Project description:To investigate the function of FABP4 in the intestinal in vitro cultured organoids,FABP4, which located in crypt lyszyme+ Paneth cells can regulate the expression of defensins, especially HFD-mediated downregulation of defensin in Paneth cells will provide insights into factor(s) underlying modern diseases. We then performed gene expression profiling analysis using data obtained from RNA-seq of FABP4fl/flpvillinCreT (FABP4KO) mice and FABP4fl/fl (WT)mice in vitro cultured intestinal organoids.
Project description:Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Studies with germ-free or gnotobiotic animals represent the gold standard for research on bacterial-host interaction but they are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete murine intestinal microbiota and prove to have significant biologic validity. Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by approximately 400 fold while ensuring the animals’ health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer’s patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. We present a robust protocol for depleting mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion is phenotypic characteristics and epithelial gene expression profile similar to those of germ-free mice. Comparison of genome-wide gene expression of colon intestinal epithelial cells from mice subjected to microbiota depletion protocol against to control mice.
Project description:Gene expression profile of FABP4 treatment in RAW264.7 macrophages was examined to show a ligand (palmitic acid)-dependent and a ligand-independent effect of FABP4. RAW264.7 macrophages were treated with and without 200 nM recombinant FABP4 in the absence and presence of 0.2 mM palmitic acid.
Project description:Gene expression profile of FABP4 treatment in RAW264.7 macrophages was examined to show a ligand (palmitic acid)-dependent and a ligand-independent effect of FABP4.
Project description:Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Studies with germ-free or gnotobiotic animals represent the gold standard for research on bacterial-host interaction but they are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete murine intestinal microbiota and prove to have significant biologic validity. Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by approximately 400 fold while ensuring the animals’ health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer’s patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. We present a robust protocol for depleting mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion is phenotypic characteristics and epithelial gene expression profile similar to those of germ-free mice.
Project description:Using single cell RNAseq assay, we examine the effect of acute diet-swtiching on the transcriptomic profiles of various intestinal epithelial cells.
Project description:To determine the effect of prohibitin overexpression on global gene expression in Caco2-BBE intestinal epithelial cells. 4 individual wells of Caco2-BBE cells, passage 41, were transfected with either empty vector (pcDNA4) or prohibitin/pcDNA4 for 72 hours. Total RNA isolated from 4 wells of cells/per treatment were pooled together for labeling and hybridization purposes.
Project description:Aflatoxin M1 (AFM1) is a common mycotoxin in dairy milk and it is typically concurrently present with other mycotoxins that may represent a threat for food safety. However, knowledge on how AFM1, alone or in combination with other mycotoxins may affect human intestinal epithelial integrity remain to be established. We employed transcriptome and proteome analysis integrated with biological validation to reveal the molecular basis underlining the effect AFM1 and/or ochratoxin A (OTA) exposure on intestinal epithelial integrity of differentiated Caco-2 cells. Exposure to 4 μg/ml of OTA was found to disrupt human gut epithelial integrity, whereas 4 μg/ml of AFM1 did not. Integrated transcriptome and proteome analysis of AFM1 and OTA, alone or in combination, indicate synergistic effect of the two mycotoxins in disrupting intestinal integrity. This effect was mechanistically linked to a broad ranges of pathways related to intestinal integrity enriched by down-regulated genes and proteins, associated to focal adhesion, adherens junction, and gap junction pathways. Furthermore, the cross–omics analysis of mixed AFM1 and OTA compared with OTA alone suggest that kinases family members, including MLCK, MAPKs, and PKC are the potential key regulators on modulating intestinal epithelial integrity. These findings provide novel insight into the synergistic detrimental role of multiple mycotoxins in disrupting intestinal integrity, and, therefore, identify potential target to improve milk safety related to human health.