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“Omics”
Published in Kirk A. Phillips, Dirk P. Yamamoto, LeeAnn Racz, Total Exposure Health, 2020
Sun et al. (2015) and Dirks et al. (2016) have reviewed the status of genome-wide epigenetic profiling technologies. To probe the methylation status genome wide, methylated DNA is treated with sodium bisulfite which converts methylated cytosine to uracil (read as thymine when sequenced or probed by a methylation microarray such as the Illumina Methylation Epic BeadChip assay). For known human genes, the Illumina Methylation Epic BeadChip assay reports methylation status at ~853,307 CpG sites with coverage of >90% of ~26,000 CpG islands (regions of >200 bp with %GC >50%), North and South Shores of islands (2,000 bp regions upstream and downstream of CpG islands) and 80% coverage of North and South Shelves (regions between 2,000–4,000 bp upstream and downstream of CpG islands). A limitation of array-based technology is that it is difficult to incorporate single nucleotide polymorphism-type data that might be uncovered by bisulfite sequencing approaches. Whole-genome bisulfite sequencing offers the ability to probe all 28 million known CpG and correlate genotype and methylation patterns. Reduced representation bisulfite sequencing and variants offer coverage of 1.5–4 million unique CpG by using methylation-sensitive restriction digestion to enrich CpG-rich regions and sequence only these. The impact of histone modifications is frequently assayed by immunoprecipitation of chromatin using affinity tags directed against specific histone modifications followed by sequencing.
Agrochemical-mediated cardiotoxicity in zebrafish embryos/larvae: What we do and where we go
Published in Critical Reviews in Environmental Science and Technology, 2023
Yang Yang, Yue Tao, Zixu Li, Yunhe Cui, Jinzhu Zhang, Ying Zhang
Crosstalk is not limited to that between the Wnt/β-catenin and Notch signaling pathways. For example, using chromatin immunoprecipitation, Baik et al. (2016) that Smad1 can bind to known targets of the BMP signaling pathway; unexpectedly, Smad1 was also found to bind to wif1 and Tbx5 (key transcription factors of cardiac specification). Specifically, Smad1 can bind to the promoter and intron of wif1 to enhance its transcription level and thereby inhibit activation of the Wnt/β-catenin signaling pathway, whereas binding of Smad1 to Tbx5 inhibits its normal expression, due to the fact that the binding effect of Smad1 disruption of the interaction of Tbx5 with the chromosome remodeling complex and leads to abnormal cardiac development (Takeuchi et al., 2011). Moreover, phosphorylation of Smad1 is dependent on the interaction of BMP with its corresponding ligand, and GSK-3β phosphorylates and degrades Smad1 and ensure normal activation of the Wnt/β-catenin signaling pathway. The crosstalk mentioned in this paper is only a relatively minor part of the early cardiac development in zebrafish. Most studies of cardiotoxicity induced by agrochemicals focused on changes in individual signaling pathways; however, phenotypic changes in cardiac defects are often mediated by multiple pathways, and precise identification of the first responders can reveal the mechanisms of toxicity induced by a specific agrochemical or class of agrochemicals.
Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells
Published in Preparative Biochemistry & Biotechnology, 2023
Jiayang Jiang, Xiaoman Zhou, Chunliu Gao, Rongqin Ke, Qiwei Guo
In the present study, approximately 108 interstitial cells were obtained from two testes of a mouse and 1.7 × 105, 3.9 × 105, and 11.9 × 105 LCs were extracted using the regular method, modified method 1, and modified method 2, respectively. When compared to those of previous studies based on continuous and discontinuous Percoll gradients, the purities of the regular method and modified method 1 were comparatively satisfactory, but the yields of our methods were lower. Two reasons could be attributed to this finding. First, the volume of the LC-concentrated solution collected from Percoll gradients was relatively small (e.g. 0.5 mL). Secondly, the wash steps in our methods could decrease the yields.[11] Nevertheless, the yields of LCs in the present study were sufficient for further analysis with most molecular technologies, including single-cell sequencing,[25] assay for transposase-accessible chromatin using sequencing,[26] and chromatin immunoprecipitation assay.[27]
Xenobiotic metabolism and transport in Caenorhabditis elegans
Published in Journal of Toxicology and Environmental Health, Part B, 2021
Jessica H. Hartman, Samuel J. Widmayer, Christina M. Bergemann, Dillon E. King, Katherine S. Morton, Riccardo F. Romersi, Laura E. Jameson, Maxwell C. K. Leung, Erik C. Andersen, Stefan Taubert, Joel N. Meyer
Besides the NR1J group NHRs, several HNF4-related NHRs have recently emerged as regulators of detoxification gene programs in C. elegans. NHR-86 is required to express phase I detoxification in worms exposed to the immunomodulatory toxin RPW24, including four cyp genes, two gst genes, and 14 ugt genes. As in the case of NHR-8, however, the requirement for nhr-86 is not universal, as the highly RPW24-inducible cyp-35A2, cyp-35A3, cyp-35B1, and cyp-35B2 genes remained chemical-responsive in nhr-86 mutants; whether these genes are instead regulated by other NHRs such as NHR-8 which was not tested (Peterson et al. 2019). It is noteworthy that Peterson et al. (2019) used chromatin immunoprecipitation followed by sequencing to identify direct NHR-86 targets. The 32 genes whose promoters were bound by this TF and that required its activity for RPW24 induced expression included cyp-35A1 as the most strongly NHR-86 bound gene, as well as three ugt genes and gst-5. This provides first evidence that detoxification regulatory NHRs might directly bind critical response genes in live worms. Further, NHR-86 binding to several promoters increased upon exposure to RPW24. This suggests that NHR-86, and perhaps other NHRs, might display toxin-induced binding to relevant detoxification promoters as a mechanism to enhance their expression on demand.