Measuring oxidative DNA damage and DNA repair using the yeast comet assay

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair

Density-Dependence as a Size-Independent Regulatory Mechanism

The growth function of populations is central in biomathematics. The main dogma is the existence of density dependence mechanisms, which can be modelled with distinct functional forms that depend on the size of the population. One important class of regulatory functions is the $\theta$-logistic, which generalises the logistic equation. Using this model as a motivation, this paper introduces a simple dynamical reformulation that generalises many growth functions. The reformulation consists of two equations, one for population size, and one for the growth rate. Furthermore, the model shows that although population is density-dependent, the dynamics of the growth rate does not depend either on population size, nor on the carrying capacity. Actually, the growth equation is uncoupled from the population size equation, and the model has only two parameters, a Malthusian parameter $\rho$ and a competition coefficient $\theta$. Distinct sign combinations of these parameters reproduce not only the family of $\theta$-logistics, but also the van Bertalanffy, Gompertz and Potential Growth equations, among other possibilities. It is also shown that, except for two critical points, there is a general size-scaling relation that includes those appearing in the most important allometric theories, including the recently proposed Metabolic Theory of Ecology. With this model, several issues of general interest are discussed such as the growth of animal population, extinctions, cell growth and allometry, and the effect of environment over a population.Comment: 41 Pages, 5 figures Submitted to JT

Oxidative stress induces degradation of mitochondrial DNA

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar–phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions

Buried alive: Aquatic plants survive in ‘ghost ponds’ under agricultural fields

The widespread loss of wetlands due to agricultural intensification has been highlighted as a major threat to aquatic biodiversity. However, all is not lost as we reveal that the propagules of some aquatic species could survive burial under agricultural fields in the sediments of ‘ghost ponds’ - ponds in-filled during agricultural land consolidation. Our experiments showed at least eight aquatic macrophyte species to germinate from seeds and oospores, following 50–150 years of dormancy in the sediments of ghost ponds. This represents a significant proportion of the expected macrophyte diversity for local farmland ponds, which typically support between 6 and 14 macrophyte species. The rapid (< 6 months) re-colonisation of resurrected ghost ponds by a diverse aquatic vegetation similarly suggests a strong seed-bank influence. Ghost ponds represent abundant, dormant time capsules for aquatic species in agricultural landscapes around the globe, affording opportunities for enhancing landscape-scale aquatic biodiversity and connectivity. While reports of biodiversity loss through agricultural intensification dominate conservation narratives, our study offers a rare positive message, demonstrating that aquatic organisms survive prolonged burial under intensively managed agricultural fields. We urge conservationists and policy makers to consider utilizing and restoring these valuable resources in biodiversity conservation schemes and in agri-environmental approaches and policies

Role of PCNA-dependent stimulation of 3′-phosphodiesterase and 3′–5′ exonuclease activities of human Ape2 in repair of oxidative DNA damage

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis

Functional biodiversity in the agricultural landscape: relationships between weeds and arthropod fauna

We reviewed studies aimed at understanding functional relationships between weeds and arthropods in agroecosystems as influenced by biodiversity at different scales, with the main goal of highlighting gaps in knowledge, research methods and approaches. We first addressed: (i) the regulation of arthropod communities by weed diversity at genetic, species and habitat levels, (ii) the regulation of weed communities by arthropods through seed predation and dispersal and (iii) belowground weed-insect interactions. We then focussed on methodologies to study weed–arthropod interactions in agricultural landscapes and discuss techniques potentially available for data analysis and the importance of joint weed–arthropod trend detection. Lastly, we discuss the implications of research findings for biodiversity conservation policies (agri-environmental schemes) and suggest some priorities for future work. Results showed that to date research has largely ignored weed–arthropod interactions in agricultural landscapes. No information is available on the role of weed genetic diversity as driver of weed–arthropod interactions, whereas studies on effects of species and habitat diversity often lack a functional perspective and ⁄ or a spatial component. Also, information on how management of the wider agricultural biotope might express positive weed– arthropod functional interactions is scarce. Another area worth being explored is the relationship between weed-leaf ⁄ root herbivores and beneficial arthropods. Tools for spatial data analysis might be useful for elucidating weed–arthropod interactions in agricultural landscapes, but some methodological aspects, e.g. the definition of the most appropriate experimental design and sampling scale ⁄ frequency, must be refined. New studies on weed–arthropod interactions should encompass an explicit spatial component; this knowledge is particularly important for improving IPM ⁄IWM systems and designing more targeted agri-environmental schemes

Evaluation of a task-based community oriented teaching model in family medicine for undergraduate medical students in Iraq

BACKGROUND: The inclusion of family medicine in medical school curricula is essential for producing competent general practitioners. The aim of this study is to evaluate a task-based, community oriented teaching model of family medicine for undergraduate students in Iraqi medical schools. METHODS: An innovative training model in family medicine was developed based upon tasks regularly performed by family physicians providing health care services at the Primary Health Care Centre (PHCC) in Mosul, Iraq. Participants were medical students enrolled in their final clinical year. Students were assigned to one of two groups. The implementation group (28 students) was exposed to the experimental model and the control group (56 students) received the standard teaching curriculum. The study took place at the Mosul College of Medicine and at the Al-Hadba PHCC in Mosul, Iraq, during the academic year 1999–2000. Pre- and post-exposure evaluations comparing the intervention group with the control group were conducted using a variety of assessment tools. RESULTS: The primary endpoints were improvement in knowledge of family medicine and development of essential performance skills. Results showed that the implementation group experienced a significant increase in knowledge and performance skills after exposure to the model and in comparison with the control group. Assessment of the model by participating students revealed a high degree of satisfaction with the planning, organization, and implementation of the intervention activities. Students also highly rated the relevancy of the intervention for future work. CONCLUSION: A model on PHCC training in family medicine is essential for all Iraqi medical schools. The model is to be implemented by various relevant departments until Departments of Family medicine are established

Presence of Epstein-Barr virus latency type III at the single cell level in post- transplantation lymphoproliferative disorders and AIDS related lymphomas

AIMS: To investigate the expression pattern of Epstein-Barr virus (EBV) latent genes at the single cell level in post-transplantation lymphoproliferative disorders and acquired immunodefiency syndrome (AIDS) related lymphomas, in relation to cellular morphology. METHODS: Nine post-transplantation lymphoproliferative disorders and three AIDS related lymphomas were subjected to immunohistochemistry using monoclonal antibodies specific for EBV nuclear antigen 1 (EBNA1) (2H4), EBNA2 (PE2 and the new rat anti-EBNA2 monoclonal antibodies 1E6, R3, and 3E9), and LMP1 (CS1-4 and S12). Double staining was performed combining R3 or 3E9 with S12. RESULTS: R3 and 3E9 anti-EBNA2 monoclonal antibodies were more sensitive than PE2, enabling the detection of more EBNA2 positive lymphoma cells. Both in post-transplantation lymphoproliferative disorders and AIDS related lymphomas, different expression patterns were detected at the single cell level. Smaller neoplastic cells were positive for EBNA2 but negative for LMP1. Larger and more blastic neoplastic cells, sometimes resembling Reed-Sternberg cells, were LMP1 positive but EBNA2 negative (EBV latency type II). Morphologically intermediate neoplastic cells coexpressing EBNA2 and LMP1 (EBV latency type III), were detected using R3 and 3E9, and formed a considerable part of the neoplastic population in four of nine post-transplantation lymphoproliferative disorders and two of three AIDS related lymphomas. All samples contained a subpopulation of small tumour cells positive exclusively for Epstein-Barr early RNA and EBNA1. The relation between cellular morphology and EBV expression patterns in this study was less pronounced in AIDS related lymphomas than in post-transplantation lymphoproliferative disorders, because the AIDS related lymphomas were less polymorphic than the post-transplantation lymphoproliferative disorders. CONCLUSIONS: In post-transplantation lymphoproliferative disorders and AIDS related lymphomas, EBV latency type III can be detected by immunohistochemistry in a subpopulation of tumour cells using sensitive monoclonal antibodies R3 and 3E9. Our data suggest that EBV infected tumour cells in these lymphomas undergo gradual changes in the expression of EBV latent genes, and that these changes are associated with changes in cellular morphology