Mycobacterium tuberculosis can measure epitope-specific antibody levels in a competition assay. Immunodominant epitopes were defined from clinical samples and related to the clinical spectrum of disease. Antibody to the immunodominant epitopes was associated with HLA-DR15. Occupational exposure showed a different response and was consistent with recognition of dormancy-related proteins and protection despite exposure to tuberculosis (TB). Studies in leprosy revealed the importance of immune deviation and the relationships between T and B cell epitopes. During treatment, antibody levels increased, epitope spreading occurred, but the affinity constants remained the same after further antigen exposure, suggesting constraints on the process of epitope selection. Epitope-specific antibody levels have a potential role as biomarkers for new vaccines which might prevent the progression of latent to active TB and as tools to measure treatment effects on subpopulations of tubercle bacilli.Monoclonal antibodies restricted to  Mycobacterium tuberculosis (Mtb). It has long been clear that BCG vaccination is excellent at preventing primary forms of TB and that the immunodeficiencies caused by HIV infection or by increasing age give rise to these same forms of TB. On the other hand, BCG does not prevent post-primary disease, particularly sputum smear-positive pulmonary tuberculosis (S + PTB). Many animal models which attempt to elucidate the nature of reactivation of latent infection merely recapitulate the same pattern of immunodeficiency found in primary disease.There are many unanswered questions in tuberculosis (TB) for which an understanding of both clinical aspects and the adaptive immune response is critical. Most research has concentrated on the processes of infection and the initial, innate immune response to Post-primary TB is characterized by an immune response to both cross-reactive antigens, as in the tuberculin response, and species-restricted antigens, such as those found in the RD1 sequence, namely esat-6 and cfp-10. Destructive caseation is an essential feature of post-primary disease and much has been made of the difference between apoptotic and necrotic cell death as the pathogenetic mechanism . Sette eThis paper will describe the data available on antibody responses to species-restricted B cell epitopes according to clinical parameters. It will explore whether these immunological markers can discriminate among the clinical states of TB infection and disease. Underlying this discussion remain the problems of why some B cell epitopes are immunodominant, how antibody diversity becomes fixed, whether conformational epitopes are more important than linear epitopes, and the relationship between T and B cell epitopes.A soluble extract from irradiated Mtb, prepared by crushing with glass beads or ultrasonic degradation, gave a better range of antigens than tuberculin . Mouse mThe importance of lipoproteins to the immune response has been demonstrated by deleting the prolipoprotein signal peptidase (IspA) . S + PTBEpitope-specific (TB23 Mab) and antibody levels to the purified 19-kDa antigen  contributed most to improving the serological sensitivity after the 38-kDa antigen in a study of six Mabs and human sera . AntibodHIV–TB co-infection has demonstrated three facts about the role of CD4+ T cells in TB . FirstlyChildren with TB and those with extra-pulmonary (EP) TB did not have antibody to the 38-kDa antigen . PatientOne of the surprising findings in measuring epitope-specific antibody levels was the presence of antibody to the TB68 epitope of the 16-kDa antigen  in nurses on a TB ward with frequent exposure to infectious TB . The bacMycobacterium leprae, and one series which bound predominantly to M. leprae  were able to define two groups, one which reacted equally with LAM derived from both Mtb and . leprae . The str. leprae  and usinhydrates , 30, 31.M. leprae might overlap the binding site of the Mtb-specific Mabs sufficiently to inhibit binding, there being no homolog of the M. leprae 35-kDa antigen in Mtb. Bystander stimulation of B cells seems less likely although the probability of exposure to leprosy would have been less than TB  was found in TB patients . Two exp than TB . Conform than TB . Thus, a than TB .Antibody levels are proportional to antigen levels and strong T follicular helper cell responses can often initiate bystander B cell activation hypergammaglobulinemia , 33. PatIn an acute inflammation, the immune response tends to be focused on a few immunodominant antigens and a phase of chronic inflammation is associated with epitope spreading . AntigenFollowing epitope-specific antibody levels during treatment, despite changes in antibody levels, the affinity constant for the antibody or antibodies to an individual epitope did not change . In HIV Can the changes in epitope-specific antibody levels predict cure or relapse? Preliminary evidence suggests that antibody to the dormancy antigen α-crystallin (TB68 epitope of the 16-kDa antigen) might be helpful in predicting relapse during treatment, but as antibody levels persisted beyond successful treatment, a biomarker of cure still eludes us .Antigen recognition varies across the TB spectrum. Antigen concentration is likely responsible for the immunodominance of epitopes of secreted proteins in S + PTB. There may be a role for measuring antibody/T cell responses to dormancy antigens and some lipoproteins as predictors of disease and biomarkers of protection and response to treatment.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this article, some fundamental topics related to the initial steps of organic film growth are reviewed. General conclusions will be drawn based on experimental results obtained for the film formation of oligophenylene and pentacene molecules on gold and mica substrates. Thin films were prepared via physical vapor deposition under ultrahigh-vacuum conditions and characterized in-situ mainly by thermal desorption spectroscopy, and ex-situ by X-ray diffraction and atomic force microscopy. In this short review article the following topics will be discussed: What are the necessary conditions to form island-like films which are either composed of flat-lying or of standing molecules? Does a wetting layer exist below and in between the islands? What is the reason behind the occasionally observed bimodal island size distribution? Can one describe the nucleation process with the diffusion-limited aggregation model? Do the impinging molecules directly adsorb on the surface or rather via a hot-precursor state? Finally, it will be described how the critical island size can be determined by an independent measurement of the deposition rate dependence of the island density and the capture-zone distribution via a universal relationship. Various organic electronic devices are already on the market, e.g. solar cells, light emitting diodes, displays and radio frequency identification tags, to name just a few. The advantages of organic electronics in comparison to silicon-based technology are manifold. Properties such as low-cost of fabrication, material flexibility, large-scale fabrication by roll-to-roll printing at moderate temperatures and even possibly biocompatibility and biodegradability of the electronic material are scientifically and technologically attractive. Although for many purposes polymers are used, the application of small organic semiconducting molecules becomes increasingly important. In this case, organic thin films are mainly fabricated by physical vapor deposition. While this method weakens some of the above-mentioned advantages, it allows a very precise fabrication and tailoring of the organic films, which is an inevitable prerequisite for detailed investigations of the growth mechanism. However, despite those inherent advantages of physical vapor deposition techniques, many of the fundamental physical processes taking place during adsorption and organic thin film formation are still poorly understood.22H14), p-quaterphenyl (C24H18) and p-hexaphenyl (C36H26) on gold and mica substrates. These molecules are commonly used and increasingly relevant for applications in organic electronics. Pentacene (5A) is the most frequently used material to fabricate organic transistors, due to its high charge carrier mobility  or the Au[112] direction   direction .6P deposited on KCl(001) exhibits a similar temperature dependent layer formation as described above. However, the diffusion probability at equivalent temperatures is much higher than on Au(111). While at and below room temperature only small crystallites form, the deposition at 450 K leads to extremely long needles . The AFM image in 2(AlSi3O10)(OH)2). Generally, well-defined mica substrates are produced by cleaving a mica sample along the basal plane, which produces an atomically flat crystalline surface. Cleavage proceeds along a potassium layer, which yields 0.5 monolayers of potassium in ionized form on each cleavage plane. Since in the lower lying silicon plane every fourth Si4+ ion is substituted by an Al3+ ion, the negative net charge in the subsurface layer and the same number of K+ ions on the surface form a dipole field with components parallel to the substrate i is an ii et al.  is that


Multiple sclerosis (MS) can affect the visual system at all anatomical sites from the retinal nerve fiber layer (RNFL) to the visual cortex. MS demyelinating lesions most commonly occur at the level of the optic nerve, causing acute episodes of optic neuritis (ON) . ON usuain vivo – even applying advanced structural MRI techniques such as diffusion tensor imaging (DTI) – the second hypothesis has been repeatedly tested by functional MRI (fMRI) during visual stimuli or, more recently, resting conditions.While the first hypothesis is extremely difficult to test Adaptive neuroplasticity is formally defined as the reorganization of distributed patterns of brain activity that accompany action, perception, and cognition, and that compensate for impaired function resulting from disease or brain injury . On the Using visual-stimulated fMRI, MS patients in the early stages of ON have consistently shown a reduced response to visual stimuli in the primary visual (striate) cortex when compared to healthy controls (HCs) –6. TheseAlthough visual-stimulated fMRI studies have allowed to comprehend relevant aspects of functional changes occurring in visual cortical areas during ON, they are intrinsically limited by the fact that the measured signal changes mostly reflect the degree of anterior visual pathway damage rather than intrinsic activity of visual cortical areas. Moreover, since optic nerve damage is highly variable between subjects with ON, there is a wide inter-subject variability, which further undermines the results and interpretation of visual-stimulated fMRI studies.Given these premises, resting-state (RS) fMRI has a relevant role as it overcomes most of the abovementioned limitations, and allows to explore spontaneous (self-generated) activity of cortical visual areas.In the study by Roosendaal et al., – the first to explore the visual RS network (V-RSN) together with other major RSNs – the authors did not find any significant changes in both clinical isolated syndrome (CIS) and relapsing remitting (RR) MS patients when compared to each other and HCs. Such findings were not surprising since the study was not designed to explore the relationship between V-RSN changes and ON. Indeed, no historical data on previous ON, no clinical/instrumental evaluation of the visual function, and no measures of optic nerve damage were acquired .Using a similar approach, Faivre et al. explored all main RSNs, including the V-RSN, to compare early RRMS patients with HCs. Similarly to the Roosendaal study, data acquisition and analysis did not take into account previous episodes of ON. Nevertheless, the authors were able to demonstrate functional rearrangements inside multiple regions of the V-RSN , thus showing significant neuroplastic changes of this RSN in MS patients .In order to specifically explore the effect of previous ON on intrinsic V-RSN connectivity, we investigated a population of 30 RRMS patients [16 without (nON-MS) and 14 with (ON-MS) previous ON] and 15 HCs . For thiSince our patients with RRMS showed a significant damage of the anterior visual pathways and until now there have been no other RS studies conducted on ON, we compared our results with those obtained in non-MS diseases affecting the anterior visual pathways, such as Leber hereditary optic neuropathy and early blindness , 15. In Our data, therefore, confirm and complement previous photic-stimulated fMRI studies, showing that visual recovery after ON might be associated with cortical reorganization within extra-striate visual areas , 7, 10. Although the study of the V-RSN has certainly added relevant information on ON-related neuroplastic changes taking place at the level of the striate and extra-striate cortex, the current lack of longitudinal studies do not allow to discriminate between adaptive ad non-adaptive changes.To date, visual plasticity of the visual system in MS has been investigated especially after ON.Relevant cortical functional changes have been consistently described at the level of striate and extra-striate areas using either photic-stimulated or RS-fMRI studies.Available evidences also suggest that some of the observed neuroplastic changes, in particular, those occurring at the level of the occipital extra-striate cortex , might act as adaptive neuroplasticity, thus impacting positively on visual recovery after ON.Once the functional plasticity of the visual system related to ON in MS will be further elucidated it might become a useful model to evaluate the effect of new neuroprotective or (adaptive) plasticity-promoting molecules.Future investigations will have to further assess adaptive and non-adaptive neuroplasticity of the visual system in MS patients. More specifically, cross-sectional and longitudinal studies will have to address, which cortical functional changes are associated to functional recovery after ON. Once the methodology will be set up and standardized in a multicenter context will be able to assess the effect of old or new MS drugs on mechanisms of visual recovery after ON.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aim: This study was undertaken to evaluate the percentage of gutta-percha-filled area (PGFA) using microscopic analysis of the cross-sections in the apical third of root canals when filled either with Thermafil technique, Warm Vertical Condensation technique and Cold Lateral Condensation technique without using sealers.Materials and methods: Sixty single rooted extracted per-manent teeth were collected. After crown amputation, the teeth were randomly divided into three experimental groups of 20 specimens each. Group I–Thermafil obturation technique, group II–warm vertical condensation obturation technique and group III–cold lateral condensation obturation technique. Obturation was performed by specific techniques without using sealers. After obturation, the teeth were cross-sectioned horizontally at 2 to 3 mm from apex with the help of double sided diamond disk. Sections were digitally photographed and measured under Stereomicroscope at magnification of 50×.Using a KS 100 imaging system the area of canals and the gutta-percha was recorded, also the percentage of gutta-percha filled area (PGFA) was calculated. The observations thus ob-tained were subjected to statistical analysis using ANOVA and student ‘t’ test.Results: Maximum group difference was observed between groups I and III (3.558 ± 0.138) while minimum difference was observed between groups I and II (1.223 ± 0.137). Thus, all the between group differences were statistically significant.Conclusion: This study supports the belief that the Thermafil Obturation technique produces significantly higher percentage of gutta-percha filled area (PGFA) than the warm vertical condensation technique or cold lateral condensation technique.How to cite this article: Samadi F, Jaiswal JN, Saha S, Garg N, Chowdhary S, Samadi F, Tripathi VP. A Comparative Evaluation of Efficacy of Different Obturation Techniques used in Root Canal Treatment of Anterior Teeth: An in vitro Study. Int J Clin Pediatr Dent 2014;7(1):1-5. The Thermafil obturation technique produces higher radiopacity, excellent viscosity and fluidity and produces a high degree homogenous mass of gutta-percha in the canal unlike lateral condensation.6In recent years, a number of plasticized gutta-percha techniques have been introduced that have purported to seal the root canal better, like Warm Vertical Compaction technique (WVC) and Thermafil obturation technique which incorporate the use of thermal or frictional heat to plasticize the gutta-percha, allowing for better adaptation to canal walls, higher degree of homogeneity and provide optimum apical and coronal sealing when compared to lateral condensation.Hence, the present study was undertaken to evaluate the percentage of gutta-percha-filled area (PGFA) using microscopic analysis of the cross-sections in the apical third of root canals when filled either with Thermafil technique, WVC technique and CLC technique.Sixty single rooted extracted permanent teeth with single straight canals were used for this study . Teeth wThe teeth were randomly divided into three groups accordingly to the filling technique used (n = 20), CLC tech-nique, WLC technique and Thermafil technique. Obturation was performed by specific techniques without using sealers.A standardized 50 number gutta-percha master cone was fitted in the root canal at the working length and checked for tug-back criteria. Cold lateral condensation was done using standardized finger spreaders. The finger spreader was inserted between the master cone and the canal wall to within 1 mm of the working length. Cold lateral condensation was performed using accessory gutta-percha points until the canals were completely filled. The filling was judged to be completed when size 15 finger spreader could not penetrate beyond the coronal third of the canal. Excess gutta-percha was removed with the hot instrument.A size 50 Thermafil verifier was used to check the size of the canal and thus, the correct Thermafil obturator. The Thermafil plastic obturator (Dentsply) was heated in a Thermaprep Oven (Dentsply) for 30 seconds according to the manufacturer's instructions. Firm apical pressure was used to insert the Thermafil obturator to the working length. A round diamond bur in a turbine handpiece was used to cut the plastic shaft at 1 to 2 mm within the access cavity and the excess gutta-percha was removed with an instrument.For this technique, various hand held pluggers were used. Three pluggers whose diameter were just slightly less than that of canal preparation at any prior level were selected for working on the coronal, middle and apical 1/3rd of canal. After master cone insertion, a spreader was heated to 60°C in a glass bead sterilizer and allowed to plunge 3 to 4 mm into the apical most extent of gutta-percha and was allowed to remain there till it began to cool. Then it was removed and largest prefitted plugger was used to vertically pack gutta-percha mass apically. This procedure was repeated till the canal was fully filled.After obturation, the teeth were cross-sectioned horizon-tally at 2 to 3 mm from apex with the help of double-sided diamond disk. Color studies of sections were taken using a Stereomicroscope at magnification of 50× . The sliThe observations thus obtained were subjected to statistical analysis using analysis of variance (ANOVA), Post hoc and Student ‘t’ test were performed to know the effect of each variable and to reveal the statistical significance. The confidence level of study was proposed to be 95% hence p-value < 0.05 has been considered significant, p-value < 0.01 has been considered highly significant and p-value < 0.001 has been considered very highly significant.It was observed that there was no overlapping in inter-quartile ranges of any of the three study groups.Group I (Thermafil) had significantly higher mean value as compared to the two study groups (groups II and III) and group II (WVC) had significantly higher mean value as compared to group III (CLC). Thus, the differences among all the groups were statistically significant.5The main purpose of endodontic treatment is to clean, shape and fll the root canal space thoroughly and prevent any interchange between the oral cavity, the root canal system and periradicular tissues, providing a barrier to reinfection. The success of endodontic treatment depends on adequate mechanical debridement of root canal and quality obturation with biocompatible material.67 According to various investigators 10 the apical root canal has been found to be less clean than the middle and coronal portions of the root canal suggesting that bacteria remain in the apical canal. In the present study no sealer was used although generally recommended during conventional root canal filling procedure. Peters (1986) and Georgopoulou et al (1995) stated that sealer shrinks upon setting while others are susceptible to dissolution in contact with tissue fluids leading to increase in leakage along the root filling over time.1112 If sealers had been used there would have been variations regarding the width of root canal, the depth of the heat application and also the size of the sealer filled canal area. Although including sealer may facilitate gutta-percha movement, the reasons to leave sealers out were more important.Fogel (1995) and Wu et al (1998) proposed that the quality of root canal filling at the apical third is important because after post space preparation only the apical root filling of 3 or 4 mm in length is left and the cemented post may leak.In the present study maxillary anterior teeth were used in attempts to avoid the presence of isthmus or fattening area, commonly found in molars and mandibular incisors.The prepared teeth were randomly divided and obturated in following three groups (n = 20 per group) namely Thermafil technique (group I), WVC technique (group II) and CLC technique (group III).1314Cold lateral condensation technique is a common method for obturation. According to Gordon et al (2005) and Xu et al (2007) CLC technique serves as the gold-standard against which new techniques are compared. In the present study, though the density of gutta-percha was found to be relatively good, but obvious voids and spreader tracts were apparent in the cross-sections.The other two groups (WVC technique and Thermafil technique) with PGFA greater than that of CLC utilized some form of heat to plasticize the gutta-percha.15 It is possible that heated gutta-percha simply fows and expands around the pluggers instead of being forced in to the irregularities. These could be the reasons why WVC technique showed less percentage of gutta-percha filled area compared to the Thermafil technique.Warm vertical condensation technique should result in the plasticizing of the gutta-percha apical to the heat carrier. It can be concluded that after WVC the quality of the adaptation of gutta-percha to the wall of the apical root canal varies and the sufficient amount of gutta-percha present in the apical canal and sufficient heating are essential in achieving a good adaptation in canals of widely varying diameter. Venturi and Breschi (2004) reported that multiple heating has shown to produce high levels of gutta-percha shrinkage.The group filled by the Thermafil system at the apical third  revealed the best results. Thermafil samples presented a more homogeneous mass which included gutta-percha and the plastic carrier. Thermafil obturators are flexible plastic carrier coated with alpha phase gutta-percha while extruder of element obtu-rating unit contains beta phase gutta-percha. Alpha phase gut-tapercha has low melting temperature and good adhesiveness whereas beta phase gutta-percha has high melting points and no properties of adhesion. Hence the potential for shrinkage of thermoplasticized gutta-percha as used in Thermafil should be lesser than element obturation unit it has excellent viscosity, fluidity and enhanced adherence replicating the root better and have fewer voids.The thermoplastic gutta-percha techniques are simpler than the lateral condensation technique which means the operator is less subjected to fatigue, but such consideration must be subordinate to the primary goal of achieving the best prognosis for the patients.After obturation, each sample was sectioned horizontally at 2 to 3 mm from the apical foramen using low-speed saw  with a diamond disk (125 × 0.35 × 12.7 mm – 330c) and continuous water irrigation in order to prevent overheating. The specimens were polished before they were examined under stereomicro-scope.This study was designed to quantify the gutta-percha component on percentage basis in order to provide a measure of quality. As observed in the present study, the mean PGFA was found to be maximum in group I (Thermafil technique) followed by group II (WVC) and group III (CLC technique).On the basis of the results and statistical analysis, it can be concluded that: Cold lateral obturation technique resulted in definite voids and gaps between the gutta-percha and canal interface at apical third of root canals. Warm vertical condensation technique produce a homogenous mass of gutta-percha with reduced voids and increased adaptation as compared to cold lateral obturation technique. Thermafil obturation technique exhibited least voids and gaps compared to other tested techniques. Obturation of root canals with alpha-phase gutta-percha on a plastic core-carrier, Thermafil, resulted in a more dense and well adapted root canal fll at the apical third in comparison to the cold lateral obturation technique and WVC technique. None of the tested obturation techniques provided a gap-free or void-free root canal filling.


Reproducible definition and quantification of imaging biomarkers is essential. We evaluated a fully automatic MR-based segmentation method by comparing it to manually defined sub-volumes by experienced radiologists in the TCGA-GBM dataset, in terms of sub-volume prognosis and association with VASARI features. MRI sets of 109 GBM patients were downloaded from the Cancer Imaging archive. GBM sub-compartments were defined manually and automatically using the Brain Tumor Image Analysis (BraTumIA). Spearman’s correlation was used to evaluate the agreement with VASARI features. Prognostic significance was assessed using the C-index. Auto-segmented sub-volumes showed moderate to high agreement with manually delineated volumes (range (r): 0.4 – 0.86). Also, the auto and manual volumes showed similar correlation with VASARI features . The auto-segmented contrast-enhancing volume and post-contrast abnormal volume showed the highest AUC , comparable to manually defined volumes . BraTumIA and manual tumor sub-compartments showed comparable performance in terms of prognosis and correlation with VASARI features. This method can enable more reproducible definition and quantification of imaging based biomarkers and has potential in high-throughput medical imaging research. Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults and it is among most lethal primary tumors, characterized by an unfavorable prognosis with a 5-year survival rate of 5%Studies have shown that phenotypic features, captured non-invasively by structural magnetic resonance (MR) imaging, are able to stratify GBM patients into distinct prognosis groups356Also radiogenomic analyses have linked specific MR phenotypes to gene expression profiles in GBM, where molecular characteristics of tumors can be predicted by extraction of characteristic MR features of the necrotic core and the contrast-enhanced tumor rim8910To elucidate meaningful associations between imaging features and survival or genomic alterations of GBM, accurate and reproducible measurement of MR features is critical121415et al. proposed an automated segmentation algorithm to define proportions of MR-GBM sub-volumes, which Mazurowski et al. associated with survival on 68 TCGA patients, however without comparison with expert defined sub-volumes or external validation of the segmentation algorithm19Recently, a dedicated fully automated segmentation tool for brain tumors (BraTumIA), has shown to provide statistically equivalent tumor volumes to those manually defined by medical experts, in terms of volume and spatial overlapWe hypothesize that automatic MR based sub-volume tumor segmentation of GBM will result in comparable prognostic power and association with VASARI data, compared to manually defined sub-volumes.The purpose of this study is to evaluate the prognostic value of GBM tissue compartments segmented fully automatically using BraTumIA as compared to those manually delineated by expert physicians in the TCGA-GBM datasetFurthermore, we compared the resulting automatically segmented tumor sub-volumes with the VASARI2324MRI sets were downloaded from the Cancer Imaging archive (TCIA) for 109 patients. This is a subset of the patients that were included in the Cancer Genome Atlas projectThe Brain Tumor Image Analysis (BraTumIA) software was employed for automatic segmentation of MR sets. The software requires the user to load the four respective MRI modalities . First, the images are preprocessed, which encompasses skull stripping followed by multimodal rigid image registration (where the post-contrast T1-weighted image serves as template). Second, segmentation is performed via voxel-wise tissue classification. Every voxel is classified into either unaffected tissue  or tumor tissue . This process can be subdivided into three subsequent stages: a feature extraction yielding a voxel-wise feature vector, a supervised classification step and a spatial regularization. The feature vector is composed of appearance-sensitive  and context-sensitive features . The classifier is a decision forest that outputs for every voxel and tissue type a corresponding probability. The predicted tissue class label of a voxel is chosen as the one with the highest probability. The regularization serves as a refinement of previously generated label maps and is conducted in a hierarchical manner27et al.Prior to the application to new patient data, the segmentation method was trained on 56 high-grade glioma patient image sets not included in the present study. These image sets encompassed the training data used in the study of Porz The segmentation method of BraTumIA was ranked among the top performing methods at the MICCAI Brain Tumor Segmentation Challenges (BRATS) 2012 and 2013The software platform Velocity AI  was used to manually delineate, slice-by-slice, a region containing the abnormal signal detected in the post-contrast T1w images, which included the contrast enhancing and necrotic regions for 109 patients.Similar regions of interest were manually drawn for FLAIR images, encompassing the tumor’s total abnormal signal detected by FLAIR, including all the bright areas indicative for edema as well as abnormal regions. The FLAIR region of interest was restricted to the ipsilateral side of the tumor and did not include signals that may be originated due to ventricular spread, aiming to estimate the overall tumor involvement.Contrast enhancement and necrosis volumes were determined from the contours drawn on the post-contrast T1w images on an assisted mode using FASTA post contrast abnormal volume (PCAV) was defined as the sum of the necrotic and contrast enhancing volumes. The total abnormal volume (TATV) was defined as the entire abnormal signal in the FLAIR images. The edema region (FLAIR envelope) was defined as the difference of the PCAV from the TATV .Volumetric agreement between the automatically segmented volumes and manual contours was assessed using Spearman rank correlation for all volumes. Because the non-enhancing tumor region was only defined by the automatic segmentation method, we re-defined the automatic edema volume as the sum of auto edema and auto non-enhancing regions, for comparison with the manual edema volume.We also compared proportion features derived from the automatically segmented volumes against semi-quantitative measurements scored using the VASARI visual feature guideNext, the prognostic performance of the automatic or manually segmented volumes on pre-operative MRI was assessed using the concordance index (CI), which is the probability that among two randomly drawn samples, the sample with the higher risk value has also the higher chance of experiencing an event  and takes time to event and censoring into account.As endpoints we used overall survival and 1 year survival calculated from the start of treatment, a clinically important endpoint for GBM patients. For the 1 year survival endpoint, data were excluded if patients were alive and their follow-up time was less than one year (n = 10). 1 year survival predictive performance was evaluated using the area under the curve (AUC) of the receiver operator characteristic (ROC). Additionally, we reported the lower and upper bounds of the confidence interval for all CI/AUC values.To assess if a CI/AUC is significantly different than random we used the “noether” method as implemented in the R survcomp packageTo evaluate if the different sub-volumes had complementary information in a multivariate setting, we used a minimum redundancy maximum relevance (mRMR) algorithm implemented in the mRMR R packageThe mRMR algorithm returns a univariate rank of features; we trained logistic regression models adding features one by one starting by the highest ranked feature. We cross-validated the performances of each model with resamples and retained the model with the highest mean AUC.Statistical analyses were performed in R .We investigated the correlation between the automatic and manual techniques to define the tumor sub-compartments: necrosis volume, contrast enhancing volume, edema volume (FLAIR), and the composed post-contrast abnormal volume and the total abnormal tumor volume. Scatter plots depicting these correlations are shown in r = 0.4. High Spearman’s correlations were observed for the contrast enhancing and FLAIR envelope regions, r = 0.79 and r = .77, respectively. Because the non-enhancing tumor was not manually defined, we compared the manual edema region with the sum of the auto-segmented non-enhancing and edema regions. This resulted in an increased correlation of r = 0.80, and suggests that in terms of volumetric association, the sum of auto edema and non-enhancing regions reflects more closely the manually defined edema sub-volume.The Spearman’s rank correlation between both segmentation techniques for defining the necrosis volume was r = 0.86 and r = 0.86, respectively.Strong correlations were observed for both tumor-definition methods for the PCAV (necrosis + enhancing regions) and the TATV , The VASARI features were available for 80 patients. To evaluate the correlation between the auto-segmented volumes and the VASARI features, four relevant percent statistics were calculated: proportion of enhancing tumor, proportion of non-enhancing tumor, proportion of necrosis and proportion of edema.We used the TATV as denominator to determine the sub-volume relative proportions.The Spearman correlations for the proportion of contrast-enhancing, necrosis and edema with their respective VASARI features were: r = 0.35, 0.43 and 0.36 (p < 0.001). The non–enhancing proportion of the tumor did not show a correlation with its corresponding VASARI feature .Additionally we evaluated if the manual volumes are associated with the VASARI features. The Spearman’s rank correlations with the VASARI features were 0.17 (p = NS), 0.67 (p < 0.001) and 0.41 (p < 0.001), for the percentage of contrast enhancing, percentage of necrosis and percentage of edema, respectively.The median follow up of all patients was 10.9 months (range: 0.2–56.2 months). At the time of last follow-up, 81.5% of the patients had deceased and 18.5% were alive. One year survival rate was 44.4%. One case was excluded as survival and follow-up times were not available.The prognostic performance of the automatic and manual volumes was determined using the C-index. For the 1 year survival endpoint, we used the area under the curve (AUC) of the receiving operating characteristic (ROC), which is identical to the C-index but can be used for binary classifications.Contrast enhancing volume and PCAV were significantly prognostic for both segmentation methods. Necrosis was significantly prognostic only for the manual contours. For necrosis, both prognostic performance measures were significantly higher for the manual volume as compared to the automatic volume.Edema (FLAIR) did not show significant prognostic value for any of the segmentation techniques. Interestingly, the TATV which encompasses all tumor sub-compartments and the edema did not show significant prognostic power, except for the manual TATV C-Index. This underlines the importance of performing a meaningful sub-division of the entire GBM lesion into distinct sub-compartments with distinct prognostic implications.p < 0.001) and PCVA  as defined by the auto-segmentation algorithm. However, these AUCs were not significantly higher than those estimated for the manual contrast-enhancing and PCVA volumes. The non-enhancing region, only segmented by the automatic technique, did not show significant CI or AUC values, 0.54 (p = 0.2) and 0.60 (p = 0.08), respectively.The highest AUC values were observed for the contrast enhancing region  did not improve the logistic regression model accuracy.Medical imaging is moving towards a quantitative computational science, where automated methods for tumor definition and characterization are increasingly being developed and appliedet al. proposed an automatic segmentation algorithm developed and evaluated on 73 TCGA patients, which was compared with manual references in terms of volumetric correlationet al. applied this method to 68 TCGA patients and evaluated automatically defined GBM features for prediction of survival. This study did not compare the automatically defined volumes with those defined by medical experts in terms of prognosisExtensive literature has reported on the inter-observer variability with respect to tumor definition in GBM12356This is to our knowledge, the first study comparing the prognostic performance of automatically segmented GBM sub-compartments with their manually defined equivalents. We showed that the contrast enhancing volume and the post contrast abnormal volume (necrosis + contrast enhancing) had the highest ability to predict overall and one year survival. However these predictive values were not significantly higher than those obtained with the manual sub-volumes. Interestingly, the total abnormal tumor volume, encompassing necrotic, enhancing and non-enhancing tumor portions plus edema did not show a significant prognostic power, except for the manually defined TATV CI. This drop in prognostic performance may be driven by the edema, which was not prognostic for both methods. This emphasizes the importance of distinguishing the GBM tumor sub-compartments in terms of prognosis.Necrosis was prognostic for the manually defined volume only. We observed as well that the necrotic region was significantly smaller for the auto-segmentation algorithm, as compared to the manual volumes. However the manual definition of necrosis was done by a binary classification of the initial manually delineated region encompassing the necrotic core and the contrast-enhancing tumor region. This binary classification was performed by applying an intensity threshold that varies across patients and inevitably introduces variability to the definition of the necrotic and contrast-enhanced sub-volumes. This may also explain why the manual necrotic volumes are significantly larger than the automatic ones, while conversely the contrast-enhanced volumes are significantly smaller for the manual method.Overall, the sub-volumes segmented automatically appeared to be comparable in terms of prognosis to the manually delineated sub-volumes. The mRMR-based multivariate feature selection indicated that the auto contrast-enhancing sub-volume had the highest predictive accuracy and that adding other volumes  did not improve the prediction accuracy.We compared the auto-segmented volumes with relevant VASARI features. The results showed that the non-enhancing relative proportion of the tumor showed no correlation with the VASARI non-enhancing proportion. This perhaps due to the difficulty, even for expert raters, to define the non-enhancing tumor volume. The correlations for the percent of necrosis, contrast-enhancing and edema with their corresponding VASARI features ranged from weak to moderate, however statistically significant in all cases. This correlation was not significant for the manual contrast-enhancing portion, while it was stronger for the manual necrotic portion and its corresponding VASARI feature.The dataset evaluated in this study is a subset of a larger cohort of patients included in the TCGA consortiumA limitation of this algorithm is that it has been developed for pre-operative MR sets only. Future developments should involve its extension to longitudinally acquired imaging sets that would enable volumetric measurements of tumor response to treatment, i.e. evaluation of radiological response rates and the quantification of the extent of tumor resectionThe end goal of auto-segmentation algorithms is to enable reproducible definition and quantification of imaging based markers that have prognostic implications. Thus further evaluation of this algorithm will involve larger patient cohorts, evaluation of its influence in defining intra-tumoral imaging metrics as well as longitudinal analyses of tumor response to treatment.How to cite this article: Velazquez, E. R. et al. Fully automatic GBM segmentation in the TCGA-GBM dataset: Prognosis and correlation with VASARI features. Sci. Rep.5, 16822; doi: 10.1038/srep16822 (2015).


Meta-analyses have become an essential tool in synthesizing evidence on clinical and epidemiological questions derived from a multitude of similar studies assessing the particular issue. Appropriate and accessible statistical software is needed to produce the summary statistic of interest.Metaprop is a statistical program implemented to perform meta-analyses of proportions in Stata. It builds further on the existing Stata procedure metan which is typically used to pool effects  but which is also used to pool proportions. Metaprop implements procedures which are specific to binomial data and allows computation of exact binomial and score test-based confidence intervals. It provides appropriate methods for dealing with proportions close to or at the margins where the normal approximation procedures often break down, by use of the binomial distribution to model the within-study variability or by allowing Freeman-Tukey double arcsine transformation to stabilize the variances. Metaprop was applied on two published meta-analyses: 1) prevalence of HPV-infection in women with a Pap smear showing ASC-US; 2) cure rate after treatment for cervical precancer using cold coagulation.The first meta-analysis showed a pooled HPV-prevalence of 43% (95% CI: 38%-48%). In the second meta-analysis, the pooled percentage of cured women was 94% (95% CI: 86%-97%).metaprop, no studies with 0% or 100% proportions were excluded from the meta-analysis. Furthermore, study specific and pooled confidence intervals always were within admissible values, contrary to the original publication, where metan was used.By using The online version of this article (doi:10.1186/2049-3258-72-39) contains supplementary material, which is available to authorized users. Meta-analyses combine information from multiple studies in order to derive an average estimate. Different meta-analysis procedures exist depending on the statistic to be reported. Examples of statistics of interest include association measures such as risk difference, risk ratio, odds ratio, difference in means, or simply one-dimensional binomial or continuous measures such as proportions or means.There are three important aspects in meta-analysis: a) the analysis framework, b) the model and c) the choice of the method to estimate the heterogeneity parameter. These aspects interact with each other. A meta-analyst has a choice between the fixed- and random-effects model.In the fixed-effects model, it is assumed that the parameter of interest is identical across studies and the difference between the observed proportion and the mean is only due to sampling error. In the random-effects model, the observed difference between the proportions and the mean cannot be entirely attributed to sampling error and other factors such as differences in study population, study designs, etc. could also contribute. Each study estimates a different parameter, and the pooled estimate describes the mean of the distribution of the estimated parameters. The variance parameter describes the heterogeneity among the studies and in the case where the variance is zero, this model simply reduces to the fixed-effects model.There are three frameworks in modeling of binomial data. The most popular framework uses approximation to the normal distribution by use of transformations and is known as the approximate likelihood approach , 2. SomeThe second approach recognises the true nature of the data and is known as the exact likelihood approach. In this framework, the special relationship between the mean and the variance as characterised by binomial data is captured by the binomial distribution . The betThe third approach is a compromise between approximate and exact likelihood. In the first stage, the data is modeled using the binomial distribution. In the second stage, the normal distribution is used after the logit transformation to model the heterogeneity among the studies. This is an emerging approach and is often recommended by statisticians . Most stThere are three popular methods to estimate the parameters. The non-iterative method popularised byDersimonian and Laird . The othmetan interval which is automatic when the Freeman-Tukey double arcsine(ftt) option is enabled. We used the score confidence intervals for the individual studies.We extracted data that generated Figure two in Dolman et al.  see Fig. Since t. metaprop nb_cured nb_treated, random by(region)ftt cimethod(score)/* */label(namevar = study) graphregion(color(white)) plotregion(color(white))/* */ xlab /* */ xtick force/* */ xtitle) nowt stats /* */ olineopt(lcolor(black) lpattern(shortdash)) /* */ diamopt(lcolor(black)) /* */ boxopt(msymbol(S)) rcols(col)/* */ astext(70) texts(80) nohet notablemetaprop) presents the study-specific proportions with 95% score confidence intervals, the regional and overall pooled estimates with 95% Wald confidence intervals, I2 statistic, and test of significance of the overall pooled estimates. In contrast with Figure metan), all the confidence intervals have admissible values.Figure metaprop_one which requires Stata 13 to fit the generalized linear mixed model (GLMM).We extracted data that generated Figure two in Dolmanet al.  see Fig. We fit . metaprop_one nb_cured nb_treated, random logit groupid(study) /// label sortby(year author) /// xlab xline) /// ti color(blue)) /// subti color(blue)) /// xtitle) nowt nostats /// olineopt(lcolor(red) lpattern(shortdash)) /// diamopt(lcolor(red)) pointopt(msymbol(s) msize(2)) /// astext(70) texts(100)Chi2 statistic of Likelihood ratio (LR) test comparing the random- and fixed-effects model, the estimated between-study variance and test of significance testing if the estimated proportion is equal to zero. The P-value for the LR is 0.022 indicating presence of significant heterogeneity. From the previous command, the Q-statistic is analogous to the LR statistic. In contrast with Figure metan), all the confidence intervals have admissible values. The estimated pooled mean and the corresponding 95% intervals are similar to those obtained earlier  option avoids exclusion of studies with 0%. or 100% prevalence. While this ensures that the studies are retained, the confidence intervals for the pooled estimate may yield inadmissible values.Furthermore, use of Wald confidence intervals for the individual studies when the estimated proportion is close to zero often yields inadmissible values. This is because the Wald confidence intervals are always symmetric around an estimate. In contrast to the Wald, the exact or score confidence intervals can be asymmetric especially near the extreme values. By computing the exact or score confidence intervals for the individuals studies, we are guaranteed of admissible values. While the exact confidence are regarded as the ‘gold’ standard, we recommend the use of score confidence intervals because the coverage is close to the nominal level, whereas the coverage is always higher than the nominal level for the exact method. By using the Freeman-Tukey double arcsine transformation, all the studies are retained, furthermore, we are guaranteed to have admissible confidence intervals for each individual study as well as for the pooled proportion. While the distribution of the Freeman-Tukey double arcsine statistic is more normal for sparse data, the procedure breaks down with extremely sparse data and should thus be used with caution . Whenevemetaprop enables epidemiologists to pool proportions in Stata, avoiding problems encountered with metan. metaprop allows inclusion of studies with proportions equal to zero or 100 percent, and avoids confidence intervals exceeding the 0 to 1 range. The logistic-normal random-effects model draws the users a step closer towards the use of exact methods recommended for binomial data.


Currently, clinical examination, ultrasound scanning (with or without fine needle aspiration cytology), preoperative CT-scan and MRI are available for the differential diagnosis of parotid gland swelling. A preliminary non-invasive salivary diagnostic tool may be helpful in the clinical decision making process. Altered salivary micro-RNA (miRNA) expression levels have been observed in saliva from patients with various cancers. Therefore, we investigated miRNA expression levels in saliva samples from patients with a parotid gland neoplasm using Human miRNA cards in comparison to controls.In the discovery phase, eight miRNAs were identified having different expression levels in patients compared to controls. In the validation phase, the differences in miRNA expression levels between patients and controls were confirmed for seven out of eight discovered miRNAs (p < 0.001). A combination of two miRNAs yielded a receiver-operator-characteristics curve with an AUC of 0.94 . Validation of discovered miRNAs in segregated collected parotid saliva revealed that expression of these miRNAs differ between whole saliva and parotid saliva.A two miRNA combination can predict the presence of a parotid gland neoplasm. Furthermore, this study suggested that the identified, patient-specific, salivary miRNAs were not derived from the parotid gland itself. MicroRNAs (miRNAs) are small non-coding RNAs consisting of 19–25 nucleotides. They are involved in various cellular processes such as cell differentiation, proliferation and survival. MiRNAs inhibit translation by binding to complementary sequences in the 3’ UTR of multiple target mRNAs, usually resulting in their silencing .Many studies have demonstrated the differential expression of miRNAs between cancer and the related normal surrounding tissue. Specific miRNA expression profiles were identified in a number of cancers ,5–9. FluFor clinical decision making, it is important to determine whether salivary gland swelling is due to a neoplasm or a reactive process. Nowadays, clinical examination and invasive methods such as ultrasound scanning (with or without fine needle aspiration cytology), preoperative CT-scan and MRI are the most common diagnostic methods used .In the present study, we have investigated whether salivary miRNA expression profiles of whole saliva from patients with a parotid gland neoplasm differ from those of healthy controls. We found that the expression levels of seven validated miRNAs were higher in whole saliva from patients compared to controls. Using a combination of two of these, it was possible to discriminate between patients with a parotid gland neoplasm and controls. Furthermore, we found differences in the expression of the validated miRNAs between whole saliva and parotid saliva from the same patient.For the discovery phase, unstimulated whole saliva samples from 20 patients with a parotid gland neoplasm, obtained from the Salivary Gland Tumor Biorepository (SGTB) at the MD Anderson Cancer Clinic in Houston, TX, USA were tested. Data with regard to the miRNA expression in this group has already been described . Ten unsThe miRNAs emerging from the discovery phase were validated in an independent sample set of whole saliva. This set consisted of 32 randomly selected unstimulated whole saliva samples from patients with a parotid gland neoplasm and was obtained from the SGTB; 14 were collected at the Department of Otolaryngology and Head-Neck Surgery, VU University medical center, Amsterdam, The Netherlands.Unstimulated whole saliva samples from 14 healthy controls were collected at the Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands .Besides whole saliva, we also collected parotid saliva from 12 patients with a parotid gland neoplasm. Parotid saliva samples were collected separately from both the affected and the contralateral  parotid gland using salivettes  at the VU University medical center. As a control, parotid saliva was collected from 14 healthy controls. All parotid saliva was collected upon stimulation with 2% citric acid .MiRNA profiling of saliva samples from our previous study  was re-eThe Ct value is defined as the cycle number in the fluorescence emission which exceeds that of a fixed threshold. A Ct of 15–30 was considered high expression, and a Ct of 35 was considered low expression. A Ct value above 40 was considered as undetectable miRNA. We calculated ∆Ct by subtracting the Ct value of the reference gene, RNA polymerase III transcribed U6 snRNA , from thEight miRNAs which showed the highest difference in expression between the samples from patients with a parotid tumor and normal controls and were overlapping between malignant and benign neoplasms were selected for further testing in the validation phase. The selected miRNAs were validated by RT-qPCR using Taqman miRNA assays in an independent sample set consisting of 46 saliva samples from patients with a parotid salivary gland tumor and 14 saliva samples from controls. RT-qPCR was performed as previously described . BrieflyRT-qPCR was used to analyze parotid saliva for the presence of the validated miRNAs. Total RNA was isolated from parotid saliva as described above. Parotid saliva samples were analyzed for the presence of miRNAs using Taqman microRNA assays as described above for validation. All qPCRs were performed in duplicate for all candidate miRNA and negative controls (in which RNA was omitted). For miRNA qPCR experiments, U6 snRNA was used as the reference miRNA.http://www.r-project.org/).In the discovery phase, miRNA profiling data was analyzed for miRNAs that were absent and/or present in whole saliva from patients with a parotid gland neoplasm in comparison to controls. Wilcoxon rank-sum test was performed to compare the expression of miRNAs in samples from patients with a parotid gland neoplasm to controls in order to determine the level of significance for the miRNA biomarkers. The miRNAs emerging from the discovery phase were validated in an independent set of 60 saliva samples from 46 patients with a parotid gland neoplasm and 14 controls. Wilcoxon rank-sum test was used to compare the ∆Ct values for miRNAs between the groups. A two-sided p-value less than 0.05 was deemed statistically significant. Next, multivariate logistic regression analysis was used to construct a classification model to discriminate between patients with a tumor and healthy controls. Backward-stepwise model selection criterion was used to obtain a final model. Specifically, we started fitting a prediction model with all the miRNA biomarkers that differentially expressed between the two groups. In each model reduction step, the miRNA that was the least significant was removed. We continued by successively re-fitting reduced models and applying the same rule until all remaining miRNAs were statistically significant. ROC curves were constructed to determine the diagnostic/predictive values of individual and combined biomarkers from the logistic model. Area under the curve was computed via numerical integration of the ROC curves. The performance of the model for classification was assessed by identifying the cut-off point of the predicted probability which yielded the largest sum of sensitivity and specificity. Leave-one-out cross-validation was used to validate the logistic regression model and the cross-validation error rate was reported. All statistical analyses were performed by statistical software R Version 2.15.0 , for patients and healthy controls from VU university medical center and the Academic Centre for Dentistry Amsterdam. Also, permission was obtained from the SGTB at MD Anderson Cancer Clinic (IRB: LAB07-0383), the Samsung Medical Center (SMC IRB file #: 2008-01-028-016) and the UCLA Institutional Review Board (UCLA IRB) (ref number: FWA00004642). All participants were adults (> 18 yrs of age) and gave their written informed consent.ΔCt) in whole saliva samples from patients with a parotid gland neoplasm compared to their expression levels in whole saliva from healthy controls. In addition, 3 miRNAs  had a significantly higher expression level in whole saliva from controls compared to patients with a parotid gland neoplasm  and miRNA expression profiles in whole saliva from controls were established using Taqman Human MicroRNA cards. 742 miRNAs were analyzed . Of thesneoplasm .The expression levels of the 8 miRNAs identified in the discovery phase were validated by RT-qPCR in a separate independent sample set consisting of whole saliva samples from 60 individuals: 46 patients with a parotid gland neoplasm and 14 healthy controls . Four ofMultivariate logistic regression models with these miRNA biomarkers were constructed for the classification of patient samples into parotid gland neoplasm and healthy categories. The initial full model consisted of the seven significant miRNAs from A box-and-whisker plot predicting the probability of neoplasm  was consNext, we investigated whether the 7 validated neoplasm-associated miRNAs in whole saliva from both patients and controls were derived from the affected parotid gland. From 12 patients, saliva from both the affected as well as the healthy parotid gland were collected separately. Similarly, saliva from both parotid glands was collected from 14 healthy controls. Whole saliva samples from these patients and controls had already been analyzed in the validation phase. Two of the 7 validated miRNAs (hsa-miR-425-5p and hsa-miR-1825) were expressed in parotid saliva. The expression levels of these miRNAs differed only marginally between parotid saliva of patients and controls, or, within patients, between saliva from the affected and the non-affected parotid glands. However, the expression level of miRNAs in parotid saliva samples from patients was much lower than in their corresponding whole saliva samples . The expIn this study, we investigated and validated differences of miRNA expression levels in saliva from patients with a parotid gland neoplasm and healthy controls. The expression of miRNA in whole saliva from patients with a parotid gland neoplasm differs from the expression of miRNA in whole saliva from controls. The expression level of 7 miRNAs was higher in saliva from patients compared to controls and could be confirmed in an independent sample set. A combination of two of these miRNAs (hsa-miR-1233 and hsa-miR-211) enabled a predictive value with a sensitivity of 91%, and a specificity of 86% for predicting the presence of a parotid gland neoplasm.Today, the invasive fine-needle aspiration cytology (FNAC) technique is one of the most commonly used techniques for the diagnosis of a swelling of the salivary gland. A systematic review with regard to the performance of FNAC in parotid gland lesions concluded that FNAC had a specificity of 97% and a sensitivity of 80%. However the performance variability was relatively large . TherefoStrikingly, we observed differences in the expression of miRNA between the discovery and validation phases. In the discovery phase, hsa-miR-103a-3p, hsa-miR- 211 and hsa-miR-425-5p were expressed higher in whole saliva samples from controls compared to whole saliva samples from patients. In contrast, these miRNAs had a higher expression level in whole saliva from patients compared to controls in the validation phase. Such differences in miRNA expression have been shown before, highlighting the importance and necessity of validating biomarkers in an independent sample set . Inconsiwww.miRBase.org) as to whether the discovered miRNAs had validated targets. Three of the discovered miRNAs  had validated targets. It has been shown that hsa-miR-211 targets tumor suppressor cadherin 5, which promotes colorectal cancer cell growth in vitro and in vivo [BCL2) a member of the Bcl2-family of apoptosis regulator proteins. A high expression of hsa-miR-296-5p may result in suppression of BCL2 transcription which, in turn, could lead to higher rates of cell death. One of the many targets of hsa-miR-425-5p is Dicer1, a RNase endonuclease III enzyme that is necessary for miRNA maturation. A study describing the expression of Dicer1 in mucoepidermoid carcinoma found that Dicer1 was expressed higher in MEC compared to surrounding normal tissue [Because miRNAs are known to have a gene regulatory role, we conducted an unbiased search  were expressed in parotid saliva from patients and controls. The expression of hsa-miR-103a-3p, hsa-miR-211, hsa-miR-296-5p, hsa-miR-1233 and hsa-miR-1267 in whole saliva and the absence of these miRNAs in parotid saliva suggest that these miRNAs are not specifically expressed and/or secreted from the parotid gland tumor. The differences in miRNA expression which we observed between unstimulated whole saliva  and stimThe initial data presented in this study are a first step toward developing a clinical application to distinguish parotid salivary gland tumors from healthy parotid glands. Future studies should be directed to further unravel the differences between whole saliva and parotid saliva. Although whole saliva is more easily accessible, we cannot conclude, at this moment, which source gives more robust data for tumor diagnostics. Moreover, in the future, differences in miRNA expression in different histological tumor types should also be taken into consideration. Our results justify and urge further investigation towards the discovery of potentially relevant miRNAs in saliva.S1 TableIn the discovery phase, the expression of miRNAs in whole saliva from patients with a parotid gland neoplasm (n = 20) were compared to the expression of miRNAs in whole saliva from controls (n = 10).(XLSX)Click here for additional data file.S2 TableData of the validation of 8 miRNAs in an independent sample set consisting of 46 whole saliva samples from patients with a parotid gland neoplasm and 14 whole saliva samples from controls.(XLSX)Click here for additional data file.


In addition, MVPA with a whole-brain searchlight approach was implemented and classification maps were entered into a second-level permutation based random effects models using statistical non-parametric mapping  is known to give robust estimates of the temporal voice areas (TVAs). However, there is growing interest in employing multivariate analysis approaches to fMRI data . The aim of the current study was to localize voice-related areas in both female and male listeners and to investigate whether brain maps may differ depending on the gender of the listener. After a univariate analysis, a random effects analysis was performed on female ( Prior functional magnetic resonance imaging (fMRI) findings suggest a robust brain response to vocal vs. non-vocal sounds in many regions of the human auditory cortex in particular in the superior temporal gyrus (STG). Vocal sounds, including but not restricted to speech sounds, evoke a greater response than non-vocal sounds with bilateral activation foci located near the anterior part of the STG extending to anterior parts of the superior temporal sulcus (STS) and posterior foci located in the middle STS . Instead of modeling individual voxels independently , MVPA considers the information of distributed pattern in several voxels  . In this study, we were interested in the effect of gender on the results of the voice localizer and we asked an explorative research question of whether brain activation and/or classification accuracy maps in response to vocal (speech and non-speech) and non-vocal sounds differ between female and male listeners without prior assumptions about the strength of voice-specific activity.n = 149 females and n = 123 males. Thus, this study provides a large sample size, a well-established experimental design and the direct comparison of two different fMRI data analysis approaches applied on the exact same data.The voice localizer paradigm is often used in the literature  and 123 male  with self-reported normal audition were analyzed. This study was conducted at the Institute of Neuroscience and Psychology (INP) in Glasgow and approved by the ethics committee of the University of Glasgow. Volunteers provided written informed consent before participating and were paid afterwards.fMRI data of 272 healthy participants, 149 female  and non-vocal (20 blocks) conditions. Vocal blocks contained only sounds of human vocal origin  and consisted of speech  or non-speech . The vocal stimuli consisted of recordings from 7 babies, 12 adults, 23 children, and 5 elderly people. Half of the vocal sounds (speech and non-speech) consisted of vocalizations from adults and elderly people (women and men) with comparable proportions for both genders . The other half of the vocal sounds consisted of infant vocalizations (speech and non-speech) which also included baby crying/laughing. Recorded non-vocal sounds included various environmental sounds . A total number of 40 blocks were presented. Each block lasted for 8 s with an inter-block interval of 2 s. Stimuli  were normalized for RMS and are available at TR = 1900 ms; TE = 2.52 ms; inversion time (TI) = 900 ms; slice thickness = 1 mm; FoV = 256 mm; matrix size = 256 × 265; excitation angle = 9°; 192 axial slices).Scanning was carried out in a 3T MR scanner  and all data were acquired with the same scanner at the INP in Glasgow. Functional MRI volumes of the whole cortex were acquired using an echo-planar gradient pulse sequence  = 2000 ms; Echo Time (TE) = 30 ms; slice thickness = 3 mm; inter-slice gap = 0.3 mm; field of view (FoV) = 210 mm; matrix size = 70 × 70; excitation angle = 77°). A total number of 310 volumes  were collected with a total acquisition time of 10.28 min. Anatomical MRI volumes were acquired using a magnetization-prepared rapid gradient echo sequence (MPRAGE) . After reorientation of functional and anatomical volumes to the AC/PC line (anterior- and posterior commissure), functional images were motion corrected . Since, subjects may have moved between anatomical and functional data acquisition, the anatomical volumes were co-registered to the mean functional image produced in the realignment above. Anatomical volumes were segmented in order to generate a binary gray matter template at threshold probability level of 0.5 for each individual participant. This template was applied during model specification in both univariate analysis und MVPA. For the univariate processing, realigned functional volumes were normalized to a standard MNI template  and spatially smoothed with a 6 mm full-width at half mean (FWHM) Gaussian Kernel.Pre-processing was performed using the statistical parametric mapping software SPM8  brain maps were calculated. To investigate whether brain activity significantly differs between genders in response to vocal vs. non-vocal sounds, contrasts between females vs. males  were computed in a second level RFX analysis . This analysis was restricted to voxels with classification accuracy significantly above theoretical chance (p < 0.01 uncorrected) in both females and males .The design matrix was defined such that each block of the experimental paradigm correlated to one condition, yielding a design matrix with 20 onsets for each condition . Analysis was based on the conventional general linear model (GLM) and stimuli were convolved with a boxcar hemodynamic response function provided by SPM8. Contrast images of vocal vs. non-vocal conditions were generated for each individual subject and entered into a second-level random effects analysis (RFX). To declare at the group-level whether any difference between the two conditions was significantly larger than zero, a one-sample Multivariate pattern classification was performed on unsmoothed and non-normalized data using Matlab  and in-house utility scripts , where the default linear support vector machine (SVM) classifier was applied. The classifier was trained and separately tested following a leave-one out cross validation strategy applied on the 40 beta parameter estimates obtained from the univariate analysis (GLM).SD)  with 10,000 permutations  in both females and males. The resulting mask included 3783 voxels . The same mask was applied for both, the univariate analysis and MVPA.The between-group analysis was restricted to a mask defined by voxels with classification accuracy significantly above theoretical chance (http://www.mccauslandcenter.sc.edu/mricro/mricron/).Separate brain maps of vocal vs. non-vocal contrast in female and male participants as well as brain maps of contrasts between genders for both, univariate analysis and MVPA were generated using the program MRIcoGL , highly similar for male and female subjects Figure . These rT-values of each hemisphere. The brain activation differences between vocal and non-vocal response was consistent across maxima in females  and in males . Female listeners showed one large cluster covering the temporal lobes and subcortical parts of the brain. By contrast male listeners showed two separate voxel clusters in the left and right temporal lobes and no subcortical cluster connecting the two hemispheres  as well as for male listeners  were distincly above the theoretical chance level of 0.5  for either analysis methods. The reverse contrast , however, revealed significant voxel clusters showing greater parameter estimates for univariate analysis and higher classification accuracy for MVPA in female participants  or classification accuracy maps (multivariate approach) from males and females revealed no significant voxels with greater parameter estimates for males > females at the chosen statistical significance threshold  showed a positive response for females in both hemispheres and for the left hemisphere in males. The Cohen's d effect size values (d = 0.48 and 0.49) suggested a moderate difference at the peak voxel  suggested a small difference at the peak voxel  in both hemispheres, in which classification accuracy was greater for female than male participants . A significant difference in success of the MVPA between female and male listeners was apparent in the middle part of the MTG in both hemispheres and in the middle part of the STG in the left hemisphere. Effect sizes showed a small difference at the peak voxels. Despite the large sample size used in this study, the univariate analysis showed no major activation differences between genders. Only two small clusters with one to four voxels were significant in the posterior and anterior part of the STG. In the univariate analysis, the overall activation difference between vocal vs. non-vocal sounds was stronger in female as compared to male listeners and effect sizes showed a moderate difference at the peak voxels.The distinct gender differences located in the middle part of MTG and middle part of STG between genders revealed by the MVPA survived our applied criteria (FWE-correction). In these regions, the classifier successfully distinguished between the vocal and non-vocal condition with better overall accuracy in females as compared to males across the peak voxels. Thus, BOLD signal in parts of auditory cortex seem to carry less information for discriminating vocal from nonvocal sounds in male than females listeners. We do not make any inference on the nature of the underlying processing differences in terms of mental states or cognitive mechanisms, but possible explanations for our findings are discussed below.MVPA may overall be more sensitive to detect small differences in the activation patterns to vocal and non-vocal sounds. Thus, differences between genders appear significant only when analyzed with MVPA  and to perform a behavioral task in order to link differences in brain activation to behavior of the listener. Furthermore, it would be interesting for future studies to take into account more specific aspects of voice quality, which were not considered in the current study. Even subtle differences in phonation , articulation  and or prosody  are critical aspects of voice processing and could be investigated using similar methodical approaches. Apart from studying differences between women and men, also other listener characteristics, such as differences between young and elderly participants, different nationalities and/or familiarity with the presented voices/stimuli should be considered.Male and female participants were similar in their pattern of activity differences in response to vocal vs. nonvocal sounds in the TVA of the auditory cortex. Yet, MVPA revealed several regions of significant gender differences in classification performance between female and male listeners: in these regions the distributed pattern of local activity from female participants allowed significantly better vocal/nonvocal classification than that of male participants; no region showed the opposite male > female difference. The neuronal mechanims underlying the observed differences remain unclear.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Diabetes Health Plan (DHP) is a disease-specific health plan for patients with diabetes and pre-diabetes which offers reduced cost sharing for pharmacy, office visits and disease management. The DHP was purchased by some employers and not by others, creating a unique opportunity to conduct a rigorous evaluation of a real-world, naturally occurring intervention, i.e., a natural experiment.Examine the association of employer purchase of the DHP with emergency room (ER) and inpatient hospital utilization among employees and covered dependents with diabetes and pre-diabetes.A quasi-experimental design with the employer as the unit of analysis, comparing changes in mean ER and hospital utilization over a 3-year period. We used inverse probability weighting (IPW) to adjust for differences between employers that purchased the DHP and employers that purchased standard plans. We report estimated differences as Average Treatment Effects on the Treated (ATET). Setting: Large, national private insurer that offers health plans to public and private employers. Participants: We aggregated eligibility and claims data from covered employees and dependents with diabetes and pre-diabetes  to the employer level. The analysis included 9 employer groups that purchased the DHP in 2009 or 2010  and 183 control employer groups that purchased standard plans in 2009 or 2010 . Main Outcome Measure: Mean rates of ER and inpatient hospital utilization.DHP purchase was associated with 2.4 and 1.8 percentage-point reductions in adjusted mean rates of any ER utilization, representing 13% and 10% relative reductions at 1 and 2-years post-DHP . There was no evidence of significant associations between DHP purchase and adjusted hospital utilization.Employer groups purchasing diabetes-specific health benefit designs such as the DHP may experience lower rates of resource-intensive services such as ER utilization.This study was jointly funded by the Centers for Disease Control and Prevention (Division of Diabetes Translation) and the National Institute of Diabetes and Digestive and Kidney Diseases as part of the Natural Experiments for the Translation of Diabetes (NEXT-D) Study (Grant number DP002722).


P < 0.01), anxious/depressed mood (P < 0.05), and aggressive behavior (P < 0.01). Additionally, SSR sleep problem subscale scores indicating lower sleep quality  associated significantly with attention problems (P < 0.05). These results point out that sleep trouble should be recognized and treated in adolescents with IBD to possibly avoid the emerging of psychosocial symptoms.The current study assessed the associations between sleep and psychosocial symptoms in 157 Finnish adolescents with inflammatory bowel disease (IBD). Sleep trouble was self-rated in Sleep Self-Report (SSR) and in Youth Self-Report (YSR). Psychosocial symptoms of the adolescents were assessed by the YSR and Child Behavior Checklist (CBCL). Patients reporting sleep trouble had significantly more psychosocial symptoms than their counterparts without sleep trouble. This was shown in the CBCL and YSR scales of total problems ( Adequate sleep in adolescence is important for healthy development and proper daytime functioning. The evidence suggests that disturbances in the quantity and quality of sleep are associated with emotional and behavioral problems, somatic complaints, problems in academic and social performance, and overall quality of life in adolescents from normal populations –4. SleepIBD, which includes subtypes of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic relapsing inflammatory condition of the gastrointestinal tract and is most typically diagnosed in late adolescence and early adulthood . PrimaryThe present study aims to extend our previous findings with the same sample by comparing daytime emotional well-being and behavior in IBD patients reporting sleep trouble and patients not reporting sleep trouble. We also studied how sleep quality reported by the adolescents with IBD associated with their emotional and behavioral symptoms. To the best of our knowledge, no earlier reports exist on the associations of psychosocial symptoms and sleep in adolescent patients with IBD.Detailed subject recruitment has been described in our recent publication on psychosocial symptoms in adolescents with IBD . BrieflyStandardized questionnaires (see below) translated into Finnish were sent to the 287 patients and their parents. After one reminder, completed Youth Self-Report (YSR)  and SleeThe CBCL and YSR questionnaires include 113/112 problem items, respectively, rated on a three-point scale from 0 to 2  . The queT-scores for all broad-band and narrow-band scales are reported to ease the valuation of clinical significance of psychosocial symptoms and to enable comparison with other parallel studies [T-score above 63. Scores from 60 to 63 are considered to be in the subclinical range. For narrow-band syndrome scales the clinical and subclinical cut-off points are higher  [ studies . The cli, resp.) . In thisThe sleep trouble item (item 100 = Trouble sleeping?) in the YSR was used to define whether the patient had  or did not have (does not apply) sleep trouble. The sleep quality was measured by self-rated SSR questionnaire . The firThe Ethics Committee of Helsinki University Central Hospital approved the study protocol.t-test for independent samples (T-scores used). The mean group differences for narrow-band syndrome scales were tested with nonparametric Mann-Whitney U test (raw scores used). When analysing differences in the frequencies of adolescents with/without sleep trouble scoring over the subclinical cut-off points on the broad-band (T-score ≥ 60) and narrow-band scales (T-score ≥ 65) in the CBCL and YSR questionnaires, cross tabulation and the nonparametric chi-squared test were used. For correlation analyses we used two-tailed Pearson correlation. Statistical significance was set at P ≤ 0.05.Statistical analyses were performed using SPSS 17.0 software for Windows. CBCL and YSR items were scored using ADM (version 6.5) scoring software (Achenbach 1999–2006). Mean group differences for total, emotional, and behavioral problems broad-band scales were compared with Those 32 (20%) adolescents who reported having trouble sleeping sometimes or often were classified as sleep-troubled. The sleep-troubled patients reported significantly more total problems, anxiety/depression, somatic complaints, and aggressive behavior than the adolescents with no sleep trouble . Addition = 13) of the sleep-troubled adolescents reported a subclinical/clinical number of emotional problems which is significantly more compared to the non-sleep-troubled adolescents . Of the narrow-band syndrome scales included in the emotional problems scale, sleep-troubled adolescents scored significantly more often over the subclinical cut-off point on the scales of anxious/depressed mood  and somatic complaints  while the difference on the scale of withdrawn/depressed was nonsignificant (P > 0.05). No group differences between sleep-troubled and non-sleep-troubled patients were found for the frequencies of adolescents scoring over the subclinical cut-off point on the scales of total problems and behavioral problems.Nearly half  of adolescents with sleep trouble and in 13% (n = 16) of adolescents with no sleep trouble (P < 0.01). Significantly more adolescents with sleep trouble had a subclinical/clinical number of emotional problems  compared with the adolescents without sleep trouble . Of note, the group difference in the frequency of adolescents scoring over the subclinical limit on the scale of emotional problems was caused mainly by the reports in the scale of somatic complaints. The T-score for somatic complaints scale was significantly more often above the subclinical limit in adolescents with  than without sleep trouble ; the difference was nonsignificant in the syndrome scales of anxious/depressed and withdrawn/depressed and in the behavioral problems scale (P = 0.063).In parental reports, the P < 0.01), behavioral symptoms (P < 0.05), and attention problems (P < 0.05) according to the parent reports  and emotional symptoms  in this patient sample  was simiCommunity-based studies in adolescents show a robust association between subjective sleep trouble and emotional problems –21. SimiStrength of this study was that the number of patients was high, both the adolescents and their parents served as a source of information, and the psychosocial symptoms of the patients were measured by standardized CBCL/YSR questionnaires. The presence of sleep trouble was defined by a self-rated question of the YSR, which can easily be used as a screening instrument in clinical practice. Additionally, the standardized Sleep Self-Report, SSR, was used here to assess sleep trouble more thoroughly. We have reported earlier that there is some discrepancy in reporting about the presence of sleep trouble by adolescents with IBD and their parents . In the The present results suggest that a further evaluation of psychosocial symptoms is required, especially, among those adolescents with IBD, whose sleep is disturbed according to their own opinion. Sleep troubled adolescents with IBD seem to be at a greater risk for anxious/depressed mood and behavioral and attention problems than those whose sleep is not disturbed. Also, early interventions to improve sleep quality among adolescents with IBD are needed. Due to a cross-sectional study design, conclusions of causal connections here are limited. In the future, longitudinal studies are required to further assess causal connections of subjectively and objectively measured sleep amount/quality and psychosocial symptoms in adolescents with IBD. Also, future studies should find out if improving sleep quality lessens the emotional or behavioral symptoms or if, vice versa, treating the emotional symptoms improves sleep quality in adolescents with pediatric IBD.


Median survival of advanced malignant pleural mesothelioma (MPM) is less than 2 years, and novel treatments are urgently needed. Immunotherapy with adoptive T cell transfer is an attractive approach that could be added to current therapeutic concepts. Fibroblast activation protein (FAP) is expressed by all subtypes of MPM  and can in human application of FAP-specific autologous re-directed T cells in patients with newly diagnosed, inoperable MPM in the malignant pleural effusion. We report here on the first patient treated in February 2015. At that time, he was 74 years old and had received the diagnosis of MPM in November 2014. After 2 cycles of standard chemotherapy (Cisplatin/Pemetrexed), 250 ml of peripheral blood were collected (day -21), CD 8 T cells extracted and retro-virally transduced with a FAP-specific CAR, while the patient was receiving the 3rd cycle of chemotherapy. On day 0 1x106 CAR T cells were injected under sterile conditions into the pleural effusion through a pleural drainage on the intensive care unit (ICU), followed by 48 hours ICU surveillance. Simultaneously, cytokine levels were measured in the serum and in the pleural effusion.This is a Phase I study with the primary clinical endpoint of safety and the secondary endpoints of feasibility and immune monitoring. Immune monitoring includes measurement of T cell subpopulations, cytokines and inflammatory parameters. This is the first Ex vivo analysis showed antigen-specific activity and function of re-directed T cells.In this first patient, we demonstrated safety and technical feasibility of the application of re-directed T cells. No adverse events due to the injection were observed, in particular no immune-mediated toxicity . Re-directed T cells against FAP could be a safe and feasible treatment option for patients with MPM in the future. Further studies are needed to demonstrate anti-tumor efficacy.