Project description:Untargeted Proteomics of full protein extract of Listeria monocytogenes cultures with sublethal concentrations of nisin and fengycin.
Project description:Background: Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in animal models of diabetes. Human studies have also revealed loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages in full thickness gastric biopsies from gastroparesis patients. Aim: We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in full thickness gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying and symptoms. Methods: Full-thickness gastric antrum biopsies were obtained from nine DG, seven IG patients and five non-diabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1300 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity Pathway Analysis and correlations were carried out. Multiple-testing corrected p-values <0.05 were considered statistically significant. Results: 73 proteins were differentially expressed in DG, 132 proteins in IG and 40 proteins were common to DG and IG. In both DG and IG, “Role of Macrophages, Fibroblasts and Endothelial Cells” was the most statistically significant altered pathway (DG FDR: 7.9x10-9; IG FDR: 6.3x10-12). In DG, properdin expression correlated with GCSI-bloating (r: -0.99, FDR: 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C zeta type and complement C2 correlated with 4 hr gastric retention (r: -0.97, FDR: 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Conclusions: Protein expression changes suggest a central role of macrophage-driven immune dysregulation and complement activation in gastroparesis.
Project description:The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Wild-type strain and spx mutant cells exposed or not to diamide stress were subjected to tiling array gene expression analysis. To distinguish direct and indirect effects, the genomic sites bound by the Spx-RNAP complex were mapped using a chromatine immunoprecipitation approach. This allowed the identification of 144 transcription units comprising 275 genes under direct Spx regulation. This data set contains 4 samples. Immunoprecipitated (IP) DNA from Bacillus subtilis Bas044 strain (BSB1, a tryptophan-prototrophic derivative 168 strain expressing a SPA-tagged Spx protein) were extracted from bacterial cultures in absence or presence of diamide. IP and whole cell extract DNA were hybridized on tiling array to generate ChIP-on-chip profiles. Two biological replicates were analyzed.
Project description:Investigation of whole genome gene expression level changes in sporulating Bacillus subtilis 168 delta-prpE mutant, compared to the wild-type strain. The mutation engineered into this strain results in impaired germination of spores. A six chip study using total RNA extracted from three separate wild-type cultures of sporulating Bacillus subtilis 168 and three separate cultures of sporulating mutant strain, Bacillus subtilis 168 delta-prpE, in which prpE (yjbP BSU11630) gene coding for a protein phosphatase is deleted entirely. Each chip consists of four fields able to measure the expression level of 4,104 genes from Bacillus subtilis subsp. subtilis strain 168 NC_000964 with eight 60-mer probe pairs (PM/MM) per gene, with two-fold technical redundancy.
Project description:The intestinal mucus layer consists of secreted and transmembrane (TM) mucins that are expressed on the apical surface of enterocytes and goblet cells. The TM mucin has a complex domain structure consisting of an extended highly O-glycosylated extracellular domain (ED) and a cytoplasmic tail (CT) with signaling potential. The enteropathogenic bacterium Salmonella expresses a giant adhesin called SiiE that interacts with the MUC1, resulting in invasion at the apical surface of the intestinal epithelium. Here, we determined the contributions of the MUC1 ED and CT to Salmonella invasion and subsequent immune responses. To investigate the role of the MUC1 ED in Salmonella invasion, the mucin-selective protease StcE was used to remove the glycosylated ED from the surface of intestinal HT29-MTX cultures. StcE-mediated removal of the MUC1 ED blocked Salmonella invasion to levels comparable to MUC1 knockout cells. To study the contribution of the MUC1 CT, a targeted CRISPR/Cas9 approach was used that resulted in the removal of the CT but left the ED and transmembrane domain intact. Salmonella invasion into these MUC1-ΔCT cultures was comparable to wild type cells. These results demonstrate that MUC1 ED, but not the MUC1 CT, is essential for apical Salmonella invasion. To determine the contributions of MUC1 domains, we performed a transcriptomics analysis of uninfected MUC1-WT, MUC1-ΔCT, and ΔMUC1 cultures and cultures that were infected with Salmonella for 6-hour. Deletion of the full MUC1 protein led to a large number of differentially expressed genes, while a smaller group of 132 genes was differentially expressed in infected MUC1-WT cultures compared to MUC1-ΔCT cultures. Amongst these genes were many NFκB-regulated genes that showed reduced expression in the absence of the MUC1 CT. Immunoblot analysis demonstrated that expression of the NFκB inhibitory cytoplasmic subunits p100, p105, and IκBα was significantly reduced in MUC1-WT cultures compared to MUC1-ΔCT and ΔMUC1 cultures. We conclude that the MUC1 ED is essential for Salmonella invasion, and that the MUC1 CT plays a role in activation of the NFκB pathway.
Project description:Gene expression profiling of pre-adipocytes 3T3-L1 reveals anti-adipogenic potential to metabolic associated diseases through whole transcriptomic analysis. We evaluated the effects of Tsuruazuki extract on pre-adipocytes 3T3-L1. We performed an untargeted whole-genome transcriptome analysis to explore functionality of Tsuru on 3T3-L1 cells.
Project description:Human iPSCs and NSCs were engineered by AAVS1 and/or C13 safe-harbor TALENs which mediated targeted integration of various reporter genes at single or dual safe-harbor loci. Multiple clones of targeted human iPSCs were used to compare with parental untargeted NCRM5 iPSCs. Polyclonal targeted human NSCs were used to compare with their parental untargeted NCRM1NSCs or H9NSCs. Total RNA obtained from targeted human iPSCs or NSCs compared to untargeted control iPSCs or NSCs.
Project description:The transcriptional factor Zur plays a key role in regulating zinc homeostasis in Bacillus subtilis. The genomic sites bound by Zur were mapped using a chromatine immunoprecipitation approach. This allowed the identification of 80 inter- and intragenic chromosomal sites bound by Zur. This data set contains 2 samples. Immunoprecipitated (IP) DNA from Bacillus subtilis BSAS36 strain (BSB1, a tryptophan-prototrophic derivative 168 strain expressing a SPA-tagged Zur protein) were extracted from bacterial cells in the exponential growth phase. IP and whole-cell extract DNA were hybridized on tiling array to generate ChIP-on-chip profiles. Two biological replicates were anlyzed.