Project description:In order to better understand the functional properties of this type of cells under different circumstances, we integrated the single-cell RNA-seq data of peripheral blood CD8+ T cells from healthy subjects, MS patients and COVID-19 patients generated from the 10X Genomics platform using the Seurat package. KIR+CD8+ T cells from different conditions (healthy, MS, and COVID-19) formed a distinct cluster with high expression of effector genes (GZMB and PRF1) as well as KIR transcripts. These findings reveal the commonality of KIR+CD8+ T cells across physiological and diseased status as well as their uniqueness relative to other CD8+ T cells.
Project description:We combined the Single-probe single cell MS(SCMS) experimental technique with a bioinformatics software package, SinCHet-MS (Single Cell Heterogeneity for Mass Spectrometry), to characterize changes of tumor heterogeneity, quantify cell subpopulations, and prioritize the metabolite biomarkers of each subpopulation.
Project description:We identify cellular heterogeneity during in MS patient samples from the CNS Multiple sclerosis (MS) is an autoimmune neurologic disease leading to demyelination and neurologic dysfunction controlled by both genetic and environmental factors. In addition to CNS-infiltrating immune cells, CNS-resident cells, such as astrocytes, are thought to play an important role in MS pathogenesis. However, a comprehensive understanding of the extent to which gene expression is disrupted in astrocytes is not known. Here, we implement single-cell RNA sequencing, in vivo genetic perturbations, cell-specific RNA profiling by Ribotag, as well as single-cell RNA sequencing of human MS patient samples to identify a transcriptional regulatory network in astrocytes that controls the pathogenesis of EAE and potentially, MS. We defined an astrocyte subpopulation characterized by expression of the small Maf protein, MAFG, which represses NRF2-driven antioxidant mechanisms and promotes EAE pathogenesis. Mechanistically, MAFG suppresses NRF2-dependent antioxidant genetic programs by cooperating with its cofactor, MAT2a, to promote DNA methylation in the context of CNS inflammation, which in turn increases pathogenic signaling processes in astrocytes. MAFG/MAT2a astrocytes are controlled by GM-CSF signaling, which drives EAE pathogenesis and MAFG expression. MAFG is activated in astrocytes derived from MS patients, which are characterized by DNA methylation programs, pro-inflammatory signaling processes including GM-CSF signaling, and repressed NRF2 activation. Together, these data create a transcriptional and epigenetic framework to analyze CNS inflammation in MS and may provide new therapeutic targets.
Project description:Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (~1,000 cells) and demonstrated 0-9 intact cells injected with a dependency on the duration of spray voltage application. Application of spray voltage for 2 min injected an average of 7±2 cells and resulted in 33-57 protein and 40-88 proteoform identifications (N=4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single cells (N=4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17-40 proteins and 23-50 proteoforms identified. Furthermore, an additional cell line, THP-1, was analyzed at the single-cell level and proteoform abundances were compared to show the capabilities of single-cell top-down proteomics (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.
Project description:This research clinical trial studies high definition single cell analysis in blood and tissue samples from patients with colorectal cancer which has spread to the liver. High definition single cell analysis allows doctors to study the properties of cancer cells that are sometimes found in the blood of patients and to determine how the genes and proteins in them may change over time. Studying samples from patients with colorectal cancer in the laboratory may help doctors learn more about how cancer spreads, as well as how to predict the disease outcomes in patients with cancer.