CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter

To elucidate the antibacterial efficacy of chemically synthesized and custom-made sulphur nanoparticles (SNPs) of two different sizes and surface modifications against a number of multidrug-resistant Gram-negative bacilli (GNB) harbouring the New Delhi metallo-β-lactamase 1 enzyme (NDM-1). Antimicrobial susceptibility of the isolates was determined. The strains were evaluated for the presence of carbapenemases, β-lactamases, 16S rRNA methylases and integrons. Chemically synthesized, polyethylene-glycol (PEG)-stabilized SNPs of 10 nm and custom-made non-capped SNPs of 60 nm were physicochemically characterized and evaluated for their antibacterial efficacy against multidrug-resistant GNB using the agar dilution method (ADM) and the broth microdilution method (BMD). The cytotoxicity of the chemically synthesized SNPs was evaluated with a human-derived hepatoma (HepG2) cell line using a WST-1 assay kit. All isolates were multidrug-resistant and possessed NDM-1 along with other β-lactamases, 16S rRNA methylases and integron 1. Chemically synthesized PEGylated SNPs showed a bactericidal effect against all tested strains at a concentration between 9.41 and 18.82 mg/L using BMD. The ADM data revealed that SNPs had uniform MICs (18.82 mg/L) for all tested strains. On the other hand, custom-made SNPs failed to impart any antibacterial effect at the equivalent concentrations of chemically synthesized SNPs. The WST-1 assay revealed no significant cytotoxicity of the PEGylated SNPs even at the highest concentration (94.08 mg/L). To the best of our knowledge, this is the first attempted study to show the effectiveness of nanoparticles against multidrug-resistant GNB harbouring NDM-1.

In this study we evaluated and correlated the cytotoxic effects of zinc oxide nanoparticles (ZnO-NPs) to epigenetic modifications, using human embryonic kidney (HEK-293) cells as a model system. Imaging of singlet and total reactive oxygen species (ROS) in ZnO-NPs treated live cells was performed followed by the evaluation of its effects on cytoskeletal, mitochondrial, and nuclear integrity, and on the expression of ROS responsive genes. Next, we determined the global and locus specific changes in DNA-methylation at the three genomic repeat sequences namely global LINE-1, subtelomeric D4Z4 and pericentromeric NBL2, and at the promoter of selected ROS responsive genes (AOX1, HMOX1, NCF2, SOD3). Our studies indicated severe actin depolymerization, increased release of mitochondrial cytochrome C, and nuclear enlargement in ZnO-NPs treated cells. At the epigenetic level we observed global reduction in 5-methylcytosine and increase in 5-hydroxymethylcytosine content. Additionally, we observed significant increase in the expression of TET-methylcytosine dioxygenase genes but not in the expression of DNA-methyltransferases (DNMTs). Based on our findings, we suggest ZnO-NPs induces abundant increase in ROS to promote multimodal structural and functional anomalies in cells. Most importantly, ZnO-NP induced ROS may promote global hypomethylation in cells by triggering the expression of TET-enzymes, avoiding DNMT interferences. Global DNA demethylation is considered to be the hallmark of majority of cancers and once acquired this could be propagated to future progenies. The present study, hence can be used as a platform for the assessment of epigenomic toxicity of ZnO-NPs in humans in the light of its use in commercial products.