Project description:The severe harm of depression to human life has attracted great attention to neurologists, but its pathogenesis is extremely complicated and has not yet been fully elaborated. Here, we provided a new strategy for revealing the specific pathways of abnormal brain glucose catabolism in depression, which from the supply of energy substrates and the evaluation of mitochondrial structure and function. By using stable isotope-resolved metabolomics technique, we discovered the tricarboxylic acid cycle (TCA cycle) is blocked and the gluconeogenesis is abnormally activated in chronic unpredictable mild stress (CUMS) rats. In addition, our results showed an interesting phenomenon that the brain attempted to activate all possible metabolic enzymes in energy-producing pathways, but CUMS rats still exhibited a low TCA cycle activity due to impaired mitochondria. Depression caused mitochondrial structure and function impaired, and then led to abnormal brain glucose catabolism. The combination of the stable isotope-resolved metabolomics and mitochondrial structure and function analysis can accurately clarify the mechanism of depression. The mitochondrial pyruvate carrier and acetyl-CoA maybe the key targets for depression treatment. The strategy provides a unique insight for exploring the mechanism of depression, the discovery of new targets, and the development of ideal novel antidepressants.
Project description:To better understand proteostasis in health and disease, determination of protein half-lives is essential. We improved the precision and accuracy of peptide-ion intensity based quantification in order to enable accurate determination of protein turnover in non-dividing cells using dynamic-SILAC. This enabled precise and accurate protein half-life determination ranging from 10 to more than 1000 hours. We achieve good proteomic coverage ranging from four to six thousand proteins in several types of non-dividing cells, corresponding to a total of 9699 unique proteins over the entire dataset. Good agreement was observed in half-lives between B-cells, natural killer cells and monocytes, while hepatocytes and mouse embryonic neurons showed substantial differences. Our comprehensive dataset enabled extension and statistical validation of the previous observation that subunits of protein complexes tend to have coherent turnover. Furthermore, we observed complex architecture dependent turnover within complexes of the proteasome and the nuclear pore complex. Our method is broadly applicable and might be used to investigate protein turnover in various cell types.
Project description:To better understand proteostasis in health and disease, determination of protein half-lives is essential. We improved the precision and accuracy of peptide-ion intensity based quantification in order to enable accurate determination of protein turnover in non-dividing cells using dynamic-SILAC. This enabled precise and accurate protein half-life determination ranging from 10 to more than 1000 hours. We achieve good proteomic coverage ranging from four to six thousand proteins in several types of non-dividing cells, corresponding to a total of 9699 unique proteins over the entire dataset. Good agreement was observed in half-lives between B-cells, natural killer cells and monocytes, while hepatocytes and mouse embryonic neurons showed substantial differences. Our comprehensive dataset enabled extension and statistical validation of the previous observation that subunits of protein complexes tend to have coherent turnover. Furthermore, we observed complex architecture dependent turnover within complexes of the proteasome and the nuclear pore complex. Our method is broadly applicable and might be used to investigate protein turnover in various cell types.
Project description:To better understand proteostasis in health and disease, determination of protein half-lives is essential. We improved the precision and accuracy of peptide-ion intensity based quantification in order to enable accurate determination of protein turnover in non-dividing cells using dynamic-SILAC. This enabled precise and accurate protein half-life determination ranging from 10 to more than 1000 hours. We achieve good proteomic coverage ranging from four to six thousand proteins in several types of non-dividing cells, corresponding to a total of 9699 unique proteins over the entire dataset. Good agreement was observed in half-lives between B-cells, natural killer cells and monocytes, while hepatocytes and mouse embryonic neurons showed substantial differences. Our comprehensive dataset enabled extension and statistical validation of the previous observation that subunits of protein complexes tend to have coherent turnover. Furthermore, we observed complex architecture dependent turnover within complexes of the proteasome and the nuclear pore complex. Our method is broadly applicable and might be used to investigate protein turnover in various cell types.
Project description:The goal of this study was to compare the transcriptome between wild type strain of Listeria monocytogenes and delete nmlR mutant strain of L. monocytogenes using NGS. Method: Duplicate samples of rRNA depleted RNA from wild type and mutants were used to study transcriptomes by ion torrent platform. Transcriptomes of wild type and nmlR mutant were compared by EDGE-pro program. Result: Differential expression by EDGE-pro showed 74 genes with differential expressions between wild type and nmlR null mutant (46 genes were negatively regluated and 28 genes were positively regulated by NmlR). rRNA-depleted RNA samples from stationary phase wilde type and nmlR null mutant cultures were used to compare transcriptomes. Some affected genes from RNAseq result were selected for confirmation by quantitative reverse transcriptase PCR.
Project description:Genome-wide identification of transcription factor (TF) binding sites in the genome of the fission yeast Schizosaccharomyces pombe. The ChIP-nexus method was used. TFs included were: Cbf11-TAP and Cbf12-TAP (and their DBM mutants with impaired DNA binding), TAP-Mga2, and Fkh2-TAP (as an irrelevant control TF). IPs from an untagged WT strain were also analyzed. Cbf11-related IPs were performed from exponential cultures, while Cbf12-related IPs were performed from stationary cultures. YES complex medium was used for all cultivations.
Project description:Gene expression profiling of EHEC and its luxS-deficient mutant strain which cannot produce autoinducer-2 molecule at the late log-phase in 0.6M NaCl LB broth (osmotic stress condition) against controls grown under normal osmotic condition (LB broth)
Project description:To cause disease, Salmonella enterica serovar Typhimurium requires two type-III secretion systems, encoded on Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver virulence proteins, termed effectors, into the host cell cytosol. While the importance of these effector proteins to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded effector, SseL. Deletion of the sseL gene resulted in bacterial filamentation and elongation and unusual localization of Salmonella within infected epithelial cells. Infection with the ?sseL strain also caused dramatic changes in lipid metabolism and led to massive accumulation of lipid droplets in infected cells. Some of these changes were investigated through metabolomics of gallbladder tissue. This phenotype was directly attributed to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine resulted in the same phenotype as the deletion mutant. Excessive buildup of lipids due to the absence of a functional sseL gene was also observed in S. Typhimurium-infected livers. These results demonstrate that SseL alters host lipid metabolism in infected epithelial cells by modifying ubiquitination patterns of cellular targets. Female C57BL/6 mice were infected with the indicated strain of Salmonella enterica serovar Typhimurium by oral gavage. Four gallbladders were collected and pooled per sample group and metabolites extracted using a mixture of methanol and chloroform. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities, monoisotopic neutral masses of interest were queried against MassTrix (http://masstrix.org). Masses were searched against the Mus musculus database within a mass error of 3 ppm.