Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitrowith the DNA-damaging agents, methyl methanesulfonate （MMS） or hydrogen peroxide （Hz02）, and then embedded in agarose gel on glass sl ides. The slides were immersed in alkaline solution （〉pH 13） for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period （1 min） of the alkaline DNA unwinding time.

Iron-Mediated Oxidative DNA Damage Detected by Fluorometric Analysis of DNA Unwinding in Isolated Rat Liver Nuclei

Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.

SARS-CoV-2 helicase NSP13 hijacks the host protein EWSR1 to promote viral replication by enhancing RNA unwinding activity

Background:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)emerged in December 2019 and has led to a global coronavirus disease 2019(COVID-19)pandemic.Currently,incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs.Results:In this study,we found that SARS-CoV-2 hijacks the host protein EWSR1(Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1)to promote the activity of its helicase NSP13 to facilitate viral propagation.NSP13 is highly conserved among coronaviruses and is crucial for virus replication,providing chemical energy to unwind viral RNA replication intermediates.Treatment with different SARS-CoV-2 NSP13 inhibitors in multi-ple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection.Using affinity-purification mass spectrometry,the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13.Furthermore,silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels.Mechanistically,EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability.Conclusions:Our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.

Protective Effect of Distillate and Redistillate of Cow’s Urine in Human Polymorphonuclear Leukocytes Challenged With Established Genotoxic Chemicals

Objective From the ancient period cow’urine has been used as a medicine. In Veda, cow’urine was compared to the nectar. In Susrut, several medicinal properties of cow’ urine have been mentioned and are known to cause weight loss, reversal of certain cardiac and kidney problems, indigestion, stomach ache, edema, etc. However, the literature and scripture did not mention the antigenotoxic properties of cow’urine. Methods In the present investigation, the antigenotoxic/ antioxidant properties of cow’ urine distillate and redistillate were studied in vitro. The antioxidant status and volatile fatty acid levels were determined. Actinomycin-D (0.1ol/L) and hydrogen peroxide (150 mol/L) were used for inducing DNA strand break with 0.1% DMSO as negative control. Dose for the antigenotoxic effect of cow’ urine was chosen from the dose response study carried out earlier. Results Both actinomycin-D and H2O2 caused statistically significant DNA unwinding of 80% & 75% respectively (P<0.001) as revealed by fluorimetric analysis of DNA unwinding (FADU), and the damage could be protected with the redistilled cow urine distillate (1, 50 & 100 ) in simultaneous treatment with genotoxic chemicals. Conclusion The redistillate of cowurine was found to possess total antioxidant status of around 2.6 mmol, contributed mainly by volatile fatty acids (1500 mg/L) as revealed by the GC-MS studies. These fatty acids and other antioxidants might cause the observed protective effects.

Naringin ameliorates acetic acid induced colitis through modulation of endogenous oxido-nitrosative balance and DNA damage in rats

The aim of this study was to evaluate the effect of naringin on experimentally induced inflammatory bowel dis- ease in rats. Naringin （20, 40 and 80 mg/kg） was given orally for 7 days to Wistar rats before induction of colitis by intrarectal instillation of 2 mL of 4% （v/v） acetic acid solution. The degree of colonic mucosal damage was analyzed by examining mucosal damage, ulcer area, ulcer index and stool consistency. Intrarectal administration of 4% acetic acid resulted in significant modulation of serum alkaline phosphatase, lactate dehydrogenase, superoxide dismutase （SOD）, glutathione （GSH）, malondialdehyde （MDA） and myeloperoxidase （MPO） content along with colonic nitric oxide （NO）, xanthine oxidase （XO） level and protein carbonyl content in the colonic tissue as well as in blood. Naringin （40 and 80 mg/kg） exerted a dose dependent （P 〈 0.05） ameliorative effect, as it significantly increased hematological parameter as well as colonic SOD and GSH. There was a significant （P 〈 0.05） and dose dependant inhibition of macroscopical score, ulcer area along with colonic MDA, MPO activity by the 7 days of pretreatment of naringin （40 and 80 mg/kg）. Biochemical studies revealed a significant （P 〈 0.05） dose dependant inhibition in serum alkaline phosphatase （ALP） and lactate dehydrogenase （LDH） levels by pretreatment of naringin. Increased levels of colonic NO, XO, protein carbonyl content and DNA damage were also sig- nificantly decreased by naringin pretreatment. The findings of the present investigation propose that naringin has an anti-inflammatory, anti-oxidant and anti-apoptotic potential effect at colorectal sites as it modulates the production and expression of oxidative mediators such as MDA, MPO, NO and XO, thus reducing DNA damage.

Genotoxicity of Pesticide Waste Contaminated Soil and Its Leachate

Objective Improper land disposal of hazardous waste can result in leaching of hazardous constituents which may contaminate ground and surface water leading to adverse impact on human health and environment consequences. The present study utilized mammalian cell culture for the genotoxicity assessment of waste and its leachate. Methods Genotoxic potential and chemical analysis of pesticide derived tarry waste contaminated soil extract and its leachate was assessed using in vitro human lymphocyte cultures and GC-MS. Results The investigation revealed that the soil extract could cause significant to highly significant genotoxicity in the form of DNA strand break at 25 mL (P<0.01), 50 mL, 100 mL and 200 mL (P<0.001) and chromosomal aberration at 25 mL (P<0.01) and 50 mL and 100 mL (P<0.001). The leachate could cause significant DNA strand break and chromosomal aberration only at 100 mL and 200 mL (P<0.01) dose levels. Conclusion The genotoxicity observed is attributed to carbaril and tetra methyl naphthyl carbamate, the major ingredients of the extracts, as revealed by GC-MS.

DNA Damage Caused By Pesticide-contaminated Soil

Objective To determine the DNA damaging potential and the genotoxicity of individual compounds in pesticide contaminated soil. Methods In the present study, DNA damaging potential of pesticide-contaminated soil and the genotoxicity of individual compounds present in the soil were assessed using fluofimetdc analysis of DNA unwinding assay. Results The contaminated soil sample showed 79% （P〈0.001） of DNA strand break, whereas technical grade of major catbaryl and α-naphthol constituents of the contaminated soil showed 64% （P〈0.01） and 60% （P〈0.02） damage respectively. Conclusion Our results indicate that the toxicity caused by contaminated soil is mainly due to carbatyl and α -napthol, which are the major constituents of the soil sample analyzed by CrC-MS.

Adaptive Force Control of in Web Handling Systems

Winding/unwinding system control is a very important issue to web handling machines. In this paper, a novel adaptive H∞ control strategy is developed for winding process control. A gain scheduling scheme is proposed based on a neural fuzzy approximator to improve the transient response and enhance tension control;the controller’s convergence and adaptive capability can be further improved by an efficient hybrid training algorithm. The effectiveness of the proposed adaptive H∞ control is verified by experimental tests. Test results show that the developed gain approximator can adaptively accommodate parameter variations in the system and improve the control performance.

Helicase activity and substrate specificity of RecQ5β

RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β～（1-467）, we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.

Cloning, expression, purification and characterization of replication protein from plasmid pGP2 from Acetobacter estunensis

The Acetobacter estunensis Rep34 protein participates in the replication of bacterial plasmid pGP2. The Rep34 protein of the A. estunensis, was cloned to the expression vector, that ensure fusion with a His-tag sequence (Rep34 His-tagged), over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. On this purified protein number different activities and motifs were detected. DNA band-shift assays showed that the Rep34 His-tagged protein bound to the regulation region for replication on the linear double-stranded DNA. In the protein was determined phosphatase activity, ATPase activity and protein is possible to unwind double strand DNA.

A class of iterative greedy algorithms related to Blaschke product

Mobius transforms,Blaschke products and starlike functions as typical conformal mappings of one complex variable give rise to nonlinear phases with non-negative phase derivatives with the latter being de ned by instantaneous frequencies of signals they represent.The positive analytic phase derivative has been a widely interested subject among signal analysts(see Gabor(1946)).Research results of the positive analytic frequency and applications appears in the literature since the middle of the 20th century.Of the positive frequency study a directly related topic is positive frequency decomposition of signals.The mainly focused methods of such decompositions include the maximal selection method and the Blaschke product unwinding method,and joint use of the mentioned methods.In this paper,we propose a class of iterative greedy algorithms based on the Blaschke product and adaptive Fourier decomposition.It generalizes the Blaschke product unwinding method by subtracting constants other than the averages of the remaining functions,aiming at larger winding numbers,and subtracting n-Blaschke forms of the remaining functions,aiming at generating larger numbers of zero-crossings,to fast reduce energy of the remaining terms.Furthermore,we give a comprehensive and rigorous proof of the converging rate in terms of the zeros of the remainders.Finite Blaschke product methods are proposed to avoid the in nite phase derivative dilemma,and to avoid the computational diculties.