Mycobacterium tuberculosis antigens in urine is attractive as a potential means of diagnosing tuberculosis (TB) regardless of the anatomical site of disease. The most promising candidate antigen is the cell wall lipopolysaccharide antigen lipoarabinomannan (LAM), which has been used to develop commercially available enzyme-linked immunosorbent assays. Although highly variable diagnostic accuracy has been observed in different clinical populations, it is now clear that this assay has useful sensitivity for diagnosis of HIV-associated TB in patients with advanced immunodeficiency and low CD4 cell counts. Thus, this assay is particularly useful when selectively used among patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards. These are the very patients who have the highest mortality risk and who stand to gain the most from rapid diagnosis, permitting immediate initiation of TB treatment. A recently developed low-cost, lateral-flow (urine ‘dip-stick’) format of the assay provides a result within 30 minutes and is potentially a major step forward as it can be used at the point-of-care, making the possibility of immediate diagnosis and treatment a reality. This paper discusses the likely utility of this point-of-care assay and how it might best be used in combination with other diagnostic assays for TB. The many further research studies that are needed on this assay are described. Consideration is particularly given to potential reasons for the variable specificity observed in existing field evaluations of LAM ELISAs. Whether this might be related to the assay itself or to the challenges associated with study design is discussed.Detection of  Tuberculosis (TB) remains a major challenge to global health . There wDespite the fact that low-income and middle-income countries account for over 90% of the worldwide burden of TB, these regions still rely heavily on sputum smear microscopy and chest radiology for TB diagnosis ,4. TheseMycobacterium tuberculosis antigen detection has long been viewed positively as an option for TB diagnosis as this has the potential advantage of reflecting mycobacterial burden while remaining unimpaired by immune status. Moreover, analysis of urine rather than sputum samples is a very attractive option as urine is simple to collect without generating hazardous bioaerosols, it is safe to handle in the laboratory, it has relatively few bacterial contaminants and sample quality is unlikely to be highly variable.A number of mycobacterial antigens can be detected in the urine of patients with pulmonary TB ,8, but tLAM is one of three major groups of interrelated lipopolysaccharides within the mycobacterial cell wall -16. All M. tuberculosisMycobacterium leprae and Mycobacterium bovis. Th. Th31]. Specificity of Determine TB-LAM was assessed in patients with negative sputum cultures regardless of clinical status (n = 431) and was 98.6% overall and exceeded 98% in all analyses stratified by CD4 cell count and other patient characteristics . The posA second study by Peter and colleagues from South Africa has evaluated Determine TB-LAM among hospital in-patients . Two groData were analysed in a number of different ways. One key finding was that when the faintest positive bands on the test strips were used (grade 1 cut-point) as recommended by the manufacturer, specificity in culture-negative patients was very poor  but improved to 96%  using the grade 2 cut-point . Inter-oSensitivity was assessed using two different analyses, first using a culture-based gold-standard and, second, a gold standard that additionally incorporated clinico-radiological diagnoses with a positive response to TB treatment . Data weThe attractive features of Determine TB-LAM are that it can be used at the point-of-care within an out-patient clinic setting or on a hospital ward. Thus, patients can be rapidly diagnosed (30 minutes per test) and treated at a single clinic visit. It is low-cost (approximately $3.50 per test) compared to approximately $18 per cartridge for an Xpert MTB/RIF assay or over $20 for automated liquid culture. The assay can be used by health care workers with no laboratory training and without need for any diagnostic hard-ware or electricity supply. It does not generate large volumes of biohazardous waste and the test strips can be stored at room temperature.The assay only has utility in those with HIV infection and advanced immunodeficiency (CD4 cell counts <200 cells/μL and/or WHO stage 3 or 4 disease) and therefore should not be used to test unselected TB suspects attending medical out-patient clinics or TB clinics. Instead, the assay should be targeted for use in those with known HIV infection being screened for TB prior to starting ART or requiring admission to hospital medical in-patient wards. Since the assay sensitivity is so strongly associated with blood CD4 cell count, it would best be used in patients with CD4 cells counts known to be <200 cells/μL. This is a draw-back for resource-limited settings where CD4 count testing may not routinely be available.Determine TB-LAM would ideally be used in combination with other diagnostic assays, providing incremental diagnostic sensitivity and/or expediting diagnosis to permit TB treatment to be started at a single clinic visit. The potential advantages of various combinations of assays are shown in Table Chest radiography permits rapid patient evaluation, but the radiological features of HIV-associated TB in those with CD4 cell counts <200 cells/μL are often very non-specific  and the Mycobacterial culture is available in very few resource-limited settings, but is invaluable for accurate diagnosis of smear-negative TB. The very long time to culture-based diagnosis in such patients (3 weeks in a study in South Africa ) is a maIn some countries such as South Africa, the Xpert MTB/RIF assay is being implemented nationwide to replace sputum smear microscopy as the first diagnostic assay. Determine TB-LAM provides little incremental sensitivity when used in combination with Xpert MTB/RIF  Figure , and yetThere is currently much interest in development of strategies to reduce early mortality in ART programmes in sub-Saharan Africa , and TB Many more studies on the Determine TB-LAM assay are needed and a list of research questions that need to be addressed is given in Table Studies have consistently reported that the LAM assays (ELISAs or point-of-care assay) have useful sensitivity in HIV-infected patients with low CD4 cell counts ,24,31,34There are a range of possible reasons for low specificity in some studies:i) Sensitivity of the gold standard assay for TB diagnosis. The best gold standard diagnostic assay for TB is liquid culture, which has greater sensitivity than solid media culture. However, the sensitivity of any culture method may also vary between laboratories depending, for example, on the intensity of the sputum decontamination procedure, the supplements added to the growth media and other variables. Thus, over-decontamination of sputum samples, for example, may lead to apparent false-positive LAM-antigenuria.ii) Reliance on sputum samples for gold standard diagnosis. All studies to date have relied on sputum culture as the diagnostic gold standard except for the study by Shah and colleagues which also included mycobacterial blood cultures . This isiii) Use of clinical follow-up. The use of clinical follow-up for suspecting or refuting a TB diagnosis in the presence of negative microbiology is problematic in HIV-infected patients with advanced immunodeficiency. Clinical follow-up may not be possible due to death, the risk of which is high in this patient population. Early ART initiation may alter the clinical picture. In addition, a high rate of new incident TB and frequent co-pathology often confuses the clinical picture.M. tuberculosis strains were the most strongly reactive, pre-clinical studies in vitro did find cross-reactivity between a range of mycobacterial strains  when using the prototype ELISA developed by Chemogen [Mycobacterium avium, M. kansasii and M. fortuitum[iv) Non-tuberculous mycobacteria. A key question is whether non-tuberculous mycobacterial species in vivo could cause LAM-antigenuria. As discussed earlier, LAM encompasses large family of related molecules which are expressed by all mycobacterial species. Although Chemogen . Similarfortuitum. Thus, tfortuitum to 4 outfortuitum. Of notev) Urine sample contamination. It is known that the anti-LAM antibodies used in the commercial LAM assays cross-react with a number of non-mycobacterial species. Dheda and colleagues reported that cultures containing common oral flora such as various species of Actinobacteria (strains of Nocardia and Streptomyces) and Candida showed reactivity in the Clearview TB ELISA . It is nThe Determine TB-LAM assay is the first point-of-care assay for TB and this has been found to have specific utility in those with advanced HIV-associated immunodeficiency. These are the very patients who could potentially gain the most from rapid point-of-care diagnosis, permitting initiation of TB treatment at a single clinic visit. The first clinical evaluation in the field  was veryThe author has no conflicts of interest what so ever to declare.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/103/prepub


Ras oncogene family  occur in up to 30% of human cancers. Due to this high mutation frequency and the recent observation that survival of KrasG12D-driven cancer cells depends on the continuous expression of the oncogene in vivo [in vivo. Whereas Craf is essentially involved in the development of Kras-driven lung cancer [V600E is even more aggressive than oncogenic Kras to induce preneoplastic lesions and cancer in the pancreas , it imp0αH1047R . FurtherAlso 30 years after the description of the human Ras oncogenes, cure for most Ras-driven cancers is not available. However, new sophisticated tumor models allow definition of essential Ras effectors in a tumor entity specific context. Thus, novel and important target's with the potential to offer cure emerge for Kras-driven tumor entities. Therefore, we are confident that science will improve the outcome of Ras-mutated tumors in the next decade.


Plasmodium falciparum malaria.Pregnancy is associated with an increased risk of developing a malaria infection and a higher risk of developing severe malaria. The pharmacokinetic properties of many anti-malarials are also altered during pregnancy, often resulting in a decreased drug exposure. Piperaquine is a promising anti-malarial partner drug used in a fixed-dose combination with dihydroartemisinin. The aim of this study was to investigate the population pharmacokinetics of piperaquine in pregnant and non-pregnant Sudanese women with uncomplicated Symptomatic patients received a standard dose regimen of the fixed dose oral piperaquine-dihydroartemisinin combination treatment. Densely sampled plasma aliquots were collected and analysed using a previously described LC-MS/MS method. Data from 12 pregnant and 12 non-pregnant women were analysed using nonlinear mixed-effects modelling. A Monte Carlo Mapped Power (MCMP) analysis was conducted based on a previously published study to evaluate the power of detecting covariates in this relatively small study.A three-compartment disposition model with a transit-absorption model described the observed data well. Body weight was added as an allometric function on all clearance and volume parameters. A statistically significant decrease in estimated terminal piperaquine half-life in pregnant compared with non-pregnant women was found, but there were no differences in post-hoc estimates of total piperaquine exposure. The MCMP analysis indicated a minimum of 13 pregnant and 13 non-pregnant women were required to identify pregnancy as a covariate on relevant pharmacokinetic parameters (80% power and p=0.05). Pregnancy was, therefore, evaluated as a categorical and continuous covariate  in a full covariate approach. Using this approach pregnancy was not associated with any major change in piperaquine elimination clearance. However, a trend of increasing elimination clearance with increasing gestational age could be seen.The population pharmacokinetic properties of piperaquine were well described by a three-compartment disposition model in pregnant and non-pregnant women with uncomplicated malaria. The modelling approach showed no major difference in piperaquine exposure between the two groups and data presented here do not warrant a dose adjustment in pregnancy in this vulnerable population. Plasmodium falciparum malaria is a major contributor to maternal mortality in Sudan; around 37% of all maternal deaths between 1985 and 1999 at the Medani Teaching Hospital in Medani City, Sudan, were attributed to malaria .Pi is the individual estimate for a model parameter  in the ith individual. θp is the population mean of parameter P and ηi,P is individual ith deviation from the population mean. BSV is estimated from a normal distribution with variance ω2 and zero mean. Between dose occasion variability (BOV) was evaluated on absorption parameters [Equation 2].where ij is the individual parameter estimate for the ith patient on the jth dose occasion, κ is the deviation from the population mean after each dose occasion, taken from a normal distribution with variance Π2 and zero mean. An additive residual error model was assumed since data were transformed into their natural logarithms . Body weight was tried in the model as a simultaneous incorporation of an allometric function on all clearance (power of 0.75) and volume parameters (power of 1), considering the strong biological prior of this covariate relationship [Where Ptionship -49.th, 50th and 95th percentile of observed data were plotted over the simulated 95% confidence interval of the same percentiles to evaluate the models predictive performance. Bootstrap diagnostics were performed using 1000 re-sampled datasets, stratified on pregnancy.Basic goodness-of-fit plots and simulation-based diagnostics were used to evaluate the final model. Visual and numerical predictive checks  were perA Monte Carlo Mapped Power analysis (MCMP)  was condPregnancy was investigated in the final pharmacokinetic model utilizing a full covariate approach where pregnancy was included as a categorical covariate on all pharmacokinetic parameters. Estimated gestational age (EGA) was evaluated as a continuous covariate on individual parameters using a linear and a power function, and the most appropriate covariate relationship (lowest OFV) was incorporated into a full covariate model for EGA. These two full covariate models were bootstrapped (n=200) to investigate the impact of pregnancy. A pregnancy related change in the parameter estimate of more than 20% was deemed to have clinical relevance.Fourteen non-pregnant and twelve pregnant women were recruited into the study but two non-pregnant women withdrew their consent and were excluded from the analysis. Full demographics are given in Table a) and the transit-compartment rate constant (ktr) were set equal to increase the stability of the model. An additive residual error model was adequate in describing the residual random variability. BSV could be estimated reliably on elimination clearance, one inter-compartmental clearance parameter, and one peripheral volume parameter. The addition of BSV on the bioavailability resulted in a significant improvement in model fit (ΔOFV=−70.9). BOV had considerable impact on both mean transit absorption time and bioavailability (ΔOFV=−340). The final parameter estimates and a schematic picture of the final structural model is presented in Table Five hundred sixty-four (564) post-dose plasma samples of piperaquine were used in the pharmacokinetic analysis. A total of 7 1.2%) of these samples were measured to be below the limit of quantification and omitted. A three-compartmental disposition model resulted in a significantly better model fit compared with a two-compartment model (ΔOFV=−32.8). An additional peripheral compartment (four-compartment disposition model) did not further improve the fit (ΔOFV=0). A transit-compartment (n=3) absorption model was superior to all other absorption models (ΔOFV≥−44.5). In the final model the absorption rate constant  were analysed using the Mann–Whitney U-test to identify differences between pregnant and non-pregnant women Table . There wThe MCMP analysis (5% significance level and 80% power) indicated that 8+8 and 13+13 women are needed to detect pregnancy as a covariate on elimination clearance and bioavailability, respectively Figure . A formaTwo full covariate models were constructed from the final model to investigate the clinical relevance of pregnancy and EGA separately. Pregnancy had a relatively large impact on mean transit absorption-time, volume of distributions and inter-compartment clearances but no significant effect on elimination clearance Figure . The incThe final model resulted in good model diagnostic performance and reliable parameter estimates Figure . CalculaP. falciparum malaria. Few studies have been performed to evaluate the effect of pregnancy on the population pharmacokinetics of piperaquine, and this is the first study conducted in an African population. The treatment was well-tolerated and none of the participating women had recrudescent malaria infections.In this study, the pharmacokinetic properties of piperaquine have been investigated using nonlinear mixed-effects modelling in pregnant and non-pregnant Sudanese women treated with piperaquine-dihydroartemisinin for uncomplicated Previous studies of piperaquine pharmacokinetics have presented both two- and three-compartment disposition models depending on the amount of data included in the analysis ,19,20,53a and ktr, and they were therefore set to be equal. This is a common limitation and the same approach has been used in previous studies [The absorption phase was best described with a transit compartment model with three transit compartments including random effects on bioavailability (BSV and BOV) and mean transit absorption time (BOV). The transit-compartment model provides a more physiological representation of the absorption process compared to the absorption models used in previous studies  ,17. Rece studies ,20. Inco studies . This miPregnant women had a shorter terminal half-life compared to non-pregnant women, which is in agreement with the non-compartmental analysis , and higThe main aim of this study was to investigate the pharmacokinetic differences between pregnant and non-pregnant women, but the sample size was not large enough to make a conventional covariate search. The MCMP analysis resulted in a minimum of 13 patients needed in each group in order to identify the previously described covariate relationships with 80% power and a significance level of 0.05 Figure . HoweverThe model presented in this study was built on data from few patients and a single individual can therefore have a considerable impact on the results. Piperaquine population pharmacokinetic parameter estimates from the final model are in agreement with previous reports Table . HoweverP. falciparum malaria in Sudan. The terminal half-life was shorter in pregnant compared to non-pregnant women, but the total drug exposure was comparable between the two groups. This supports previous findings that no dose adjustments are needed on account of altered piperaquine pharmacokinetics in pregnancy.In conclusion, this study presents the population pharmacokinetic properties of piperaquine in pregnant and non-pregnant women with uncomplicated The Welcome Trust is a UK-based medical research charity and is independent of all drug companies. It has no financial links with the manufacturers of either the diagnostics tests or the drugs used in this study. The authors declare no conflict of interest.IA, FN, ND, and NW conceived the project. WH, NL and JT quantified the drug concentrations. RH and JT performed the pharmacokinetic analysis and wrote the first draft of the manuscript. All authors revised the manuscript critically for important intellectual content and approved the final manuscript.


The failure of current strategies to provide an explanation for controversial findings on the pattern of pathophysiological changes in Alzheimer's Disease (AD) motivates the necessity to develop new integrative approaches based on multi-modal neuroimaging data that captures various aspects of disease pathology. Previous studies using [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) and structural magnetic resonance imaging (sMRI) report controversial results about time-line, spatial extent and magnitude of glucose hypometabolism and atrophy in AD that depend on clinical and demographic characteristics of the studied populations. Here, we provide and validate at a group level a generative anatomical model of glucose hypo-metabolism and atrophy progression in AD based on FDG-PET and sMRI data of 80 patients and 79 healthy controls to describe expected age and symptom severity related changes in AD relative to a baseline provided by healthy aging. We demonstrate a high level of anatomical accuracy for both modalities yielding strongly age- and symptom-severity- dependant glucose hypometabolism in temporal, parietal and precuneal regions and a more extensive network of atrophy in hippocampal, temporal, parietal, occipital and posterior caudate regions. The model suggests greater and more consistent changes in FDG-PET compared to sMRI at earlier and the inversion of this pattern at more advanced AD stages. Our model describes, integrates and predicts characteristic patterns of AD related pathology, uncontaminated by normal age effects, derived from multi-modal data. It further provides an integrative explanation for findings suggesting a dissociation between early- and late-onset AD. The generative model offers a basis for further development of individualized biomarkers allowing accurate early diagnosis and treatment evaluation. Establishing an accurate diagnosis of Alzheimer's disease has been a major challenge in the past decades. With an increasing amount of studies aiming at detection and validation of imaging biomarkers for this disease, many apparently controversial findings have been reported over the time. The failure of current strategies to provide a consistent explanation for these differential findings motivates the necessity to develop new integrative approaches based on multi-modal data that capture various aspects of disease pathology. In our study we propose such a generative model providing a comprehensive approach towards integration of previously published differential findings in early- and late-onset AD. We believe that our analytical strategy not only provides the link between imaging biomarkers and clinical phenotype considering the effects of aging, but could also lead to new areas of research in terms of creation of new, individualized biomarkers for a more accurate diagnosis of Alzheimer's disease. Neuroimaging studies using [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) and structural magnetic resonance imaging (sMRI) provide substantial evidence of high sensitivity for early detection and progression assessment in Alzheimer's disease (AD) at a group and single subject level The question that needs to be answered is whether age and symptom severity indeed account for most of these discrepancies. In other words, what are the relative contributions of age and disease to the anatomical patterns of abnormality of structure and function? Investigation of these relationships may also provide clues to another long-time controversy – can the observed differences between young and aged AD patients be regarded as a continuum or is there a clear separation into two cohorts dependent on separate pathological mechanisms?Recent studies have suggested that AD related brain changes may be similar to those associated with healthy aging. If so, this could explain age overestimation determined from sMR images from AD patients Another controversially discussed issue is the relative capability and sensitivity of FDG-PET and sMRI to detect AD related pathology. Recent studies provided evidence for the superiority of each of the two imaging modalities as compared to the other to detect AD related pathology To address these issues and questions we generate group level anatomical models of pathophysiological changes observed in AD using FDG-PET and sMRI data. In these models we account for PVE and integrate disease-, age- and symptom severity-associated changes in AD patients. We further dissociate them from healthy aging related changes using a combination of voxel-based general linear models (GLMs). We additionally assume that AD-induced changes are added to changes observed in healthy aging. We use the models to generate age- and symptom severity- specific whole-brain patterns of glucose hypometabolism and atrophy. To validate the obtained model and to address the questions described above, we contrapose the models' predictions in terms of anatomical plausibility to findings reported in previous studies investigating age- and AD-related changes. Thereby, we aim to provide at a group level an integrative explanation for the controversial findings described above regarding spatial characteristics and magnitude of glucose hypometabolism and atrophy in AD. We further make conclusions on the relative capability of FDG-PET and sMRI to predict AD related pathology at a group level.We hypothesize, on the basis of the above considerations www.adni-info.org) sMRI and FDG-PET data from multiple centres of 80 patients with a clinical diagnosis of AD and 79 healthy controls  database , the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration (FDA), private pharmaceutical companies and non-profit organizations, as a $60 million, 5- year public-private partnership. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment (MCI) and early Alzheimer's disease (AD). Determination of sensitive and specific markers of very early AD progression is intended to aid researchers and clinicians to develop new treatments and monitor their effectiveness, as well as lessen the time and cost of clinical trials. The Principal Investigator of this initiative is Michael W. Weiner, MD, VA Medical Center and University of California – San Francisco. ADNI is the result of efforts of many co-investigators from a broad range of academic institutions and private corporations, and subjects have been recruited from over 50 sites across the U.S. and Canada. The initial goal of ADNI was to recruit 800 adults, ages 55 to 90, to participate in the research, approximately 200 cognitively normal older individuals to be followed for 3 years, 400 people with MCI to be followed for 3 years and 200 people with early AD to be followed for 2 years.” For up-to-date information, see http://www.loni.ucla.edu/ADNI/Data/ADNI_Data.shtml).The sMRI dataset included standard T1-weighted images obtained with different scanner types using a 3D MP-RAGE (magnetization-prepared 180 degrees radio-frequency pulses and rapid gradient-echo) sequence varying in TR and TE (repetition and echo time) with an in-plane resolution of 1.25×1.25 mm and 1.2 mm slice thickness acquired at 1.5T magnetic field strength. All raw data were pre-processed to correct for distortion and B1 non-uniformity as described on the ADNI webpage  dynamic: a 30 min six-frame acquisition (6 five-minute frames), with scanning from 30 to 60 min post FDG injection; 2) static: a single-frame, 30 min acquisition with scanning 30–60 min post injection; and 3) quantitative: a 60 min dynamic protocol consisting of 33 frames, with scanning beginning at injection and continuing for 60 min. The majority of the scans in the ADNI study were acquired with the first acquisition protocol. Images further differed in resolution, orientation, voxel and image dimensions and count statistics. The frames from 30 to 60 minutes post injection were spatially realigned to minimize inter-frame motion artefacts and a mean image of these frames was calculated for each subject. These mean images were used for further analysis.http://www.fil.ion.ucl.ac.uk/spm/) implemented in Matlab 7.7 . The same pre-processing algorithm was used for sMRI and FDG-PET data, as described elsewhere 3, bias correction for inhomogeneity artefacts for sMRI data, segmentation of sMRI data into different tissue classes , and masking of non-GM voxels in FDG-PET data. PVE correction using the modified Müller-Gärtner method All data processing steps were carried out using the SPM5 software package  were calculated on a voxel-wise basis . To calce.g., quadratic relationship between age or symptom severity in the AD cohort we calculated further regression analyses after removing variance explained by healthy aging. These comprised a) including only linear relationships for both age and symptom severity; b) additionally modelling quadratic relationships with age; and c) additionally modelling a quadratic relationship for symptom severity. Gender was included as a covariate for both imaging modalities and total intracranial volume was additionally included as a covariate for the sMRI data. A significance threshold of 0.001 uncorrected at voxel-level and 0.05 family-wise error (FWE)-corrected for non-stationarity of smoothness at cluster level was used for statistical analyses All three voxel-based models are based on a previously validated assumption of linearity between healthy aging- and AD symptom severity- related changes with FDG-PET and sMRI data. http://www.spss.com/statistics/). A Pearson's correlation coefficient was calculated (p<.05) in the AD group to investigate the relationship between age and symptom severity.Group comparisons of AD patients and control subjects for age and symptom severity were carried out using T-tests with a significance threshold of p<.05. Group differences regarding gender were evaluated using a chi-square test for independent samples. The statistical analyses were performed with SPSS 17.0 (2(1) = 0.06;p = .811].AD patients and control subjects did not differ in age . As expected MMSE differed significantly between the groups . The comparison of AD patients and control subjects in relation to sex showed no statistical differences [χThe generative model for healthy aging based on sMRI reveals a widespread pattern of grey matter volume reductions sparing only bilateral dorsal primary sensorimotor regions, brainstem, lateral thalamus and the dorsal part of caudate nucleus . We obseThe equivalent FDG-PET based model demonstrates a specific pattern of age-related metabolic changes with an age related decrease in glucose utilization of more than 10% per decade in bilateral parietal, occipital, sensorimotor, premotor, dorsolateral prefrontal and anterior insular cortices. Additionally, we see a major reduction in glucose metabolism in bilateral posterior putamina and in the left dorsal caudate nucleus .We report a negative relationship between symptom severity and both metabolism and GM volume  and Fig. Lesser Symptom severity related GM volume changes at age 60 years were detected throughout the brain with greatest degrees of atrophy seen in bilateral parietal, temporal, occipital, dorsolateral prefrontal, posterior cingulate and premotor cortices, the precuneus, dorsal caudate nucleus, amygdala and hippocampus. At 80 years of age, the greatest, bilateral, symptom severity related atrophy was found in parietal, temporal, occipital, primary sensorimotor and dorsolateral prefrontal cortices, the hippocampus and thalamus.We show symptom severity- related glucose metabolism reductions bilaterally in posterior temporal, parietal, lateral occipital, dorsolateral prefrontal and premotor cortices and in the precuneus. Symptom severity related hypometabolism is less extensive at higher than lower ages.In general, regional decreases in metabolism and grey matter volumes relative to healthy aging are significantly more pronounced in the lower compared to higher age range in AD. In the model, we observe substantial age-dependant differences in terms of hypometabolism and atrophy in AD patients compared to a healthy aging baseline even at an MMSE score of 30. At the age of 60 years these differences are bilaterally restricted to inferior frontal gyrus, premotor regions, inferior and medial temporal gyrus, cerebellum, rectal gyrus and to left parietal regions. Regions showing an initial difference in this age range do not correspond well to the anatomical pattern observed in later symptom severity stages and remain rather less affected compared to other regions. Initial differences observed at the age of 80 years are located in bilateral parietal, bilateral hippocampal and left sensorimotor regions and in both caudate nuclei.We observe a more consistent anatomical pattern of initial differences in hypometabolism at a MMSE score of 30. For the whole age range of 60–80 years, initial glucose hypometabolism is observed in bilateral parietal, inferior temporal and posterior cingulate cortices, posterior thalamus and the precuneus. Additionally, at 80 years of age we demonstrate significant differences in bilateral primary sensorimotor and premotor regions and in the anterior temporal lobes. All regions showing initial glucose hypometabolism, except for the posterior thalamus, also show the steepest symptom severity-related metabolic decline. Regions hypometabolic only at age 80 show no specific symptom severity-related decline.To dissociate brain changes related to healthy aging from AD pathology at specific ages we computed the intersection age of models for healthy aging and aging in AD at the voxel level . With sMWith sMRI, the linear regression model describing the relationship between age, symptom severity and structural changes in AD showed a significant correlation for each factor with atrophy . After rWith FDG-PET we found a significant positive correlation between age and glucose metabolism in bilateral temporal and parietal regions. There were no other significant correlations with age or symptom severity.Inclusion of a quadratic relationship with age or MMSE into the models revealed significant positive correlations of glucose metabolism with a quadratic age coefficient only in left dorsal parietal cortex . No otheIn this study we demonstrate that a generative model captures the anatomical and metabolic features associated with AD and healthy aging in the age range 60 to 80 years accurately and robustly. The model differentiates between the effects of aging and symptom severity in AD patients. It also provides a means to test for interactions between them, as exemplified here with disease progression and patient age. The age-dependant differential sensitivity of structural and metabolic scanning for the detection of AD pathology we demonstrate emphasises the ability of our generative model to infer expected age- and symptom severity- specific changes recorded with both imaging modalities.Our study's three main findings support and extend observations made previously. Firstly, the spatial location of changes related to different ages and symptom severities in AD was mostly consistent with regions reported in the literature e.g., early- and late-onset AD, was suggested Our second result is that in general, the magnitude of healthy aging related imaging changes between 50 and 80 years is comparable to that of changes associated with an increase in AD symptom severity at any age. Substantial regional overlap in both hypometabolism and atrophy was observed between a network of areas affected by healthy aging and that identified in AD. To interpret this overlap it is important to note that all changes related to age and symptom severity in AD we report here were calculated after removing the variance explained by healthy aging. That means that all age-related differences can be interpreted as an add-on required to normal age-related changes to induce a predefined symptom severity at the corresponding age. In previous research comparing differently aged AD groups with age-matched healthy control subjects a split of AD into different subgroups A third result is in the dissociation of age- and disease- related processes inferred from the intersection ages of healthy aging and AD models. While in previous research some studies successfully applied whole-brain approaches to discriminate dementia patients from control subjects The obtained models could be used to improve early AD detection e.g. by training automated classifiers on age- and symptom severity- specific pathological AD patterns (features) extracted by thresholding the obtained AD model. They could also be applied directly in clinical assessment by evaluating the similarity of observed pathology in any individual to age- and symptom severity- specific patterns generated using the AD model. Thereby, to enable individual assessment, percent signal difference maps could be calculated between each subject's imaging data and imaging data generated using the healthy aging model. However, both of these approaches require careful evaluation in future studies prior to clinical application.Nonetheless, a valid interpretation of our results needs to consider the effects of several other assumptions and limitations. First, the sensitivity of the generative model for detecting and predicting AD pathology depends on the accuracy of the model of healthy aging. The age- related, widespread patterns of brain atrophy and hypometabolism we find are consistent with previous findings A major advantage of our approach is the generalizability of our model to any constellation of age and symptom severity. A further difference to conventional approaches is that the method we propose relies on a voxel-wise group mean, ignoring the variance and so allowing detection of quite minor group differences. This type of analysis, though more flexible than conventional statistics, nevertheless requires caution in the interpretation of results. Minor differences between groups of healthy subjects and AD patients could still be due to random effects unrelated to AD. However, as we model disease progression  we would expect the magnitude of differences to increase with higher symptom severity. This assumption suggests that local differences between AD patients and control subjects that are not so correlated with it are more likely to be due to random artefacts than disease related pathology.For all three models used in our study, we made the assumption that healthy aging affects AD patients in the same way as healthy subjects in terms of hypometabolic and atrophic changes, with AD pathology additive to changes associated with healthy aging. This assumption is in line with current views that AD as a pathological process is unrelated to healthy aging.vs. older or in mildly vs. severely impaired patients. Any bias towards an alternative diagnosis or co-diagnosis with another dementing condition could lead to a different anatomical or hypometabolic pattern. Such a differential pattern of glucose hypometabolism was seen, for example, in sensorimotor regions only in old AD patients with low symptom severity.A further issue of interpretation of interactions is the recognised inaccuracy of clinical diagnosis of AD, which may differ in younger Most of these problems are also common to standard statistical methods of evaluation of group differences. In group statistics one often uses a high significance threshold to avoid false positive results thus minimising their effect on between group differentiations. By contrast, our approach also provides an opportunity to evaluate the impact of possible confounding effects, such as age in this case, on the discrimination between AD patients and control subjects.Text S1Acknowledgment list for ADNI publications.(PDF)Click here for additional data file.


A role for Epstein Barr virus (EBV) in multiple sclerosis (MS) has been postulated. Previous systematic reviews found higher prevalences of anti-EBV antibodies in MS patients compared to controls, but many studies have since been published, and there is a need to apply more rigorous systematic review methods.We examined the link between EBV and MS by conducting a systematic review and meta-analysis of case-control and cohort studies that examined the prevalence of anti-EBV antibodies in the serum of cases and controls. We searched Medline and Embase databases from 1960 to 2012, with no language restriction. The Mantel-Haenszel odds ratios (OR) for anti-EBV antibodies sero-positivity were calculated, and meta-analysis conducted. Quality assessment was performed using a modified version of the Newcastle Ottawa scale. Thirty-nine studies were included. Quality assessment found most studies reported acceptable selection and comparability of cases and controls. However the majority had poor reporting of ascertainment of exposure. Most studies found a higher sero-prevalence of anti-EBNA IgG and anti-VCA IgG in cases compared to controls. The results for anti-EA IgG were mixed with only half the studies finding a higher sero-prevalence in cases. The meta-analysis showed a significant OR for sero-positivity to anti-EBNA IgG and anti-VCA IgG in MS cases  3.3 to 6.6, p<0.00001] and 4.5  respectively). However, funnel plot examination suggested publication bias for the reporting of the anti-EBNA IgG. No significant difference in the OR for sero-positivity to anti-EA IgG was found .These findings support previous systematic reviews, however publication bias cannot be excluded. The methodological conduct of studies could be improved, particularly with regard to reporting and conduct of laboratory analyses. Multiple Sclerosis (MS) is a complex, chronic, inflammatory, neurological disease that affects the central nervous system The aetiology of the disease is still not well understood. Both genetic and environmental factors play roles in the development of the disease Of all infectious agents, Epstein Barr virus (EBV) has been most strongly associated with MS The odds ratio (OR) for MS patients to be EBV sero-negative was found to be 0.06 in a review by Ascherio and Munger et al. from 2010 The most recent systematic review that updated the association between MS and sero-positivity for different anti-EBV antibodies was that of Santiago The methods of the systematic review followed a pre-specified protocol developed by the authors.Medline and Embase were electronically searched using a comprehensive list of MeSH and Emtree headings and text words that were informed by previous reviews and from search tools in Ovid Medline and Embase . The seaFor a study to be included in the review, it had to be a case-control or a cohort study, recruited patients with MS diagnosis (confirmed or probable) and controls with no MS diagnosis . Participants could be from any age group and studies measured serum anti-EBV IgG antibodies (using any method) in both cases and controls. We had few exclusion criteria in order to be as generalisable as possible. Exclusion criteria were non-human studies, very small studies with less than 20 cases or 20 controls and studies that only measured anti-EBV IgM (indicating recent infection).Titles and abstracts for inclusion were reviewed by one author. A second author reviewed part of the search results (140 out of 1056 reports) and a kappa statistic for the degree of agreement between the two reviewers was calculated and was high at 0.76. Full texts of potentially relevant articles were then screened by one author and uncertainties discussed with a second author. Reference lists from included studies were hand searched to identify any further related studies. Where extra data were required, the author of the article was contacted to obtain further information. Relevant data from each study were initially extracted independently by two authors (23 out of 39 studies). After sufficient agreement was reached, data were extracted by one author and checked by a second author.The quality assessment of included studies was based on the Newcastle-Ottawa assessment scale (NOS) 2 was used to assess heterogeneity between studies The Mantel-Haenzsel odds ratio (OR) of sero-positivity to EBV was calculated for each anti-EBV antibody (anti-Epstein-Barr Nuclear Antigen 1 (anti-EBNA1) or EBNA complex, anti-Viral Capsid Antigen (anti-VCA) and anti-Early Antigen (anti-EA)), and for sero-negativity to EBV using Review Manager Version 5.1 We conducted subgroup and sensitivity analysis testing of the meta-analyses in Review Manager version 5.1 to compare the OR of sero-positivity for the three anti-EBV antibodies in the following categories:Paediatric versus (vs.) adult studies. We considered studies with median participant's age of 20 or less as paediatric,Studies above median latitude vs. those below median latitude. All studies were assigned to latitude according to area of recruitment of participants. If the area of recruitment was not clear, studies were assigned to latitude according to the address of the lead author,Studies controlling for age vs. those that did not,Studies controlling for gender vs. those that did not,Studies using McDonaldStudies that included only cases with confirmed MS diagnosis vs. those that included confirmed and probable cases,Studies that used Immunofluorescence assays (IFA) vs. those which used Enzyme-linked immunosorbent assays (ELISA),Studies with overall quality assessment scores above the median in the modified NOS scale compared to those scoring below the median.To minimise the effect of multiple comparisons, we used 99% CI for all subgroup and sensitivity analyses.After removing duplicate reports, the search through Medline and Embase yielded 1056 reports . We thenThe characteristics of participants in the included studies are summarised in et al. There were 30 studies that tested for anti-EBNA IgG (anti-EBNA1 or EBNA complex IgG) sero-prevalence . Removing the two studies The meta-analysis generated an overall OR of 4.47  . There wThe funnel plot revealed an asymmetrical distribution of studies around the line of identity, indicating the possibility of publication bias .et al. There were 24 studies that tested for anti-VCA IgG sero-prevalence. After dividing the study Sundstrom 2 = 59% (p = 0.0001). A funnel plot revealed a slightly less asymmetrical distribution of studies than for anti-EBNA IgG  . Again tEBNA IgG .There were 14 studies that tested for anti-EA IgG sero-prevalence. The median sero-positivities in the 14 studies in cases and controls were similar (18% and 16% respectively).2 = 70% (p<0.0001). The funnel plot revealed a more symmetrical distribution of studies, suggesting publication bias is less likely  . The hets likely .We considered sero-negativity data as adequate if the authors mentioned explicitly that cases and controls were negative for all anti-EBV anti-bodies  or mentioned that they were EBV negative without specifying which anti-EBV antibody they were negative for. Based on this, only seven studies provided data for the analysis 2 = 56% (p = 0.03). Due to the small number of studies in the analysis, a funnel plot was not used to assess the risk of publication bias.The meta-analysis generated an overall OR of 0.13  . Heterogpost hoc  . SubgrouStudies that matched for age or sex had higher ORs for all anti-EBV IgG sero-positivities compared to those that did not  and 5. Tet al., Levin, et al., and Ponsonby, et al., with score of 11 stars In our modified NOS scale the maximum score that could be achieved by a study was 12 stars. The majority of studies scored below half this with an overall median of five. The highest scoring studies were by Ascherio, For selection criteria, the majority of studies had adequate definitions of cases and controls. However only a quarter of the studies recruited an obviously representative or a consecutive sample of cases, and only six studies recruited adequate community controls without an obvious source of bias. For the comparability criteria, 28 out of 39 studies matched cases and controls for at least one factor, with half of the studies matching for age and at least one additional factor. For the exposure criteria, only a quarter of studies reported blinding sample analysts, with three studies reporting conducting the serology in clinical laboratory (distant from the investigators). Half of the studies reported explicitly serological cut off values with only eight studies reporting whether there were missing data .post hoc analysis. The OR of sero-positivity was higher for all anti-EBV IgG outcomes in studies scoring above the median  with studies scoring at or below the median in a e median , althougThis review has again found an association between EBV sero-positivity, based on anti-EBNA and anti-VCA IgG testing. We also found no evidence for a difference in sero-prevalence of anti-EA IgG, indicative of recent infection.et al. et al. There was considerable between study heterogeneity, and we examined different factors that might have been influential. Although none of the subgroup analyses reached statistical significance, several trends emerged. There was a slightly higher OR in paediatric studies compared to adult studies. This might be connected to higher overall exposure of adults to EBV infection than paediatric controls. However, Pakpoor post hoc analysis according to the diagnosis status of MS participants revealed a higher combined OR in studies recruiting only confirmed vs. confirmed and probable/CIS patients for exposure to anti-EBNA and VCA IgG. This finding is plausible as unconfirmed MS patients are more likely to be misclassified and therefore have a lower prevalence of EBV exposure than true MS patients.We did not find a difference in combined OR between studies using more recent criteria  compared to older criteria or failure to report criteria (which may simply reflect lack of reporting). The et al. Studies matching for age and/or sex were found to have higher ORs for anti-EBNA and VCA IgG sero-positivity. Other important confounders are geographical location and socio-economic status. We could not test for the effect of controlling for these factors because few studies explicitly mentioned controlling for them. Comparison of studies according to their latitude did not reveal a trend. In some studies the exact area of recruitment was not mentioned and an estimate had to be used. A recent study by Disanto et al. Comparing the OR in studies that used IFA to detect anti-EBV antibodies vs. those that used ELISA, revealed that the former had double the OR for anti-VCA and anti-EA IgG sero-positivity but similar results for anti-EBNA. These results are consistent with the analysis by Pakpoor Other factors that may contribute to the differences in estimates of ORs are the selection and source of cases and controls. Few studies clearly described the method of selection of cases . Ideally, the community would be the best source for controls At least half of the studies scored poorly in our modified NOS scale for quality assessment. This may partly relate to poor reporting rather than poor conduct. This highlights the importance of following a standardised method for reporting to ensure that all relevant information is reported and to facilitate interpretation and validity assessment of studies. One such method is the STROBE statement , which was developed to address standardised reporting of observational studies et al. One major strength of this review is a more thorough search strategy with no language restriction. Despite this, we only found a single published study from areas other than Europe, North America and Australia Click here for additional data file.Checklist S1MOOSE Checklist.(DOCX)Click here for additional data file.


SPECT allows registration of regional cerebral blood flow (rCBF) which is altered in a characteristic temporoparietal pattern in Alzheimer's Dementia. Numerous studies have shown the diagnostic value of reduced cerebral blood flow and metabolic changes using perfusion SPECT and FDG-PEPT in AD diagnosis as well as in differential diagnosis against frontotemporal dementia, dementia with Lewy bodies and vascular disease. Recently more pathophysiology-based biomarkers in CSF and Amyloid-PET tracers have been developed that probably have a higher diagnostic accuracy than the more indirect rCBF changes seen in perfusion SPECT. In the paper review, we describe recent advances in AD biomarkers as well as improvements in the SPECT technique. Memory problems in elderly patients are common and can be due to a wide range of conditions. Alzheimer's Disease (AD) accounts for a large amount of cases, especially when gradual onset and further cognitive problems like aphasia, apraxia, or reduced activities of daily life are present. AD originally has been a pathologic diagnosis. The “golden standard” of AD are comprehensive clinical criteria . These criteria have been validated against pathology with a wide range of sensitivity (65%–100%) and specificity (65%–90%) [99mTc-HMPAO) in AD versus normal volunteers, patients without neurodegenerative dementia (like depression) or other forms of dementia like frontotemporal dementia (FTD), dementia with Lewy bodies (DLB), or vascular dementia [Since 1991 more than 50 studies have been performed to compare the diagnostic value of SPECT . FDG has long been the traditional method to measure secondary glucose metabolism changes downstream the neurodegenerative process in AD. Since cerebral metabolism and blood flow are coupled, there is a similar pattern of regional disturbed glucose metabolic rates (rCMRGlu) in the temporal-parietal cortex, less in the prefrontal area as mentioned before. The first description appeared in 1982 by Farkas et al. [The standard radionuclide in clinical PET centers is FDG  in AD pathophysiology, there have been efforts to find appropriate APP-based CSF tests. However, initial assays measured all amyloid peptides and were not specific enough. After the pioneering work of Motter et al. in 1995  who founHyperphosphorylation of tau  occurs solely in AD and is therefore theoretically a more specific biomarker. However, studies showed large differences in sensitivity and specificity. A meta-analysis of 51 single-center studies by Mitchell  showed a11C-PiB) was the first of a new family of PET tracers that have shown to be of high diagnostic value in AD. Specific binding of PiB to brain Amyloid has been extensively studied in case-control studies and longitudinal observations since 2004 [11C-based PET tracers is their short half-life (appr. 20 minutes) which restricts their use to dedicated research sites with on-site cyclotrons and radiochemistry labs. 18F-based tracers have a longer half-life (appr. 110 minutes). The first 18F tracer for AD was FDDNP and developed to recognize both plaques and tangles. This could overcome the problem that some patients have extensive plaque formation but no dementia. The AUC for global rating of the FDDNP-PET could be up to 1.0 for AD versus control, 0.95 for MCI versus control and 0.98 for AD versus MCI. These promising results suggest that Amyloid-PETs or combined Amyloid-Tau-PETs may have a diagnostic value superior to other imaging tools like FDG-PET or volumetric MRI [18F-PET markers like Flutemetamol (PiB with 18F) are in phase II clinical studies [18F Amyloid imaging agent [The Pittsburgh Compound-B  and Dementia with Lewy-Bodies (DLB) is sometimes difficult. FTD is a heterogeneous disease and in contrast to AD no established specific and validated biomarkers are available. In the above-mentioned review of Dougall et al. 2004 , the autThe image acquisition technique in SPECT is prone to signal degradation due to different physical phenomena like depth-dependent blurring, photon attenuation and scattering.SPECT images are evaluated similar to other imaging techniques like MRI or CT, inferring bias due to subjective image interpretation. Technical limitations in the past made iterative image reconstruction necessary . In receIt is still unclear whether the “AD pattern” in perfusion SPECT merely reflects cortical neurodegeneration or the secondary cholinergic deficit following degeneration of the basal nucleus of Meynert. Studies that both investigated CSF biomarkers and SPECT showed no correlation , 38. SimIn perfusion SPECT usually lobar hypoperfusion is registrated. More recently, MRI techniques have shown that there is also compensatory hyperperfusion in AD and FTD . PossiblPerfusion SPECT and FDG-PET are metabolic based and were the first methods of “functional” brain imaging in Alzheimer's disease showing hypoperfusion in the temporo-parietal regions with the highest load with plaques and tangles in postmortem brains with AD.11C PiB). Now, there is development of longer-lasting 18F PET tracers as well first evidence that plaques and tangles can be imaged with a single PET compound, but the results have to be confirmed in independent studies.This has lead to a widespread use of perfusion SPECT and FDG-PET as a diagnostic tool in AD. Since specificity exceeds sensitivity the diagnostic value of perfusion SPECT to rule out AD usually is higher than to confirm it. However, accuracy of any diagnostic tool is critically dependent on disease severity and the population under investigation. In addition, perfusion SPECT is useful in discriminating vascular dementia, FTD, and DLB in the absence of validated specific biomarkers for these conditions. More recently pathophysiology-based CSF-biomarkers, especially beta-amyloid peptide-42 and tau protein, have been investigated with a diagnostic accuracy that seems to be superior to perfusion SPECT and FDG-PET. In addition, due to their direct reflection of the underlying process, CSF biomarkers  are also suited for early diagnosis, for example, in patients with MCI, where the diagnostic value for SPECT is even lower than in prevalent AD. In contrast to CSF assays, PET biomarkers have the advantage of showing the anatomic distribution of pathology and were first available for amyloid plaques (


Fibrosis is thought to play an important role in the pathogenesis of the adverse sequelae of hypertrophic cardiomyopathy (HCM). Late gadolinium enhancement (LGE) cardiovascular magnetic resonance detects replacement fibrosis but cannot presently detect interstitial fibrosis. The latter is thought to be a very early feature of HCM and can be triggered by inflammation, which can also expand the size of the myocardial interstitium. We sought to determine whether extracellular volume (ECV) mapping by T1-relaxometry in concert with T2-mapping can identify interstitial changes in HCM and whether these were related to myocardial edema.Twenty HCM patients  and 13 healthy volunteers  were studied. Left ventricular T1 and T2 maps were acquired at the base, mid-ventricle and apex in patients and at the mid-ventricle in controls using a modified Look-Locker inversion recovery sequence and a T2-prepared steady-state free precession mapping sequence. Post-contrast T1 maps were acquired a minimum of 15 min after 0.1 mmol/kg gadolinium together with conventional LGE images. After motion correction, pre- and post-contrast T1 parameter maps were co-registered to generate ECV parameter maps. Parameter maps and LGE images were viewed side-by-side and regions of interest (ROI) were drawn in areas with and without LGE. Data were analysed using a linear mixed effects model to account for clustering of ROI within patients.ROI analysis revealed a significantly higher ECV in regions with LGE relative to controls [Figure ECV mapping in HCM detects replacement fibrosis, but was unable to detect interstitial fibrosis. There was no evidence of edema in areas of LGE suggesting that this was not a major contributor to ECV expansion in our HCM cohort.This work is supported by the NIHR Cardiovascular Biomedical Research Unit at Royal Brompton and Harefield NHS Foundation Trust, and Imperial College, London, UK. Dr Ismail is supported by the British Heart Foundation. Dr Kellman is supported by the National Institutes of Health, USA.


Alexithymia has been a familiar conception of psychosomatic phenomenon. The aim of this study was to investigate whether there were subtypes of alexithymia associating with different traits of emotional expression and regulation among a group of healthy college students.1788 healthy college students were administered with the Chinese version of the 20-item Toronto Alexithymia Scale (TAS-20) and another set of questionnaires assessing emotion status and regulation. A hierarchical cluster analysis was conducted on the three factor scores of the TAS-20. The cluster solution was cross-validated by the corresponding emotional regulation.The results indicated there were four subtypes of alexithymia, namely extrovert-high alexithymia (EHA), general-high alexithymia (GHA), introvert-high alexithymia (IHA) and non-alexithymia (NA). The GHA was characterized by general high scores on all three factors, the IHA was characterized by high scores on difficulty identifying feelings and difficulty describing feelings but low score on externally oriented cognitive style of thinking, the EHA was characterized by high score on externally oriented cognitive style of thinking but normal score on the others, and the NA got low score on all factors. The GHA and IHA were dominant by suppressive character of emotional regulation and expression with worse emotion status as compared to the EHA and NA.The current findings suggest there were four subtypes of alexithymia characterized by different emotional regulation manifestations. Alexithymia has been a familiar conception as "no words for feeling" in psychiatry and psychosomatic medicine since it was first termed by Sifneos . Now itsResearchers ,10 foundThe purpose of this study was to examine whether there might be subtypes of alexithymia characterized by different behavioural manifestations. In so doing, the current study adopted a cluster analytical approach to examine whether there were natural grouping of people characterized by different psychological features associating with alexithymia. Cluster analysis is a statistical procedure for determining cases can be placed into groups if they share properties in common, while the cases in different clusters are as dissimilar as possible. It was hypothesized that there were various subtypes of alexithymia characterized by different psychological features associating with alexithymia.Individuals on various level of alexithymia would adopt different ways to express and regulate their emotion. The higher alexithymia groups would perform more serious level of depressive or anxious emotional status and more possible to adopt improper regulation strategy.1788 college students (freshmen and sophomore) were recruited from three regional universities in Guangzhou, south China. 1071 were males and 616 were females, aged 20.44 ± 1.40 years and 20.51 ± 1.39 years respectively, 101 individual did not mention their gender or age. Economic status was also recorded by a multiple-choice question in the checklist for monthly income per person a month. Among all subjects, 110 individuals did not mention their economic status.All subjects were literarily informed the aim of current study was to examine about psychological status of Chinese youngsters and were voluntarily attended this study. All of them would receive a feedback on the assessment results via email. This study was approved by the ethics committee of the Sun Yat-Sen University.Alexithymia was assessed by the 20-item Toronto alexithymia scale (TAS-20) to assess the severity of alexithymia,14. It iEmotion expression tendency was assessed by the Chinese version of the Emotional Expressivity Scale (EES) . It is aEmotion Regulation Questionnaire (ERQ)  was usedDepression was measured with the Beck depression inventory (BDI) ,22. It iAnxiety was assessed with the Chinese version of the state portion of State-Trait Anxiety inventory (STAI-T) . This isThe Statistical Package for Social Sciences (SPSS) 15.0  was used for all statistical analyses reported.Independent sample t-tests were conducted to analyze the gender effect on the total scores of TAS-20. ANOVA was conducted to evaluate the potential effect of economic status upon the total scores of TAS-20, whereas correlation analyses were used to explore any association of education and age with the TAS-20 scores.Cluster analyses were conducted in two phases. First, a hierarchical cluster analysis was conducted using the 3 factors: difficulty identifying feelings; difficulty describing feelings; externally oriented cognitive style of thinking scores of TAS-20 as the clustering variables and the between-group linkage method with a squared Euclidean distance measure to discriminate clusters. Second, the cluster solution was validated with analysis of variance (ANOVA) on scores of Emotional Expressivity Scale with its subscales, Emotion Regulation Questionnaire, Beck depression inventory and the state portion of State-Trait Anxiety inventory of the identified groups.No significant difference was found in the total mean TAS-20 scores between boys and girls . Age was significantly correlated the total mean TAS-20 , whereas there was no significant association between education and TAS-20 total score . No significant different in the total mean TAS-20 scores between economic status was found . Given the effect of age upon the TAS-20 score was negligible, it was not controlled for subsequent analyses between cluster comparisons.Table The four clusters did not differ significantly in terms of gender propotion  and economic status . However there showed significant difference in terms of age  and education .An ANOVA showed that the four subtypes of alexithymia differed significantly in terms of emotional status, emotional expression and regulation as our estimation Table . The genThe major findings of this study showed there were four subtypes of alexithymia and were consistent with previous studies. For example, Vorst and Bermond  suggesteValidation of the cluster solution suggested that these subtypes of alexithymia were characterized by different emotional expression and regulation abilities. The general-high alexithymia (GHA) and introversive-high alexithymia (IHA) were characterized by poorer emotional regulation and expression with worse emotion status. In more details description, the extroverted-high alexithymia (EHA) seemed to be modest in emotion status, with emotion regulating more efficiently as compared to the general-high alexithymia (GHA). These results suggest the potential functional outcome of these different subtypes. Mattila  found thIt should be noted that the EHA cluster includes the 77.3% of the sample. The EHA cluster shows alterations mostly on externally oriented cognitive style of thinking (EOT). Some studies showed difficulty identifying feelings (DIF) and difficulty describing feelings (DDF) had good internal reliability but not EOT . Some reThe current study has several limitations. First, participants were recruited from a convenient sample pool was the main limitation of this study. Whether these cluster groups could discovered in more broad population needs future study adopted a more rigorous epidemiological approach to improve its representativeness. Second, the findings were based on subjective self-report measures. More rigorous methodologies adopting experimental designs or neurophysiological approaches such as ERP or imaging paradigms should be enforced in the near future in order to validate potential differential neural bases of these subtypes of alexithymia. Finally, the current cross-sectional design could not examine the stability of the cluster solutions across different time points. Future study should adopt a longitudinal design to test the stability of the cluster solution.The current findings suggest there were four subtypes of alexithymia characterized by different emotional regulation manifestations.ANOVA: analysis of variance; BDI: Beck Depression Inventory; BVAQ: Bermond-Vorst Alexithymia Questionnaire; DDF: difficulty describing feelings; DIF: difficulty identifying feelings; EES: Emotional Expressivity Scale; EHA: extrovert-high alexithymia; EOT: externally oriented cognitive style of thinking; ERP: Event-related Potential; ERQ: Emotion Regulation Questionnaire; FFM: Five-Factor Model; GHA: general-high alexithymia, IHA: introvert-high alexithymia; NA: non-alexithymia; SPSS: Statistical Package for Social Sciences; STAI-T: Trait portion of the State-Trait Anxiety inventory; TAS-20: 20-item Toronto alexithymia scale.The authors declare that they have no competing interests.JC designed the study, collected and analyzed data, and wrote the first draft of the manuscript. RCKC conceived and designed the study, and wrote the first draft of the paper. TX analyzed data and participated in writing up the manuscript. JJ participated in writing up the manuscript. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/33/prepub


To report the microstructural features of Meesmann corneal dystrophy (MCD) in two patients.The first patient was a 10-year-old boy who presented with bilateral visual loss, diffuse corneal epithelial microcystic changes, high myopia and amblyopia. With a clinical impression of MCD, automated lamellar therapeutic keratoplasty was performed in his left eye. Histopathologic examination of the corneal button disclosed epithelial cell swelling and cyst-like intracytoplasmic inclusions. The cells contained moderate amounts of periodic acid-Schiff-positive and diastase-sensitive material (glycogen). Transmission electron microscopy revealed numerous vacuoles and moderate numbers of electron-dense membrane-bound bodies in the cytoplasm, similar to lysosomes, some engulfed by the vacuoles. The second patient was a 17-year-old female with a clinical diagnosis of MCD and episodes of recurrent corneal erosion. On confocal scan examination of both corneas, hyporeflective round-shaped areas measuring 6.8 to 41.4 μm were seen within the superficial epithelium together with irregular and ill-defined high-contrast areas in the sub-basal epithelial region. The subepithelial nervous plexus was not visible due to regional hyperreflectivity.This case report further adds to the microstructural features of Meesmann corneal dystrophy and suggests confocal scan as a non-invasive method for delineating the microstructural appearance of this rare dystrophy. Meesmann corneal dystrophy (MCD) is a rare bilateral corneal epithelial disorder which appears in the first or second year of life and was first described by Pameijer in 1935.3in vivo diagnosis of corneal disorders.Confocal scan is a non-invasive diagnostic tool for rapid evaluation of all corneal layers and A 10-year-old boy presented with bilateral decreased vision since the age of three. His parents were consanguineous but normal on ophthalmic examinations. Best-corrected visual acuity (BCVA) was 20/160 and 20/800 in his right and left eyes with correction of −12.00 and −13.00 sphere, respectively. On slitlamp biomicroscopy, diffuse intra-epithelial microcystic changes were present within the entire corneal epithelium . IntraocThe corneal button was sent in 10% formalin to the pathology laboratory. After bisecting the specimen, one half was processed and embedded in paraffin wax. Sections were prepared and stained with Hematoxylin and Eosin (H&E) for studying the general morphology and by PAS sequence with and without diastase to identify glycogen.A 17-year-old female presented with foreign body sensation and pain in both eyes since 3 years ago without any significant medical or family history. BCVA in her right and left eyes was 20/40 and 20/50, respectively with refractive error of −0.5 sphere in both eyes. On retroillumunation by slitlamp biomicroscopy, there were diffuse (limbus to limbus) intra-epithelial microcystic lesions together with regional haze in both corneas  but otheAfter topical anesthesia, confocal scan 3.0  was performed on both eyes using methylcellulose as a coupling agent between the front lens  and the surface of the cornea. The automatic full thickness and epithelial modes were used to capture images from all corneal layers with particular attention to the anterior parts of the involved cornea. The manual analytic software of the confocal scan was utilized to measure the abnormal confocal findings.Confocal scan examination of both corneas disclosed scattered and well-defined round to oval hyporeflective intracytoplasmic areas measuring 6.8 to 41.4 μm in their largest diameter within the superficial corneal epithelium , diffuseThe histopathologic features of the excised corneal button in our first case were similar to those described by Chiou et al:We observed hyporeflective cystic structures of various sizes in the superficial corneal epithelium on confocal scan examination of the second case, which is similar to findings previously reported for MCD.3in vivo confocal microscopy can provide a non-invasive method for confirming the diagnosis.The diagnosis of MCD is based on clinical findings such as bilateral limbus to limbus microcystic intraepithelial changes on high power slitlamp biomicroscopy,In conclusion, this report could further add to the microstructural information available on Meesmann corneal dystrophy and present new confocal microscopic features of this rare dystrophy.


Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization.To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function.Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. Proteins are not static entities, but exist as a dynamic ensemble of inter-converting conformations. These ensembles exhibit a wide range of spatial and temporal scales of internal motions; localized protein motions involving bond vibrations and fluctuations within a group of few atoms are fast  where as large-scale concerted, collective fluctuations involving sub-domains or entire protein are typically slow  hills and valleys of varying heights and depths, populated by conformations of the protein. Within each valley, the population of conformations share significant similarity in terms of their structures as well as internal energies. The sub-population of protein conformations within each of these valleys represent a sub-state. The multiple levels in the hierarchy stem from the energetic differences (and energy barriers) between the various sub-states. Internal protein motions driven by thermodynamical energy fluctuations allow the protein to transition from one sub-state to another. In cases where several sub-states are separated by small energy barriers from each other but collectively by a larger barrier from other sub-states, together the collection of these sub-states can be viewed as a new sub-state in the multi-level hierarchy.In the familiar representation, one can imagine the potential energy landscape to be rugged and be formed of hidden) conformations and sub-states that critically alter the active site environment for catalysis Internal protein motions correspond to the inter-conversion of protein conformations as they move within a sub-state or as they visit from one sub-state to another Experimental techniques revealed a wealth of information about the inter-connection between conformational fluctuations and protein function. X-ray studies and nuclear magnetic resonance (NMR) methods have provided information about the most populated states  for an increasing number of proteins Computational simulations allow bridging multiple time-scales and provide detailed atomistic insights into protein motions Computational tools to analyze and identify conformational sub-states in the multi-level hierarchy that will enable to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function are still limited. The conformations sampled during MD simulations correspond to a highly multi-dimensional data set due to the large number of degrees of freedom associated with the protein. Characterizing the high-dimensional multi-variate data, which is embodied in these MD simulations, is a long standing problem in statistics and related fields QAA is based on pursuing higher order statistics of positional deviations associated with the conformational data sampled during the MD simulations. Using three different proteins - human ubiquitin, T4 lysozyme and enzyme cyclophilin A - we show that QAA identifies and characterizes the conformational sub-states relevant to function. Based on the inspection of the conformation populations in the sub-states using parameters such as internal energy or other biophysically relevant order parameters, we observe that the identified sub-states contain crucial structural and dynamical elements relevant to promoting the designated function of each of these proteins. A recursive application of QAA yields a multi-level motion hierarchy with global modes dominating the top level and subsequent levels revealing progressively localized motions within the proteins. Additionally, the rare-conformational transitions associated with the interconversion between the identified sub-states allows vital insights into these protein's structure, motions and function.This section is organized as follows. First, the conformational diversity observed in computational (MD) simulations is examined for anharmonic motions. Then, the theoretical details behind QAA are presented. Finally, QAA is illustrated on three different model systems: (1) human ubiquitin, (2) T4 lysozyme, and (3) enzyme human cyclophilin A. The results provide insights into how intrinsic fluctuations in each of these proteins enable the functionally important conformations to be sampled. In the discussion section, we compare QAA with other computational techniques that are also used to characterize the conformational diversity.A common measure for exploring anharmonicity (or non-Gaussianity) is the fourth-order statistic kurtosis, Human ubiquitin is used as a prototypical example to examine the nature of atomic fluctuations. For comparison, we use In e-scales . Side-chUsing a Gaussian fit to the ribution . Ubiquitibutions . Long-tautions  From a biophysical perspective, the joint distributions in positional deviations illustrate a potential and more serious limitation of QHA. Considering the same residues illustrated in quasi-anharmonic analysis (QAA), a method based on diagonalizing a tensor of fourth-order statistics describing positional fluctuations and their couplings. We use an efficient algebraic technique called joint-diagonalization of cumulant matrices (JADE), a well known algorithm in the machine learning literature for analysis of multi-variate data To address the issues of both higher-order correlations and non-orthogonality, as well as address the limitations of QHA, we propose quasi-anharmonic for two reasons: first, we study anharmonicity explicitly whereby the sources are fully decorrelated and higher-order dependencies are minimized; second, we impose a linear model which ignores any non-linear coupling that may exist in the fluctuations between different parts of a protein.We model the observable positional deviation vector, To derive The harmonic modes For exposition, we will set the QHA bases The covariance matrix The fourth order cumulant tensor Since We can reduce the fourth order dependencies by minimizing the sum of the cross-cumulant terms, which is equivalent to diagonalizing the tensor Substituting for Thus, We first illustrate that QAA works correctly in the pairwise distributions considered in Ubiquitin is universally expressed in eukaryotes and plays a fundamental role in the proteosomal degradation pathway by labeling specific proteins. The protein's three-dimensional structure is highly conserved over evolution Here, we considered the  in blue  and cons1 and R2 . In clusWe next examine if these conformational wells exhibit any similarity in terms of their internal energies, defined as the sum of van der Waals and electrostatic energy over all interactions in the protein and computed using the program NAMDEnergy The separation between the high- and low-energy conformations from each cluster, as identified by QAA, provides a unique opportunity to examine the biophysical relevance of the relative populations and its impact on ubiquitin binding. Note that at any given level of the conformational hierarchy, the presence of a minor population of conformations sharing either high- or low- internal energy. These minor populations deviate from the largest heterogenous cluster in exhibiting motions along functionally relevant regions. As one descends the conformational hierarchy, it becomes clear that the flexible regions of the protein do not change; only the amplitude of the actual conformational change changes . These changes in both motions and energetics allow ubiquitin to sample conformations that may in fact exceed the observed diversity in all of its bound conformations. Observe that the top 3 anharmonic modes of motion covers all of the conformational heterogeneity exhibited by the bound X-ray ensemble  as it catalyzes cis/trans isomerization catalyzed by cyclophilin A  , and the0°–100°) , a furthThe comparison of enzyme conformations between these clusters (both at Level 1 and 2) provide insights into the intrinsic dynamical features of the enzyme. The movement along the vectors between these clusters  show that the largest motions is located in the protein regions that are colored in the cis/trans isomerization reaction. The decomposition of the landscape in sub-states allows identification of the conformations that have features relevant to the transition state, and therefore, allows identification of the subtle changes in various dynamically relevant residues. Ongoing analysis of reactive trajectories as they visit these sub-states will allow us to quantify the rates of interconversion and its connection to the reaction kinetics.A careful analysis at Level 2 also indicates that the conserved active-site Phe113 switches conformation from one cluster to another cluster. This induces an important change in the hydrophobic environment in the active-site. Similarly on the other side, the region 13–16 is interconnected to 141–156 and 55–60 eventually allowing catalytically important Arg55 to mediate the substrate orientation through two important hydrogen-bonds . As prevBased on the results from three different proteins, we have illustrated the ability of QAA to delineate events linked to molecular recognition of binding partners and enzyme catalysis under equilibrium and non-equilibrium conditions respectively. In each case, QAA identified energetically coherent conformational sub-states and functionally relevant global motions.The energetic homogeneity in the sub-states discovered by QAA is a consequence of pursuing super- and sub-Gaussian fluctuations explicitly. Gaussian fluctuations arise when atoms are moving under the influence of an harmonic potential well, whereas super- and sub-Gaussian fluctuations are sampled from wells that could have non-harmonic shapes including square well, double-well/multi-well. This is consistent with previous studies that evaluated the nature of atomic fluctuations from picosecond time-scale MD simulations Unlike QHA, the anharmonic modes from QAA need not be orthogonal. Hence, it is possible for these anharmonic modes to activate each other depending on their intrinsic coupling. The coupling coefficient or the interaction strength can be measured as QAA has clear advantages over established methods in segmenting a protein's energy landscape into multi-scale, energetically coherent conformational sub-states, and in identifying novel reaction coordinates. It is also important to note that choice of non-orthogonality amongst the basis vectors in QAA does not limit its ability to define suitable order parameters. Indeed, as we have shown, the zyme see . Furtherme see ; . Thus, iWith QAA we emphasized two statistical properties of internal protein motions: anharmonicity and non-orthogonality. Previous work characterizing anharmonicity in MD simulations used picosecond length trajectories For investigating protein dynamics in collective coordinate space, a number of techniques have been developed for identifying biophysically meaningful directions of the conformational landscape using orthogonal motion basis In the results section, we have motivated how QAA differs from QHA in terms of interpreting overall motions using the joint distributions of positional fluctuations in two dimensions. In higher dimensions  based decoupling approach called full correlations analysis (FCA) for detecting higher-order correlations Overall, by pursuing higher-order statistics and anharmonicity of protein motions, it has been possible to obtain novel insights into the conformational sub-states and transitions between these sub-states that would have been otherwise difficult . Further, examining the non-orthogonal dependencies in atomic fluctuations delineates energetic differences within and between various sub-states in the landscape . The nonProteins are not rigid structures but intrinsically capable of exploring an ensemble of conformations, enabled by a wide range of internal motions. The role of these conformational fluctuations, if any, in the designated functions of the proteins including biomolecular recognition and enzyme catalysis has been challenging to characterize. The challenge partly arises from the fact that the internal protein motions occur on a wide range of time-scales, while the individual experimental instruments only provide access to information corresponding to narrow windows of resolution. Computational methodology recently provided vital insights, due to its ability to provide atomistic level information on a wide range of time-scales. Emerging evidence has indicated the possibility that certain parts of the conformational ensembles (or sub-states) may posses structural features that could be relevant and even vital for the mechanism of designated function. Unfortunately, due to the low probability of finding these conformations in the multi-level hierarchy of a protein's conformational landscape, makes the identification and characterization of these sub-states rather difficult.In this paper, a new methodology QAA is described that allows automated discovery of a hierarchy of sub-states associated with the conformational ensemble of proteins. Utilizing atomistic level MD simulations of proteins or protein in association with other molecules (such as binding partners or enzyme-substrate complex) as input, this methodology pays close attention to the anharmonic nature of internal protein motions and pursues the higher-order statistics of the internal motions. One of the most important advantages of this approach is that it allows clean separation between the conformational sub-states, by projecting the conformations sampled during the MD simulations in a lower dimensional space represented by QAA vectors. Characterization of the populations in these sub-states for any relevant properties  allows the detailed characterization. In addition, to identifying these sub-states, the motions associated within the sub-states and inter-conversion between the sub-states provide new insights in to the inter-relationship between protein structure, motions and function.The use of QAA shows the equilibrium motions of human ubiquitin at the For reactive systems such as the enzyme cyclophilin A, QAA allows characterization of conformational sub-states along the reaction pathway. A hierarchical description of the sub-states along the reaction pathway identifies sub-states with structural and dynamical features critical for attainment of the transition state. Inspection of conformational transitions that allow the enzyme to move from one sub-state to another represents rare-conformational transitions that are intrinsic properties of cyclophilin A. In each of these functionally relevant transitions provides further biophysical insights into the previously identified network of coupled vibrations parm98parm98 force-field for investigating protein dynamics has been verified previosuly − ions, the protein was immersed in a pre-equilibrated SPC MD simulation for T4 lysozyme were initiated from the crystal structure 2LZM All production runs were performed using NVE conditions with periodic boundary conditions. Bond lengths to hydrogens were maintained through out the simulations with SHAKE algorithm. Electrostatic interactions were evaluated using Particle Mesh Ewald (PME) method and the long-range interactions were truncated at 10 Å. A single continuous MD production run of lysozyme was carried out for a total of 120 ns with snapshots being saved every 10 ps, resulting in a total of 12,000 snapshots.His–Ala–Gly–Pro–Ile–Ala based on the PDB structure 1AWQ The human cyclophilin A was modeled as previously described with peptide substrate Figure S1Long-tail distributions at shorter time-scales; side-chains have greater anharmonicity than backbone atoms. Anharmonic distribution of positional deviations (Å) from ubiquitin MD simulations at 5 ns and 50 ns. For each atom, the positional displacement from the time-averaged position was calculated at 50 ps intervals. The same bin size (0.54 Å) was used for all histograms. Distributions correspond to: C(TIF)Click here for additional data file.Figure S2QAA captures intrinsic non-orthogonal directions pointing towards energetically coherent directions in the landscape; QHA and FCA do not. For the ubiquitin simulation (0.5 (TIF)Click here for additional data file.Figure S3Projections of ubiquitin simulation  onto eight top quasi-anharmonic modes  from QAA illustrate distinct separation in energy distributions. Structures are colored according to scaled  non-bonded energies, that is, the sum of electrostatic and van der Waals energy terms. Color bins are thresholded at (TIF)Click here for additional data file.Figure S4Lysozyme simulation projected onto eight top quasi-anharmonic modes  from QAA illustrate distinct separation in energy distributions. Structures are colored according to scaled internal energies, as explained in the main text. Color bins are thresholded at (TIF)Click here for additional data file.Figure S5Lysozyme simulation projected onto six QAA coordinate systems. Axis labels correspond to mode indices ranked by fluctuation magnitude, and were chosen sequentially. Structures are colored according to d(TIF)Click here for additional data file.Figure S6Lack of homogeneity in the internal energy distributions of QHA. For the 0.5 (TIF)Click here for additional data file.Figure S7Ubiquitin landscape represented by the first three basis vectors using dihedral PCA  from the 0.5 s simulations . Projected conformations show the presence of spatial clusters. However, when colored by the scaled internal energy, energetic homogeneity is lacking, unlike in the analogous QAA-based clusters Click here for additional data file.Figure S8Lysozyme simulations projected onto FCA basis. (A) We follow the protocol used in B) Comparison of FCA and negentropy for top 100 FCA modes. Circles indicate the modes selected for the projection coordinates in panel (A) and are sized according to the variance of the associated modes. Note that variance is not a reliable indicator of anharmonicity. (C) Correlation between negentropy and kurtosis for the top 100 FCA modes. Of these modes, 85 display Gaussian statistics ((TIF)Click here for additional data file.Figure S9Analysis of Lysozyme simulations using Full Correlation Analysis. (A) Lysozyme simulation projected onto six full correlation analysis (FCA) coordinate systems  according to procedure in (TIF)Click here for additional data file.Movies S1For ubiquitin, the movies depict the motions of C(MPG)Click here for additional data file.Movies S2For T4 lysozyme the large-scale motions for Level 1 and Level 2 (shown in (MPG)Click here for additional data file.Movies S3For the enzyme cyclophilin A, the movies depict movements of the highlighted regions in (MPG)Click here for additional data file.Text S1Intuition for why QAA finds energetically coherent sub-states(PDF)Click here for additional data file.Text S2Comparison of QAA with dihedral PCA(PDF)Click here for additional data file.Text S3Comparing QAA with Full-Correlation Analysis(PDF)Click here for additional data file.Text S4Movies from QAA for Ubiquitin, Lysozyme and Cyclophilin A(PDF)Click here for additional data file.