Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells

DNA polymerase eta (Pol η) is one of several Y family translesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV-induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased with empty vector control (EVC), wild-type POLH and Y52E-POLH over 48 hours post-UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV-induced DNA lesions in HaCaT cells.     

Forensic utility of the feline mitochondrial control region – A Dutch perspective

Different portions of the feline mitochondrial DNA control region (CR) were evaluated for their informative value in forensic investigations. The 402 bp region located between RS2 and RS3 described most extensively in the past is not efficient for distinguishing between the majority of Dutch cats, illustrated by a random match probability (RMP) of 41%. Typing of the whole region between RS2 and RS3, and additional typing of the 5’portion of the feline CR decreases the RMP to 29%, increasing the applicability of such analyses for forensic investigations.The haplotype distribution in Dutch random bred cats (N = 113) differs greatly from the distributions reported for other countries, with a single haplotype NL-A1 present in 54% of the population. The three investigated breeds showed haplotype distributions differing from each other and the random bred cats with haplotype NL-A1 accounting for 4%, 29% and 32% of Maine Coon, Norwegian forest cats and Siamese & Oriental cats. These results indicate the necessity of validating haplotype frequencies within continents and regions prior to reporting the value a mtDNA match. In cases where known purebred cats are involved, further investigation of the breed may be valuable.     

Genetic variants of DNA repair pathway genes on lung cancer risk

As is commonly perceived, polymorphisms in genes of deoxyribonucleic acid (DNA) repair pathway plays a fundamental role in defective DNA repair and mutagenesis prevention and serves to contribute to the individual susceptibility to the development of a variety of cancers. Recently, an increasing number of studies have been dedicated to the contentious and ambiguous links between polymorphisms in genes of DNA repair pathway and lung cancer (LC) risk. In response, a comprehensive updated meta-analysis has been proposed herein to assess the correlation between polymorphisms of DNA repair pathway genes and susceptibility to LC. This paper has identified and retrieved eligible articles from PubMed, Google Scholar, Web of Science, and CNKI databases till February 20, 2019. Finally, 295 case-control studies as to the fourteen polymorphisms of DNA repair pathway genes were enrolled. When the results have been pooled, we have brought to light the conclusion that ERCC2-rs13181 polymorphism has an elevated association with LC risk under allele, heterozygote, and dominant comparisons. In the subgroup analysis by ethnicity, we have found that the Caucasian individuals with “B” variant possess risk of LC which was more than twice as much as allele, homozygote, and recessive models. In comparison, Asian carriers of rs13181 polymorphism in ERCC2 gene are more susceptible to LC in heterozygote, dominant models. To sum up, ERCC2-rs13181 polymorphism could be a critical factor in stimulating LC evolvement. Future studies with a larger sample size and multivariate factors are needed to vindicate these findings.     

Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation,Stemness, and BrafV600E-Induced Tumorigenesis

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U.S. initiatives to strengthen forensic science & international standards in forensic DNA

A number of initiatives are underway in the United States in response to the 2009 critique of forensic science by a National Academy of Sciences committee. This article provides a broad review of activities including efforts of the White House National Science and Technology Council Subcommittee on Forensic Science and a partnership between the Department of Justice (DOJ) and the National Institute of Standards and Technology (NIST) to create the National Commission on Forensic Science and the Organization of Scientific Area Committees. These initiatives are seeking to improve policies and practices of forensic science. Efforts to fund research activities and aid technology transition and training in forensic science are also covered.The second portion of the article reviews standards in place or in development around the world for forensic DNA. Documentary standards are used to help define written procedures to perform testing. Physical standards serve as reference materials for calibration and traceability purposes when testing is performed. Both documentary and physical standards enable reliable data comparison, and standard data formats and common markers or testing regions are crucial for effective data sharing. Core DNA markers provide a common framework and currency for constructing DNA databases with compatible data. Recent developments in expanding core DNA markers in Europe and the United States are discussed.     

Protective activity of a novel resveratrol analogue,HS-1793, against DNA damage in 137Cs-irradiated CHO-K1 cells

Resveratrol has received considerable attention as a polyphenol with anti-oxidant, anti-carcinogenic, and anti-inflammatory effects. Radiation is an important component of therapy for a wide range of malignant conditions. However, it causes damage to normal cells and, hence, can result in adverse side effects. This study was conducted to examine whether HS-1793, a novel resveratrol analogue free from the restriction of metabolic instability and the high dose requirement of resveratrol, induces a protective effect against radiation-induced DNA damage. HS-1793 effectively scavenged free radicals and inhibited radiation-induced plasmid DNA strand breaks in an in vitro assay. HS-1793 significantly decreased reactive oxygen species and cellular DNA damage in 2 Gy-irradiated Chinese hamster ovary (CHO)-K1 cells. In addition, HS-1793 dose-dependently reduced the levels of phosphorylated H2AX in irradiated CHO-K1 cells. These results indicate that HS-1793 has chemical radioprotective activity. Glutathione levels and superoxide dismutase activity in irradiated CHO-K1 cells increased significantly following HS-1793 treatment. The enhanced biological anti-oxidant activity and chemical radioprotective activity of HS-1793 maintained survival of irradiated CHO-K1 cells in a clonogenic assay. Therefore, HS-1793 may be of value as a radioprotector to protect healthy tissue surrounding tumor cells during radiotherapy to obtain better tumor control with a higher dose.     

Assessment of polycyclic aromatic hydrocarbon exposure,lung function,systemic inflammation,and genotoxicity in peripheral blood mononuclear cells from firefighters before and after a work shift

Firefighting is regarded as possibly carcinogenic, although there are few mechanistic studies on genotoxicity in humans. We investigated exposure to polycyclic aromatic hydrocarbons (PAH), lung function, systemic inflammation and genotoxicity in peripheral blood mononuclear cells (PBMC) of 22 professional firefighters before and after a 24‐h work shift. Exposure was assessed by measurements of particulate matter (PM), PAH levels on skin, urinary 1‐hydroxypyrene (1‐OHP) and self‐reported participation in fire extinguishing activities. PM measurements indicated that use of personal protective equipment (PPE) effectively prevented inhalation exposure, but exposure to PM occurred when the environment was perceived as safe and the self‐contained breathing apparatuses were removed. The level of PAH on skin and urinary 1‐OHP concentration were similar before and after the work shift, irrespective of self‐reported participation in fire extinction activities. Post‐shift, the subjects had reduced levels of oxidatively damaged DNA in PBMC, and increased plasma concentration of vascular cell adhesion molecule 1 (VCAM‐1). The subjects reporting participation in fire extinction activities during the work shift had a slightly decreased lung function, increased plasma concentration of VCAM‐1, and reduced levels of oxidatively damaged DNA in PBMC. Our results suggest that the firefighters were not exposed to PM while using PPE, but exposure occurred when PPE was not used. The work shift was not associated with increased levels of genotoxicity. Increased levels of VCAM‐1 in plasma were observed. Environ. Mol. Mutagen. 59:539–548, 2018. © 2018 Wiley Periodicals, Inc.