Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification.We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin “states”, individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse “exceptions” from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin.These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.The online version of this article (doi:10.1186/1471-2164-15-988) contains supplementary material, which is available to authorized users. Chromatin compactness is the key feature of chromatin that reflects its accessibility to transcription machinery. Tightly packed closed chromatin is considered a hallmark of gene silencing, and chromatin opening precedes lineage-specific gene expression thus providing an excellent indicator of cell fate commitment , 2. HoweDrosophila genome and on previous genome-wide analyses of chromatin marks in Drosophila Schneider-2 (S2) cell line, we have created the map of chromatin compactness in these cells and cross-referenced it to known patterns of chromatin modifications and gene expression.Here, we sought clarification on two important topics in chromatin biology: first, whether there is a straightforward and immutable association between chromatin compactness and certain chromatin modifications or combinations thereof, and second – what is the relation between chromatin compactness and gene expression. Toward this objective we designed and validated a high throughput assay for general chromatin sensitivity to DNase I (GCSDI) as a powerful approach to determine chromatin compactness across genome. Previous low-throughput studies have established the power of GCSDI to detect domains of both open and closed chromatin in specific genomic loci –8. This To measure GCSDI across genome we combined a brief DNase I treatment of permeabilized cells with random amplification of the DNase I-nicked genomic DNA, followed with analysis of sequence representation in amplified material by a high-throughput method. DNase I preferentially nicks DNA in open chromatin, rendering these regions inefficient template for amplification and thus predisposing them for under-representation in amplified material. The difference in representation between known open and closed chromatin loci can be reliably detected by the GCSDI assay after treatment with diverse amounts of DNase I, and thus is a reliable analysis outcome that is not overly sensitive to DNase I treatment conditions  were hybridized with tiling Affymetrix microarrays, signal intensities for each probe were averaged within the experimental groups and fold differences between the groups were calculated. Positive log2 values were assigned to the sequences underrepresented in DNase I-treated sample (open) and negative values - to the sequences overrepresented in DNase I-treated sample (closed). The identified open and closed chromatin regions were extensive and contiguous, consistent with previous low-throughput studies –8 and inTwo segmentation models of chromatin compactness were created using the genome-wide GCSDI profiles. (i) We used a sliding window algorithm to identify transition points and to segment genome into contiguous series of open or closed chromatin domains, with the mean size of 15 kb and ranging up to 500 kb .We also analyzed distribution of individual modifications which have been traditionally linked to certain chromatin structure predictions, and intriguingly found that while open chromatin was associated with numerous abundant chromatin modifications, closed chromatin was largely unmodified. This was true in whole-genome analysis Figure  and alsoDrosophila genome [Taking into account that morphologically dense heterochromatin is often situated at nuclear periphery, we proposed that a significant proportion of closed chromatin is included in lamina-associated domains (LADs). Indeed, comparison of our GCSDI analysis with the LAD map of a genome  revealeda genome  these fiWhile intergenic spacers were mostly closed or neutral, actively expressed genes were predominantly open and silent genes were generally closed across the genome   and open chromatin in silent gene promoters (one-third in 2CM and 17% in 3CM). We first sought to rule out the trivial explanations such as frequent presence of alternative inactive promoters in active genes, as well as imprecision of chromatin analysis or incorrect selection of the promoter regions. In these cases, even though a promoter may appear in “odd” configuration, the chromatin structure of the gene body would match its expression status. We found just the opposite - the chromatin configuration of the gene body followed that of the promoter Figure A indicattau was signIntriguingly, we also found that active genes with closed promoters showed lower transcript levels than conventionally expected active gene with open promoters, indicating that chromatin compactness may serve to modulate active gene expression. The mechanisms underlying this type of regulation warrant further inquiry, as do the other unexpected trends identified in our study, such as the general paucity of known chromatin marks positively identifying closed chromatin and the tendency of large introns to stay in closed configuration even when the genes are expressed. We expect that the novel analysis of epigenomic regulation with a straightforward and sensitive assay described here will contribute an empirical approach supplementing predictive chromatin structure assessments, thereby advancing both basic and biomedical research in chromatin biology.6 S2 cells were permeabilized with 0.05% NP40 and resuspended in DNase I Buffer . Part of each sample was set aside and later used as non-digested control. DNase I (Promega) digestion was performed at 37°C for 10 or 15 minutes with diverse amounts of the enzyme to optimize the procedure ; for further analysis cells were treated with 0.5U DNase I for 10 minutes. 20 ng of DNA purified from the treated cells using the DNeasy Blood & Tissue Kit (Qiagen) were used as a template for whole genome amplification. The library preparation step using GenomePlex WGA2 Kit was followed by amplification with the GenomePlex WGA 3 Reamplification Kit (Sigma). dUTP was incorporated at the amplification step to enable the probe fragmentation procedure according to Affymetrix recommendations. The amplification product was purified with the Wizard SV Gel and PCR Clean-Up System (Promega), fragmented and labeled using the GeneChip WT Double-Stranded DNA Labeling Kit (Affymetrix), and hybridized with GeneChip Drosophila Tiling 2.0R Array following the manufacturer’s instructions.DNase I treatment of cells was performed as previously described  with minRaw data from microarray CEL files were normalized using CisGenome  and log2To generate two-configuration model (2CM), probes with log2 differential signal within 1 standard deviation of the mean were discarded and the signals for the remaining probes were capped at 1 for positive and -1 for negative. A sliding window was used to determine transition points between open (positive) and closed (negative) segments. The difference (d) between the mean log2 differential signals of flanking regions was calculated for every probe. The cutoff d values were established by analyzing 100 permutations of probes, with the requirement that real genome data is significantly different from permuted models (p < 0.05). A series of significant d values and flanking region sizes (n) were tested to determine the model most discriminating between real genome and random permutation. The results presented here are based on analysis using n = 48 and d = 0.8, which identified 2244 transition probes in the real dataset and only 72 on average in permutated controls.To generate three-configuration model (3CM), all probes were analyzed for presence of differential signal peaks using an HMM algorithm built in CisGenome, with a posterior probability greater than 0.5. An FDR value of 0.1 was used to filter the detected peaks which were further consolidated into domains as following: two adjacent peaks are joined if the distance between them is less that threshold value , and if they are of the same sign (either both positive or both negative). Otherwise, the segment between the peaks is assigned neutral state. 2CM and 3CM domain coordinates in BDGP5 genome annotation are provided as tables in Additional files http://www.modencode.org) [http://compbio.med.harvard.edu/flychromatin/data.html[LAD coordinates  were dowode.org) ; specifidata.html. Coordindata.html. All datWe downloaded short-read (Illumina) sequences for 5 massively parallel mRNA sequencing experiments on S2 cells from two GEO datasets (GSM390063 and GSM390064), aligned these reads to the Drosophila reference genome (BDGP5) using TopHat , and calactin and Letm1 representing open chromatin, and Crtp and Yu representing closed chromatin [Additional file 1: Figure S1: Evaluation of the amplified DNA from DNase I-treated and untreated cells. Low amplification bias evident by the absence of discrete bands in agarose gel (A), and selective depletion of open chromatin in sample from cells treated with DNase I under diverse conditions . Four genome regions with known chromatin compactness were analyzed: hromatin .  and in more detail for the lower size ends of distribution histograms (left panels). (JPEG 59 KB)D. melanogaster. (JPEG 44 KB)Additional file 3: Figure S3: Distribution of open (green), neutral (yellow), and closed (red) chromatin domains detected by 3CM analysis on chromosomes of Additional file 4: Figure S4: Representation of detected open ,neutral, and closed chromatin domains in genome and their association with predicted chromatin states. Proportions of open and closed chromatin detected by 3CM are shown for individual chromosomes (A) and for the genome regions predicted as 9 chromatin states [Additional file 5: Figure S5: Contributions of the predicted chromatin states to open, neutral, and closed chromatin detected by 3CM. Results are shown for the whole genome, and separately for major autosomes and chromosomes X and 4. (JPEG 49 KB)Additional file 6: Figure S6: Enrichment and depletion of chromatin modifications in open, neutral, and closed chromatin. Heat maps show percent proportions of regions enriched with (red) or depleted of (blue) particular chromatin modifications in open and closed chromatin domains detected by 2CM and 3CM. Data are cumulative for the entire genome, euchromatin of major autosomes and chromosome X, the entire chromosome 4, and combined pericentromeric heterochromatin of major autosomes and chromosome X. (JPEG 104 KB)Additional file 7: Figure S7: Link between lamina-associated domains (LADs)  and closAdditional file 8: Figure S8: Relationship between open and closed chromatin and gene structure. Proportions of open, closed, and neutral chromatin detected by 3CM are shown for intergenic spacers and active or silent genes (A) and for structural elements of active and silent gene (B). Analysis of relationship between chromatin structure and the size of gene (C) and intron (D) shows that proportion of open chromatin diminishes as the gene and intron size increases for both active and silent genes. (E), Distribution of open and closed chromatin along large (>10 kbp) active gene introns. (JPEG 58 KB)GCSDI domains 2CM.(XLSX 64 KB)Additional file 9:GCSDI domains 3CM.(XLSX 645 KB)Additional file 10:


Cdc20) during meiosis I progression until all kinetochores come under tension [for review see: Resumption of oocyte meiotic maturation is driven by activation of cyclin-dependent kinase 1 (Cdk1), which phosphorylates target proteins that are important for nuclear envelope breakdown, chromosome condensation, and remodelling of the actin and microtubule cytoskeleton. As a consequence, a bipolar spindle is formed and condensed chromosomes become aligned at the metaphase plate. The spindle assembly checkpoint (SAC) prevents the activation of the anaphase-promoting complex/ cyclosome APC/CCdc0 during Although Cdk1/cyclin B complexes have long been considered sufficient for driving mitosis and meiosis, recent studies in various systems have shown that the simultaneous suppression of the antagonizing protein phosphatase 2A (PP2A), which is mediated by the Greatwall (Gwl) kinase , is also required for mitotic entry or progression -5. AlthoMeiosis in oocytes represents a specialized cell division whereby a sharp increase in Cdk1 activity after completion of meiosis I prevents them from entering S phase and oocytes transit directly to meiosis II. Moreover, the paired sister chromatids remain condensed during the meiosis I−meiosis II transition and form typical bipolar metaphase spindles and they remain arrested at metaphase II until fertilization . We founMeiosis I in mammalian oocytes is a lengthy process, and the prometaphase of meiosis I  takes about 8 hours, which is much longer than the approximately 30 minutes of prometaphase in most mitotic cells. We found that wild type mouse oocytes progressed through prometaphase I without suppressing PP2A thus the lack of Mastl did not cause a further increase in PP2A activity during this process. At the same time, the kinetics of Cdk1 activation, meiosis resumption and prometaphase I progression were not affected in Mastl-null oocytes. However, higher PP2A activity after meiosis I in Mastl-null oocytes prevented the activation of Cdk1 and entry into meiosis II . Based oThus, our results indicate that Mastl mediated PP2A inhibition is crucial for the timing of anaphase I and for the entry into meiosis II in mouse oocytes, however, it also raises many new questions. For instance, if Mastl is dispensable for prometaphase I progression, is Mastl activity normally suppressed during this process? If so, what are the possible mechanisms that inactivate Mastl during prometaphase I? Following chromosome segregation in meiosis I in Mastl-null oocytes, we found that the oocyte chromosomes were rapidly decondensed and nuclear envelope was reassembled. Thus, Mastl appears to mediate an additional surveillance mechanism after chromosome segregation in meiosis I that prevents chromosome decondensation and nuclear envelope reassembly. Similarly, how Mastl regulates APC/C in meiosis I remains unknown and will require further studies.


Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis, is a complex multifactorial disease that is characterized by the persistent presence of fatigue and other particular symptoms for a minimum of 6 months. Symptoms fail to dissipate after sufficient rest and have major effects on the daily functioning of CFS sufferers. CFS is a multi-system disease with a heterogeneous patient population showing a wide variety of functional disabilities and its biological basis remains poorly understood. Stable alterations in gene function in the immune system have been reported in several studies of CFS. Epigenetic modifications have been implicated in long-term effects on gene function, however, to our knowledge, genome-wide epigenetic modifications associated with CFS have not been explored. We examined the DNA methylome in peripheral blood mononuclear cells isolated from CFS patients and healthy controls using the Illumina HumanMethylation450 BeadChip array, controlling for invariant probes and probes overlapping polymorphic sequences. Gene ontology (GO) and network analysis of differentially methylated genes was performed to determine potential biological pathways showing changes in DNA methylation in CFS. We found an increased abundance of differentially methylated genes related to the immune response, cellular metabolism, and kinase activity. Genes associated with immune cell regulation, the largest coordinated enrichment of differentially methylated pathways, showed hypomethylation within promoters and other gene regulatory elements in CFS. These data are consistent with evidence of multisystem dysregulation in CFS and implicate the involvement of DNA modifications in CFS pathology. Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis, is a complex multifactorial disease that is characterized by an unexplained fatigue lasting for a minimum of 6 months as well as the presence of at least 4 of the following symptoms: muscle or joint pain, lack of refreshing sleep, headache, sore throat, post-exertional malaise, tender cervical and axillary lymph nodes, and impaired memory and concentration Studies examining gene regulation using whole blood and peripheral blood mononuclear cells (PBMCs), composed primarily of lymphocytes and monocytes, point towards abnormalities in lymphocyte function in CFS. CFS sufferers exhibit disrupted homeostasis between the Th1- (cell-mediated) and Th2-  immune response, where CFS is associated with a predominantly Th2-mediated immune response An accumulating number of studies have examined epigenetic modifications associated with immune responses in the context of disease For this exploratory study, we selected 12 female CFS patients and 12 healthy control females from a total of 231 patients recruited from 4 clinical centers in order to match CFS patients and control subjects for age and body mass index (BMI), excluding obese subjects and subjects with a history of exposure to immunomodulatory medications, as these conditions may alter epigenetic and immune profiles This study adhered to the human experimentation guidelines as outlined by the Helsinki Declaration of 1975. The collection and analysis of clinical information and biological samples by the SolveCFS BioBank was ethically approved by the University of Toronto (IRB #27391) and the Genetic Alliance ethics review board (IRB # IORG0003358), which approved all procedures for obtaining written informed consent from all subjects to participate in this study. Consent forms were signed in duplicate, with one copy provided to subjects and one copy securely stored at the SolveCFS Biobank.Volunteers diagnosed with CFS and healthy controls were recruited by the SolveCFS BioBank. Comprehensive medical histories of the volunteers were recorded, including demographic data, age of CFS onset (if diagnosed with CFS) and medication use. The RAND-36, a validated 36 item self-report inventory, was used to assess health-related quality of life 6 cells were stored at –80°C after centrifugation.Whole blood in heparanized tubes collected at 4 clinical sites was sent to the Rutgers University Cell and DNA Repository at ambient temperature via overnight shipping. Plasma was collected after centrifugation and the remaining blood was diluted with DPBS. Processing of samples followed guidelines approved by Rutgers University . Briefly, PBMCs were isolated using Ficoll gradient centrifugation and resuspended in 1X DPBS +1% fetal bovine serum. A sample was taken for automatic cell counting using a ViCell XR Viability Analyzer. Dry pellets of 10×106 cells using the Omega E.Z.N.A. Tissue DNA Kit  according to the manufacturer’s instructions. Genomic DNA was eluted in Tris-EDTA buffer  and a NanoDrop 2000c spectrophotometer  was used to check the quantity and quality of the DNA. All samples were prepared to a minimum final concentration of 100 ng/µl, A260/A280 = 1.8–2.0, and A260/A230>2.0. When required, DNA cleanup was performed using the MinElute Reaction Cleanup Kit  according to the manufacturer’s instructions.For each subject, genomic DNA was extracted from approximately 2.50×10http://genome.ucsc.edu/), where CpG island Shores were defined as the 2 kb regions immediately upstream (North) or downstream (South) of a CpG island, and CpG island Shelves were defined as the 2 kb regions immediately upstream or downstream of a CpG island shore www.ncbi.nlm.nih.gov/geo/) under the accession number GSE59489.Approximately 1.5 µg of DNA was sent to Genome Québec  on dry ice. Samples were processed according to Genome Québec and Illumina protocols for the Infinium HumanMethylation450 BeadChip (450 K) array. This microarray offers coverage of more than 480,000 CpG methylation sites including promoters, untranslated regions (UTRs), first exons, gene bodies, and CpG islands. Annotations in relation to genic regions and CpG islands were based on annotation available from the UCSC Genome Browser . Extracted DNA was sodium bisulfite converted using the EZ DNA Methylation Kit according to the manufacturer’s protocol . EpigenDX performed pyrosequencing with the following primer sets: HIPK3 (ADS1419-FS2), LCN2 (ADS1074-FS2), LY86 (ADS2397-FS).http://david.abcc.ncifcrf.gov/) using differentially methylated probes as the gene list and the 450 K Illumina microarray as the background. This analysis examines the statistical association between genes in the gene list provided and GO terms and determines the enrichment of GO terms relative to the background gene set, as performed in several previous epigenomic studies using the Illumina methylation microarrays http://baderlab.org/Software/WordCloudPlugin). GO analysis was also performed with the GeneMANIA plugin in Cytoscape 2.8.2 (http://www.genemania.org/plugin/) using the 2012-08-12 human genome GeneMANIA build as the background. An application with similar functionality to WordCloud was not available for the GeneMANIA results.An analysis examining all GO terms was performed with DAVID , genic location, location relative to a CpG island, and according to the 3 broad functional categories: gene promoters within 1500 bp and 200 bp of the transcription start site , regulatory elements , and within the coding regions of genes (gene body).DNA methylation analysis was performed using R software with the Illumina Methylation Analyzer (IMA) package IBM SPSS software (Version 22) was used to perform statistical analysis on average age and BMI (Student’s t-test), RAND-36 scores (Student’s t-test), and average methylation according to pyrosequencing (Wilcoxon rank-sum test) using Bonferroni correction for multiple comparisons where applicable. GO results from both DAVID and GeneMANIA were considered to be significant if they survived an FDR≤0.10 cutoff. DAVID GO results were clustered using default settings of the Enrichment Map plugin After microarray normalization of the raw DNA methylation data, 327,409 probes remained for subsequent analysis. Applying the probe selection criteria, 1,192 CpG sites were identified as differentially methylated between CFS patients and healthy control subjects, corresponding to 826 genes. The list of differentially methylated CpG sites and their respective genes can be found in st exons or CpG islands.Region-level analysis was performed by grouping probes within their respective annotations, and comparing average methylation values for all CpG sites at each location between CFS patients and healthy controls using the IMA regionswrapper function. A total of 934 differentially methylated CpG sites were found within or proximal to genes  and 448 differentially methylated CpG sites were mapped with respect to their location relative to CpG islands (i.e. CpG island locations). As shown in Three CpG sites that were identified as significantly different on the array were selected for validation using pyrosequencing. These CpG sites were mapped to the following genes : LY86 , HIPK3 , and LCN2 . Analysis by pyrosequencing confirmed the direction of methylation differences between CFS and control subjects identified by 450 K Illumina microarray , with similar methylation levels detected by pyrosequencing Click here for additional data file.Table S2CpG sites identified as differentially methylated in CFS patients compared to healthy controls.(XLSX)Click here for additional data file.Table S3Region-level analysis of differentially methylated genes grouped by location in CFS patients compared to healthy controls.(XLSX)Click here for additional data file.Table S4Significant GO terms and their cluster groups in CFS compared to healthy controls as determined by DAVID and Enrichment Map.(XLSX)Click here for additional data file.Table S5Significant GO terms in CFS compared to healthy controls as determined by GeneMANIA.(XLSX)Click here for additional data file.Table S6CpG sites in cluster group genes identified as differentially methylated between CFS patients and healthy controls.(XLSX)Click here for additional data file.Table S7CpG sites in genes within the immune cell regulation cluster identified as differentially methylated between CFS patients and healthy controls.(XLSX)Click here for additional data file.


High nuclear expression of the RNA-binding motif protein 3 (RBM3) has previously been found to correlate with favourable clinicopathological characteristics and a prolonged survival in several cancer forms. Here, we examined the clinicopathological correlates and prognostic significance of RBM3 expression in tumours from a consecutive cohort of upper gastrointestinal adenocarcinoma.Immunohistochemical RBM3 expression was analysed in tissue microarrays with primary radiotherapy- and chemotherapy-naive adenocarcinoma of the esophagus, gastroesophageal junction and stomach (n = 173). In addition paired samples of normal squamous epithelium (n = 53), gastric mucosa (n = 117), Barrett’s esophagus/gastric intestinal metaplasia (n = 61) and lymph node metastases (n = 71) were analysed. Kaplan-Meier analysis and Cox proportional hazards modelling was applied to assess the impact of RBM3 expression on overall survival (OS) and recurrence-free survival (RFS).RBM3 expression was similar in primary tumours and lymph node metastases, but significantly higher in primary tumours and metastases arising in a background of intestinal metaplasia compared with cases without intestinal metaplasia (p < 0.001). RBM3 expression was significantly reduced in more advanced tumour stages (p = 0.006). Low RBM3 expression was significantly associated with a shorter OS in cases with radically resected (R0) tumours  and RFS in curatively treated patients with R0 resection/distant metastasis-free disease . These associations remained significant in adjusted analysis .High expression of RBM3 may signify a subset of upper gastrointestinal cancers arising in a background of intestinal metaplasia and independently predicts a reduced risk of recurrence and death in patients with these cancer forms. These findings are of potential clinical utility and merit further validation. Esophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of death from cancer , relatedBoth esophageal and stomach cancer often present with diffuse symptoms causing a delay in diagnosis, with the majority of cases being detected in more advanced disease stages leading to an impaired prognosis. Therefore, there is an urgent need to identify new prognostic and treatment predictive biomarkers for improved diagnostics and clinical management of patients with these cancer forms.in vitro and in vivo[The RNA binding motif protein 3 (RBM3) has been identified as a promising prognostic and potentially treatment predictive biomarker in several major cancer forms e.g. breast, ovarian, prostate, bladder and colorectal cancer as well as malignant melanoma -11. Specd in vivo. While td in vivo, and thed in vivo.To our best knowledge, the prognostic significance of RBM3 in adenocarcinoma of the upper gastrointestinal tract has not yet been described. Therefore, in this study, the expression and prognostic impact of RBM3 was examined in a consecutive cohort of radiotherapy- and chemotherapy-naive adenocarcinoma of the esophagus and stomach (n = 173). In addition, RBM3 expression was analysed in a subset of matched normal squamous epithelium (n = 53), gastric mucosa (n = 117), intestinal metaplasia (IM) , and lymph node metastases (n = 71).The study cohort encompasses a consecutive cohort of 175 patients with esophageal and gastric adenocarcinomas who had been surgically treated in the University hospitals of Lund and Malmö from Jan 1st 2006 – Dec 31st 2010. The original and included cohort has been described in detail previously . All tumEthical permission was received from the regional ethical board of Lund University (ref nr 445/07).Tissue microarrays (TMAs) were constructed using a semi-automated arraying device . Duplicate tissue cores (1 mm) were obtained from viable, non-necrotic areas from all primary tumours. In addition, matched lymph node metastases were sampled from 81 cases, IM (gastric IM or Barrett’s esophagus) from 73 cases, normal squamous esophageal epithelium from 96 cases and normal gastric mucosa from 131 cases. Duplicate cores were obtained from different blocks of the primary tumour and different lymph node metastases in cases with more than one metastasis. Normal squamous epithelium and gastric mucosa was represented in single cores, and IM in 1–3 cores.For immunohistochemical analysis, 4 μm TMA-sections were automatically pre-treated using the PT Link system and then stained in an Autostainer Plus  with the mouse monoclonal anti-RBM3 antibody AAb030038  diluted 1:1000. The specificity of the antibody has been validated previously ,7.The immunohistochemical staining was evaluated by two independent observers who were blinded to clinical and outcome data (LJ and AG). Scoring differences were discussed to reach consensus. For assessment of nuclear RBM3 expression, the estimated percentage of cells with nuclear RBM3 expression was recorded (NF), as well as the predominant nuclear intensity (NI) as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A combined nuclear score (NS) was constructed by multiplying fraction and intensity. Cytoplasmic staining was not as evident, and recorded as 0 (absent) or 1 (present).Immunohistochemical staining for Ki67 was performed in the Ventana BenchMark ULTRA system  with a monoclonal antibody . The staining was evaluated by two independent observers who were blinded to clinical and outcome data (CH and DK). The fraction of staining was categorized as follows: 0-1%, 2-10%, 11-20%, 20-50%, and >50%. For statistical analysis three categories were applied: 0-20%, 21-50% and >50%.U test was applied for distribution analyses between the total nuclear RBM3 score and clinicopathological characteristics. Classification and regression tree (CRT) analysis [Mann Whitney analysis ,15 was uRBM3 expression could be evaluated in 173/175 (98.9%) primary tumours and 71/75 (94.7%) metastases. There was no obvious heterogeneity between tissue cores. Sample immunohistochemical images are shown in Figure As demonstrated in Table In primary tumours, there was no significant association between RBM3 expression and proliferation assessed by Ki67 expression  and at 0.440 for RFS  .Inclusion of tumour location  in the multivariable model did not alter the independent prognostic significance of RBM3 expression and tumour location Neither cytoplasmic RBM3 expression nor Ki67 expression were found to be prognostic (data not shown).The results from this study demonstrate that reduced RBM3 expression is an independent factor of a shorter overall and recurrence free survival in patients with adenocarcinoma of the upper gastrointestinal tract. These findings add to the increasing number of cancer forms in which RBM3 expression has proven to be an independent prognostic and treatment predictive biomarker -12. In lIn the here analysed consecutive cohort, all cases receiving neoadjuvant chemotherapy had been excluded and only a minor proportion, 9.6%, had received adjuvant treatment. Thus, the association of RBM3 expression with a favourable prognosis is not likely explained by a treatment predictive effect. Nevertheless, given that RBM3 expression has previously been shown to correlate with sensitivity to cisplatin treatment in epithelial ovarian cancer cells , has becIn general, RBM3 expression seems to be upregulated in preinvasive and cancerous tissues compared to their normal counterparts ,20,21. IOf note, RBM3 expression was significantly higher in tumours with associated Barrett’s esophagus or intestinal metaplasia compared to tumours without the presence of these pre-neoplastic lesions. These findings are of potential interest as they implicate a role for RBM3 in the intestinal pathway of carcinogenesis of upper gastrointestinal adenocarcinoma. Given that high RBM3 expression was demonstrated to be an independent favourable prognostic factor, its association with intestinal-type epithelium is also in line with findings from a previous comprehensive analysis of adenocarcinoma of the distal esophagus and esophagogastric junction, in which cases with associated intestinal-type mucosa were demonstrated to exhibit more favourable histopathological characteristics and a significantly prolonged survival compared to cases with associated cardiac-type mucosa .In contrast to our previous study on malignant melanoma, where RBM3 was found to be downregulated in metastases compared to primary tumours , RBM3 exin vitro as well as in various normal proliferating and malignant tissues [In the present study, we also examined the associations of RBM3 expression with the proliferation marker Ki67 in primary tumours and metastases. While no correlation was found in primary tumours, there was a significant association between higher RBM3 expression and proliferation in metastases. The relevance of this finding, which may well be coincidental, is however less evident, as neither RBM3 expression in the metastases nor Ki67 expression in primary tumours or metastases were significantly associated with clinical outcome in the here analysed cohort. Some studies have demonstrated an association between RBM3 expression and proliferation  tissues , however tissues . Moreove tissues ,24.A limitation to the present study is the retrospective setting, where curative intent may be difficult to determine. However, loss of RBM3 expression was found to be prognostic in all cases with radically resected primary tumours, with our without inclusion of the small number of cases having distant metastases. The reasons for surgical removal of the primary tumour in cases with R0 resection/distant metastasis were either bleeding of the primary tumour or presence of non-locoregional lymph node metastases and, of note, RBM3 remained an independent factor also after adjustment for distant metastasis in this category.Another possible limitation is the potential sampling bias and heterogeneity issue associated with use of the TMA technique. It should however be pointed out that these issues will remain even after analysis of full-face sections, as these also represent only a part of the tumour. Moreover, immunohistochemical analysis of multiple sections is not a feasible approach in biomarker studies. Therefore, a relative strength of the TMA design used in the present study is that duplicate cores were, in the majority of cases, obtained from different blocks of the primary tumour and different lymph node metastases in cases with more than one metastasis. Moreover, TMA-based biomarker analyses have been demonstrated to give equal or even improved prognostic information compared to analysis of full-face sections .In conclusion, the results from this study demonstrate that reduced RBM3 expression is an independent factor of poor prognosis in patients with adenocarcinoma of the upper gastrointestinal tract. Moreover, RBM3 expression was found to be significantly higher in tumours with associated Barrett’s esophagus or gastric intestinal metaplasia compared to tumours without the presence of these pre-neoplastic lesions. These findings are of potential interest as they implicate a role of RBM3 in different pathways of the pathogenesis of adenocarcinomas of the esophagus, esophagogastric junction and stomach. The value of RBM3 for prognostication and treatment stratification in patients with upper gastrointestinal adenocarcinoma should be pursued in future studies.RBM3: RNA-binding motif protein 3; OS: Overall survival; RFS: Recurrence free survival; R0: Radical resection; HR: Hazard ratio; CI: Confidence intervall; TMA: Tissue micro array; NF: Nuclear fraction; NI: Nuclear intensity; NS: Nuclear score; CRT: Classification and regression tree; IM: Intestinal metaplasia; MCM3: Minichromosome maintenance 3.The authors declare that no competing interests exist.LJ evaluated the immunohistochemical stainings, performed the statistical analyses and drafted the manuscript. CH collected and re-examined clinicopathological data and assisted with TMA construction. AG evaluated the immunohistochemical stainings and assisted with the statistical analysis. DC evaluated the immunohistochemical stainings. BN constructed the tissue micro array and performed the IHC stainings. MU contributed with antibody validation. JE assisted with collection of clinical data. KJ conceived of the study, evaluated the immunohistochemistry, and helped draft the manuscript. All authors read and approved the final manuscript.Patient and tumour characteristics for the entire cohort, esophagus, cardia and stomach.Click here for fileClassification regression tree analysis for selection of prognostic cutoffs regarding RBM3 expression in (A) OS in the entire cohort and (B) RFS in patients with R0 resection.Click here for file


To examine the comparative efficacy and safety of antithrombotic treatments  at a standard adjusted dose , acetylsalicylic acid (ASA), ASA and clopidogrel) for non-valvular atrial fibrillation and among subpopulations.Systematic review and network meta-analysis.A systematic literature search strategy was designed and carried out using MEDLINE, EMBASE, the Cochrane Register of Controlled Trials and the grey literature including the websites of regulatory agencies and health technology assessment organisations for trials published in English from 1988 to January 2014.Randomised controlled trials were selected for inclusion if they were published in English, included at least one antithrombotic treatment and involved patients with non-valvular atrial fibrillation eligible to receive anticoagulant therapy.For stroke or systemic embolism, dabigatran 150 mg and apixaban twice daily were associated with reductions relative to standard adjusted dose VKA, whereas low-dose ASA and the combination of clopidogrel plus low-dose ASA were associated with increases. Absolute risk reductions ranged from 6 fewer events per 1000 patients treated for dabigatran 150 mg twice daily to 15 more events for clopidogrel plus ASA. For major bleeding, edoxaban 30 mg daily, apixaban, edoxaban 60 mg daily and dabigatran 110 mg twice daily were associated with reductions compared to standard adjusted dose VKA. Absolute risk reductions with these agents ranged from 18 fewer per 1000 patients treated each year for edoxaban 30 mg daily to 24 more for medium dose ASA.Compared with standard adjusted dose VKA, new oral anticoagulants were associated with modest reductions in the absolute risk of stroke and major bleeding. People on antiplatelet drugs experienced more strokes compared with anticoagulant drugs without any reduction in bleeding risk. To fully elucidate the comparative benefits and harms of antithrombotic agents across the various subpopulations, rigorously conducted comparative studies or network meta-regression analyses of patient-level data are required.PROSPERO registry—CRD42012002721. This network meta-analysis includes data on edoxaban from the recently published ENGAGE AF-TIMI 48 trial.The studies pooled in this meta-analysis include antiplatelet agents in addition to newer oral anticoagulants and standard adjusted dose vitamin K antagonists.We present findings on the relative and absolute scale, and graphically illustrate absolute risks using icon arrays.We report detailed a priori subgroup analyses for stroke or systemic embolism and major bleeding using data derived from FDA Public Summary Documents.Comparisons between antithrombotic treatments were largely comprised of single, albeit large, studies.There is notable heterogeneity and the small number of studies limits the analyses that can be conducted to account for heterogeneity in the absence of patient-level data.Icon arrays only consider the primary efficacy and safety endpoints of underlying studies and do not account for the different clinical consequences associated with each of the outcomes.Atrial fibrillation (AF) is a common cardiac arrhythmia associated with increased morbidity and mortality.22 score <2 or ≥2; time in therapeutic range (TTR) <66% or ≥66%; Age <75 or ≥75 years. Therefore, the objective of this paper was to compare antithrombotic agents for the prevention of stroke and major bleeding in patients with non-valvular AF and among subpopulations.Existing guidelines recommend anticoagulant therapy for patients at intermediate or high risk of stroke.The published literature was identified by searching MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations. EMBASE, BIOSIS Previews, PubMed and the Cochrane Central Register of Controlled Trials were searched through the Ovid interface to identify English-language clinical articles published from 1988 to 23 January 2014. Regular alerts were also established. Websites of regulatory agencies, Canadian and major international health technology assessment agencies, clinical practice guidelines as well as The Cochrane Library  and University of York Centre for Reviews and Dissemination databases were searched. Complete details of the electronic search strategy, including any limits used, are reported in online supplementary appendix 1. The protocol was published online and was registered in the PROSPERO International Prospective Register of Systematic Reviews (PROSPERO registry—CRD42012002721).The population of interest was individuals with non-valvular AF requiring anticoagulation . The following treatments were included in the review: apixaban, dabigatran, edoxaban, rivaroxaban, standard adjusted dose VKA, acetylsalicylic acid (ASA), ASA and clopidogrel. Numerous outcomes were considered and results are reported elsewhereActive and placebo-controlled randomised controlled trials (RCTs) were selected for inclusion if they were published in English, included at least one antithrombotic treatment under review (using pre-specified doses), reported data for stroke/SE or major bleeding, and involved patients with non-valvular AF eligible to receive anticoagulant therapy, regardless of the level of stroke risk. Trials that included patients with contraindication to anticoagulant treatment were excluded. VKA trials were included if the dose was adjusted to a target international normalised ratio (INR) 2.0–3.0. Any dose of ASA was considered for inclusion, but ASA dose was stratified in the analysis as low (≤100 mg daily), medium (>100 mg to ≤300 mg daily) or high (>300 mg daily). We only included new oral anticoagulants which had at least one large phase III study. Three reviewers independently extracted the data on baseline characteristics, intervention(s) evaluated, including dose, duration and relevant co-medication, and results for each included article, using a standardised template. All extracted data were checked for accuracy by three independent reviewers. Any disagreements in the assessment of these data were resolved through discussion until consensus was reached. Quality assessment of RCTs was also performed independently by two reviewers using a standardised table based on major items from the SIGN 50 instrument for internal validity.2 <2 and CHADS2 ≥2 and had detailed data available from the Food and Drug Administration (FDA) Public Summary Report.2), were assigned for basic parameters throughout, although informative priors were considered.14Bayesian network meta-analyses were conducted using WinBUGS software . A binomial likelihood modelA key assumption behind network meta-analysis is that the analysed network is consistent, that is, there is no conflict between direct and indirect evidence.2 score).2 score <2 or ≥2; Age <75 or ≥75 years.Network meta-analysis also requires that studies are sufficiently similar in order to pool their results.2 score was reported in eight trials, encompassing the vast majority of patients included in the systematic review. Reported CHADS2 scores were consistent with a high-risk population (CHADS2 ≥2), with patients from ROCKET-AF2=3.5).The systematic review included 16 individual RCTs (reported in 32 publications and FDA reports); all evaluated the efficacy and safety of antithrombotic agents: apixaban (5 mg twice daily), dabigatran (150 or 110 mg twice daily), edoxaban (30 or 60 mg daily), rivaroxaban 20 mg daily), standard adjusted dose VKA, ASA (low dose (<100 mg daily), medium dose (100–300 mg daily)), or low-dose ASA plus clopidogrel (75 mg daily) in patients with non-valvular AF. None of the included studies directly compared one new oral anticoagulant drug with another (see online supplementary appendix 4). Of the 82 396 randomised patients included in the primary analysis, five large multicentre trials account for 78 296 patients (96%): ENGAGE AF-TIMI 48 n=21 105), mg daily 105, mg Mean age across the included trials ranged from 62 to 83 years (see online supplementary appendix 3). All trials included patients of both gender and had a higher proportion of male participants in most studies. However, the proportions of patients with a prior stroke or TIA varied substantially across the included trials, ranging from 3% in JAST to 55% in ROCKET-AF.The studies were critically appraised individually and the details are reported elsewhere.et al,et alThe evidence network  for the Dabigatran (150 mg twice daily) and apixaban were associated with reductions in stroke or SE relative to standard adjusted dose VKA . The use2, TTR and age . Furthermore, the posterior mean residual deviance 29.41) is close to the number of unconstrained data points (27), which is an indication of reasonable model fit. A number of subgroup analyses and alternative modelling strategies were conducted on the primary analysis, including adjusting for CHADS and age . We also.41 is clet al,et alThe evidence network for the primary analysis for major bleeding was comprised of 15 RCTs representing eight treatments in addition to placebo/observation (N=83 015). The evidence network for major bleeding is similar to the evidence network for stroke or SE  but inclEdoxaban 30 mg daily, apixaban, edoxaban 60 mg daily and dabigatran 110 mg twice daily were associated with reductions in the risk of major bleeding compared with standard adjusted dose VKA . No diffThe estimates of effect derived from the direct pairwise comparisons aligned well with those obtained from the network meta-analysis in direction and magnitude (see online supplementary appendix 7). Furthermore, the posterior mean residual deviance (38.0) is close to the number of unconstrained data points (35), which is an indication of reasonable model fit. A number of subgroup analyses and alternative modelling strategies were conducted on the primary analysis . We alsoet alet al,We identified 16 RCTs comparing antithrombotic agents for the prevention of all cause stroke or SE and major bleeding in patients with AF. To the best of our knowledge, this is the most up-to-date systematic review and network meta-analysisFor stroke or SE, dabigatran 150 mg and apixaban twice daily were associated with reductions in stroke or SE relative to standard adjusted dose VKA. In contrast, low-dose ASA and the combination of clopidogrel plus low-dose ASA that appeared were associated with increases in stroke or SE relative to standard adjusted dose VKA. No differences were detected between standard adjusted dose VKA and each of the following interventions: dabigatran 110 mg twice daily, edoxaban 30 and 60 mg daily, rivaroxaban, medium-dose ASA and no treatment/placebo. The absolute risk reduction of stroke or SE relative to standard adjusted dose VKA ranged from 6 fewer to 15 more events per 1000 patients treated per year, with dabigatran 150 mg twice daily yielding the largest absolute risk reduction and clopidogrel plus low-dose ASA the largest absolute risk increase.For major bleeding, edoxaban 30 mg daily, apixaban, edoxaban 60 mg daily and dabigatran 110 mg twice daily were associated with a decreased risk of major bleeding relative to standard adjusted dose VKA. No differences for major bleeding were detected between standard adjusted dose VKA and each of the remaining interventions: dabigatran 150 mg twice daily, rivaroxaban, clopidogrel plus low-dose ASA and all ASA dosages. The absolute risk difference of major bleeding relative to standard adjusted dose VKA ranged from 18 fewer to 24 more events per 1000 patients treated, with edoxaban 30 mg daily yielding the largest absolute risk reduction and medium-dose ASA the largest absolute risk increase. Results of network meta-analyses reflect the relative efficacy and safety profile of antiplatelet and anticoagulant drugs as documented by large clinical trials. Thus, international guidelines22, age and TTR (see online supplementary appendices 3 and 4). For example, patients using rivaroxaban had a higher risk of stroke because ROCKET-AF2 ≥2. As such, we conducted subgroup analysis to adjust for differences in patient and study-level characteristics. We derived data for subgroup analyses from FDA Public Summary documents,Network meta-analysis involves pooling of trials. We observed variability across studies included in the evidence network in terms of CHADS2 <2 and CHADS2 ≥2 and had detailed data made publicly available following an FDA review of dabigatran. Use of a different study  for baseline event rate data is unlikely to change the results substantially, given that the event rates in standard adjusted dose VKA arms were similar across studies. Fifth, we present findings using graphics that have been largely applied in the network meta-analysis literature,There are several limitations with the analysis we employed. First, there is notable heterogeneity in patients and study characteristics (see online supplementary appendices 3 and 4). However, the small number of studies limits the analyses that can be conducted to account for heterogeneity in the absence of patient-level data. Second, there is insufficient subgroup data available for other outcomes reported in the RCTs included. As a consequence, detailed analysis was limited to two outcomes—stroke and SE and major bleeding. Third, we reported the results from the fixed-effects model in the main text. We felt that this was appropriate as the nodes in evidence networks are connected largely by single studies. Effect estimates derived from the fixed-effects model aligned more closely with the direct estimates. Reporting results from the fixed-effects model in the main text most likely biases results in favour of the new oral anticoagulants, as treatments that achieved statistically significant results in the primary RCTs retained statistically significant findings when using the fixed-effects model, but not always when using the random-effects model due to the priorCompared with standard adjusted dose VKA, dabigatran 150 mg and apixaban twice daily were associated with reductions in the incidence of stroke or SE in patients with AF, whereas low-dose ASA and the combination of clopidogrel plus low-dose ASA increased the risk relative to standard adjusted dose VKA at preventing stroke or SE. Edoxaban 30 mg daily, apixaban, edoxaban 60 mg daily and dabigatran 110 mg twice daily were associated with reduced risk in major bleeding compared with standard adjusted dose VKA. Although the results of the current review revealed that there were differences in stroke or SE and major bleeding among antithrombotic agents in the management of patients with AF in relative terms, it is unclear whether the absolute risk reductions associated with these differences translate into clinically meaningful benefits in practice, especially in subpopulations of patients with a lower baseline risk of stroke or SE. Rigorously conducted comparative studies or network meta-regression analyses of patient-level data are required to fully elucidate the comparative benefits and harms of antithrombotic agents across the various subpopulations.


It has been recently reported that telocytes, a stromal  cell subset involved in the control of local tissue homeostasis, are hampered in the target organs of inflammatory/autoimmune disorders. Since no data concerning telocytes in minor salivary glands (MSGs) are currently available, aim of the study was to evaluate telocyte distribution in MSGs with normal architecture, non-specific chronic sialadenitis (NSCS) and primary Sjögren’s syndrome (pSS)-focal lymphocytic sialadenitis. Twelve patients with pSS and 16 sicca non-pSS subjects were enrolled in the study. MSGs were evaluated by haematoxylin and eosin staining and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germinal center (GC)-like structures. Telocytes were identified by immunoperoxidase-based immunohistochemistry for CD34 and CD34/platelet-derived growth factor receptor α double immunofluorescence. Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and encircled both the excretory ducts and the secretory units. In NSCS, despite the presence of a certain degree of inflammation, telocytes were normally represented. Conversely, telocytes were markedly reduced in MSGs from pSS patients compared to normal and NSCS MSGs. Such a decrease was associated with both worsening of glandular inflammation and progression of ectopic lymphoid neogenesis, periductal telocytes being reduced in the presence of smaller inflammatory foci and completely absent in the presence of GC-like structures. Our findings suggest that a loss of MSG telocytes might have important pathophysiological implications in pSS. The specific pro-inflammatory cytokine milieu of pSS MSGs might be one of the causes of telocyte loss. Previous studies reported a marked reduction in telocytes in the skin and in other organs targeted by the fibrotic process including myocardium, lung and gastrointestinal tract of systemic sclerosis (SSc) patients As far as SGs are concerned, to date the only study available described telocyte distribution in normal parotid glands reporting that these peculiar stromal cells surround secretory and excretory structures, namely acini and ducts, and are also in close contact with blood vessels i.e. presence of scattered lymphocyte aggregates that do not reach the number of 50 and therefore cannot be classified as foci) and FLS Taken the lack of data in normal and pSS MSG tissue, the aims of the present study were to evaluate telocyte distribution in MSGs with normal architecture, non-specific chronic sialadenitis  were deparaffinized, rehydrated and stained with haematoxylin and eosin. Focus score and Tarpley biopsy score  Cellular infiltrate and lymphoid organization were assessed by immunofluorescence staining of serial sections with antibodies against human CD3 (T-cell marker), CD20 (B-cell marker) and CD21 (marker of follicular dendritic cells characteristic of GC-like structures). T/B cell segregation was assessed by CD3/CD20 double immunofluorescence staining. Telocytes were identified by CD34 and PDGFRα immunofluorescence, according to previously published protocols 2O2 in methanol for 15 min. at room temperature to block endogenous peroxidase activity. Sections were then washed and incubated with Ultra V block  for 10 min. at room temperature according to the manufacturer’s protocol. After blocking non-specific site binding, slides were incubated overnight at 4°C with mouse monoclonal anti-human CD34 antibody  diluted in 1% BSA in PBS. The day after, tissue sections were washed three times in PBS and incubated with biotinylated secondary antibodies  for 10 min. at room temperature. Subsequently, the slides were washed three times in PBS and incubated with streptavidin peroxidase  for 10 min. at room temperature. Immunoreactivity was developed using 3-amino-9-ethylcarbazole  as chromogen (brownish-red colour). MSG sections were finally counterstained with Mayer’s haematoxylin , washed, mounted in an aqueous mounting medium and observed under a Leica DM4000 B microscope equipped with fully automated transmitted light axes (Leica Microsystems). Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune mouse IgG (Sigma-Aldrich) were included as negative controls for antibody specificity. Light microscopy images were captured with a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).To further identify telocytes in MSG sections with an independent method, indirect immunoperoxidase-based immunohistochemistry was also performed. After deparaffinization and rehydration, MSG tissue sections (3 μm thick) were boiled for 10 min. in sodium citrate buffer  for antigen retrieval and treated with 3% Hn = 5) or severe FLS (n = 7). As compared with mild FLS, patients with severe FLS displayed more severe disorganization of the glandular architecture with larger periductal inflammatory aggregates replacing the secretory units and reduced structural integrity of ducts with prominent epithelial cell apoptosis and loss (Fig.+ follicular dendritic cell network (Fig.+ cells could be detected (Fig.Findings of histopathological analysis of MSGs from patients with pSS and control cases are shown in Figurework Fig., such stwork Fig.. As expework Fig. and onlywork Fig.. Both incted Fig. and J.According to previously published studies +/PDGFRα+ and, at higher magnification, showed a slender nucleated body and long, thin and varicose processes, the telopodes.Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and formed an almost continuous layer encircling both the excretory ducts and the secretory units Figs, E and IIn MSG sections from NSCS cases, the distribution of telocytes was similar to that observed in normal MSGs Fig., F and J+/CD31−, and therefore they could be clearly distinguished from CD34+/CD31+ vascular endothelial cells (Fig.The findings obtained using the immunoperoxidase-based methodology were confirmed by immunofluorescence analysis of MSG sections Fig.–P. In adTo detect possible differences in telocyte distribution according to the presence of GC-like structures, we also performed CD34/CD21 double immunofluorescence on pSS MSG sections. Interestingly, no telocytes could be detected around ducts surrounded by inflammatory foci displaying GC-like structures, while some periductal telocytes were still present in the absence of GC-like structures Fig.–C.+/PDGFRα+ periacinar and periductal stromal cells displaying a slender nucleated body and long, thin and varicose processes consistent with the description of telocytes in the human gastrointestinal tract + dendritic interstitial cells previously described in SGs In the last decades, the pathogenic role of stromal cells in systemic inflammatory/autoimmune disorders including pSS has been extensively investigated A growing number of studies have described telocytes in different mammalian tissues and organs via intercellular contacts or by the release of microvesicles, exosomes and paracrine mediators As far as MSG inflammation is concerned, the evidence that telocytes are markedly reduced in FLS but not in NSCS deserves some consideration. In this setting, the formation and maintenance of FLS as well as the development of ectopic lymphoid structures during chronic inflammation are dependent on the expression of lymphotoxins, cytokines and chemokines by several cell types In conclusion, our data demonstrate an overall impairment of telocyte network in MSGs during pSS. In line with the proposed functions of telocytes


The woring (QS) . In conting (QS) .Enterococcus faecalis grown over an implantable polymeric material (PLGA-HA). Inhibition assays of adhesion and biofilm formation, and SEM observation were used to quantify and qualify E. faecalis' biofilm formation. To assay for biofilm inhibition, bacterial strains were grown statically for 20 h at 37°C in TSB. Polystyrene 96-well microtitre plates were inoculated with 100 μL/well of bacterial suspension previously diluted to 5 × 108 CFU/mL in TSB supplemented with 4% sucrose (w/v) and 3.5% (v/v) dimethyl sulphoxide (DMSO). Compounds to be tested for antibiofilm activity were prepared at a concentration of 175 μg/ml, in the same media as before, and added to the test wells. Biofilm inhibition capacity was assessed by CFU counting after bacterial disaggregation from the surfaces and microscopic analysis of the polymers. Cytotoxicity of the lactams against human fibroblasts and keratinocytes was tested, at four different concentrations , by MTT assay [Here we analysed the effects of novel synthetic lactams, compounds derived from furanones which display recognized antibiofilm activity. Three synthetic lactams were tested against biofilm of All compounds showed biofilm inhibition rates higher than 99%, as accessed by CFU counting and in agreement with images obtained by SEM. At the MIC (minimum inhibitory concentration) human cells viability indexes of all compounds were higher than those recommended for approval in clinical use. Results presented here indicate that lactams have excellent potential for use in the treatment of periodontal disease and in association with PLGA-HA can be used in the prevention of peri-implantitis disease.


Mutations in the protein MKS1 cause severe developmental disorders such as Meckel-Gruber syndrome (MKS). Dysfunctional MKS1 caused a panel of cellular defects ranging from abortive centriolar migration to ciliary instability and defective ciliary signaling . Most anParamecium, we identified a new function of MKS1 in cell polarity.By combining two complementary cell models -a mammalian epithelial cell line and the unicellular We show that MKS1 displays a typical pattern of membrane-associated protein, being localised to exocytotic vesicles, the plasma and the ciliary membrane and to cell junctions during epithelial differentiation. Based on RNAi experiments of MKS1 which leads to impairment of ciliary sensory functions, defective vesicle transport and plasma membrane distension, we propose that MKS1 knockdown impairs interactions between actin and membranes.We finally show how MKS1 depletion interferes with epithelial differentiation and cell organogenesis in 3D cultures.


Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated  An increased demand for food with improved freshness, sensorial, and nutritional values has directed food processing toward the use of milder heat treatments that require secondary mild preservation hurdles to assure stability and safety of the products . As a reBacillus subtilis, is determined by a number of factors including the spore structural components  but also the sporulation conditions  affect its resistance  on blood agar  in food before ingestion resulting in an emetic syndrome. B. cereus associated diseases are usually mild and self-limiting but in rare instances they can lead to fatal outcomes , a role in spore damage repair was further confirmed using a targeted deletion mutant  in combination with single spore sorting and a Most Probable Number (MPN) approach. To quantify the effect of matrix conditions on recovery capacity of wild type and mutant spores BHI and rice media, both in solid and liquid form were included. This approach allows for identification of candidate genes that may contribute to recovery capacity of heat treated B. cereus spores.To this end, Bacillus cereus ATCC 14579 obtained from the American Type Culture Collection (ATCC), and its mutant derivatives used in this study (Table 1) were cultured in Bacto Brain Hart Infusion broth  at 30°C with aeration at 200 rpm. A nutrient-rich, chemically defined sporulation medium (MSM medium) described previously  were constructed using the temperature-sensitive suicide plasmid pAUL-A  to select for the desired transformants. Two erythromycin resistant colonies were selected and grown overnight in BHI at 30°C in the presence of E10. The resulting culture was diluted (1:200) in fresh LB with E10 and grown o/n at 42°C to select for plasmid integration. Selected strains resulting from a single cross-over integration event were grown overnight in BHI at 30°C to induce double crossover events and subsequently plated and grown at 30°C. Resulting colonies were replica plated on BHI with and without E10 and incubated at 37°C. Colonies sensitive to E10 were selected. PCR analyses  (Supplementary Table Deletion mutants (d pAUL-A . Flankind pAUL-A  for upstcdnL1/ΔcdnL2) was obtained as described above with the exception that the cdnL2 knock out plasmid was transformed into a B. cereus ΔcdnL1 (BC4714) mutant strain constructed previously . The PCR tubes were kept for 1 min at 4°C followed by a step at 95°C for 45 s and finally cooled for 1 min at 4°C in a thermal cycler . The duplicates were pooled and 100 μl of this pooled fraction was used as sample for spore sorting experiments. The same pooled fraction was diluted decimally in suspension buffer and 50 μl samples were used for spore enumeration on BHI plates (in duplicate) and incubated at 30°C up to 3 days with daily enumeration of resulting colonies. For each strain, from the same spore preparation, at least four independent heating experiments were performed.One hundred micro liter of spore suspension containing approximately 10To evaluate the degree of spore damage, the method previously reported by Flow cytometry was performed with a FACSAriaIII cell sorter (BD Biosciences) using a fiber launched solid state air-cooled laser operating at 488 nm. Only forward scatter (FCS) and side scatter (SSC) functionality was used. The machine was calibrated using standard Cytometer Setup & Tracking beads and Accudrop beads (BD Biosciences). All parameters were measured using logarithmic amplification. During the procedures a 85 micron nozzle (drop driving frequency was ∼45 kHz/s) was used with flow rate one and during sorting a maximum event rate of 2000 events/s was used. Cells and spores were discriminated from electronic noise using both SSC and FSC. Sorting criteria and gating strategy were based on FSC and SSC populations  excluding remaining doublets. In order to achieve high purity and recovery, the “Single Cell” precision mode (Purity mask 32 and Phase mask 16) was used for sorting. Cells or spores were sorted on solid and in liquid media.B. cereus ATCC 14579 and the mutant derivatives were spotted in duplicate on a single BHI and rice agar plate , and in case of BHI values from OD600 0.2 to 0.8 (OD600 of fresh media 0.1). The MPN values and their upper and lower limits were calculated using MPN Calculator1.One hundred micro liter of unheated or heat treated spore suspension  was diluted in 1.5 ml of HEPES buffer (pH 7.4) in 5 ml polystyrene falcon tube  and loaded into the flow cytometer. For heat treated spores, a series of 1, 10, and 100 individual spores were sorted either into wells of 384-well plates  containing 50 μl of BHI or rice broth or on one of the 52 available locations on standard BHI or rice agar plates. The resulting growth data representing three consecutive decimal dilutions were used as input for the MPN quantification method . For heaA rice based medium was prepared according to the method reported previously  by boiliB. cereus spores was evaluated using FCM in combination with single spore sorting. The single untreated B. cereus spores were sorted into four different media namely BHI broth, rice broth, BHI agar plates and rice agar plates. Besides BHI, a rice media was selected as this food matrix was shown previously to support growth from B. cereus spores on agar plates . In general, smaller colonies were formed on rice agar plates compared to BHI agar plates for both outgrowing untreated spores (data not shown) and vegetative cells .The impact of the liquid and solid media composition on the growth of r plates  and on Ar plates  that resr plates . All foucdnL1 mutant was affected in the ratio between mildly and severely damaged spores . This indicated that not only the composition of the media but also its liquid or solid state has an effect on the recovery of the spores.To evaluate recovery of sorted heat treated spores in a high throughput format, a combination of single spore sorting with MPN method was applied. Sorting of spores in series of 1, 10, and 100 of heat treated spores at individual locations (well or spot) increased the resolution of the measurements allowing to observe significant differences within expected 2 log inactivation range. Interestingly, for the heat treated spores a comparable recovery in BHI broth and on rice agar plates was observed while rice broth and BHI agar plate supported recovery of approximately 50% of the surviving spores compared to BHI broth , BC4834, BC5038, and BC5242 (cdnL1 (BC4714)), a putative transcriptional regulator, was slightly but significantly affected in repair and outgrowth of heat treated B. cereus spores  is encoded on the B. cereus ATCC 14579 genome and it was hypothesized that its gene product masked effects on spore damage recovery in the cdnL1 deletion mutant. Therefore, a cdnL2 (BC3648) mutant and a combined cdnL1/cdnL2 mutant were included in the present study.Previously, genes expressed during germination and outgrowth of heat treated ith qPCR  to confid BC5242 . A mutans spores . A paralTable 1), four displayed various sporulation defects, mutants either did not sporulate (ΔBC4834), displayed an incomplete sporulation process (ΔBC1312) or the resulting spores were not fully released form the mother cell (ΔBC3437 and ΔBC3921). Therefore, these mutants were excluded from further analysis.Attempts to construct mutants in BC3438 and BC5038 were unsuccessful. Of the 13 successfully constructed mutants . Deletion of ΔBC5242 and ΔBC0853 led to the highest reduction in recovery for all tested media  suggesting that effects of these genes on recovery and possibly damage repair were media independent. In contrast, deletion of BC0690 resulted in higher recovery compared to the wild type in both BHI broth (50% increase) and on BHI agar plates (150% increase) . Deletion of cdnL1 (BC4714) resulted in a recovery in BHI broth comparable to that of the wild type, albeit that time to growth was delayed in BHI broth and to a lesser extent on BHI agar plates  resulted in a slight reduction in recovery compared to wild type in BHI broth, while recovery of the cdnL1/cdnL2 double mutant (ΔBC4714/ΔBC3648) was reduced by approximately 50% in all tested media compared to wild type . BC0852 and BC0460 mutants displayed a comparable reduction in recovery as the cdnL1/cdnL2 double mutant. Finally, the recovery of ΔBC1314 depended on the recovery media, in rice broth the recovery was comparable to wild type  while in BHI broth deletion led to over 50% reduction in recovery compared to wild type. Recovery of heat treated spores of all but two (ΔBC0460 and ΔBC0690) deletion mutants was higher on rice agar plates, compared to BHI broth . This mutation resulted in reduction in recovery down to 3.6% of the wild type recovery in BHI broth. The function of BC5242 is unknown, but orthologs of its gene product can be found in many  sensing . In prok sensing , howeverild acid .B. cereus spores (B. cereus ATCC 14579 genome sequence suggested that BC1314 (and BC1315) result from a frame-shift mutation in the phaQ gene  synthesis cluster, and PHB was previously shown to be accumulated in cells in the form of granules that serve as a carbon and energy source during the late sporulation process in B. cereus , phaQ (encoding a repressor of phaP expression), phaB (acetoacetyl-CoA reductase), phaR and phaC (subunits of PHB synthase) , lacking genes encoding both CdnL transcriptional regulators present in B. cereus ATCC 14579 showed 60% reduction in recovery in BHI broth compared to the wild type. Deletion of cdnL1 (BC4714) was shown previously to increase the fraction of severely damaged spores in the surviving population after a heat treatment of 1 min at 95°C (cdnL1 (BC4714). However, outgrowth from heat treated ΔcdnL1 spores was delayed in BHI broth compared to the wild type spores, eventually reaching comparable recovery efficiency. The ΔcdnL2 mutant (BC3648) showed lower recovery compared to ΔcdnL1 (BC4714), and this was most pronounced in liquid media. Nevertheless, both ΔcdnL1 and ΔcdnL2 in media other than BHI broth show improved recovery compared to wild type. It remains to be determined whether the observed increase in recovery of the individual cdnL mutants could be explained by cross regulation of the counterpart. Both cdnL1 and cdnL2 genes are induced in vegetative cells in response to various environmental stresses, including salt and cold stress, whereas acid and oxidative stress specifically induced expression of cdnL1 and not cdnL2  orthologs to ykkC (BSU13090) and ykkD (BSU13100) of B. subtilis 168. ykkC and ykkD are a paired small multidrug resistance (PSMR) members, and their co-expression in Escherichia coli led to a multidrug-resistant phenotype . At the moment it cannot be excluded that differences in spore recovery in BHI broth are due to lack of one or more specific proteins in spores of tested deletion mutants that makes them more or less resistant and/or susceptible to heat damage. However, application of rice media and BHI agar plates compared to BHI broth for sorted spores also revealed differences in recovery between media suggesting different requirements for recovery. Particularly deletion of BC0460 or BC0690 resulted in reduced recovery on rice plates while spores of remaining seven deletion mutants showed improved recovery on rice plates compared to BHI broth (Supplementary Figure B. cereus spores is affected by the matrix composition with highest recovery of wild type spores in BHI broth or on rice agar plates, followed by BHI agar plates and rice broth. The comparative analysis of the wild type and newly constructed deletion mutants provided new insights in the putative role of the deleted genes in the recovery of heat treated B. cereus spores.We have shown that recovery of heat treated Conceived and designed the experiments: AW, MT, TA, MNG. Performed the experiments: AW, MT. Analyzed the data: AW, MT. Wrote the paper: AW, MT, TA, MNG.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.


Muscle stem cells termed satellite cells are essential for muscle regeneration. A central question in many neuromuscular disorders is why satellite cells are unable to prevent progressive muscle wasting. We have analyzed muscle fiber pathology and the satellite cell response in Pompe disease, a metabolic myopathy caused by acid alpha-glucosidase deficiency and lysosomal glycogen accumulation. Pathology included muscle fiber vacuolization, loss of cross striation, and immune cell infiltration.The total number of Pax7-positive satellite cells in muscle biopsies from infantile, childhood onset and adult patients (with different ages and disease severities) were indistinguishable from controls, indicating that the satellite cell pool is not exhausted in Pompe disease. Pax7/Ki67 double stainings showed low levels of satellite cell proliferation similar to controls, while MyoD and Myogenin stainings showed undetectable satellite cell differentiation. Muscle regenerative activity monitored with expression of embryonic Myosin Heavy Chain was weak in the rapidly progressing classic infantile form and undetectable in the more slowly progressive childhood and adult onset disease including in severely affected patients.These results imply that ongoing muscle wasting in Pompe disease may be explained by insufficient satellite cell activation and muscle regeneration. The preservation of the satellite cell pool may offer a venue for the development of novel treatment strategies directed towards the activation of endogenous satellite cells.The online version of this article (doi:10.1186/s40478-015-0243-x) contains supplementary material, which is available to authorized users. Healthy skeletal muscle has a remarkable capacity to repair both minor and severe damage. Extensive muscle damage, such as caused by exercise or in muscular disease, requires a stem cell-mediated response. The muscle stem cells or muscle satellite cells are located at the myofiber periphery underneath the basal lamina . SatelliGenetic diseases affecting skeletal muscle pose a continuous challenge to the muscle regenerative system. More than 700 neuromuscular disorders are known . While tOne hypothesis to explain this is depletion of the satellite cell pool due to continuous regeneration attempts. This idea has been derived from in vitro characterization of myoblasts from Duchenne Muscular Dystrophy (DMD) patients, which showed reduced proliferative capacity . In agreA different group of neuromuscular disorders are the metabolic myopathies . Recent We have analyzed skeletal muscle biopsies from classic infantile patients and from patients with childhood and adult onset Pompe disease and compared the extent of muscle damage, satellite cell activation, immune cell infiltration, and muscle regenerative activity.2-chilled isopentane and cryosectioned (5–10 μm). Medical Research Council (MRC) sumscores were calculated from the assessment of the strength of 26 muscle groups essentially as described [Muscle biopsies were taken from the Vastus Lateralis using a standard open surgery or needle biopsy procedure as described previously  from Pomescribed .HE staining and Periodic Acid Schiff (PAS) staining were performed on tissue that was processed into glycolmetacrylate (GMA) . Acid PhScoring of muscle damage was based on a system previously described by . All sec5–10 μm cryosections of patient and control biopsies were stored until analysis at −80 °C. Sections were thawed before staining. Primary antibodies were directed against CD68, clone KP1 , eMyHC , Ki67 , Laminin , MyoD , Myogenin , Pax7 , CD3 , CD20Cy; clone L26; Dako, 1:400). Following the primary antibody incubation, sections were rinsed and incubated with biotin-conjugated anti-mouse antibody  and then with Alexafluor594-conjugated streptavidin . Sections incubated with rabbit primary antibodies were subsequently incubated with Alexafluor 488-conjugated goat-anti rabbit antibodies . Finally chicken anti-Laminin was detected with Alexafluor647-conjugated goat anti-chicken antibodies . All sections were counterstained with Hoechst33258 . The slides were mounted with Mowiol . For some biopsies, limited amounts of sections of sufficient quality were available for immunofluorescent analysis, and not all stainings could be performed for all patients.For digital imaging complete histological sections were scanned on a Hamamatsu NanoZoomer 2.0 (Hamamatsu Photonics). Images were analyzed using NDP view software . Sections for immunofluorescence were scanned on a Zeiss LSM700  using tile-scan modality. Image analysis was performed using Fiji (fiji.sc/Fiji).P <0.05. All calculations were performed using Graphpad 5.0 .All data are expressed as means ± SD. Multiple groups were compared using a one way non-parametric ANOVA. Bonferroni correction was applied as Post-hoc test to adjust for multiple testing. Statistical significance was set at Biopsies of Pompe patients before the start of enzyme replacement therapy were taken from the Quadriceps Femoris (QF) and were used in this study. Patients were categorized into 4 groups based on age of disease onset and disease severity: 1) Classic infantile Pompe disease, with disease onset shortly after birth; 2) Childhood onset Pompe disease—disease onset ranging from 1 to 18 years; (3) Adult Pompe disease—mildly affected (>18 years old and <15 years disease symptoms); (4) Adult Pompe disease—severely affected . Mildly affected adults (group 3) on average appeared younger as compared to severely affected adults (group 4)  at 1–2 years of age, followed by a decline to about 2 % in adults . Pompe pBoth satellite cell proliferation, monitored from the percentage of Ki67-positive satellite cells, and differentiation, indicated by the number of MyoD- and Myogenin-positive cells, were similar to controls for all Pompe patient groups. This indicated a failure of a robust satellite cell response in Pompe disease irrespective of disease severity. A previous report described low levels of satellite cell activation in classic infantile Pompe patients monitored 1 year after enzyme replacement therapy . Two patAn important question is whether the failure of satellite cell activation in Pompe disease is caused by the absence of an activating signal or the presence of an inhibitory factor. In muscle injury induced experimentally or by exercise, sarcolemmal damage triggers satellite cell activation by disturbing the niche of the satellite cell in between the sarcolemma and basal lamina. In Pompe disease, no gross disruption of the sarcolemma can be observed even in classic infantile patients, as shown using electron microscopy  and lighSubsequent signals involved in both activation and inhibition of muscle regeneration are derived from the immune system. Following injury, a transient immune response triggers satellite cell activation and muscle regeneration. However, chronic immune responses can potently inhibit muscle regeneration and induce TGFβ-signaling-mediated muscle fibrosis as is the case for the muscular dystrophies. An immune response in Pompe patients is a hereto understudied factor considering the paucity of published reports on this aspect. One previous report has also detected inflammatory cells in skeletal muscle of an adult Pompe patient , but theAnother factor that may contribute to the deregulation of satellite cell activation is impaired autophagy. It has been suggested that hematopoietic stem cells depend on autophagy as energy source for proper activation , and recThe positive news for Pompe patients is that their satellite cell pool is intact and not exhausted. This may open novel treatment options targeted at enhancement of satellite cell activation and improved muscle regeneration. A prerequisite for successful implementation of such an approach is that the intrinsic properties of satellite cells in Pompe patients are intact. Electron microscopy studies of satellite cells from Pompe patients showed normal morphology . StudiesIn conclusion, despite the extensive muscle damage satellite cells do not become robustly activated during Pompe disease progression. In line with this, the regenerative response in skeletal muscle of classic infantile Pompe patients was weak and absent in childhood- and adult-onset Pompe patients. The preservation of the satellite cell pool may offer a venue for the development of novel treatment strategies directed towards the activation of endogenous satellite cells.