) , we recorded the interactions between the males from the time the intruder was introduced into the arena . a total of 71 recordings of the contests were observed for up to 15 min from the time when the intruder initiated movement . when the intruder initiated grappling with the owner ( for details of this behavior , see yasuda et al . 2012 ) , we considered that the contest had escalated . in the escalated contests , we recorded whether the intruder succeeded in taking the female from its original owner . after the contests , we measured the shield length ( sl , calcified anterior portion of the cephalothorax ) of all crabs as an index of their body size . we also recorded whether the intruder had a major cheliped ( intruders without a major cheliped ; n = 15 , intact intruders ; n = 56 ) . mean sl in weaponless intruders ( 3.91 0.19 sd mm ) was significantly larger than that in intact intruders ( 3.76 0.37 sd mm ; t - test , t = 2.16 , p = 0.035 ) . except for data on contests involving a male without a major cheliped , the data in this study were also used in yasuda and koga ( in submission ) , in which we examined the assessment strategy of intruders during male male contests . previous studies have reported that the lack of chelipeds in crustaceans is due to escape from a predator ( e.g. , juanes and smith 1995 ) , and the hermit crab p. middendorffii autotomize its major cheliped in anti - predatory behavior against a predatory crab ( matsuo et al . since there is a significant highly positive correlation between shield length and major cheliped length in males of p. minutus ( yasuda and koga in submission ) , weaponless intruders may also have possessed a large major cheliped prior to its loss and might have autotomized the cheliped to escape from attracted predators . on the other hand , since few pagurus males are injured in intraspecific aggression ( e.g. , yasuda et al . 2011 ) , weaponless or not is expected to be independent of the relative strength to conspecifics . a fouled molting event is another explanation of the lack of cheliped ( maginnis 2006 ) . although no data exist , these possibilities might explain why weaponless males in p. minutus had autotomized their major cheliped before collection in this study . all statistical analyses were performed using version 3.2.2 of the r software ( r core team 2015 ) . we used a generalized linear model ( glm ) with a binomial error distribution to examine whether weaponless intruders gave up the contest without grappling . the response variable was a binary variable that defined whether the intruder gave up the contest without grappling ( yes = 1 , no = 0 ; n = 71 ) . the explanatory variables were whether the intruder had lost its major cheliped ( loss = 1 , intact = 0 ) and the difference in sl between the intruder and the owner . the sl of females guarded by the owners was also treated as an explanatory variable in the glm . we then examined the effect of lacking a major cheliped on the success of takeover in intruders after escalation ( n = 61 ) . another response variable was whether intruders succeeded in takeover ( yes = 1 , no = 0 ) . the explanatory variables were the same as in the analysis of whether males gave up the contests . regardless of the presence or absence of a major cheliped , few intruders gave up the contest without escalation ( n = 10 , 14.1 % ) , so most intruders initiated grappling ( n = 61 , 85.9 % ) . there was no significant difference in the probability of giving up between intruders without a major cheliped and intact intruders ( table 2 ; fig . 1 ) . intruders with a smaller sl than the opponents were significantly less likely to grapple with the owner ( table 2 ; fig . 1 ) . in the escalated contests , intruders that lacked their major cheliped had a significantly lower frequency of success of takeover than intact intruders ( table 2 ; fig . 2 ) . intruders with a large sl relative to that of their opponents had a significantly higher chance of success of takeover ( table 2 ) . in intruders that lacked their major cheliped , all three of the males that succeeded in takeover had a larger sl than their opponents ( fig . 2 ) . the sl of the female had no significant effect on either escalation of the contest or the intruder s success in takeover ( table 2).table 1summary of male male contests of pagurus minutus status of intruders n before escalationafter escalationgiving upescalationsuccess in takeoverfailure in takeover n ( % ) n ( % ) n ( % ) n ( % ) loss of major cheliped151(6.7)14(93.3)3(21.4)11(78.6)intact569(16.1)47(83.9)29(61.7)18(38.3 ) table 2results of male male contests in pagurus minutus estimatese z p whether intruders gave up the contest ( n = 71 ) intercept0.062.590.020.98 loss of major cheliped0.621.190.520.61 difference in shield length3.181.033.10<0.01 shield length of female0.870.960.910.37whether intruders succeeded in takeover of the female from the owners ( n = 61 ) intercept0.652.380.270.78 loss of major cheliped2.760.903.07<0.01 difference in shield length3.861.243.11<0.01 shield length of female0.410.860.460.64analysis was based on a glm with a binomial error distribution fig . 1logistic regression results for whether intruders gave up the contest without escalation ( i.e. , without grappling ) in male male contests of pagurus minutus . values of 0 and 1 represent intruders that escalated or gave up the contest , respectively . shield length of the females guarded by owners was treated as the average value in the curves . note that there was no significant difference in frequency of escalation between intact intruders and intruders without a major cheliped ( table 2 ) fig . 2logistic regression results for whether pagurus minutus intruders succeeded in takeover of females guarded by the owners after escalation . intruders without a major cheliped had significantly decreased frequency of takeover compared with intact intruders . values of 0 and 1 represent intruders who failed or succeeded in takeover after escalation , respectively . shield length of the females guarded by owners was treated as the average values in the curves . the vertical dotted line indicates no body size difference between males summary of male male contests of pagurus minutus results of male male contests in pagurus minutus analysis was based on a glm with a binomial error distribution logistic regression results for whether intruders gave up the contest without escalation ( i.e. , without grappling ) in male male contests of pagurus minutus . values of 0 and 1 represent intruders that escalated or gave up the contest , respectively . shield length of the females guarded by owners was treated as the average value in the curves . note that there was no significant difference in frequency of escalation between intact intruders and intruders without a major cheliped ( table 2 ) logistic regression results for whether pagurus minutus intruders succeeded in takeover of females guarded by the owners after escalation . intruders without a major cheliped had significantly decreased frequency of takeover compared with intact intruders . values of 0 and 1 represent intruders who failed or succeeded in takeover after escalation , respectively . shield length of the females guarded by owners was treated as the average values in the curves . the lack of a major cheliped had no effect on the decision of intruders to escalate during male male contests in p. minutus . intruders of this species showed a lower frequency of escalation when they were smaller than their opponent ( based on sl ) , but loss of their major cheliped did not affect this decision . this suggests the smaller intruders might be assessing any potential costs ( e.g. , energetic or injury ) before escalation independent of the status of their own major cheliped . after escalation , however , the success of takeover was significantly lower in intruders without a major cheliped ( n = 3 out of 14 escalations , 21.4 % ) than in intact intruders ( n = 29 out of 47 escalations , 61.7 % ) . given that the intact intruders use their major cheliped to take a female away from their opponents during male male contests in pagurus species ( yasuda et al . 2014 ) , intruders without a major cheliped undoubtedly have a lower rhp . a significant disadvantage caused by loss of the major cheliped is common in dyadic contests by decapods ( e.g. , neil 1985 ; smith 1992 ; abello et al . p. minutus intruders without a major cheliped are , therefore , willing to initiate contests despite their lower chance of success , which seems to be an irrational choice . first , intruders without a major cheliped might fail to assess their actual rhp relative to that of their opponent because of the lack of assessment index in themselves . in contrast to theoretical predictions , weaker contestants often initiate aggression against stronger opponents even after visual and/or tactile assessment ( smith et al . this suggests that contestants can not always assess their relative rhp , even when they are weaker than their opponent . for example , if rhp assessment depends on a given trait ( i.e. , an assessment index ) , the lack of an index might decrease the accuracy of this assessment and increase the frequency of irrational decisions . in various taxa , including decapod crustaceans , weapons are used more than body size as an assessment index for relative rhp ( barki et al . contestants of p. minutus also perform mutual rhp assessment based on major cheliped size rather than body size during all phases of male male contests ( yasuda and koga , in submission ) . loss of the major cheliped by p. minutus , therefore , may cause the lack of an assessment index . if so , the intruders without a major cheliped might decrease the accuracy of mutual assessment , resulting in escalation to a fruitless contest . second , despite the importance of the major cheliped , a male s rhp and its decisions based on its rhp are also strongly affected by body size . body size differences are one of the most common factors that determine the asymmetry of rhp between males in various taxa ( andersson 1994 ; hardy and briffa 2013 ) , and the disadvantage created by loss of the major cheliped can be overcome by a ( much ) larger body size than that of an intact opponent ( e.g. , for the shore crab carcinus maenas ; abello et al . the advantage of larger body size for pagurus species during male male contests is well known ( e.g. , wada et al . , we also demonstrated that both the giving - up decision and success of takeover in intruders were affected by body size relative to their opponents in p. minutus . these results suggest that body size in males of p. minutus reflects actual rhp as well as major cheliped size ( yasuda and koga in submission ) . moreover , given that weaponless intruders tended to be larger in body size , some of them might have a relatively high rhp even without their major cheliped . all three intruders that lacked their major cheliped but that nonetheless succeeded in takeover were larger in body size than their opponents , which provides support for this possibility . together , some uncertainty about the relative rhp between contestants reflected by body size would have remained even after intruders had lost their major cheliped . third , the relationship between the cost and benefit of male male contests would affect the intruder s decision . theoretical studies have predicted that weaker individuals are likely to initiate contests that have a low cost and high benefit ( grafen 1987 ; morrell et al . 1991 ; moore 2007 ) , few pagurus males are injured during male male contests , even if physical struggles occur as a result of escalation ( yasuda et al . thus , the cost of a contest for mates is likely to be relatively low in these species . moreover , many studies have reported precopulatory mate guarding and intense male male fights for guarded females in this group ( imafuku 1986 ; goshima et al . 1998 ; wada et al . 1999 ; yasuda et al . 2011 , 2014 ; yasuda and koga , in submission ) . intruders try to take over a female guarded by an opponent , independent of the female s quality , in p. filholi ( tanikawa et al . 2012 ) , p. middendorffii ( yasuda et al . 2012 ) , and p. minutus ( present study ) . this evidence suggests a scarcity and therefore high value of mature females in these species . thus , male male contests in pagurus hermit crabs might therefore meet the assumptions of theoretical benefit - cost predictions ; that is , intruders without a major cheliped would show no difference from intact intruders in the frequency of escalation because of the high benefit to cost ratio . several studies have recently explored why potentially weaker individuals might initiate aggression against stronger competitors despite a low chance of success ( just and morris 2003 ; morrell et al . although p. minutus intruders without a major cheliped were more likely to lose a contest than intact intruders , they escalated contests rather than giving up at an early stage . this suggests that male male contests in pagurus hermit crabs may relate to a mistaken rhp assessment , a decision based on body size , and the favorable relationship between the benefits and costs of contests . however , further investigation will be needed to evaluate the relative importance of these explanations .

a 63-year - old male patient was submitted to cbct ( i - cat , imaging sciences international , hatfield , pa , usa ) of the mandible for the planning of rehabilitating treatment with dental implants . 1 ) , sequential parasagittal sections obtained every 1 mm in the distomesial direction ( fig . 3 ) revealed the presence of two mental foramina on the right side of the mandible , while only one foramen could be identified on the left side . at the level of the second premolar , before its opening in the vestibular portion of the mandible , the mandibular canal on the right side showed a ramification with a distinct and independent intraosseous course in the posterior direction and an additional opening in the vestibular cortical bone of the mandible . the mental foramen and accessory mental foramen , measuring 2.83 and 1.75 mm in diameter , respectively , were present at the same height , with the accessory mental foramen in a slightly posterior position . the mental foramen on the left side was a single structure located at the same height as the others , but with a diameter of 4.84 mm . three - dimensional reconstruction demonstrated the presence of two mental foramina on the right side and only one , of wider diameter , on the left side ( fig . precise knowledge of vital anatomical structures such as the inferior alveolar nerve and the mental nerve is fundamental to obtaining favorable results after surgical procedures such as dental implant placement.6 this also applies to the mental foramen , through which the ramifications of the inferior alveolar nerve emerge.12 although rare , variations associated with the mental foramen have been reported , 3,10,11 which might cause morbidities in the patient , such as possible injuries due to surgical procedures , if undetected . 6 regarding the mental foramen , variations are observed in its location , with the reference point being encountered more anteriorly , below the canine , or posteriorly , close to the second molar . variations in the number of foramina have also been reported , with more than one mental foramen being present on one or more sides of the mandible . 6 this variation results from the ramification of the mental nerve before emerging into the mental foramen.3,10 in the identification of anatomical variations , it is important to differentiate an accessory mental foramen from a nutrient foramen . the accessory mental foramen is an opening in the bone originating from a ramification of the mandibular canal , as observed in the present case . conversely , the nutrient foramen does not originate in the mandibular canal and its dimensions are comparatively small.3 in our case , the dimensions of the right mental foramen and of the accessory mental foramina were 2.83 and 1.75 mm , respectively . on the basis of other studies,12,13 we considered accessory mental foramina the smallest among them . an accessory mental foramen is usually less than 1.0 mm in size,5,12 but in the present case , the accessory mental foramen was relatively wide , posing a greater risk of surgical complications in this region . the mean diameter of the mental foramen is typically 3.0 mm , ranging from 1.8 to 5.1 mm.14 in the present case , the left mental foramen was 4.84 mm in diameter , which was considered larger than the mean , but within normal limits . katakami et al.,12 in a study of 150 patients , observed the presence of 17 accessory mental foramina by cbct , with 59% of them being located posteriorly to the mental foramen . another study conducted on 157 patients demonstrated the presence of 15 accessory mental foramina , nine of them located posteriorly to the mental foramen.13 on the basis of these findings , the present study agreed with the literature since the accessory mental foramina detected here were slightly dislocated in a posterior direction with respect to the mental foramen . the location of the accessory mental foramen would directly affect the planning of rehabilitating treatment since it might interfere with the procedures performed for a dental implant . if the accessory mental foramina would be located inferiorly to the mental foramen , for example , the interference caused by their presence , will be lower or practically nonexistent since the planning would already be based on the location of the mental foramen . when an accessory mental foramen is identified , more attention should be paid to obtaining successful treatment . damage to neurovascular bundles may cause hemorrhage , paresthesia , or dysesthesia , among other consequences.6 thakur et al.11 emphasized the importance of identifying the accessory mental foramen before the treatment of neuralgia , since in patients with this variation , incomplete neurectomy of the mental nerve results in treatment failure and symptom recurrence . some studies on accessory mental foramina are available in the literature , but most of them were conducted on dry mandibles.1,15 sawyer et al.1 conducted an anthropological study on different ethnic groups and observed an accessory mental foramen frequency of 1.5% to 12.5% , with no clear predilection for gender or age . in general , only one accessory mental foramen is observed in a hemimandible , although the presence of three such foramina has also been reported.1,15 clinical and radiographic studies of this condition are less frequent,10 and an even smaller proportion of cases involve ct and cbct.3,4,5,12,13,16 although cairankaya and kansu10 observed an accessory mental foramen using conventional radiographs , these exams could not precisely identify the presence and the course of the mandibular canal , the mental foramen , and the accessory mental foramina due to the distortion of the image or the low contrast between the foramen and the mandibular trabecular bone.6 naitoh et al.17 compared the identification of an accessory mental foramen in panoramic radiographs and in reconstruction images ( cbct ) . after examining 365 patients , the authors detected 37 accessory mental foramina with the aid of cbct and only 18 accessory mental foramina on panoramic radiographs . further , kulkarni et al.18 reported a case in which the accessory mental foramen and its nervous component were discovered accidentally during surgery since the presurgical panoramic radiography had not revealed any variation . thus , a precise accessory mental foramen analysis is only possible with ct / cbct , which provides an image of better quality and lower distortion than conventional exams do.11 the presence of anatomical variations in the maxilla and the mandible is still frequently neglected . it is important to underscore that these variations can be identified in presurgical imaging exams , permitting more accurate planning and contributing to successful treatment . since conventional radiographs may fail in the identification of variations , cbct is of fundamental importance in this process . the recognition of accessory mental foramina contributes to the use of an appropriate surgical technique , preventing eventual damage to the nerves and vessels in the lower premolar and molar regions .

based on numerous studies , sex ratio at birth ( srb , male proportion ) varies with many variables ( james 1987 ; ansari - lari et al . , 2004 ; saadat 2005 , 2008 ; james 2006 , 2009 ; masoudi and saadat 2007 ; saadat and khalili 2009 ) . the subject of changing in srb during or shortly after wars was investigated by several researchers ( james 2009 ) . it is reported that conflicts or exposure to stressful conditions may have a negative effect on the srb ( lyster 1974 ; fukuda et al . , 1998 ; ansari - lari and saadat 2002 ; catalano et al . , 2005 , 2006 ; saadat 2006 , 2008 ; james 2009 ) . the srb decreased in iran during iran / iraq war ( ansari - lari and saadat 2002 ) , during civil war in the balkan ( zorn et al . , 2002 ) several months after earthquakes in kobe ( japan ) ( fukuda et al . , 1998 ) and bam ( iran ) ( saadat 2008 ) , and in the few months after 11 september events in california and new york city ( catalano et al . , 2005 , 2006 ) . several studies have been indicated that srb increased in bosnia - herzegovina during civil war ( polasek et al . , 2005 ; polasek 2006 ) , after the first and the second world wars ( james 2009 ) , during civil war in tajikistan ( hohmann et al . , 2010 ) and in sardasht ( north - west iran ) after chemical weapons attack by saddam 's regime on 27 july 1987 ( saadat 2006 ) . other studies indicating that the srb did not alter during or after wars ( mcmahon and pugh 1954 ; polasek et al . , 2005 ; polasek 2006 ; abu - musa et al . , 2008 , 2009 ; james 2009 ) previously we reported that the srb dramatically decreased in iran during iran / iraq war ( ansari - lari and saadat 2002 ; saadat 2006 ) . it is suggested that stressful conditions may have some role(s ) for the alteration of srb in iran ( saadat and ansari - lari 2004 ) . there is no data on the secondary sex ratio in iraq during its war with iran . between january 20 and february 20 2010 a team of 11 researchers visited houses in an area of fallujah , iraq ( some 50 miles west of baghdad ) . they visited 711 houses and obtained responses to a questionnaire on cancer , birth defects , infant mortality , and sex of live births . the authors summarized the sex of participants in 5-years age groups in table 1 of their article ( busby et al . , 2010 ) . the war between iran and iraq began at september 1980 and ended in july 1988 . to determine the possible effect of the war on the srb , the total studied period was divided to three distinct parts as before , during , and after the war . the odds ratio ( or ) ( the odds of being male ) was calculated , considering the exposure to the war as a risk factor . an or>1.0 shows an increase and an or<1.0 shows a decrease in male proportion in comparison with the srb in reference periods ( before and/or after war ) . statistical analysis was performed using the statistical package for social sciences ( spss inc . , according to data presented in the article of busby et al . , ( 2010 ) , we extracted 785 births ( 348 males , 437 females ) during 1980 - 1989 , 1144 births ( 655 males , 489 females ) , before 1980 , and 2914 births ( 1496 males , 1418 females ) after 1989 ( table 1(tab . 1 ) the srb in fallujah were 0.5725 , 0.4433 and 0.5133 before 1980 , during 1980 - 1989 and after 1989 , respectively . the srb decreased during 1980 - 1989 in comparison with the ratios before 1980 ( or=0.595 , 95 % ci : 0.495 - 0.714 , p<0.001 ) and after 1989 ( or=0.755 , 95 % ci : 0.644 - 0.884 , p=0.001 ) . considering that the iran - iraq wartime was 1980 - 1988 , therefore it seems that the srb decreased in fallujah ( iraq ) during the war . this finding is consistent with our previous finding indicating decrease in srb in iran during the iran / iraq war ( ansari - lari and saadat 2002 ; saadat 2006 ) and other studies ( zorn et al . , 2002 ; catalano et al . , 2005 , 2006 ) it is possible that stressful conditions ( war ) have effect on decreasing the srb . first of all there is obviously no guarantee that the survivors living in fallujah in 2010 had the same sex ratio as the births 20 to 29 years before . it should be noted that men could have been killed in larger numbers during the us assault in 2004 , or could have out - migrated because of the invasion . second , we have no data on sex ratio at birth in other parts of iraq . third , the significant difference may occur by chance because the sample size was small . this study was supported by a grant - in - aid for promotion of education and science in shiraz university , provided by the ministry of sciences , research , and technology of iran .

several key reactions in a cell involve proteins interacting with the phosphate moiety , either as an isolated phosphate ion or as part of a phosphorylated ligand . the phosphate group has been observed to interact with more than half of the known proteins ( 1 ) . moreover many phosphate binding proteins are involved in pathways whose malfunction causes important human diseases ( 2,3 ) . the binding of the phosphate group usually gives a significant contribution to the overall binding energy in the interaction between proteins and phosphate - containing ligands ( 4 ) . the ability to bind the phosphate group evolved multiple times , as evidenced by its occurrence in several non - homologous protein families . however some recognition motifs such as the p - loop ( 5 ) and the rossmann - type fold ( 6 ) are extremely frequent . several methods for the prediction of ligand binding sites are available as web servers . tools like 3dligandsite ( 7 ) , probis ( 8,9 ) and site hound - web ( 10 ) use information derived from protein structures to predict binding sites irrespective of the interacting ligand . other web - based methods are focused on the prediction of binding sites for specific classes of ligands . for instance metaldetector ( 11 ) , predicts metal binding sites using only the sequence of the protein . similarly protedna ( 12 ) is a dna binding site predictor based on the analysis of the sequence of known transcription factors that also takes into account the alignment of the predicted secondary structure elements . there is also a small number of web servers devoted to the prediction of binding sites for specific ligands using structural information . rnabindr ( 13 ) predicts rna binding sites using a naive bayes classifier trained on solved rna protein complexes ; pepsite ( 14 ) predicts peptide binding sites using spatial position - specific scoring matrices that describe the preferred protein environment of each amino acid in the peptide . given the importance of the phosphate group in several biological processes ( see above ) , we developed pfinder ( 15 ) , the only available method for the prediction of phosphate binding sites in protein structures . this method is based on the observation that the same phosphate binding structural motifs occur in evolutionarily unrelated proteins , irrespective of the identity of the ligand as a whole ( 16,17 ) . our approach therefore consists in using a previously constructed dataset of phosphate binding motifs ( 16 ) to scan a structure of interest with the superpose3d ( 18 ) structural comparison algorithm . the residues in the query structure that match one of these motifs are predicted as phosphate binding . moreover the phosphate group is placed on the query protein according to its position in the template motif . the predictions are then filtered to exclude those found in the interior of the protein . residues which are not conserved in the family of the query protein are also discarded . in the present work , we describe phosfinder ( http://phosfinder.bio.uniroma2.it ) , a web server interface for the pfinder method that makes it accessible to a broader audience . the phosfinder server is based on the pfinder method ( 15 ) for the prediction of phosphate binding sites in protein structures . pfinder uses the superpose3d structural comparison software ( 18,19 ) to scan a structure of interest against a data set of 215 phosphate binding motifs identified in a previous work ( 16 ) . each one of these motifs is composed of at least three amino acids binding a phosphate group , either in its ion form or as part of a bigger ligand , and it is present in at least two different scop folds ( 20 ) . the structural comparison is governed by two parameters : the root mean square deviation ( rmsd , a measure of geometric similarity ) between corresponding residues ( the c and side - chain geometric centroid are considered for this calculation ) , and the blosum62 ( 21 ) substitution value of paired residues . whenever a known phosphate binding motif matches with residues of the query protein , its bound phosphate group is roto - translated onto the query structure . the phosphate group represents the predicted phosphate position and the protein residues in the match represent the corresponding inferred phosphate binding site . predictions that result in the phosphate group being placed inside the protein solvent - excluded surface are discarded since they are unlikely to represent real binding sites . the remaining sites are then clustered with a hierarchical - clustering ( centroid - linkage ) procedure . for each cluster , the predicted phosphate binding position that is closer to the cluster centroid is retained as representative of the cluster . finally a conservation score is assigned to each predicted site using the available pfam ( 22 ) multiple alignments . for each protein residue , the percentage of similar ( blosum62 score 1 ) residues in the corresponding multiple alignment column is calculated . in order to normalize and compare values from different multiple alignments , the percentile corresponding to each value with respect to the distributions of values in the alignment is calculated . the conservation score of the predicted phosphate binding site is calculated as the average of the conservation scores of its constituent residues . the core programs of phosfinder are written in python , c and c and are linked to the web interface using cgi . the structure can be provided as a pdb code ( 23 ) , or as a user - submitted pdb format file . using the advanced search , the user can specify different parameters : ( i ) the rmsd threshold of the structural comparison ( ranging from 0.6 to 0.9 ) ; ( ii ) the blosum62 substitution threshold ( that can be set to 1 , 0 or + 1 , making the search less or more stringent ) ; and ( iii ) the conservation value ( ranging from 0 to 100 , the default value is set to 66 ) used to rank the predictions . the user can also upload a phosphate binding motif that is not comprised in the selected data set . the server outputs a list of predicted phosphate binding sites ranked and colored according to the calculated conservation score ( figure 1 ) . detailed information about the structural matches between the query protein residues and the known phosphate binding motifs are also displayed ; these include the number and position of the residues in the protein , the rmsd of the structural match , the conservation score and a button that allows the user to inspect the known phosphate binding motif , involved in the structural match , in its original protein . the phosphate binding sites are also displayed in a jmol ( an open - source java - based viewer for 3d chemical structures available at http://www.jmol.org/ ) graphical applet : the query protein structure is shown , in ribbon style , together with the predicted sites , represented as spheres and colored according to the conservation score ( figure 2 ) . the user can highlight the analyzed chains , show / hide the predictions , the prediction labels , the protein surface and any ligand bound by the protein . a group of buttons allows the user to view in detail the pfam alignment of the protein and to retrieve the results as a parsable text file . a pdb - formatted file can be downloaded , containing the query protein structure together with the coordinates of the predicted phosphate binding sites . the website also includes help pages that guide the user with worked examples ( a complete and interactive output page is given as example ) . the phosfinder web site is freely available at http://phosfinder.bio.uniroma2.it and does not require any registration . seven phosphate binding sites have been predicted on the oryctolagus cuniculus phosphorylase kinase ( pdb code 1phk ) with 0.7 and + 1 as the rmsd and blosum62 thresholds , respectively . the extensible table , on the left , reports all the predicted phosphate binding sites in detail , ranked according to their conservation score . a color scheme ( top - right ) helps the user to visually discriminate binding sites with a score higher than a specified threshold ( in this case the threshold has a value of 70 ) . the top - ranked phosphate binding site has a high - conservation score ( 97.0 ) . the prediction derives from a structural match involving three residues of the query protein and a known phosphate binding motif belonging to the sulfobulus tokodaii fructose-1,6-bisphosphatase ( pdb code 1umg ) with an rmsd of 0.43 . a jmol graphical applet displays the query protein structure ( grey ) in ribbon style with the predicted binding sites represented as spheres and colored according to the conservation score ( gold , silver and bronze ) . the top - ranked and highly conserved prediction is colored in gold and is located extremely close to the -phosphate of the crystallized atp molecule . two groups of buttons , located above the results table and below the applet , respectively , allow the user to view / download the results , and to interact with the 3d visualization of the protein with the predicted binding sites . figure 2.the graphical jmol applet showing a known phosphate binding motif on a phosphate binding protein from escherichia coli ( pdb code 1ixi ) . this motif is composed of five amino acids binding a dihydrogenphosphate ion : ala9 , asn56 , ser139 , gly140 and thr141 . this visualization is accessed from the results table and shows in more detail the original structure from which the motif responsible for the prediction was derived . an example phosfinder output page . seven phosphate binding sites have been predicted on the oryctolagus cuniculus phosphorylase kinase ( pdb code 1phk ) with 0.7 and + 1 as the rmsd and blosum62 thresholds , respectively . the extensible table , on the left , reports all the predicted phosphate binding sites in detail , ranked according to their conservation score . a color scheme ( top - right ) helps the user to visually discriminate binding sites with a score higher than a specified threshold ( in this case the threshold has a value of 70 ) . the top - ranked phosphate binding site has a high - conservation score ( 97.0 ) . the prediction derives from a structural match involving three residues of the query protein and a known phosphate binding motif belonging to the sulfobulus tokodaii fructose-1,6-bisphosphatase ( pdb code 1umg ) with an rmsd of 0.43 . a jmol graphical applet displays the query protein structure ( grey ) in ribbon style with the predicted binding sites represented as spheres and colored according to the conservation score ( gold , silver and bronze ) . the top - ranked and highly conserved prediction is colored in gold and is located extremely close to the -phosphate of the crystallized atp molecule . two groups of buttons , located above the results table and below the applet , respectively , allow the user to view / download the results , and to interact with the 3d visualization of the protein with the predicted binding sites . the graphical jmol applet showing a known phosphate binding motif on a phosphate binding protein from escherichia coli ( pdb code 1ixi ) . this motif is composed of five amino acids binding a dihydrogenphosphate ion : ala9 , asn56 , ser139 , gly140 and thr141 . this visualization is accessed from the results table and shows in more detail the original structure from which the motif responsible for the prediction was derived . phosfinder was trained on a set composed of 59 high - quality , non - redundant ( 30% sequence identity level ) , structures of proteins binding nucleotides and other non - nucleotide phosphorylated ligands in a 2:1 proportion ( as in the whole protein data bank ) . non - nucleotide phosphorylated ligands are randomly chosen from a set of 1273 phosphate - containing ligands occurring in < 10 pdb structures in the whole protein data bank . for each of these ligands , the training phase allowed us to find optimal values for the method parameters : the rmsd , blosum62 substitution value and conservation thresholds . the best results were obtained with an rmsd threshold of 0.9 , 1 as the blosum62 substitution threshold and 66 for the conservation score . this combination of parameters resulted in the identification of at least one correct prediction in 69% of the training proteins , with an average of 3.7 0.4 false positives per structure . the method was then tested on an independent set of 52 proteins , culled from the ligasite database ( 25 ) , which bind phosphorylated ligands . all the test proteins ( i ) are checked for redundancy with the training set ( at 30% of sequence identity ) ; ( ii ) do not contain mutations ; ( iii ) bind a phosphorylated ligand . pfinder identified at least one correct prediction in 63% of the holo and in 62% of the apo structures with an average of 4.8 0.7 false positives per structure in both cases . previous works showed that binding site residues have lower b - factors ( 26 ) and even though they can not be located in extremely stable regions of the proteins they can not be disordered either ( 27 ) . this is encouraging for our method because it implies that when we compare two binding sites of similar shape and in the same conformation ( i.e. both apo or holo ) , there should not be too many differences in the coordinates of the residues due to thermal motion . moreover , it has been observed ( 28,29 ) that the variability in terms of binding site conformation is correlated with the size and flexibility of the ligand . therefore , we investigated whether the performance of phosfinder , in terms of the results on the holo versus apo conformation of the same protein , is correlated with the size or the number of hydrogen bond donors and acceptors of the cognate ligand . we first considered the average distance between the best prediction and the position of the phosphate in the crystal . this measure does not differ significantly between the holo and apo sets ( paired wilcoxon test p > 0.1 ) . moreover the difference between the distance of the best prediction in the holo and apo structure of the same protein has only very weak correlations with the size of the ligand ( 0.15 ) , and the number of hydrogen bond donors ( 0.15 ) and acceptors ( 0.19 ) . there are also no significant differences between the average size of the ligand or the number of hydrogen bond donors and acceptors for the apo proteins for which no correct prediction is made versus the ones for which phosfinder correctly predicts the location of the phosphate ( wilcoxon test p > 0.9 for size , > 0.3 for number of h - bond acceptors , > 0.5 for h - bond donors ) . we think that the performance of our method is not affected when analyzing large , flexible ligands because we only consider small structural motifs mostly composed of three residues only . therefore , even if the overall shape of the binding pocket varies when the ligand is bound , the local conformation of small groups of residues is mostly preserved . moreover , we also showed that our data sets are not biased in favor of nucleotide binding folds since 34 out of 59 training proteins and 35 out of 52 test proteins have a fold that is not a wide - spread nucleotide binding folds such as the rossmann - type folds and the p - loop - containing nucleotide hydrolases . we also demonstrated that 105 out of 111 structures have at least one correct prediction made by a phosphate binding motif from a non - common nucleotide binding fold . this means that our set of template phosphate binding motifs is not biased in favor of a particular group of folds and that phosphate binding sites can be identified by motifs belonging to the different folds . the structure can be provided as a pdb code ( 23 ) , or as a user - submitted pdb format file . using the advanced search , the user can specify different parameters : ( i ) the rmsd threshold of the structural comparison ( ranging from 0.6 to 0.9 ) ; ( ii ) the blosum62 substitution threshold ( that can be set to 1 , 0 or + 1 , making the search less or more stringent ) ; and ( iii ) the conservation value ( ranging from 0 to 100 , the default value is set to 66 ) used to rank the predictions . the user can also upload a phosphate binding motif that is not comprised in the selected data set . the server outputs a list of predicted phosphate binding sites ranked and colored according to the calculated conservation score ( figure 1 ) . detailed information about the structural matches between the query protein residues and the known phosphate binding motifs are also displayed ; these include the number and position of the residues in the protein , the rmsd of the structural match , the conservation score and a button that allows the user to inspect the known phosphate binding motif , involved in the structural match , in its original protein . the phosphate binding sites are also displayed in a jmol ( an open - source java - based viewer for 3d chemical structures available at http://www.jmol.org/ ) graphical applet : the query protein structure is shown , in ribbon style , together with the predicted sites , represented as spheres and colored according to the conservation score ( figure 2 ) . the user can highlight the analyzed chains , show / hide the predictions , the prediction labels , the protein surface and any ligand bound by the protein . a group of buttons allows the user to view in detail the pfam alignment of the protein and to retrieve the results as a parsable text file . a pdb - formatted file can be downloaded , containing the query protein structure together with the coordinates of the predicted phosphate binding sites . the website also includes help pages that guide the user with worked examples ( a complete and interactive output page is given as example ) . the phosfinder web site is freely available at http://phosfinder.bio.uniroma2.it and does not require any registration . seven phosphate binding sites have been predicted on the oryctolagus cuniculus phosphorylase kinase ( pdb code 1phk ) with 0.7 and + 1 as the rmsd and blosum62 thresholds , respectively . the extensible table , on the left , reports all the predicted phosphate binding sites in detail , ranked according to their conservation score . a color scheme ( top - right ) helps the user to visually discriminate binding sites with a score higher than a specified threshold ( in this case the threshold has a value of 70 ) . the top - ranked phosphate binding site has a high - conservation score ( 97.0 ) . the prediction derives from a structural match involving three residues of the query protein and a known phosphate binding motif belonging to the sulfobulus tokodaii fructose-1,6-bisphosphatase ( pdb code 1umg ) with an rmsd of 0.43 . a jmol graphical applet displays the query protein structure ( grey ) in ribbon style with the predicted binding sites represented as spheres and colored according to the conservation score ( gold , silver and bronze ) . the top - ranked and highly conserved prediction is colored in gold and is located extremely close to the -phosphate of the crystallized atp molecule . two groups of buttons , located above the results table and below the applet , respectively , allow the user to view / download the results , and to interact with the 3d visualization of the protein with the predicted binding sites . figure 2.the graphical jmol applet showing a known phosphate binding motif on a phosphate binding protein from escherichia coli ( pdb code 1ixi ) . this motif is composed of five amino acids binding a dihydrogenphosphate ion : ala9 , asn56 , ser139 , gly140 and thr141 . this visualization is accessed from the results table and shows in more detail the original structure from which the motif responsible for the prediction was derived . an example phosfinder output page . seven phosphate binding sites have been predicted on the oryctolagus cuniculus phosphorylase kinase ( pdb code 1phk ) with 0.7 and + 1 as the rmsd and blosum62 thresholds , respectively . the extensible table , on the left , reports all the predicted phosphate binding sites in detail , ranked according to their conservation score . a color scheme ( top - right ) helps the user to visually discriminate binding sites with a score higher than a specified threshold ( in this case the threshold has a value of 70 ) . the top - ranked phosphate binding site has a high - conservation score ( 97.0 ) . the prediction derives from a structural match involving three residues of the query protein and a known phosphate binding motif belonging to the sulfobulus tokodaii fructose-1,6-bisphosphatase ( pdb code 1umg ) with an rmsd of 0.43 . a jmol graphical applet displays the query protein structure ( grey ) in ribbon style with the predicted binding sites represented as spheres and colored according to the conservation score ( gold , silver and bronze ) . the top - ranked and highly conserved prediction is colored in gold and is located extremely close to the -phosphate of the crystallized atp molecule . two groups of buttons , located above the results table and below the applet , respectively , allow the user to view / download the results , and to interact with the 3d visualization of the protein with the predicted binding sites . the graphical jmol applet showing a known phosphate binding motif on a phosphate binding protein from escherichia coli ( pdb code 1ixi ) . this motif is composed of five amino acids binding a dihydrogenphosphate ion : ala9 , asn56 , ser139 , gly140 and thr141 . this visualization is accessed from the results table and shows in more detail the original structure from which the motif responsible for the prediction was derived . phosfinder was trained on a set composed of 59 high - quality , non - redundant ( 30% sequence identity level ) , structures of proteins binding nucleotides and other non - nucleotide phosphorylated ligands in a 2:1 proportion ( as in the whole protein data bank ) . non - nucleotide phosphorylated ligands are randomly chosen from a set of 1273 phosphate - containing ligands occurring in < 10 pdb structures in the whole protein data bank . for each of these ligands , a protein structure is chosen at random from those that bind it . the training phase allowed us to find optimal values for the method parameters : the rmsd , blosum62 substitution value and conservation thresholds . the best results were obtained with an rmsd threshold of 0.9 , 1 as the blosum62 substitution threshold and 66 for the conservation score . this combination of parameters resulted in the identification of at least one correct prediction in 69% of the training proteins , with an average of 3.7 0.4 false positives per structure . the method was then tested on an independent set of 52 proteins , culled from the ligasite database ( 25 ) , which bind phosphorylated ligands . each protein was evaluated both in its apo and holo form . all the test proteins ( i ) are checked for redundancy with the training set ( at 30% of sequence identity ) ; ( ii ) do not contain mutations ; ( iii ) bind a phosphorylated ligand . pfinder identified at least one correct prediction in 63% of the holo and in 62% of the apo structures with an average of 4.8 0.7 false positives per structure in both cases . previous works showed that binding site residues have lower b - factors ( 26 ) and even though they can not be located in extremely stable regions of the proteins they can not be disordered either ( 27 ) . this is encouraging for our method because it implies that when we compare two binding sites of similar shape and in the same conformation ( i.e. both apo or holo ) , there should not be too many differences in the coordinates of the residues due to thermal motion . moreover , it has been observed ( 28,29 ) that the variability in terms of binding site conformation is correlated with the size and flexibility of the ligand . therefore , we investigated whether the performance of phosfinder , in terms of the results on the holo versus apo conformation of the same protein , is correlated with the size or the number of hydrogen bond donors and acceptors of the cognate ligand . we first considered the average distance between the best prediction and the position of the phosphate in the crystal . this measure does not differ significantly between the holo and apo sets ( paired wilcoxon test p > 0.1 ) . moreover the difference between the distance of the best prediction in the holo and apo structure of the same protein has only very weak correlations with the size of the ligand ( 0.15 ) , and the number of hydrogen bond donors ( 0.15 ) and acceptors ( 0.19 ) . there are also no significant differences between the average size of the ligand or the number of hydrogen bond donors and acceptors for the apo proteins for which no correct prediction is made versus the ones for which phosfinder correctly predicts the location of the phosphate ( wilcoxon test p > 0.9 for size , > 0.3 for number of h - bond acceptors , > 0.5 for h - bond donors ) . we think that the performance of our method is not affected when analyzing large , flexible ligands because we only consider small structural motifs mostly composed of three residues only . therefore , even if the overall shape of the binding pocket varies when the ligand is bound , the local conformation of small groups of residues is mostly preserved . moreover , we also showed that our data sets are not biased in favor of nucleotide binding folds since 34 out of 59 training proteins and 35 out of 52 test proteins have a fold that is not a wide - spread nucleotide binding folds such as the rossmann - type folds and the p - loop - containing nucleotide hydrolases . we also demonstrated that 105 out of 111 structures have at least one correct prediction made by a phosphate binding motif from a non - common nucleotide binding fold . this means that our set of template phosphate binding motifs is not biased in favor of a particular group of folds and that phosphate binding sites can be identified by motifs belonging to the different folds . phosfinder is a web server for the prediction of phosphate binding sites in protein structures . given the biological importance of the phosphate group phosfinder represents a valuable resource for structural biologists . the web server provides a user - friendly version of the pfinder method , enriched with the possibility to visualize the predicted phosphate binding sites on the query structure . moreover , the web server incorporates a new feature : the possibility for the user to upload his own phosphate binding motifs and to search them in the query structure . one of the main advantages of phosfinder is that it predicts the actual coordinates where the phosphate group is located , as opposed to a generic surface region . the analysis of apo / holo structure pairs ( 15 ) shows that the performance of phosfinder is almost completely unaffected by ligand - induced conformational changes . therefore , phosfinder can be applied to structures of unknown function that have been crystallized without a ligand . airc ( to m.h.c . ) ; firb futuro in ricerca project ( number e81j10000030001 to g.a . ) . funding for open access charge : airc .

developmental dyslexia ( dd ) , also known as reading disability , is characterized by unexpected difficulties in reading and spelling without apparent intellectual and neurological impairment . notably , it was more prevalent among children with nonsyndromic cleft lip with or without cleft palate ( nscl / p ) , a common birth defect characterized by isolated orofacial cleft with unknown but complex etiology . the rate of moderate and severe degree of reading disability were reported to be 35 and 17% , respectively , in a group of nscl / p children . although dd and nscl / p are apparently different , they might have potential common etiologic pathways . compared with age - matched non cleft controls , children with nscl / p scored significantly lower in a series of reading measurements . short - term verbal memory is the ability to keep verbal information in conscious awareness for brief periods of time . deficiency in short - term verbal memory was known to be an important endophenotype of dd . among children with nscl / p , reading disability were also featured by short - term verbal memory defects which fitted into a classic model of dd . neuroimaging studies indicated a neurological basis of dd could be explained by aberrant structure and function of reading and language networks throughout the left hemisphere . consistent with this view , the most severely affected brain region among nscl / p patients was reported to be the left temporal lobe in which observed structural abnormalities were directly related to cognitive dysfunctions . in addition to neurological findings , dd and nscl / p might have similar etiologies at genetic level as well . both dd and nscl / p are complex developmental disorders with genetic and non - genetic components to their perspective etiologies . current genetic understanding of dd is mainly based on linkage analysis and candidate gene association study . the well recognized candidate genes of dd includes dyx1c1 in dyx1 , dcdc2 and kiaa0319 in dyx2 and robo1 in dyx5 . in recent years , some of these associated genes have been replicated in different populations through association studies . meanwhile , genetic research of nscl / p has recently identified multiple genes associated with disease susceptibility through genome - wide association study ( gwas ) . previous gwas in western populations identified a number of representative single nucleotide polymorphisms ( snps ) strongly identified as nscl / p susceptibility loci . given substantial differences in genetic backgrounds , their exact contribution to risk in chinese populations remains unknown . recently , the first gwas of nscl / p in chinese reported several risk snps of nscl / p . in particular , rs8049367 located in 16p13.3 was found to be a risk variant exclusive to chinese . we also confirmed variants within previously reported susceptibility loci including rs4791774 located in 17p13.1 and rs2235371 located in 1q32.2 . given the substantial genetic components underlying dd and nscl / p , we aimed to explore observed linkage between these two disorders from genetic perspective . in the present study , we selected nscl / p susceptibility snps from our recent gwas report and evaluated their contribution to dd in a chinese case control study . our study provided additional evidences to justify underlying genetic etiologies shared by dd and nscl / p . first , primary school students aged between 7 and 13 from shandong province of china were subjected to a chinese reading test consisting of character- , word- and sentence - level questions . the participants whose reading scores were above 87th percentile or below the 13th percentile among all students in the same grade were chosen for further evaluation . these participants were subjected to a character reading test composed of 300 chinese characters individually for the assessment of reading ability . then the raven 's standard test was performed to exclude individuals with intelligence deficiency . finally , a total of 631 children , 288 dyslexic participants and 343 controls , were selected for subsequent analysis . three snps reported to be strongly associated with nscl / p , rs2235371 , rs8049367 and rs4791774 , were genotyped on sequenom massarray platform ( sequenom , san diego , ca , usa ) at capitalbio corporation ( beijing , china ) . genomic dna samples were extracted from saliva samples using oragene dna self - collection kit ( dna genotek , ottawa , ontario , canada ) and dna quantity was determined by nanodrop spectrophotometry ( nanodrop 1000 spectrophotometer , thermo scientific , wilmington , de , usa ) . a pcr reaction based on a locus - specific primer extension reaction was designed using the massarray assay design software package ( v3.1 , sequenom , san diego , ca , usa ) . maldi - tof mass spectrometer ( sequenom ) and massarray type 4.0 software ( sequenom ) were used for mass determination and data acquisition . statistical analysis was undertaken using plink software version 1.9 ( http://pngu.mgh.harvard.edu/~purcell/plink/ ) , which is an open - source whole - genome association analysis toolset and is commonly used to perform a range of basic , large - scale analyses . weinberg equilibrium tests were undertaken for each snp , and association tests were performed using additive , dominant or recessive genetic models under a logistic regression model with covariates of age and sex . first , primary school students aged between 7 and 13 from shandong province of china were subjected to a chinese reading test consisting of character- , word- and sentence - level questions . the participants whose reading scores were above 87th percentile or below the 13th percentile among all students in the same grade were chosen for further evaluation . these participants were subjected to a character reading test composed of 300 chinese characters individually for the assessment of reading ability . then the raven 's standard test was performed to exclude individuals with intelligence deficiency . finally , a total of 631 children , 288 dyslexic participants and 343 controls , were selected for subsequent analysis . three snps reported to be strongly associated with nscl / p , rs2235371 , rs8049367 and rs4791774 , were genotyped on sequenom massarray platform ( sequenom , san diego , ca , usa ) at capitalbio corporation ( beijing , china ) . genomic dna samples were extracted from saliva samples using oragene dna self - collection kit ( dna genotek , ottawa , ontario , canada ) and dna quantity was determined by nanodrop spectrophotometry ( nanodrop 1000 spectrophotometer , thermo scientific , wilmington , de , usa ) . a pcr reaction based on a locus - specific primer extension reaction was designed using the massarray assay design software package ( v3.1 , sequenom , san diego , ca , usa ) . maldi - tof mass spectrometer ( sequenom ) and massarray type 4.0 software ( sequenom ) were used for mass determination and data acquisition . statistical analysis was undertaken using plink software version 1.9 ( http://pngu.mgh.harvard.edu/~purcell/plink/ ) , which is an open - source whole - genome association analysis toolset and is commonly used to perform a range of basic , large - scale analyses . weinberg equilibrium tests were undertaken for each snp , and association tests were performed using additive , dominant or recessive genetic models under a logistic regression model with covariates of age and sex . the allele t of rs8049367 and the allele g of rs4791774 were more frequent in cases than that in controls . snp rs2235371 was not significantly associated with dd under allelic ( p=0.1905 , odd ratio ( or)=0.8569 ) , additive ( p=0.1780 , or=0.8487 ) and recessive ( p=0.5807 , or=1.1470 ) models , except dominant model ( p=0.0293 , or=0.6998 ) . however , with the adjustment of age and sex , the association under dominant model became nonsignificant ( p=0.1659 ) . snp rs8049367 was significantly associated with dd under additive ( p=0.0085 , or=1.4100 ) , dominant ( p=0.0256 , or=1.4960 ) and recessive ( p=0.0374 , or=1.7280 ) model , after adjustment for age and sex . snp rs4791774 was not significantly associated with dd under all models ( table 2 ) . after the bonferonni correction for multiple comparisons , only snp rs8049367 ( or=1.4100 , 95% ci=1.09201.8220 ) on chromosome 17 was significantly associated with dd under additive models , indicating rs8049367 is a potential snp marker for dd . in total , three susceptibility snps of nscl / p were evaluated for their contribution to risk to dd . these snps were initially reported in a recent gwas report as novel risk variants for nscl / p specific to chinese populations . in the present study , the t allele of rs8049367 showed 41% increased risk of dd , therefore rs8049367 appears to be susceptibility snp for dd as well . however , the observed association between rs8049367 and dd should be interpreted with caution as this snp is an intergenic variant located 50 kb upstream of crebbp and 32 kb downstream of adcy9 with unknown function . the crebbp gene encodes a nuclear protein that binds to camp response element binding protein ( creb ) and acts a transcriptional coactivator with histone acetyltransferase activity . taybi syndrome , which is a genetic disorder characterized by developmental delay and abnormalities as well as intellectual disabilities . knock - out of crebbp in mice resulted in permanent impairments on both long- and short - term memory . meanwhile , upregulation of crebbp - mediated transcriptional activity improved both long- and short - term memory . short - term verbal memory has a critical role in phonological processing , comprehension , and other reading - related processes . previous studies of dd frequently observed impaired short - term verbal memory among dyslexics . in a recent study , chinese dyslexics showed reduced short - term verbal memory during recall of chinese characters than age - matched normal readers . given reported implication of crebbp in memory impairment as well as developmental and intellectual abnormalities , understanding functional effects of rs8049367 on crebbp in future studies might explain its contribution to dd . previous observed reading disabilities among nscl / p were also featured by impaired short - term verbal memory which suggested underlying etiologies shared by dd and nscl / p . as rs8049367 has been associated with nscl / p in the recent gwas , our preliminary results indicated rs8049367 as a shared risk factor for both disorders . during palate development , the crebbp - mediated transcription is likely regulated by changes in its phosphorylation rather than by changes in its expression . meanwhile , expression of adcy9 was upregulated in the dental pulp stem cultures of nscl / p patients . therefore , rs8049367 might contribute to dd and nscl / p through its pleiotropic effects on crebbp and adcy9 , respectively . indeed , rs8049367 was in strong ld with an exon variant of a long non - coding rna ( lncrna ) , namely rp11462g12.2 , which was predicted to interact with both crebbp and adcy9 . thus , future investigation on lncrna rp11462g12.2 might contribute to explain the assumed pleiotropic effects of rs8049367 . in conclusion , we performed association study of three nscl / p susceptibility snps with dd in a chinese population and observed significant association of rs8049367 . this intergenic variant might have pleiotropic effects on its two nearby genes cerbbp and adcy9 . further functional investigations are warranted to validate the assumed pleiotropic effects which might explain the shared etiology underlying dd and nscl / p .