Project description:Indole is a bacterial signal secreted by pathogenic and commensal Escherichia coli in the stationary phase at high concentrations (~600 µM). Prior work from our lab has shown that indole decreases E. coli O157:H7 (EHEC) chemotaxis, motility, attachment to epithelial cells and biofilm formation. However, its effect on epithelial cells is not known. We hypothesized that indole induces gene expression changes in epithelial cells that lead to decreased pathogen colonization and infection. Changes in gene expression with the human enterocyte cell line HCT-8 exposed to indole suggested down-regulation of Toll-like receptor signaling and coordinated changes in Jak-STAT and p38 MAPK pathways. Corresponding changes in the expression of cytokines, chemokines, and their receptors also suggested that indole functions as a modulator of inflammation in intestinal epithelial cells. In addition, the expression of genes involved in tight junction organization (claudins, and tight junction proteins) and mucin production were also up-regulated by indole. Keywords: Inter-kingdom signaling interactions between human cells and bacterial signal indole
Project description:Indole is a bacterial signal secreted by pathogenic and commensal Escherichia coli in the stationary phase at high concentrations (~600 µM). Prior work from our lab has shown that indole decreases E. coli O157:H7 (EHEC) chemotaxis, motility, attachment to epithelial cells and biofilm formation. However, its effect on epithelial cells is not known. We hypothesized that indole induces gene expression changes in epithelial cells that lead to decreased pathogen colonization and infection. Changes in gene expression with the human enterocyte cell line HCT-8 exposed to indole suggested down-regulation of Toll-like receptor signaling and coordinated changes in Jak-STAT and p38 MAPK pathways. Corresponding changes in the expression of cytokines, chemokines, and their receptors also suggested that indole functions as a modulator of inflammation in intestinal epithelial cells. In addition, the expression of genes involved in tight junction organization (claudins, and tight junction proteins) and mucin production were also up-regulated by indole. Keywords: Inter-kingdom signaling interactions between human cells and bacterial signal indole Human intestinal epithelial cells were exposed to 1 mM indole for 4 h or 24 h. RNA was isolated from the control cells and cells exposed to indole for 4 h and 24 h. The experiments were performed in triplicate. A total of 9 microarrays were performed, three for each condition. The gene expression data was analyzed using significance analysis of microarrays (SAM) where a false discovery rate cut-off of 1% was used. The genes were then classified and importnat regulated pathways were reported.
Project description:To elucidate the epithelial cell diversity within the nasal inferior turbinates, a comprehensive investigation was conducted comparing control subjects to individuals with house dust mite-induced allergic rhinitis. This study aimed to delineate the differential expression profiles and phenotypic variations of epithelial cells in response to allergic rhinitis. This research elucidated distinct subpopulations and rare cell types of epithelial cells within the nasal turbinates, discerning alterations induced by allergic rhinitis. Furthermore, by interrogating transcriptomic signatures, the investigation provided novel insights into the cellular dynamics and immune responses underlying allergic rhinitis pathogenesis
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. One-condition experment, gene expression of 3A6