Amelioration of oxidative stress-mediated cytotoxicity and genotoxicity induced by copper and flubendiamide in-vivo and in- vitro by potent antioxidants

Present study was designed to assess the toxicity of copper @ 33 mg/kg and flubendiamide @ 200 mg/kg in vivo in male Wistar rats orally once daily for 90 days and protective effect of α-tocopherol, resveratrol, curcumin and catechin and in vitro cyto-genotoxicity in primary cell culture of thymocytes. In vivo study showed significant (p<0.05) increase in AST, total bilirubin and uric acid, creatinine and BUN levels while decrease in total proteins, GSH, SOD and GST levels and increased LPO and GPx with severe degenerative changes were observed in liver and kidney tissues in intoxicated groups. In vitro thymocytes were exposed to 40 µM concentration of flubendiamide and/or showed significant increase in TUNEL+ve cells, micronuclei, DNA shearing, and comet formation per 100 cells. Concurrent treatment with α-tocopherol in xenobiotics intoxicated groups showed almost normal values of the biochemical parameters and decreased LPO production and improved antioxidant enzymes activities and histoarchitecture of liver and kidney tissues suggest ameliorative potential of α-tocopherol whereas, resveratrol, curcumin, catechin or α-tocopherol in vitro decreased TUNEL+ve cells, micronuclei induction and comet formation and effect of antioxidants was concentration-dependent and their order of potency on equimolar concentration (10 µM) basis is: curcumin > resveratrol >catechin = α-tocopherol.

An optimized ChIP-Seq framework for profiling of histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin Immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein crosslinking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

Choice of Laboratory Tissue Homogenizers Matters When Recovering Nucleic Acid From Medically Important Ticks

Ticks can vector and transmit many pathogens and pose a serious human health threat throughout the world. After collection, many diagnostic laboratories must mechanically disrupt tick specimens for diagnostic testing and research purposes, but few studies have evaluated how well-commercial tissue homogenizers perform this task. We evaluated four commercially available tissue homogenizers: The Bead Ruptor 24 Elite, the Bullet Blender Storm, the gentleMACS Dissociator, and the Precellys 24. We quantitatively compared maceration level, nucleic acid quality, quantity, amplification, and DNA shearing to determine which machines performed the best. The Bead Ruptor 24 Elite had the highest overall score when disrupting a single, uninfected adult Amblyomma americanum (Linnaeus) (Ixodida: Ixodidae) and performed well in follow-on tests including disrupting individual juvenile samples and detecting pathogens from infected samples.

Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

ABSTRACTOur earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track inward at origins, melt double-strand (ds) DNA while encircling the duplex by pulling on opposite strands and shearing DNA apart (Langston and O’Donnell (2019) eLife 9, e46515). We show here that increasing the methylphosphonate neutral DNA from 10 nucleotides in the previous report, to 20 nucleotides, reveals that CMG encircling duplex DNA only interacts with the tracking strand compared to the non-tracking strand. This significantly enhances support that CMG tracks on duplex DNA by binding only one strand. Furthermore, EMSA assays using AMPPNP to load CMG onto DNA shows a stoichiometry of only 2 CMGs on an origin mimic DNA, containing a 150 bp duplex with two 3 prime single-strand (ss) tails, one on each end, enabling assay of dsDNA unwinding by a shearing force produced by only two head-to-head CMGs. The use of non-hydrolysable AMPPNP enabled a preincubation for CMG binding the two 3 prime tailed origin mimic DNA, and gave robust unwinding of dsDNA by head-to-head CMG-Mcm10’s. With this precedent, it is possible to envision that the cell may utilize opposing dsDNA motors to unwind DNA for other types of DNA transactions besides origin unwinding.

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2Pon Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

ABSTRACTRegulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5P) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2P)-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi.IMPORTANCERegulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5Poccupancy decreased and S2Plevels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.