Project description:Interventions: Group 1: Quantitative Expression Analysis of the proteom and gene Expression of Primary Tumor, normal tissue, and metastases
Primary outcome(s): Disease associated Proteins and Genes
Study Design: Allocation: ; Masking: ; Control: ; Assignment: ; Study design purpose: basic science
Project description:Based on the consistent demonstration of fibrosis of the atrioventricular node surrounded by macrophages and multinucleated giant cells in anti-Ro antibody exposed fetuses dying with heart block, this study focuses on macrophage signaling stimulated by ssRNA associated with the Ro60 protein and the impact of antagonizing innate cell drivers such as TLR7/8. Transcriptome and epigenetic modifications which affect transcription factors, NF-κB and STAT1, were selected to evaluate the phenotype of macrophages in which TLR7/8 was ligated following treatment with either anti-Ro60/Ro60/hY3 RNA immune complexes or transfection with hY3. Based on microarray, TNF and IL6 were among the most highly upregulated genes in both stimulated conditions, each of which was significantly inhibited by preincubation with hydroxychloroquine (HCQ). In contrast, following stimulation of macrophages with either TNF-α or IFN-α, which do not signal through TLR, the resultant gene expression was refractory to HCQ. Ligation of TLR7/8 resulted in increased histone methylation as measured by increased H3K4me2, a requirement for binding of NF-κB at certain promoters, specifically the kB1 region in the TNF promoter (ChIP-qPCR), which was significantly decreased by HCQ. In summary, these results support that the HCQ-sensitive phenotype of hY3 stimulated macrophages reflects the bifurcation of TLR downstream signals involving NF-κB and STAT 1 pathways and for the former dimethylation of H3K4. Accordingly, HCQ may act more as a preventive measure in downregulating the initial production of IFN-α or TNF-α and not affect the resultant autocoid stimulation reflected in TNF-α and IFN-α responsive genes. The beneficial scope of antimalarials in the prevention of organ damage, inclusive of heart block in an anti-Ro offspring or more broadly SLE, may include in part, a mechanism targeting TLR-dependent epigenetic modification.
Project description:Mitochondria fullfill central functions in cellular metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in living cells has limited experimental access. Here, we silence the translation of specific mitochondrial mRNAs in living cells by delivering synthetic peptide-morpholino chimeras. This approach allows comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and represents an innovative strategy to address mitochondrial gene expression in living cells. The tools that we present can readily be applied to analyze the mechanisms and pathophysiology of mitochondrial gene expression in a broad range of cellular model systems.
Project description:This study aims to dissect gene-environment interactions on gene expression and regulation in immune cells using scRNA-seq data from 120 donors being stimulated with 3 pathogens, for 2 different timepoints. More specifically, we identified context- and cell-type-specific eQTLs and co-expression QTLs in PBMCs.