Project description:Evaluation of short-read-only, long-read-only, and hybrid assembly approaches on metagenomic samples demonstrating how they affect gene and protein prediction which is relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic, and metaproteomic data to evaluate the metagenomic-based protein predictions.
Project description:Inflammatory bowel diseases encompass gastrointestinal illnesseses typified by chronic inflammation, loss of epithelial integrity and gastrointestinal microbiota dysbiosis. In an effort to counteract these characteristic perturbations, we used stem cells and/or a probiotic preparation in a murine model of Dextran Sodium Sulfate induced colitis to examine both their efficacy in ameliorating disease and impact on niche-specific microbial communities of the lower GI tract. Colitis was induced in C57BL/6 mice by administering 3% DSS in drinking water for 10 days prior to administering one of three treatment plans: daily probiotic (VSL#3) supplementation for 3 days, a single tail vein injection of 1x106 murine mesenchymal stem cells, or both. Controls included DSS-untreated mice and DSS-treated mice that received no therapy. Ileal, cecal and colonic sections were collected for microbiota and histological analyses. Microbiota profiling revealed distinct bacterial community compositions in the ileum, cecum and colon of control untreated animals, all of which were predicted in silico to be enriched for a number of discrete KEGG pathways, indicating compositional and functional niche specificity in healthy animals. DSS- treatment perturbed community composition in all three niches with ileal communities exhibiting the greatest change relative to control animals. Stem cell, VSL#3 and the combination treated animals exhibited treatment-specific microbiota composition in the lower GI tract, though disease scores were only improved in VSL#3 treated animals. This VSL#3-associated shift in the ileal microbiota was characterized by significant Enterobacteriaceae enrichment compared to colitic animals (p<0.05), Mice (n=40) were randomly divided into five experimental groups, four of which received Dextran Sodium Sulfate (DSS; 3% solution in drinking water) for 10 days to induce colitis. Three of the DSS-treated groups received the following treatment modalities: VSL#3 (VSL#3, n=5), mesenchymal stem cells (MSC, n=5), or VSL#3 + mesenchymal stem cells (DUAL, n=5). The fourth DSS-treated group received no intervention (DSS; n=10). The additional fifth group of animals received neither DSS nor any therapeutic intervention and acted as untreated controls (CNTL, n=15). Following colitis induction (Day 10), DSS administration was halted and mice in the VSL#3, MSC and DUAL groups received the following interventions respectively: daily oral supplementation with 5x106 CFUs per supplement of VSL#3 in 100ul PBS (VSL#3); a single tail vein injection of 1x106 murine mesenchymal stem cells in 100_l PBS on Day 10 (MSC) or a combination of both treatments To provide control data for comparison, CNTL mice (n=5 per time point) were euthanized and sampled on days 1, 10, and 14, while DSS mice (n=5 per time point) were euthanized on days 10 and 14. All MSC, VSL#3, and DUAL mice were euthanized on Day 14. Samples collected from each animal included terminal ileum (1cm proximal to the cecum), cecum (divided transversely and stored as two separate samples), and proximal colon. All samples were added to RNAlater, prior to storage at -80C for analysis. Additional colonic samples were obtained, proximal to the initial sample site for microbiome analyses, and were preserved in paraformaldehyde for histological analyses.
Project description:Background & Aims: Irritable bowel syndrome (IBS) is a disorder characterized by chronic abdominal pain and is linked to post-inflammatory and stress-correlated factors that cause changes in the perception of visceral events. Increased evidence indicates that probiotic bacteria may be useful in treating IBS. Our aims were to evaluate the efficacy of treatment with VSL#3, a mixture of 8 probiotic bacteria strains, in the neonatal maternal separation (NMS)-induced visceral hypersensitivity rat model and to determine whether it modulates the colonic expression of pain-related genes. Methods: Male NMS pups were treated orally with placebo or VSL#3 at days 3-60, while normal, not separated rats were used as control. After 60 days from birth, perception of painful sensation induced by colorectal distension (CRD) was measured by assessing the abdominal withdrawal reflex (score 0-4). The colonic gene expression analysis was assessed by using Agilent Whole Rat Genome Oligo Microarrays. Results: NMS rats exhibited both hyperalgesia and allodynia when compared with controls. VSL#3 showed a potent analgesic effect on CRD-induced pain without modifying colorectal compliance. The microarray analysis demonstrated that NMS rats had both over- and downregulation of several genes involved in inflammatory and painful processes and VSL#3 was able to counteract these alterations. Conclusions: This study indicates that VSL#3 is effective in reducing visceral pain in an experimental model of IBS by induction or suppression of pain-modulating genes. These observations provide support for the use of VSL#3 in the treatment of painful conditions related to IBS. The dataset comprises 12 samples divided into three sample groups each representing a certain treatment condition of male rats.
Project description:Background & Aims: Irritable bowel syndrome (IBS) is a disorder characterized by chronic abdominal pain and is linked to post-inflammatory and stress-correlated factors that cause changes in the perception of visceral events. Increased evidence indicates that probiotic bacteria may be useful in treating IBS. Our aims were to evaluate the efficacy of treatment with VSL#3, a mixture of 8 probiotic bacteria strains, in the neonatal maternal separation (NMS)-induced visceral hypersensitivity rat model and to determine whether it modulates the colonic expression of pain-related genes. Methods: Male NMS pups were treated orally with placebo or VSL#3 at days 3-60, while normal, not separated rats were used as control. After 60 days from birth, perception of painful sensation induced by colorectal distension (CRD) was measured by assessing the abdominal withdrawal reflex (score 0-4). The colonic gene expression analysis was assessed by using Agilent Whole Rat Genome Oligo Microarrays. Results: NMS rats exhibited both hyperalgesia and allodynia when compared with controls. VSL#3 showed a potent analgesic effect on CRD-induced pain without modifying colorectal compliance. The microarray analysis demonstrated that NMS rats had both over- and downregulation of several genes involved in inflammatory and painful processes and VSL#3 was able to counteract these alterations. Conclusions: This study indicates that VSL#3 is effective in reducing visceral pain in an experimental model of IBS by induction or suppression of pain-modulating genes. These observations provide support for the use of VSL#3 in the treatment of painful conditions related to IBS.
Project description:Using Affymetrix data analysis, important signalling pathways and transcription factors relevant to gut inflammation and anti-inflammatory action of probiotics were identified using the clinically validated probiotic VSL#3 and the IL10-knockout mouse, an animal model for inflammatory bowel disease. VSL#3 increased expression of genes involved in PPAR signalling and metabolism of xenobiotics and decreased expression of genes involved in immune response/inflammatory response.
Project description:Down-modulation or loss-of-function mutations of the Notch 1 and 2 genes are associated with development of squamous cell carcinoma (SCC), a very frequent and therapy-resilient malignancy in skin, head/neck (H/N), lung and other surface epithelia. In this context, surprisingly little is known on the role of CSL (RBP-Jk), key effector of canonical Notch signaling endowed with intrinsic transcription repressive function. CSL expression is decreased in upper epidermal layers and differentiating primary human keratinocytes (HKCs), while it is up-regulated in premalignant and malignant SCC lesions and SCC cell lines from skin, Head/Neck and lung. Increased CSL levels enhance proliferation and self-renewal potential of HKCs and SCC cells, while its silencing induces growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels give rise to rapidly expanding tumors, while upon CSL silencing they form smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKC and SCC cells plus/minus CSL silencing reveals major modulation of apoptotic, cell cycle and pro-inflammatory genes, with no significant association with Notch or keratinocyte differentiation gene signatures. KDM6B, a histone demethylase gene with highly context dependent functions, is a direct CSL negative target, with an inverse role of CSL in HKC and SCC self-renewal and tumorigenesis, with IL6 as a target of likely significance. CSL / KDM6B protein expression could be used as biomarkers of SCC development and novel indicators of cancer treatment.
Project description:Using Affymetrix data analysis, important signalling pathways and transcription factors relevant to gut inflammation and anti-inflammatory action of probiotics were identified using the clinically validated probiotic VSL#3 and the IL10-knockout mouse, an animal model for inflammatory bowel disease. VSL#3 increased expression of genes in volved in PPAR signalling and metabolism of xenobiotics and decreased expression of genes involved in immune response/inflammatory response. IL10-knockout (IL10-KO) and wildtype (WT) mice housed under specific pathogen free (SPF) conditions were sacrificed at 24 weeks by cervical dislocation. The study is comprised of two independent Microarray experiments. Microarray experiment1 compares gene expression of IL10-KO and WT colon tissue. For microarray analysis RNA was extracted from the colon tissue of each mouse (WT n=7, IL10-KO n=6). Microarray experiment2 compares gene expression of WT and IL10-KO mice fed with either placebo or probiotic VSL#3. IL10-KO and WT mice were fed with placebo or 1.3x109 cfu of lyophilized VSL#3 bacteria post weaning for 21 weeks. For microarray analysis RNA was extracted from the caecum tissue of each mouse (WT Placebo n=6, IL10-KO Placebo n=6, IL10-KO VSL#3 n=6).
Project description:Two sets of RNA samples were generated from 3 days growth in different serum lots, lot 6999 and lot 6473. Lot 6999 has been pre-screened for embryonic stem cell maintenance and is routinely used for that purpose. Lot 6473 failed all tests for embryonic stem cell maintenance. Keywords: other
Project description:Senescence of stromal fibroblasts has been linked to establishment of cancer associated fibroblasts (CAF) and aging-associated increase of tumors. However, in clinically occurring carcinomas, density and proliferation of CAFs are frequently increased rather than decreased. We previously showed that genetic deletion or down-modulation of the canonical Notch effector CSL/RBP-J? in skin dermal fibroblasts is sufficient for CAF activation with consequent development of multifocal keratinocyte tumors. We now show that CSL deletion or knockdown induces senescence of primary fibroblasts derived from dermis, oral mucosa, breast and lung. CSL functions in these cells as a constitutive direct repressor of multiple senescence- and CAF-effector genes. At the same time, it physically interacts with p53, repressing its activity, and p53 activation provides a failsafe mechanism against compromised CSL function. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effector genes and, in vivo, promotes tumour and stromal cell expansion. Together, the findings support a CAF activation/stromal evolution model under convergent CSL/p53 control. Examination of genome-wide CSL binding sites in primary human dermal fibroblasts usinf two different antibodies against CSL