Concatemer Assisted Stoichiometry Analysis (CASA) of Cell-free Reconstituted Kinetochores
Ontology highlight
ABSTRACT: Concatemer-assisted stoichiometry analysis (CASA) addresses a common challenge in cell biology: absolute quantification of protein subunits in native protein complexes.
Project description:We generated a collection of 13 plasmids, with each plasmid containing a variant of a CRISPR protospacer targeted by spacer 8 of the E. coli CRISPR-I array. We transformed the plasmids as a pool into delta cas3 E. coli cells expressing all other cas genes constitutively, with FLAG-tagged casA. We then used ChIP to enrich for CasA-bound protospacers. DNA surrounding the protospacers was PCR-amplified from input (pre-immunocrecipitation) and ChIP (post-immunoprecipitation) samples and sequenced.
Project description:In this study, we systematically assessed the fine-tuning of limited proteolysis in the extracellular space by glycosylation, one of the most widely observed PTMs, using cells, and applying the Terminal Amine Labeling of Substrates (TAILS) approach, a quantitative proteomics workflow for the system-wide assessment of protein N termini and protease cleavage events in complex biological samples. In addtion, we complement the study with DIA analysis, PRM and glycoproteomics analysis for further validation of the initial observations
Project description:Despite the availability of effective therapies such as proteasome inhibitors, multiple myeloma (MM) patients relapse with refractory disease. To identify new therapeutic targets, we assessed RNA sequencing data from CD138+ MM patient cells (n = 813) across disease stages and found that an autophagy gene signature, and particularly ULK3 expression, was strongly associated with disease progression. Functional studies revealed that ULK3 contributes to MM cell survival as part of the ULK-ATG13-FIP200 complex. We generated inhibitors (SG3-014/MA9-060) with nanomolar potency and confirmed their binding mode through co-crystallization with ULK3. In vivo, ULK3 inhibition reduced MM burden, improved survival, and protected against cancer-induced bone disease. MA9-060 also restored sensitivity to proteasome inhibitors in resistant MM cells. This synergy was validated ex vivo in patient samples, especially those with high ULK3 expression. These findings indicate a new role for ULK3-mediated autophagy in cancer and suggest that ULK3 inhibition is an effective treatment strategy for both newly diagnosed and refractory MM disease.
Project description:Arabidopsis thaliana plant expressing 35S:WIND1 shows callus-like morphology without hormone treatment. Transcriptomes of the callus-like cell expressing 35S:WIND1, callus of T87 cultured cell, 2,4-D-induced callus and control seedling plant were compared by Agilent microarray. Comparison of four kinds of Arabidopsis thaliana plants. Biological replicates: three for each.
Project description:Seminal plasma proteins play critical roles in sperm function, motility, viability and oxidative stress regulation in stallions. In this study, label-free quantitative (LFQ) proteomics was applied to characterize seminal plasma protein signatures associated with sperm quality in Arabian stallions. Semen samples were classified into High-CASA and Low-CASA groups based on computer-assisted sperm analysis (CASA) parameters. LC–MS/MS analysis identified 817 seminal plasma proteins, of which differentially abundant proteins were associated with sperm motility, viability, mitochondrial activity and DNA fragmentation. Integrated bioinformatic analyses, including differential abundance analysis, protein–phenotype correlations, Gene Ontology enrichment and STRING network analysis, revealed oxidative stress–associated proteomic remodeling linked to sperm functional status. The dataset provides insight into seminal plasma molecular mechanisms associated with stallion reproductive performance.
Project description:We Investigate the persistence of negative impacts on sperm parameters for more than one generation in C57BL/6J and in strain FVB/N subjected to prenatal exposure to di(2-ethylexyl)phthalate (DEHP) using in utero exposures followed by Computer-Aided Sperm Analysis (CASA) sampling and all-RNA-seq analysis of germ cells transcripts.
2018-11-28 | GSE107839 | GEO
Project description:Chemical Assessment of Surfaces and Air (CASA)
Project description:au11-02_cho-thf - shoots and roots treated with methotrexate and 5-formyl-thf - Folates and 1C metabolism - The objective of this study is to investigate the changes on the plant expression profiles due to 5-CHO-THF assimilation and transformation. Additionally, plants supplemented with methotrexate (MTX) (an inhibitor of folate biosynthesis) will complement the study. Results from this work will serve to better understand the effects of folate accumulation in plants. 12 dye-swap - treated vs untreated comparison
Project description:fapesp-bra-inra-10-01_bioen_hypocotyl - dark hypocotyls tor rnai - Transcriptional comparison between 2 TOR RNAi mutants versus GUS control. - Sowing after 24h imbibition at 4M-BM-0C in the dark, on MS1/5, no sucrose, 10 mM ethanol, 8 g/l agar, vertical growth with 3h light, 6 days growth in the dark (20M-BM-0C), hypocotyls were harvested under green light ; cotyledons and root were removed. 4 dye-swap - normal vs rnai mutant comparaison