The pathophysiological mechanism(s) driving non-alcoholic fatty liver disease, the most prevalent chronic liver disease globally, have yet to be fully elucidated. Here, we identify regulator of G protein signaling 6 (RGS6), up-regulated in the livers of NAFLD patients, as a critical mediator of hepatic steatosis, fibrosis, inflammation, and cell death. Human patients with high hepatic RGS6 expression exhibited a corresponding high inflammatory burden, pronounced insulin resistance, and poor liver function. In mice, liver-specific RGS6 knockdown largely ameliorated high fat diet (HFD)-driven oxidative stress, fibrotic remodeling, inflammation, lipid deposition and cell death. RGS6 depletion allowed for maintenance of mitochondrial integrity restoring redox balance, improving fatty acid oxidation, and preventing loss of insulin receptor sensitivity in hepatocytes. RGS6 is both induced by ROS and increases ROS generation acting as a key amplification node to exacerbate oxidative stress. In liver, RGS6 forms a direct complex with ATM kinase supported by key aspartate residues in the RGS domain and is both necessary and sufficient to drive hyperlipidemia-dependent ATM phosphorylation. pATM and markers of DNA damage (γH2AX) were also elevated in livers from NAFLD patients particularly in samples with high RGS6 protein content. Unsurprisingly, RGS6 knockdown prevented ATM phosphorylation in livers from HFD-fed mice. Further, RGS6 mutants lacking the capacity for ATM binding fail to facilitate palmitic acid-dependent hepatocyte apoptosis underscoring the importance of the RGS6-ATM complex in hyperlipidemia-dependent cell death. Inhibition of RGS6, then, may provide a viable means to prevent or reverse liver damage by mitigating oxidative liver damage.
Standardization of metagenomic DNA extraction protocol is a pre-requisite for a successful metagenomic study aiming to screen and exploit the variety of microorganisms inhabiting a particular soil environment. Six methods reported earlier were used for isolation of metagenomic DNA in the present study. These methods suffered with regard to either poor yield or quality of DNA. Therefore, we developed an improved method for isolation of high-molecular weight and good quality metagenomic DNA from different soil samples. Our protocol combines the enzymatic (lysozyme and proteinase K) and chemical (CTAB and CaCl) strategies to ensure efficient cell lysis and use of PEG and isopropanol for precipitation of humic impurities-free DNA. Our improved method gave high yield of good quality metagenomic DNA from diverse soils collected from garden, domestic waste dumping site, cellulose waste dumping site, sewage site, and tannery waste site. The good quality of the metagenomic DNA was evident by spectrophotometry data, PCR amplification of 16S rRNA gene and restriction digestion.
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