Project description:Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Î¦6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Î¦6 as a model system in evolutionary biology, Pph resistance to phage Î¦6 remains poorly characterized. To investigate differences between phage Î¦6 resistant Pseudomonas syringae pathovar phaseolicola strains, we performed expression analysis of super and non piliated strains of Pseudomonas syringae to determine the genetic cause of resistance to viral infection.
Project description:Type I interferons are critical anti-viral cytokines during virus infections and have also been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The secretion of type I interferon of pDCs is modulated by Siglec-H, a DAP12 associated receptor on pDCs. We showed that Siglec-H deficient pDCs produce more of the type I interferon IFN-Î± in vitro and that Siglec-H ko mice produce more IFN-Î± after murine cytomegalovirus (mCMV) infection in vivo, leading to efficient clearance of the virus. Furthermore, ageing Siglec-H ko mice showed a mild form of systemic autoimmunity. In contrast, Siglec-H ko mice developed a severe form of systemic lupus-like autoimmune disease with strong kidney nephritis several weeks after a single mCMV infection. This induction of systemic autoimmune disease after virus infection in Siglec-H ko mice was accompanied by a type I interferon signature and fully dependent on type I interferon signaling. These results show that Siglec-H normally serves as modulator of type I interferon responses after infection with a persistent virus and thereby prevents induction of autoimmune disease. For microarray experiments gene expression profiles of total splenic cells from two wt and Siglec-H ko mice 26 weeks after infection with luciferase expressing murine Cytomegalovirus (5x105 pfu) or from two uninfected wt and Siglec-H ko control mice were analyzed
Project description:The transcriptional repressor BLIMP1 is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8 as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8 and consequently Blimp1 CKO mice had higher levels of circulating CCL8 resulting in increased neutrophils in the peripheral blood, promoting a more aggressive anti-bacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than wild type mice. While CCL8 failed to recruit neutrophils directly, it was chemotactic for Î³/Î´ T cells and CCL8-responsive Î³/Î´ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on Î³/Î´ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host-defenses by suppressing expression of the chemokine CCL8. RNA (5Î¼g) was isolated from WT and Blimp1 CKO BMDM untreated or infected for 2 hours with L.monocytogenes (MOI=5). Three biological replicates were performed for each experimental condition. The gene chip mouse genome 430 2.0 Array (Affymetrix) was used. Biological replicates were normalized using the GCRMA method and analyzed using various Bioconductor tools.
Project description:Objective: Although glucagon-secreting Î±-cells and insulin-secreting Î²-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and have overlaps in their transcriptome and epigenome profiles. Notably, destruction of one of these cell populations can stimulate repopulation via transdifferentiation of the other cell type, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both Î±- and Î²-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and Î²-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in Î±- and Î²-cell specification and plasticity. Methods: We sorted human a- and b-cells and performed the â??Assay for Transposase-Accessible Chromatin with high throughput sequencingâ?? (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. Results: We identified numerous transcripts with either Î±-cell- or Î²-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The â??group specific proteinâ?? (GC; or vitamin D binding protein) was restricted to a-cells, while CHODL (chondrolectin) immunoreactivity was only present in b-cells. Furthermore, Î±-cell- and Î²-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with common single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. Conclusions: We have determined the genetic landscape of human Î±- and Î²-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel Î±- and Î²-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion. ATAC-seq on 3 human alpha cell samples, 3 human beta cell samples, and 2 human acinar cell samples. RNA-seq on 7 human alpha cell samples and 8 human beta cell samples.
Project description:Purpose: To study the effects of disrupting the Trf5-Mtr4 interaction Methods: Analyzed TRF5 and trf5-Î?98-117 strains by transcriptome sequencing of poly(A)+ RNA Results: The set of most significantly affected genes included 71 that were overexpressed in trf5-Î?98-117 and only 5 that were down-regulated.The set of 71 overexpressed genes was predominated by known TRAMP substrates Conclusions: Disruption of the Trf5-Mtr4 interaction results in a snoRNA processing defect Duplicate RNA samples of trf4Î?, trf5Î? yeast strains expressing either TRF5 or trf5-Î?98-117 were enriched for poly(A)+ transcripts and converted to a sequencing library. Transcriptome sequencing was performed on a HiSeq 2500. Each sequenced library yielded between 10 and 14 million 50nt reads that were mapped to the annotated yeast genes using Bowtie. Significantly up- or down-regulated genes in the trf5-Î?98-117 mutant were identified via edgeR.
Project description:Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal liver development. We found a major shift in expression of Wnt and RXR-Î± pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-Î± gene signature categorized HCCs into two subtypes (high Wnt, low RXR-Î± and low Wnt, high RXR-Î±), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-Î± levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-Î± achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-Î±, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target. Total RNA from whole livers taken at different developmental timepoints (embryonic day 14, embryonic day 18, post-natal day 5 and post-natal day 56) along with hepatoblasts isolated from E14 livers and immature hepatocytes isolated from E18 livers was extracted and purified using the Qiagen RNeasy Mini Kit. RNA purity and integrity were assayed by the Bioanalyser 2100 (Agilent Technologies). For each sample, 2 Âµg of total RNA was reverse transcribed and amplified by using an RNA amplification kit from Ambion. Fifteen micrograms of amplified RNA were labeled by direct chemical coupling to the Cy5 NHS ester (Amersham Biosciences). Normal adult mouse liver (Agilent) was used as control and Cy3 labeled. Labeled RNAs were purified, fragmented, and used as probes to hybridize microarrays. Gene expression profiling was done with the 4x44k mouse Agilent platform. Expression profiling of the 23 human HCC samples was previously described
Project description:Identify genes which are differentially expressed in Î?Cop1Î² compared to control islets and are also signficantly rescued in Î?Cop1Î? Î?ETV1/4/5Î² Islets were harvested from 5 biological replicates of the followig genotypes: Control, Î?Cop1Î² and Î?Cop1Î? Î?ETV1/4/5Î². Islets were individually handpicked and total RNA was isolated and purified for library preperation and Next Generation Sequencing.
Project description:Background: In the diabetic heart the Î²-adrenergic response is altered partly by down-regulation of the Î²1-adrenoceptor, reducing its positive inotropic effect and up-regulation of the Î²3-adrenoceptor, increasing its negative inotropic effect. Statins have clinical benefits on morbidity and mortality in diabetic patients which are attributed to â??pleiotropicâ?? effects. The objective of our study was to investigate the role of statin treatment on Î²-adrenergic dysfunction in diabetic rat cardiomyocytes. Methods: Î²-adrenergic responses were investigated in vivo (echocardiography) and ex vivo (left ventricular papillary muscles) in healthy and streptozotocin-induced diabetic rats, who were pre-treated or not by oral atorvastatin over 15 days (50 mg.kg-1.day-1). Micro-array analysis and immunoblotting were performed in left ventricular homogenates. Data are presented as mean percentage of baseline Â± SD. Results: Atorvastatin restored the impaired positive inotropic effect of Î²-adrenergic stimulation in diabetic hearts compared with healthy hearts both in vivo and ex vivo but did not suppress the diastolic dysfunction of diabetes. Atorvastatin changed the RNA expression of 9 genes in the Î²-adrenergic pathway and corrected the protein expression of Î²1-adrenoceptor and Î²1/Î²3-adrenoceptor ratio, and multidrug resistance protein 4 (MRP4). Nitric oxide synthase (NOS) inhibition abolished the beneficial effects of atorvastatin on the Î²-adrenoceptor response. Conclusions: Atorvastatin restored the positive inotropic effect of the Î²-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by multiple modifications in expression of proteins in the Î²-adrenergic signaling pathway, particularly through the NOS pathway.
Project description:The source of IFN-Î³ in ovarian cancer microenvironment and its biological effect to the tumor cells is unclear. The immortalized human ovarian surface epithelial cell line, HOSE-E7/hTERT (HOSE) was treated with IFN-Î³ and expression microarray analysis was performed, and probes showing significantly higher values in IFN-Î³-added group were termed â??IFN-Î³ signature genes (295 probes)â??. We then applied this signature to our ovarian cancer microarray data, which included 75 ovarian cancer clinical samples, by means of ss-GSEA. IFN-Î³ signature score was strongly correlated to the number of infiltrating CD4-positive or CD8-positive lymphocytes in the tumors. These data suggest that the IFN-Î³ in the ovarian cancer microenvironment is derived from lymphocytes, and an IFN-Î³-rich microenvironment is strongly correlated to a lymphocyte-rich microenvironment. HOSE cells were incubated in 8 separate culture dishes, 4 dishes with and 4 dishes without 500 IU/ml recombinant human IFN-Î³ (R&D Systems) in the culture medium for 6 hours prior to the analysis.