Project description:Purpose: The objective of this study was to evaluate the transcriptomic consequence of chronic exposure to deficient and excessive arginine supply in MIN6 pancreatic beta cells and reveal the molecular mechanisms underlying beta cell function and insulin secretion to arginine administration. Methods: After overnight adhesion, the medium was removed, and the cells were incubated in fresh DMEM medium containing low-, standard- or high-arginine concentration (LA, SA and HA) for 24 h. Following the treatment, RNA was extracted from cells for transcriptome sequencing. Results: Transcriptomics analysis allowed the detection of 23,620 and 22,939 transcripts in the LA and HA samples, among which 7,591 (3,736 up- and 3,855 down-regulated) and 197 (111 up- and 86 down-regulated) genes were expressed differently compared with the SA treatment. One hundred and forty one DEGs were overlapped between LA vs. SA and HA vs. SA comparisons, which were mainly involved in DNA replication, organic hydroxy compound metabolism and cellular response to oxidative stress. GO functional enrichment analysis demonstrated that terms related to proteasomal protein catabolic process, mRNA processing and ATP metabolic process were highly disturbed by LA administration, while DEGs induced by HA were mainly enriched to ammonium ion metabolic process, mRNA export and sterol biosynthetic process. Morever, KEGG pathway analysis revealed that DEGs between LA and SA treatments were mainly functionally classified as involved in protein processing in endoplasmic reticulum and oxidative phosphorylation, while DNA replication, steroid biosynthesis and cell cycle had more counts and reached significant difference levels according to the DEGs triggered by HA. Conclusions: These results suggested that the disturbed carbohydrate, lipid and amino acid metabolisms as well as the decreased cell proliferation, increased apoptosis and elevated oxidative stress contributed to the reduced insulin secretion in beta cells induced by LA or HA administrations.

Project description:Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response (3 and 6 h of exposure to agonists). By using HEK 293 cells transfected with human pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and alpha-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling by a microarray and confirmation by RT-qPCR revealed an innate immune response which was common to both LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greated magnitude than those induced by SaS. Overall, the analysis of microarray data suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. Chemokines were among the most up-regulated genes, in particular ELRCXC chemokines that target neutrophils. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. This was in accordance with the much stronger up-regulation of Cxcl10, Ccl5 and Nos2 by LPS than by SaS. The higher upregulation of chemokines that target mononuclear leucocytes (CXCL10, CCL2, CCL5 and CCL20) by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. It is noteworthy that at the protein level, secretion of TNF-alpha and IL-1-beta was not induced by either stimulus. These results suggest that the response of MEC to diffusible bacterial stimuli is able to contribute to the onset of the response with differential leucocyte recruitment but does not account directly for the differential production of major pro-inflammatory cytokines. Transcriptional profiling of bovine mammary epithelial cells (Holstein breed) comparing untreated control with mammary epithelial cells (MECs) stimulated by Staphylococcus aureus (SA) versus Escherichia coli lipopolysaccharide (LPS). 16 microarrays (2 stimuli * 2 times * quadruplicate) in a two-color dye-swap experimental design. Untreated cells served as the control. One replicate per array. Log2-intensity of each dye was analyzed separately => 32 samples.

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