Project description:All cell lines were purchased from ATCC (LGC Standards S.r.l., Milan, Italy), except T-47D and MDA-MB-468 that were obtained from the National Cancer Institute Developmental Therapeutics Program (Frederick, MD).
Project description:Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (a-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of a-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell-derived myotubes. FKRP deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell-derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. Additionally, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis.
Project description:We use high-throughout single-cell RNA sequencing technology to analyze primary human hepatocytes, combining with LC-MS/MS for drug-metabolizing function research. We measure the enzymatic metabolic function of PHH and perform a correlation analysis with the values documented in the quality inspection report. Furthermore, we assess functions beyond cellular metabolism as specified in the PHH standards. These additional functions include bile secretion, albumin (ALB) secretion, and cell purity, which are not addressed in the quality inspection report. Additionally, we examine the impact of donor characteristics such as body weight, age, underlying health conditions, and lifestyle habits (including smoking and alcohol consumption) on cellular function. We aim to conduct a comprehensive analysis of the factors that influence cellular function. This research not only seeks to supplement and refine the standards of PHH but also aspires to provide a crucial reference for the application of PHH in the domains of disease research and drug development in the future.
Project description:All cell lines were purchased from ATCC (LGC Standards S.r.l., Milan, Italy), except T-47D and MDA-MB-468 that were obtained from the National Cancer Institute Developmental Therapeutics Program (Frederick, MD). Six untreated breast cancer cell lines grown according to the growth protocol reported below
Project description:Sample multiplexing using isobaric tagging is a powerful strategy for proteome-wide protein quantification. One major caveat of isobaric tagging is ratio compression that results from the isolation, fragmentation, and quantification of co-eluting, near-isobaric peptides, a phenomenon typically referred to as “ion interference.” A robust platform to ensure quality control, optimize parameters, and enable comparisons across samples is essential as new instrumentation and analytical methods evolve. Here, we introduce TKO-iQC, an integrated platform consisting of the Triple Knock-Out (TKO) yeast digest standard and an automated web-based database search and protein profile visualization application. We highlight two new TKO standards based on the TMTpro reagent (TKOpro9 and TKOpro16), as well as an updated TKO Viewing Tool, TVT2.0. TKO-iQC greatly facilitates the comparison of instrument performance with a straightforward and streamlined workflow.
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.