This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

Obesity is an epidemic associated with many diseases. The nutraceutical Zingiber officinale (ZO) is a potential treatment for obesity; however, the molecular effects are unknown. Swiss male mice were fed a high-fat diet (59 % energy from fat) for 16 weeks to generate a diet-induced obesity (DIO) model and then divided into the following groups: standard diet + vehicle; standard diet + ZO; DIO + vehicle and DIO + ZO. Those in the ZO groups were supplemented with 400 mg/kg per d of ZO extract (oral administration) for 35 d. The animals were euthanised, and blood, quadriceps, epididymal fat pad and hepatic tissue were collected. DIO induced insulin resistance, proinflammatory cytokines, oxidative stress and DNA damage in different tissues. Treatment with ZO improved insulin sensitivity as well as decreased serum TAG, without changes in body weight or adiposity index. TNF-α and IL-1β levels were lower in the liver and quadriceps in the DIO + ZO group compared with the DIO group. ZO treatment reduced the reactive species and oxidative damage to proteins, lipids and DNA in blood and liver in obese animals. The endogenous antioxidant activity was higher in the quadriceps of DIO + ZO. These results in the rat model of DIO may indicate ZO as an adjuvant on obesity treatment.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with reported incidences of ~800 000 cases each year. One of the critical determinants in patient response to radiotherapy, particularly for oropharyngeal cancers, is human papillomavirus (HPV) status where HPV-positive patients display improved survival rates and outcomes particularly because of increased responsiveness to radiotherapy. The increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and repair of DNA double-strand breaks. Therefore, strategies to further radiosensitise HPV-positive HNSCC, but also radioresistant HPV-negative HNSCC, have focussed on targeting key DNA repair proteins including PARP, DNA-Pk, ATM and ATR. However, inhibitors against CHK1 and WEE1 involved in cell-cycle checkpoint activation have also been investigated as targets for radiosensitisation in HNSCC. These studies, largely conducted using established HNSCC cell lines in vitro, have demonstrated variability in the response dependent on the specific inhibitors and cell models utilised. However, promising results are evident targeting specifically PARP, DNA-Pk, ATR and CHK1 in synergising with radiation in HNSCC cell killing. Nevertheless, these preclinical studies require further expansion and investigation for translational opportunities for the effective treatment of HNSCC in combination with radiotherapy.

Ultra-processed food is one of the main contributors to energy supply and consumption in food systems worldwide, and evidence of their detrimental health outcomes in humans is emerging. This study aimed to assess ultra-processed food intake and its association with urinary levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of DNA oxidative damage, in 139 healthy adolescents in Karaj City in Iran. Usual dietary intake was measured using a 168-item validated FFQ. The daily intake of ultra-processed food consumption was determined through the classification of NOVA, and general linear models were used to compare the urinary levels of 8-OHdG/creatinine (ng/mg creatinine) within tertiles of ultra-processed food intake. Adolescents in the higher tertile of ultra-processed food consumption had a significantly higher mean level of urinary 8-OHdG/creatinine in comparison with the lower tertiles in the crude model (Pfor trend: 0·003) and after adjustment for confounding variables, including total energy intake, sex, age, BMI for age Z-score, obesity and physical activity (Pfor trend: 0·004). This association was still significant after adjusting for dietary intake of whole grains, nuts, legumes, the ratio of MUFA:SFA (g/d) and Mediterranean dietary score (Pfor trend: 0·002). More studies are needed to explore the determinants of ultra-processed food supply, demand, consumption and health effects; such studies should be applied to develop evidence-informed policies and regulatory mechanisms to improve children’s and adolescents’ food environment policymaking and legislation with special attention to ultra-processed food.

Drug resistance to helminth parasites is one of the most serious problems to threaten the livestock industry. The problem also poses a major threat to public health. Therefore, novel and safe agents should urgently be investigated to control parasitic infections. The current study was conducted to evaluate the possible antiparasitic effects of zinc oxide nanoparticles (ZnO-NPs) on one of the most prevalent gastrointestinal nematodes, Teladorsagia circumcincta. The worms were incubated with various concentrations of ZnO-NPs: 1, 4, 8, 12 and 16 ppm for 24 hours. Mobility and mortality of the parasites were recorded at four-hour intervals. At the endpoint, several biomarkers of oxidative/nitrosative stress, including superoxide dismutase, glutathione peroxidase and catalase, as well as lipid peroxidation, protein carbonylation, total antioxidant status, nitric oxide contents and DNA damage, were measured in the homogenized samples. ZnO-NPs showed significant anthelminthic effects, depending on time and concentration. Furthermore, the nanoparticle induced severe oxidative/nitrosative stress and DNA damage. ZnO-NPs could be considered as a novel and potent anthelminthic agent.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease and is caused by heterozygous germ-line mutations in either PKD1 (85%) or PKD2 (15%). It is characterised by the formation of numerous fluid-filled renal cysts and leads to adult-onset kidney failure in ~50% of patients by 60 years. Kidney cysts in ADPKD are focal and sporadic, arising from the clonal proliferation of collecting-duct principal cells, but in only 1–2% of nephrons for reasons that are not clear. Previous studies have demonstrated that further postnatal reductions in PKD1 (or PKD2) dose are required for kidney cyst formation, but the exact triggering factors are not clear. A growing body of evidence suggests that DNA damage, and activation of the DNA damage response pathway, are altered in ciliopathies. The aims of this review are to: (i) analyse the evidence linking DNA damage and renal cyst formation in ADPKD; (ii) evaluate the advantages and disadvantages of biomarkers to assess DNA damage in ADPKD and finally, (iii) evaluate the potential effects of current clinical treatments on modifying DNA damage in ADPKD. These studies will address the significance of DNA damage and may lead to a new therapeutic approach in ADPKD.

Sperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to fragmentation. Routine semen analysis is unable to estimate sperm chromatin damage. Sperm DNA integrity influences sperm functional capability, therefore tests that measure sperm DNA fragmentation are important to assess fertility disorders. Actually, there is a considerable number of methods for assessing sperm DNA fragmentation and chromatin integrity, sperm chromatin stability assay (SCSA modified), sperm chromatin dispersion (SCD), comet assay, transferase dUTP nick end labelling (TUNEL); and protamine evaluation in sperm chromatin assay, such as toluidine blue, CMA3, protamine expression and evaluation of cysteine radicals. This review aims to describe the main causes of sperm DNA fragmentation and the tests commonly used to evaluate sperm DNA fragmentation.

Chemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus–oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus–oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.

In mammalian cells, the p53 pathway regulates the response to a variety of stresses, including oncogene activation, heat and cold shock, and DNA damage. Here we explore a mathematical model of this pathway, composed of a system of partial differential equations. In our model, the p53 pathway is activated by a DNA-compromising event of short duration. As is typical for mathematical models of the p53 pathway, our model contains a negative feedback loop representing interactions between the p53 and Mdm2 proteins. A novel feature of our model is that we combine a spatio-temporal approach with the appearance and repair of DNA damage. We investigate the behaviour of our model through numerical simulations. By ignoring the possibility of DNA repair, we first explore the scenario in which the cell has a very inefficient DNA repair mechanism. We find that spatio-temporal oscillations in p53 and Mdm2 may occur, consistent with experimental data. We then allow p53 to be directly involved in repairing DNA damage, since experimental evidence suggests this can happen. We find that oscillations in p53 and Mdm2 can still occur, but their amplitude damps down quickly as the DNA damage is repaired. Finally, we find that a minor change to the location of the DNA damage can notably change the spatial distribution of p53 within the nucleus. We discuss the biological implications of our results.

We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.

Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1,2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy difference of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solvent interactions in implicit solvent models. We calculated electrostatic interaction energies between explicit water molecules and the base targeted for flipping. These interaction energies were calculated over the base flipping reaction coordinate to illustrate the stabilizing effect of the explicit water molecules on the flipped-out state. It is known that nucleic base pair hydrogen bonds also influenced the free energy of flipping since these favorable interactions must be broken in order for a base to flip-out of the helix. The Watson-Crick base pair hydrogen bond fractions were calculated over the umbrella sampling simulation windows in order to determine the effect of base pair interactions on the base flipping free energy. It is shown that interaction energies between the flipping base and explicit water molecules are responsible for the lower base flipping free energy difference in the explicit solvent PMF profiles.

Pharmacological antioxidant vitamins have previously been investigated for a prophylactic effect against exercise-induced oxidative stress. However, large doses are often required and may lead to a state of pro-oxidation and oxidative damage. Watercress contains an array of nutritional compounds such as β-carotene and α-tocopherol which may increase protection against exercise-induced oxidative stress. The present randomised controlled investigation was designed to test the hypothesis that acute (consumption 2 h before exercise) and chronic (8 weeks consumption) watercress supplementation can attenuate exercise-induced oxidative stress. A total of ten apparently healthy male subjects (age 23 (sd 4) years, stature 179 (sd 10) cm and body mass 74 (sd 15) kg) were recruited to complete the 8-week chronic watercress intervention period (and then 8 weeks of control, with no ingestion) of the experiment before crossing over in order to compete the single-dose acute phase (with control, no ingestion). Blood samples were taken at baseline (pre-supplementation), at rest (pre-exercise) and following exercise. Each subject completed an incremental exercise test to volitional exhaustion following chronic and acute watercress supplementation or control. The main findings show an exercise-induced increase in DNA damage and lipid peroxidation over both acute and chronic control supplementation phases (P< 0·05 v. supplementation), while acute and chronic watercress attenuated DNA damage and lipid peroxidation and decreased H2O2 accumulation following exhaustive exercise (P< 0·05 v. control). A marked increase in the main lipid-soluble antioxidants (α-tocopherol, γ-tocopherol and xanthophyll) was observed following watercress supplementation (P< 0·05 v. control) in both experimental phases. These findings suggest that short- and long-term watercress ingestion has potential antioxidant effects against exercise-induced DNA damage and lipid peroxidation.

Population studies show that greater red and processed meat consumption increases colorectal cancer risk, whereas dietary fibre is protective. In rats, resistant starches (a dietary fibre component) oppose colonocyte DNA strand breaks induced by high red meat diets, consistent with epidemiological data. Protection appears to be through SCFA, particularly butyrate, produced by large bowel carbohydrate fermentation. Arabinoxylans are important wheat fibre components and stimulate large bowel carbohydrate SCFA production. The present study aimed to determine whether an arabinoxylan-rich fraction (AXRF) from wheat protected colonocytes from DNA damage and changed colonic microbial composition in pigs fed with a diet high (30 %) in cooked red meat for 4 weeks. AXRF was primarily fermented in the caecum, as indicated by higher tissue and digesta weights and higher caecal (but not colonic) acetate, propionate and total SCFA concentrations. Protein fermentation product concentrations (caecal p-cresol and mid- and distal colonic phenol) were lower in pigs fed with AXRF. Colonocyte DNA damage was lower in pigs fed with AXRF. The microbial profiles of mid-colonic mucosa and adjacent digesta showed that bacteria affiliating with Prevotella spp. and Clostridial cluster IV were more abundant in both the mucosa and digesta fractions of pigs fed with AXRF. These data suggest that, although AXRF was primarily fermented in the caecum, DNA damage was reduced in the large bowel, occurring in conjunction with lower phenol concentrations and altered microbial populations. Further studies to determine the relationships between these changes and the lowering of colonocyte DNA damage are warranted.

This is the first study to examine the tolerance of Antarctic springtails (Collembola) to ultraviolet radiation (UV). Survival of extended attenuated exposure to sunlight was examined for both individuals and aggregations of the species Gomphiocephalus hodgsoni Carpenter over a 10 day period. Both individuals and aggregations demonstrated significantly higher survival and moult rates from control treatments kept in the dark to those exposed to UV. A photo-inhibitive element to moulting is indicated that may function to protect post-ecdysial springtails when their emergent cuticles are more sensitive to the external environment. DNA damage was measured in springtails directly exposed to sunlight for 5 h on a clear sunny day. Significant differences were found between treated animals and controls kept in the dark. There was some reduction of damage 12 and 24 h after exposure, when springtails had been placed in the dark to recover. This indicates the up-regulation of DNA repair mechanisms, with the 12 h treatment in particular showing no significant difference with controls. In addition to providing a first look at UV tolerance in these soil arthropods, these findings recommend employing strict protocols for collections of sample material for subsequent biological analysis in order to minimize the interactive effects of photo-damage.

Elevated oxidative stress is reported to be associated with pregnancy complications in highly prolific sows. Oxidative DNA damage and the antioxidant status were determined in blood samples collected during the course of gestation and lactation in multiparous sows. Blood samples were drawn from sows (n = 5) on days 30, 60, 90 and 110 of gestation (G30, G60, G90 and G110, respectively), on day 3, 10 and 18 of lactation (L3, L10 and L18, respectively) and on day 5 of postweaning (W5). Lymphocytes were isolated from the fresh blood and cryopreserved in each time point. Lymphocyte DNA damage was analyzed by alkaline single-cell gel electrophoresis (comet assay) to determine the single- and double-strand brakes and endogenous antioxidant concentrations using an HPLC system with UV detection. The comet assay showed elevated (P < 0.05) DNA damage (between 38% and 47%) throughout the gestational and lactational periods than during early gestation (G30; 21%). Plasma retinol concentration was reduced (P < 0.05) at the end of gestation (G110) compared with G30. Plasma α-tocopherol concentrations also showed a similar trend as to retinol. This study indicates that there is an increased systemic oxidative stress during late gestation and lactation, which are not fully recovered until the weaning compared with the G30, and that antioxidant nutrients in circulation substantially reduced in the mother pig at G110.