Erythrocyte PIG-A mutant frequencies in cancer patients receiving cisplatin.

Background: Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin-induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies.

Whole-genome high-fidelity sequencing: A novel approach to detecting and characterization of mutagenicity in vivo.

Direct DNA sequencing can be used for characterizing mutagenicity in simple and complex biological models. Recently we described a method of whole-genome sequencing for detecting mutations in simple models of cultured bacteria, mammalian cells, and nematode. In the current proof-of-concept study, we expand and improve our method for evaluating a more complex mammalian biological model in outbred mice.

Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.

DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult.

A Brief Practical Guide to PCR.

Polymerase chain reaction (PCR) has been a powerful molecular biology tool since the mid-1980s. Millions of copies of specific sequence regions of DNA can be generated to allow the study of these regions. Fields that use this technology range from forensics to the experimental study of human biology.

Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation.

Genome-wide detection of ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing.

Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome-wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error-free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi).

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020).

A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation.

Quantifying mutant or variable allele frequencies (VAFs) of ≤10-3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10-8 VAF, it is not a quick, easy, or inexpensive method.

Reduced vancomycin susceptibility and increased macrophage survival in Staphylococcus aureus strains sequentially isolated from a bacteraemic patient during a short course of antibiotic therapy.

Purpose: The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin.

Methods: The S. aureus strains had been isolated and sequenced previously.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data.

Lifespan Kras mutation levels in lung and liver of B6C3F mice.

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2-year carcinogenicity assay. However, there is little information regarding the effect of age on cancer-driver gene mutations in these models.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC.

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk cells identified with next generation sequencing.

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P).

Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig-a gene Mutation: Support from an in vitro assay based on L5178Y/Tk cells and the CD90.2 antigen.

Lack of cell surface glycosylphosphatidylinositol (GPI)-anchored protein(s) has been used as a reporter of Pig-a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk .

Establishing a novel Pig-a gene mutation assay in L5178YTk mouse lymphoma cells.

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g.

Whole genome sequencing of mouse lymphoma L5178Y-3.7.2C (TK) reveals millions of mutations and genetic markers.

The mouse lymphoma L5178Y-3.7.2C (TK) cell line is extensively used in genetic toxicology to conduct the mouse lymphoma assay (MLA).

Whole genome and normalized mRNA sequencing reveal genetic status of TK6, WTK1, and NH32 human B-lymphoblastoid cell lines.

Closely related TK6, WTK1, and NH32 human B-lymphoblastoid cell lines differ in their p53 functional status. These lines are used frequently in genotoxicity studies and in studies aimed at understanding the role of p53 in DNA repair. Despite their routine use, little is known about the genetic status of these cells.

ACB-PCR quantification of somatic oncomutation.

Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) is a sensitive approach for the selective amplification of an allele. Using the ACB-PCR technique, hotspot point mutations in oncogenes and tumor-suppressor genes (oncomutations) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules.

Accumulation of K-Ras codon 12 mutations in the F344 rat distal colon following azoxymethane exposure.

Azoxymethane (AOM) administration to F344 male rats is a widely used model of human colon carcinogenesis. The current study investigates quantitatively the accumulation of K-Ras codon 12 mutations following AOM exposure. Male, 6-week-old F344 rats were treated subcutaneously with 30 mg/kg body weight of AOM, and colon tissue was collected at 1, 8, 24, and 32 weeks after treatment.

Oncomutations as biomarkers of cancer risk.

Cancer risk assessment impacts a range of societal needs, from the regulation of chemicals to achieving the best possible human health outcomes. Because oncogene and tumor suppressor gene mutations are necessary for the development of cancer, such mutations are ideal biomarkers to use in cancer risk assessment. Consequently, DNA-based methods to quantify particular tumor-associated hotspot point mutations (i.

Measurement of tumor-associated mutations in the nasal mucosa of rats exposed to varying doses of formaldehyde.

This study examined the potential induction of tumor-associated mutations in formaldehyde-exposed rat nasal mucosa using a sensitive method, allele-specific competitive blocker-PCR (ACB-PCR). Levels of p53 codon 271 CGT to CAT and K-Ras codon 12 GGT to GAT mutations were quantified in nasal mucosa of rats exposed to formaldehyde. In addition, nasal mucosa cell proliferation was monitored because regenerative cell proliferation is considered a key event in formaldehyde-induced carcinogenesis.

ACB-PCR quantification of K-RAS codon 12 GAT and GTT mutant fraction in colon tumor and non-tumor tissue.

K-RAS mutation is being developed as a cancer biomarker and tumor K-RAS is being used to predict therapeutic response. Yet, levels of K-RAS mutation in normal and pathological tissue samples have not been determined rigorously, nor inter-individual variation in these levels characterized. Therefore, K-RAS codon 12 GAT and GTT mutant fractions were measured in colonic mucosa of individuals without colon cancer, tumor-distal mucosa, tumor-proximal mucosa, normal tumor-adjacent tissues, colonic adenomas, and carcinomas.