Project description:In order to reveal context specific gene expression in pancreatic beta-cells, beta-cells were purified by flow cytometry from two days after EMC-D virus infected mice, and performed microarray analysis.
Project description:Microarray analysis of peripheral blood mononuclear cells (PBMCs), lungs, and lung lesions collected over the course of hCoV-EMC infection of 6 rhesus macaques.
Project description:Microarray analysis of peripheral blood mononuclear cells (PBMCs), lungs, and lung lesions collected over the course of hCoV-EMC infection of 6 rhesus macaques. 6 rhesus macaques were infected intratracheally with hCoV-EMC. PBMCs were collected at days 0, 1, 3, and 6, and lungs were collected from serial sacrifices of 3 animals each at day 3 and day 6. Infection produced a mild-moderate, self-limiting respiratory infection, and was not lethal. We performed microarray analysis (using Agilent Rhesus arrays) on all lungs, lung lesions, and PBMCs collected for the study.
Project description:Differential expression was determined in Calu-3 cells between mock infected and infection with either Human coronavirus EMC and SARS coronavirus at different times post infection.
Project description:Differential expression was determined in Calu-3 cells between mock infected and infection with either Human coronavirus EMC and SARS coronavirus at different times post infection. Calu-3 2B4 cells were infected with Human Coronavirus EMC 2012 (HCoV-EMC) or mock infected. Samples were collected 0, 3, 7, 12, 18 and 24 hpi. There are 3 mock and 3 infected replicates for each time point, except for 12 hpi for which there are only 2 infected replicates (one replicate did not pass RNA quality check). There were no mock sampes at 18 hpi, and therefore infected samples at 18 hpi were compared with mocks at 24 hpi. For direct comparison with SARS-CoV infected cells, raw data from HCoV-EMC experiments were quantile normalized together with the SARS-CoV dataset (GEO Series accession number GSE33267).
Project description:Pancreatic β-cells are responsible for production and secretion of insulin in response to increasing blood glucose levels. Therefore, defects in pancreatic β-cell function lead to hyperglycemia and diabetes mellitus. Understanding the molecular mechanisms governing β cell function is crucial for development of novel treatment strategies for this disease. The aim of this project was to investigate the role of Cnot3, part of CCR4-NOT complex, major deadenylase complex in mammals, in pancreatic β cell function. Cnot3βKO islets display decreased expression of key regulators of β cell maturation and function. Moreover, they show an increase of progenitor cell markers, β cell-disallowed genes and genes relevant to altered β cell function. Cnot3βKO islets exhibit altered deadenylation and increased mRNA stability, partly accounting for the increase of those genes. Together, these data reveal that CNOT3-mediated mRNA deadenylation and decay constitute previously unsuspected post-transcriptional mechanisms essential for β cell identity.
Project description:Structure formation of membrane proteins is error-prone and thus requires chaperones that oversee this essential process in cell biology. The ER membrane protein complex (EMC) is well-defined as a transmembrane domain (TMD) integrase. In this study, we characterize an additional chaperone function of the EMC. We use interactomics and systematic studies with model proteins to comprehensively define client features for this EMC chaperone mode. Based on this data, we develop a machine learning tool for client prediction. Mechanistically, our study reveals that the EMC engages TMDs via its EMC1 subunit and modulates their orientation within the lipid bilayer. Productive TMD assembly reduces binding to the EMC chaperone site. Taken together, our study provides detailed insights into an EMC chaperone function, further establishing the role of the EMC as a multifunctional molecular machine in membrane protein biogenesis.
Project description:Insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Using ChIP sequencing and RNA sequencing analysis, we determined the epigenetic and transcriptional landscape of human pancreatic α, β, and exocrine cells. We found that, compared with exocrine and β cells, differentiated α cells exhibited many more genes bivalently marked by the activating H3K4me3 and repressing H3K27me3 histone modifications. This was particularly true for β cell signature genes involved in transcriptional regulation. Remarkably, thousands of these genes were in a monovalent state in β cells, carrying only the activating or repressing mark. Our epigenomic findings suggested that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. Indeed, we show that treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. Thus, mammalian pancreatic islet cells display cell-type–specific epigenomic plasticity, suggesting that epigenomic manipulation could provide a path to cell reprogramming and novel cell replacement-based therapies for diabetes. Pancreatic islets were collected post-mortem from 6 human donors and subjected to FACS to separate populations of alpha, beta, and exocrine cells. Depending on the availability of resulting material, sorted islet cell populations were used for H3K4me3, H3K27me3 ChIP-seq, or RNA-seq analysis. All ChIP-seq samples have a corresponding input from the same sample.
Project description:To better understand the underlying mechanism of beta-cell regeneration in adult zebrafish, we performed single-cell transcriptomic profiling of the pancreatic tissue (using 10X Genomics) at various stages post beta-cell ablation.