Formamidopyrimidine DNA Glycosylase Research Articles

Arginine-Selective Bioconjugation Reagent for Effective 18F-labeling of Native Proteins.

Protein-based 18F-PET tracers offer new possibilities in early disease detection and personalized medicine. Their development relies heavily on the availability and effectiveness of 18F-prosthetic groups. We prepared and evaluated a novel arginine-selective prosthetic group, 4-[18F]fluorophenylglyoxal ([18F]FPG). [18F]FPG was radiosynthesized by a one-pot, two-step procedure with a non-decay-corrected (n.d.c.) isolated radiochemical yield (RCY) of 41 ± 8% (n = 10). [18F]FPG constitutes a generic tool for 18F-labeling of various proteins, including human serum albumin (HSA), ubiquitin, interleukin-2, and interleukin-4 in ∼30-60% n.d.c. isolated RCYs. [18F]FPG conjugation with arginine residues is highly selective, even in the presence of a large excess of lysine, cysteine, and histidine. [18F]FPG protein conjugates are able to preserve the binding affinity of the native proteins while also demonstrating excellent in vivo stability. The [18F]FPG-HSA conjugate has prolonged blood retention, which can be applied as a potential blood pool PET imaging agent. Thus, [18F]FPG is an arginine-selective bioconjugation reagent that can be effectively used for the development of 18F-labeled protein radiopharmaceuticals.

Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay

DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1–2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer’s protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer’s protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0–4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer’s protocol), using TK6 cells with MMS (100–800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7–122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

Age-related influence on DNA damage, proteomic inflammatory markers and oxidative stress in hospitalized COVID-19 patients compared to healthy controls

COVID-19 infections are accompanied by adverse changes in inflammatory pathways that are also partly influenced by increased oxidative stress and might result in elevated DNA damage. The aim of this case-control study was to examine whether COVID-19 patients show differences in oxidative stress-related markers, unconjugated bilirubin (UCB), an inflammation panel and DNA damage compared to healthy, age-and sex-matched controls.The Comet assay with and without the treatment of formamidopyrimidine DNA glycosylase (FPG) and H2O2 challenge was used to detect DNA damage in whole blood. qPCR was applied for gene expression, UCB was analyzed via HPLC, targeted proteomics were applied using Olink® inflammation panel and various oxidative stress as well as clinical biochemistry markers were analyzed in plasma.Hospitalized COVID-19 patients (n = 48) demonstrated higher serum levels of 55 inflammatory proteins (p < 0.001), including hs-C-reactive protein levels (p < 0.05), compared to healthy controls (n = 48). Interestingly, significantly increased age-related DNA damage (%-DNA in tail) after formamidopyrimidine DNA glycosylase (FPG) treatment was measured in younger (n = 24, average age 55.7 years; p < 0.05) but not in older COVID-19 patients (n = 24, average age 83.5 years; p > 0.05). Although various oxidative stress markers were not altered (e.g., FRAP, malondialdehyde, p > 0.05), a significant increased ratio of oxidized to reduced glutathione was detected in COVID-19 patients compared to healthy controls (p < 0.05). UCB levels were significantly lower in individuals with COVID-19, especially in younger COVID-19 patients (p < 0.05).These results suggest that COVID-19 infections exert effects on DNA damage related to age in hospitalized COVID-19 patients that might be driven by changes in inflammatory pathways but are not altered by oxidative stress parameters.

In vivo evaluation of the neurogenotoxic effects of exposure to validamycin A in neuroblasts of Drosophila melanogaster larval brain.

Agriculture commonly utilizes crop protection products to tackle infestations from fungi, parasites, insects, and weeds. Validamycin A, an inhibitor of trehalase, possesses antibiotic and antifungal attributes. Epidemiological evidence has led to concerns regarding a potential link between pesticide usage and neurodegenerative diseases. The fruit fly, Drosophila melanogaster, has been recognized as a reliable model for genetic research due to its significant genetic similarities with mammals. Here, we propose to use D.melanogaster as an effective in vivo model system to investigate the genotoxic risks associated with exposure to validamycin A. In this study, we performed a neurotoxic evaluation of validamycin A in D.melanogaster larvae. Several endpoints were evaluated, including toxicity, intracellular oxidative stress (reactive oxygen species), intestinal damage, larval behavior (crawling behavior, light/dark sensitivity assay, and temperature sensitivity assay), locomotor (climbing) behavior, and neurogenotoxic effects (impaired DNA via Comet assay, enhanced by Endo III and formamidopyrimidine DNA glycosylase [FPG]). The results showed that exposure to validamycin A, especially at higher doses (1 and 2.5mM), induced DNA impairment in neuroblasts as observed by Comet assay. Both larvae and adults exhibited behavioral changes and produced reactive oxygen species. Most importantly, this research represents a pioneering effort to report neurogenotoxicity data specifically in Drosophila larval neuroblasts, thus underscoring the importance of this species as a testing model in exploring the biological impacts of validamycin A. The in vivo findings from the experiments are a valuable and novel addition to the existing validamycin A neurogenotoxicity database.

Effect of Repeated Bolus and Continuous Glucose Infusion on DNA Damage and Oxidative Stress Biomarkers in Healthy Male Volunteers.

Glucose variability (GV), which describes fluctuations in blood glucose levels within the day, is a phenomenon that is increasingly becoming the target of scientific attention when it comes to increased risk of coronary heart disease. Effects of GV may contribute to the development of metabolic syndrome and type 2 diabetes. Hyperglycemia can lead to oxidative stress resulting in molecular damage due to accumulation of reactive oxygen species (ROS). To discover more about the immediate effects of GV, continuous vs. bolus intravenous glucose administration was applied to 10 healthy men aged 21-30 years over a time frame of 48 h. Whole blood and plasma were analyzed for DNA damage using a comet assay with 3 different treatments (lysis buffer, H2O2, and the lesion-specific enzyme formamidopyrimidine DNA glycosylase (FPG)) as well as for the oxidative stress markers protein carbonyls (PC), unconjugated bilirubin (UCB), and ferric reducing antioxidant power (FRAP). A significant time effect was found in the three DNA damage treatments as well as in PC and UCB possibly due to circadian changes on oxidative stress, but no intervention group effect was observed for any of the markers. In conclusion, bolus vs. continuous glucose administration had no significant acute effect on DNA damage and markers of oxidative stress in healthy men.

In Vivo Genotoxicity and Toxicity Assessment of Sterigmatocystin Individually and in Mixture with Aflatoxin B1.

Mycotoxins are natural food and feed contaminants produced by several molds. The primary mode of exposure in humans and animals is through mixtures. Aflatoxin B1 (AFB1) and sterigmatocystin (STER) are structurally related mycotoxins that share the same biosynthetic route. Few in vivo genotoxicity assays have been performed with STER. In the present genotoxicity study, Wistar rats were dosed orally with STER (20 mg/kg b.w.), AFB1 (0.25 mg/kg b.w.) or a mixture of both in an integrated micronucleus (bone marrow) and comet study (liver and kidney). STER was dosed at the highest feasible dose in corn oil. No increase in the percentage of micronuclei in bone marrow was observed at any condition. Slight DNA damage was detected in the livers of animals treated with AFB1 or the mixture (DNA strand breaks and Fpg (Formamidopyrimidine DNA glycosylase)-sensitive sites, respectively). Plasma, liver, and kidney samples were analyzed with LC-MS/MS demonstrating exposure to both mycotoxins. General toxicity parameters (organs absolute weight, biochemistry, and histopathology) were not altered either individually or in the mixture. The overall absence of individual genotoxicity did not allow us to set any type of interaction in the mixture. However, a possible toxicokinetic interaction was observed.

Profiling of Barley, Wheat, and Rye FPG and OGG1 Genes during Grain Germination.

This research is about the profiling of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.) FPG and OGG1 genes during grain germination. During seed germination, reactive oxygen species accumulate, which leads to DNA damage. In the base excision repair (BER) system, the enzymes formamidopyrimidine DNA glycosylase (FPG) and 8-oxoguanine DNA glycosylase (OGG1), among others, are responsible for repairing such damage. We decided to check how the expression of genes encoding these two enzymes changes in germinating grains. Spring varieties of barley, wheat, and rye from the previous growing season were used in the study. Expression level changes were checked using Real-Time PCR. After analyzing the obtained results, the maximum expression levels of FPG and OGG1 genes during germination were determined for barley, wheat, and rye. The results of the study show differences in expression levels specific to each species. The highest expression was observed at different time points for each of them. There were no differences in the highest expression for FPG and OGG1 within one species. In conclusion, the research provides information on how the level of FPG and OGG1 gene expression changes during the germination process in cereals. This is the first study looking at the expression levels of these two genes in cereals.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

21 Toxicity of Aircraft Engine Emissions in Calu-3 Human Cells in Air-Liquid Interface Condition.

Abstract Particulate Matter (PM) emissions from aircraft engines adversely affect air quality around airports, contributing to public health concerns for airport workers and neighbouring communities. In this research, PM was collected from the exhaust of turbine engine after combustion of various types of aircraft fuels, including conventional fuel types Jet A and Jet A1, and more sustainable alternatives with relative low levels of aromatics. PM samples’ toxicity was studied in vitro by air-liquid interface (ALI) exposure of Calu-3 cells in monoculture and co-culture with macrophages (differentiated THP-1 cells). Cells were exposed to PM suspensions (250 µg/mL) using a cloud-based ALI exposure system and analysed 24-hours after exposure. DNA damage was detected through Comet assay in cells exposed to Jet A and Jet A1 samples in absence of cytotoxicity; co-incubation with endonucleases-III and formamidopyrimidine-DNA glycosylase showed that this was not associated with oxidation of DNA bases. Real-Time PCR was performed to investigate the expression profile of genes involved in oxidative stress and DNA repair pathways. Heme Oxygenase 1 gene, Mucin 5AC, and Angiotensin-Converting Enzyme 2 were modulated in the same samples where DNA damage was present. No differences were shown in the pro-inflammatory interleukine-8 secretion among the samples. Most of the PM samples generated using jet fuel with high content of aromatic compounds induced enhanced genotoxicity, suggesting that strategies lowering aromatic content may also result in less toxic PM emissions. Funded by Horizon2020 – Research of Aviation PM Technologies, mOdelling and Regulation (RAPTOR), grant number 863969.

Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay

DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0–480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO3). The initial amount of damage induced by Ro 19–8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO3 varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T0 to an equal level can be achieved by the use of KBrO3, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure.Graphical

Induction of DNA strand breaks and oxidative base damages in plasmid DNA by ultra-high dose rate proton irradiation

Purpose Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s. Materials and Methods A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis. Results Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages. Conclusions UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.

Aluminium(III) Oxide—The Silent Killer of Bacteria

In this article, we describe the antimicrobial properties of pristine anodised aluminium oxide matrices-the material many consider biologically inert. During a typical anodisation process, chromium and chlorine compounds are used for electropolishing and the removal of the first-step aluminium oxide. Matrices without the use of those harmful compounds were also fabricated and tested for comparison. The antibacterial tests were conducted on four strains of Escherichia coli: K12, R2, R3 and R4. The properties of the matrices were also compared to the three types of antibiotics: ciprofloxacin, bleomycin and cloxacillin using the Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than the aforementioned antibiotics. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA.

Accumulation of DNA damage alters microRNA gene transcription in Arabidopsis thaliana

BackgroundMicroRNAs (miRNAs) and other epigenetic modifications play fundamental roles in all eukaryotic biological processes. DNA damage repair is a key process for maintaining the genomic integrity of different organisms exposed to diverse stresses. However, the reaction of miRNAs in the DNA damage repair process is unclear.ResultsIn this study, we found that the simultaneous mutation of zinc finger DNA 3′-phosphoesterase (ZDP) and AP endonuclease 2 (APE2), two genes that play overlapping roles in active DNA demethylation and base excision repair (BER), led to genome-wide alteration of miRNAs. The transcripts of newly transcribed miRNA-encoding genes (MIRs) decreased significantly in zdp/ape2, indicating that the mutation of ZDP and APE2 affected the accumulation of miRNAs at the transcriptional level. In addition, the introduction of base damage with the DNA-alkylating reagent methyl methanesulfonate (MMS) accelerated the reduction of miRNAs in zdp/ape2. Further mutation of FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG), a bifunctional DNA glycosylase/lyase, rescued the accumulation of miRNAs in zdp/ape2, suggesting that the accumulation of DNA damage repair intermediates induced the transcriptional repression of miRNAs.ConclusionsOur investigation indicates that the accumulation of DNA damage repair intermediates inhibit miRNAs accumulation by inhibiting MIR transcriptions.

Reduced genotoxicity of lignin-derivable replacements to bisphenol A studied using in silico, in vitro, and in vivo methods

Bisguaiacols, lignin‐derivable bisphenols, are considered promising and possibly safer alternatives to bisphenol A (BPA), but comprehensive toxicity investigations are needed to ensure safety. Most toxicity studies of BPA and its analogues have focused on potential estrogenic activity, and only limited toxicological data are available on other toxicity aspects, such as genotoxicity at low exposure levels. In this study, the genotoxicity of six lignin-derivable bisguaiacols with varying regioisomer contents and degrees of methoxy substitution was investigated using a multi-tiered method, consisting of in silico simulations, in vitro Ames tests, and in vivo comet tests. The toxicity estimation software tool, an application that predicts toxicity of chemicals using quantitative structure-activity relationships, calculated that the majority of the lignin-derivable bisguaiacols were non-mutagenic. These results were supported by Ames tests using five tester strains (TA98, TA100, TA102, TA1535, and TA1537) at concentrations ranging from 0.5 pmol/plate to 5 nmol/plate. The potential genotoxicity of bisguaiacols was further evaluated using in vivo comet testing in fetal chicken livers, and in addition to the standard alkaline comet assay, the formamidopyrimidine DNA glycosylase enzyme-modified comet assay was employed to investigate oxidative DNA damage in the liver samples. The oxidative stress analyses indicated that the majority of lignin-derivable analogues showed no signs of mutagenicity (mutagenic index < 1.5) or genotoxicity, in comparison to BPA and bisphenol F, likely due to the methoxy groups on the lignin-derivable aromatics. These findings reinforce the potential of lignin-derivable bisphenols as safer alternatives to BPA.

Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions.

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables SDR. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR starts. By varying the concentration of the blocker strand and the concentration of the enzyme, we show that we can finely tune and modulate the delayed onset of SDR. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit.

The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials.

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture, format and volume of medium on the plate—is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

Distinct Mechanisms of Target Search by Endonuclease VIII-like DNA Glycosylases.

Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA non-specifically and then slides along the DNA contour by one-dimensional diffusion. Opposite to the processive mechanism is distributive search, when an enzyme binds, samples and releases DNA without significant lateral movement. Many DNA glycosylases, the repair enzymes that excise damaged bases from DNA, use processive search to find their cognate lesions. Here, using a method based on correlated cleavage of multiply damaged oligonucleotide substrates we investigate the mechanism of lesion search by three structurally related DNA glycosylases—bacterial endonuclease VIII (Nei) and its mammalian homologs NEIL1 and NEIL2. Similarly to another homologous enzyme, bacterial formamidopyrimidine–DNA glycosylase, NEIL1 seems to use a processive mode to locate its targets. However, the processivity of Nei was notably lower, and NEIL2 exhibited almost fully distributive action on all types of substrates. Although one-dimensional diffusion is often regarded as a universal search mechanism, our results indicate that even proteins sharing a common fold may be quite different in the ways they locate their targets in DNA.

Interspecific differences in oxidative DNA damage after hydrogen peroxide exposure of sea urchin coelomocytes.

Interspecific comparison of DNA damage can provide information on the relative vulnerability of marine organisms to toxicants that induce oxidative genotoxicity. Hydrogen peroxide (H2O2) is an oxidative toxicant that causes DNA strand breaks and nucleotide oxidation and is used in multiple industries including Atlantic salmon aquaculture to treat infestations of ectoparasitic sea lice. H2O2 (up to 100 mM) can be released into the water after sea lice treatment, with potential consequences of exposure in nontarget marine organisms. The objective of the current study was to measure and compare differences in levels of H2O2-induced oxidative DNA damage in coelomocytes from Scottish sea urchins Echinus esculentus, Paracentrotus lividus, and Psammechinus miliaris. Coelomocytes were exposed to H2O2 (0-50 mM) for 10 min, cell concentration and viability were quantified, and DNA damage was measured by the fast micromethod, an alkaline unwinding DNA method, and the modified fast micromethod with nucleotide-specific enzymes. Cell viability was >92% in all exposures and did not differ from controls. Psammechinus miliaris coelomocytes had the highest oxidative DNA damage with 0.07 ± 0.01, 0.08 ± 0.01, and 0.07 ± 0.01 strand scission factors (mean ± SD) after incubation with phosphate-buffered saline, formamidopyrimidine-DNA glycosylase, and endonuclease-III, respectively, at 50 mM H2O2. Exposures to 0.5 mM H2O2 (100-fold dilution from recommended lice treatment concentration) induced oxidative DNA damage in all three species of sea urchins, suggesting interspecific differences in vulnerabilities to DNA damage and/or DNA repair mechanisms. Understanding impacts of environmental genotoxicants requires understanding species-specific susceptibilities to DNA damage, which can impact long-term stability in sea urchin populations in proximity to aquaculture farms.

Functionalised Anodised Aluminium Oxide as a Biocidal Agent.

In this article, we describe the antimicrobial properties of a new composite based on anodic aluminium oxide (AAO) membranes containing propyl-copper-phosphonate units arranged at a predetermined density inside the AAO channels. The samples were prepared with four concentrations of copper ions and tested as antimicrobial drug on four different strains of Escherichia coli (K12, R2, R3 and R4). For comparison, the same strains were tested with three types of antibiotics using the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than antibiotics such as ciprofloxacin, bleomycin and cloxacillin. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA. We have also observed that the intermolecular distances between the functional units play an important role for the antimicrobial properties of the used material. Hence, we utilised the idea of the 2D solvent to tailor them.