Ten day time course of effect of influenza vaccination on gene expression profiles of lymph node cells (unselected cells and B cells) in mice
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ABSTRACT: Study was to explore the immune and transcriptomic response to seasonal influenza vaccination (TIV) in mice. For more information see Immport study SDY583. 22 total samples, 6 mice per sample: 11 unselected cell samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost) and 11 B cell samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost)
Project description:Study was to explore the immune and transcriptomic response to seasonal influenza vaccination (TIV) in mice. For more information see Immport study SDY583. 22 total samples, 6 mice per sample: 11 PBMC samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost) and 11 B cell samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost)
Project description:Study was to explore the immune and transcriptomic response to seasonal influenza vaccination (TIV) in mice. For more information see Immport study SDY583. 22 total samples, 6 mice per sample: 11 unselected cell samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost) and 11 B cell samples (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-vaccine boost)
Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To better understand the role of Arf6 in BECs, in this experiment we have compared MACS sorted BECs from Arf6-GFP-AAV vs GFP-AAV treated 3-months-old WT mice and found 86 and 110 proteins to be significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed vesicle-mediated transport, activation of GTPase activity, and ER to Golgi vesicle-mediated transport to be among the most significantly affected biological processes.
Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To better understand the role of Arf6 in BECs, in this experiment we have compared MACS sorted BECs from Arf6-KO and WT 3-months-old mice and found 140 and 172 proteins to be significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins showed mRNA processing to be among the most affected biological processes consistent with our findings on the aged BEC proteome.
Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry based proteomics. In this experiment, first we have compared MACS sorted BECs across multiple time points between 3 and 18 months of age. Using unsupervised cluster analysis, we found a segregation of age-related protein dynamics with biological functions including a downregulation of vesicle-mediated transport. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe. Therefor in our next proteomics experiment we compared BECs from 3-months-old Apoe-KO and WT mice and found 111 and 103 proteins to be up- and downregulated, respectively. Comparing the BEC proteomic signature of young Apoe-KO mice with the signature of aged (18-months-old) WT mice we found a positive correlation suggesting an accelerating effect of Apoe deficiency on BEC aging.
Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry based proteomics. In this experiment, we have compared MACS sorted BEC and full brain tissue a massive enrichment of endothelial markers including Nos3, Cdh5, and Pecam1 in BECs compared to FT.
Project description:Preclinical studies of primary cancer cells are always done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3-10% range, with most tumors in an hypoxic or 1-2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anti-cancer agents is routinely examined at ambient O2. Here, using both mouse models and human cancers, we demonstrate that tumors collected, processed and propagated at physiologic (physioxia) O2 compared to ambient air display very distinct differences in key signaling networks including Lgr5/Wnt, Yap, and Nrf2/Keap1, nuclear reactive oxygen species levels, alternative splicing, and sensitivity to several targeted therapies including PIK3CAalpha-specific and EGFR inhibitors. Significance: Extra-physiologic oxygen shock/stress (EPHOSS), as noted in cells collected/processed under ambient air, has been demonstrated to have significant impact on numbers and engrafting ability of hematopoietic stem cells. We report deleterious effects of EPHOSS on cancer cell behavior and EPHOSS-mediated effects on cancer cells give misleading information on signaling pathway activation that could severely impact the relevance of these findings. Cancer cells under EPHOSS show higher proliferation rate compared to cells under physioxia and thus are sensitive to anti-proliferative agents. Thus, drugs that show effectiveness on cancer cells collected in ambient air and subjected to EPHOSS may not be effective or as relevant in vivo, results that could partially explain the limited clinical translation of laboratory findings. Evaluating cell signaling and effects of drugs on cancer cells under physiologic O2 prior to in vivo studies could substantially reduce cost and aid in drug discovery relevant to the actual physioxia/pathological status of the tumor cells in vivo.
Project description:Gene expression profiles of individual bone marrow cells were acquired by Drop-Seq. Subsets of bone marrow cells were isolated using magnetic cell sorting to enrich for putative skeletal stem cells.
Project description:Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. We also discovered pathological proteomic signatures consistent with defects in lysosomal function and indicative of abnormal lipid metabolism. CLN3-deficient microglia were unable to efficiently turnover myelin and metabolize the associated lipids, showing defects in lipid droplet formation and cholesterol accumulation.