Project description:The Z-disc is a protein-rich structure critically important for myofibril development and integrity. In order to monitor the quantiative changes in C2C12 myoblast during myogenesis, a quantitative dimethyl-labelling approach was performed with d0 myoblasts, d5 myotubes and electrical puls stimulated d5 myotubes.
Project description:The expression levels of uropathogenic E. coli (UPEC) UTI89 cells in YESCA broth with and without 4% dimehtyl sulfoxide (DMSO) and 2% ethanol (EtOH) were studied. To determine the up- or down-regulated expression profile in the presence of dimethyl sulfoxide (DMSO) and ethanol (EtOH), UPEC UTI89 were grown in YESCA broth with 4% dimethyl sulfoxide (DMSO) or 2% ethanol (EtOH) or no comound at 26°C with 200 rpm shaking for 24 hours were used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Endometritis, a prevalent reproductive system disease with high incidence, leads to reproductive dysfunction and potential miscarriage in both humans and animals, impacting human health and the dairy industry, resulting in significant economic loss. Gut microbiota plays an important role in the pathogenesis of endometritis. However, the specific microbial and metabolic mediators participating in the pathogenesis remain to be identified. In this study, we found that the concentration of itaconate in the feces of mice increased after endometritis induced by Escherichia coli (E. coli) infection, suggesting a potential association between itaconate and inflammatory processes. Here, we investigated the specific role of dimethyl itaconate (DI), a derivative of itaconate with potent anti-inflammatory and immunomodulatory properties on endometritis. Oral administration of DI ameliorated the symptoms of E. coli-induced endometritis in mice. The protective effect was abolished by antibiotic-induced depletion of the gut microbiota, and fecal microbiota transplantation (FMT) from DI-treated mice to recipient mice ameliorated E. coli-induced endometritis. Integrative multiomics revealed that the addition of DI led to an enrichment of Muribaculaceae and the microbial purine metabolite guanosine in the feces. Muribaculum intestinale (DSM 28989), a bacterium of Muribaculaceae or guanosine supplementation alleviated E. coli-induced endometritis in mice. Mechanistically, DI promoted the multiplication of Muribaculaceae, and supplementation of M. intestinale upregulated the level of guanosine in the uterus. Transcriptome data and antibody-neutralizing experiment demonstrated the protective effect of guanosine in E. coli-induced endometritis was mediated by activating the expression of CXCL14 in uterine epithelial cells. In conclusion, our study elucidates the significant role of the gut microbiota and its metabolites in safeguarding the uterus against pathogen infections, providing substantiation for the regulation of distal organ by the gut microbiota, and establishing a basis for preventive measures against related diseases through the gut-X axis.
Project description:Wild type (wt) SUM44 cells, modelling breast cancer at primary diagnosis, were cultured in phenol red-free RPMI supplemented with 10% FBS and 1nM estradiol (E2). Long-term oestrogen deprived (LTED) cell lines, which model resistance to endocrine therapy, were cultured in phenol red-free RPMI in the absence of exogenous E2 and supplemented with 10% dextran charcoal-stripped bovine serum (DCC). Samples were harvested at baseline and at the point of resistance (LTED). To reveal differential protein abundances between wt-SUM44 and SUM44-LTED, the peptides were labelled with dimethyl labelling and underwent fractionation using OFFGEL electrophoresis. In order to reveal the ER-interactome, RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) was conducted in wt-SUM44 and SUM44-LTED.
Project description:Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied both CsCl density gradient (CDG) centrifugation-based protein fractionation and a dimethyl-labelling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl chemical labelling with chromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-based computational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, named Stable isotope-based Quantitation-Dimethyl labelling (SQUA-D), and finally the confirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7,456 unambiguous acetylation sites were found from 2,638 different acetylproteins, and 5,250 acetylation sites, including 5,233 sites on lysine side chain and 17 sites on protein N-terminus, were identified repetitively. Out of these repetitively discovered acetylation sites, 4,228 sites on lysine side chain (i.e., 80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signalling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or called unique PTM peptide arrays (UPAs) that share the identical unique PTM site pattern (UPSP). This CDG centrifugation protein fractionation in combination with dimethyl labelling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings.
Project description:The expression levels of uropathogenic E. coli (UPEC) UTI89 cells in YESCA broth with and without 4% dimehtyl sulfoxide (DMSO) and 2% ethanol (EtOH) were studied.