Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H₂O₂ treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H₂O₂, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.     

Safety and Immunogenicity of the Malaria Vaccine Candidate MSP3 Long Synthetic Peptide in 12–24 Months-Old Burkinabe Children

Background

A Phase Ia trial in European volunteers of the candidate vaccine merozoite surface protein 3 (MSP3), a Plasmodium falciparum blood stage membrane, showed that it induces biologically active antibodies able to achieve parasite killing in vitro, while a phase Ib trial in semi-immune adult volunteers in Burkina Faso confirmed that the vaccine was safe.The aim of this study was to assess the safety and immunogenicity of this vaccine candidate in children aged 12–24 months living in malaria endemic area of Burkina Faso.

Methods

The study was a double-blind, randomized, controlled, dose escalation phase Ib trial, designed to assess the safety, reactogenicity and immunogenicity of three doses of either 15 or 30 µg of MSP3-LSP adsorbed on aluminum hydroxide in 45 children 12 to 24 months of age randomized into three equal groups. Each group received 3 vaccine doses (on days 0, 28 and 56) of either 15 µg of MSP3-LSP, 30 µg of MSP3-LSP or of the Engerix B hepatitis B vaccine. Children were visited at home daily for the 6 days following each vaccination to solicit symptoms which might be related to vaccination. Serious adverse events occurring during the study period (1 year) were recorded. Antibody responses to MSP3-LSP were measured on days 0, 28, 56 and 84.

Results

All 45 enrolled children received three MSP3 vaccine doses. No serious adverse events were reported. Most of the adverse events reported were mild to moderate in severity. The only reported local symptoms with grade 3 severity were swelling and induration, with an apparently dose related response. All grade 3 adverse events resolved without any sequelae. Both MSP3 doses regimens were able to elicit high levels of anti-MSP3 specific IgG1 and IgG3 antibodies in the volunteers with very little or no increase in IgG2, IgG4 and IgM classes: i.e. vaccination induced predominantly the isotypes involved in the monocyte-dependent mechanism of P. falciparum parasite-killing.

Conclusion

Our results support the promise of MSP3-LSP as a malaria vaccine candidate, both in terms of tolerability and of immunogenicity. Further assessment of the efficacy of this vaccine is recommended.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00452088","term_id":"NCT00452088"}}NCT00452088     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

The influence of hydrological regimes on sex ratios and spatial segregation of the sexes in two dioecious riparian shrub species in northern Sweden

River management practices have altered the hydrological regimes of many rivers and also altered the availability of regeneration niches for riparian species. We investigated the impact of changed hydrological regimes on the sex ratios and the Spatial Segregation of the Sexes (SSS) in the dioecious species Salix myrsinifolia Salisb.–phylicifolia L. and S. lapponum L. by studying the free-flowing Vindel River and the regulated Ume River in northern Sweden. We surveyed sex ratios of these species in 12 river reaches on the Vindel River and in 17 reaches on the Ume River. In addition, we surveyed the sex and location above mean river stage of 1,002 individuals across both river systems to investigate the SSS of both species. Cuttings were collected from male and female individuals of S. myrsinifolia–phylicifolia from both rivers and subjected to four different water table regimes in a greenhouse experiment to investigate growth response between the sexes. We found an M/F sex ratio in both river systems similar to the regional norm of 0.62 for S. myrsinifolia–phylicifolia and of 0.42 for S. lapponum. We found no evidence of SSS in either the free-flowing Vindel River or the regulated Ume River. In the greenhouse experiment, hydrological regime had a significant effect on shoot and root dry weight and on root length. Significantly higher shoot dry weights were found in females than in males and significantly different shoot and root dry weights were found between cuttings taken from the two rivers. We concluded that changed hydrological regimes are likely to alter dimensions of the regeneration niche and therefore to influence sex ratios and SSS at an early successional stage, making it difficult to find clear spatial patterns once these species reach maturity and can be sexed.     

Recent progress in understanding the interaction between avermectins and ligand-gated ion channels: putting the pests to sleep

Avermectins and milbemycins are an important family of anthelmintics, insecticides and acaricides. Their mode of action is as positive allosteric modulators of ligand-gated chloride channels, and at higher concentrations, they gate some channels directly. Though it has long been known that the avermectins do not compete for the ligand binding site, the actual site at which they interact with their receptors has been unclear. Recent data demonstrate the importance to drug binding of amino acid residues predicted to line a water-filled pocket in the channel domain. This pocket acts as the binding site for anaesthetics and other modulators of mammalian GABA_A and glycine receptors, suggesting similarities in the mode of action between these drugs and the avermectins/milbemycins.     

How might intensification of farming influence dung beetle diversity (Coleoptera: Scarabaeidae) in Maputo Special Reserve (Mozambique)?

There are concerns over the increasing encroachment of humans, domestic livestock, and farming onto Maputo Special Reserve because of the potential for habitat modification. Therefore, differences between an undisturbed area of the reserve and a neighbouring farming area are assessed using dung beetle as indicators. In each of the two areas, pig-dung-baited pitfall traps were used to sample dung beetle assemblages in two contrasting habitats, grassland and forest. Distributional analysis of the 57 species and 36 942 individuals that were captured, showed that species richness, species turnover, relative abundance patterns, and biogeographical composition differed strongly between both habitats and areas under different land usage. However, in analyses that apportion variation, the greatest amounts were related to habitat rather than land usage. Even so, in both habitats, the total and mean number of species per trap was higher in the farmed area than in the reserve although this was a significant trend only in grassland. Furthermore, in grassland, widespread species were better represented in the farmed area than in the reserve whereas in forest, widespread species were poorly represented compared to grassland. Also in forest, Maputaland endemics were better represented in the reserve than in the farmed area. Further work is necessary to separate the different geographical, ecological, and land usage factors responsible for the patterns detected in this preliminary study. Even so, there are clearly differences between the Maputo Special Reserve and the farmed area.     

Global analysis of gene expression in the estrogen induced pituitary tumor of the F344 rat

The F344 rat rapidly forms large prolactinomas in response to chronic estrogen treatment. To identify genes expressed in the course of this estrogen induced pituitary tumor growth, we performed microarray analysis on the F344 rat pituitary after chronic estrogen treatment and on untreated controls. At a significance level set to minimize type I error, some 72 genes were found to be differentially expressed between estrogen treated and untreated. Of those genes, 70 have not been reported previously as being affected by estrogen in the F344 rat pituitary. Since many other investigators have studied the effect of estrogen on specific gene expression in rat pituitary, we also examined the mRNA expression of the 36 genes that have been previously reported as having their expression affected by estrogen in the rat pituitary. Of these, 13 were found to have their expression affected by estrogen treatment in the same direction as had been reported by others.     

Impacts of Community-based Conservation on Local Communities in the Annapurna Conservation Area, Nepal

Approaches to the management of protected areas that involve the participation of local communities are now being widely promoted. However, the impacts of such community-based conservation initiatives on local communities remain poorly defined. This research examines the socio-economic impacts of community-based conservation within the Annapurna Conservation Area (ACA), Nepal, through semi-structured interviews and a questionnaire survey with local residents, situated both within and outside the protected area. Results indicate that local communities have received a number of benefits from conservation, including improvements in access to forest resources, improved basic infrastructure such as drinking water, trails and bridges, and improvements in health, sanitation and social services. However, relatively few people (14.9%) within ACA receive direct financial income from tourism. Local communities also experience a number of costs of being involved in conservation, the most significant of which is increased crop damage by wildlife. Eighty-four percent of respondents within ACA have experienced problems of crop damage, accounting for 6% (rice) to 23% (maize) of total production. Depredation of livestock by wildlife is also experienced; mean losses per household being the equivalent of ￡3.9 (Rs. 479.70) each year. However, 66% of respondents within ACA reported that they had never experienced this problem. These results indicate that the socio-economic benefits of community-based approaches to conservation can outweigh the costs, even though the latter are significant. However, a participatory approach to management of problematic animal species will need to be developed within ACA, if conflicts between local communities and protected area management are to be avoided in future.