Project description:Improving fibroblast characterization using single-cell RNA sequencing: an optimized tissue disaggregation and data processing pipeline
Project description:Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.
Project description:Synovial sarcoma is a deadly soft-tissue malignancy with a predilection for adolescents and young adults. Mice recapitulate synovial sarcomagenesis from expression of SS18-SSX2 in certain cells. Concomitant expression of a stabilized form of beta-catenin enhances synovial sarcomagenesis and expands the potential cells of origin.
Project description:Synovial sarcoma is a deadly soft-tissue malignancy with a predilection for adolescents and young adults. Mice recapitulate synovial sarcomagenesis from expression of SS18-SSX2 in certain cells. Concomitant expression of a stabilized form of beta-catenin enhances synovial sarcomagenesis and expands the potential cells of origin. Mice bearing conditional expression of SS18-SSX2 from the Rosa26 locus and conditional excision of the 3rd exon of beta-catenin, each activated in the leg by Cre-recombinase expressed from an adeno-associated viral vector, formed large tumors at brief latency.
Project description:In the current work, we used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. Our primary aim was to determine how the synovial fluid proteome differs between the two groups in an effort to identify candidate proteins that may be associated with the development of posttraumatic OA. We hypothesized that the development of macroscopic cartilage damage following surgical ACL transection would be accompanied by differential changes in synovial fluid proteome in untreated knees compared to repaired joints.
Project description:Proteins that are vital to cell survival can become unfolded during heat stress, leading to the formation of toxic aggregates. Molecular chaperones, proteases and autophagic pathways are required to protect cells from the accumulation of protein aggregates. Small Heat Shock Proteins (sHSPs) form a first line of defense by binding to unfolding proteins to prevent irreversible protein aggregation and the complex is rapidly sequestered in stress granules during heat stress recovery. HSP101 subsequently accumulates on the surface of these insoluble foci to mediate protein disaggregation. The dynamics of this process is consistent between different cell types but indicates that protein homeostasis varies particularly between shoot and root cells. Immunoblot analysis revealed that a substantial portion of proteins present in these foci have ubiquitin moieties and protein disaggregation is necessary prior to proteasomal degradation. Co-immuno precipitation of HSP101 revealed an interaction with the 26S proteasome and localization studies revealed that HSP101 and RPN1 (Proteasome regulatory particle) initially accumulate in distinct cytosolic foci during heat stress but co-localize in the same foci during heat stress recovery which could spatiotemporal facilitate the degradation of the aggregated ubiquitinated proteins. To determine which proteins are degraded by the proteasome after disaggregation, we catalogued 620 proteins that are resolubilized by HSP101 during heat stress recovery. GO-annotation analysis revealed a significant enrichment for RNA-binding proteins, UBC13-MMS2 complex, transcription factors and ubiquitin-protein ligases. None of these proteins were preferentially targeted by the proteasome, indicating that ubiquitination occurs on a broad range of proteins and is important for the clearance of a diverse array of proteins.
Project description:Identification of novel synovial fluid biomarkers that can differentiate early changes following ACL injury from OA using tandem-mass-spectrometry
Project description:Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-Seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell cycle arrest, neuronal differentiation and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb-group targets were reactivated including tumor suppressor CDKN2A, and pro-apoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and pro-apoptotic factors BIK, BIM and BMF were important to apoptosis induction following HDAC-inhibition in synovial sarcoma