γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

Objective

Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).

Methods

Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.

Results

While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.

Conclusion

In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.     

Repeat regions R1 and R2 in the P97 paralogue Mhp271 of Mycoplasma hyopneumoniae bind heparin, fibronectin and porcine cilia

Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the centre of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modelling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)‐like and one non‐Ig domain at the C‐terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent‐exposed module containing domains 2 and 3. This model is consistent with the dumbbell‐shaped cryo‐EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C‐terminal 25 amino acids, which are predicted to form two β‐strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent‐exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus and exposing the tail for more efficient contact with the host cell surface prior to infection.     

Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide

Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreductase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route for the synthesis of NTZ-related antibacterial analogues.     

Roles of the main olfactory and vomeronasal systems in the detection of androstenone in inbred strains of mice

We investigated the role of the main olfactory and accessory olfactory systems (MOS and AOS respectively) in the detection of androstenone. We used the following experimental approaches: behavioral, surgical removal of the vomeronasal organ (VNX) followed by histochemical verification and Fos immunohistochemistry. Using a Y-maze paradigm we estimated sensitivity of NZB/B1NJ and CBA/J mice to androstenone. CBA mice were 2,000-fold more sensitive to androstenone than NZB mice. VNX caused a 4- to 16-fold decre...     

Assessing and reporting heterogeneity in treatment effects in clinical trials: a proposal

Background

This paper presents the study protocol for a pragmatic randomised controlled trial to evaluate the impact of a school based program developed to prevent teenage pregnancy. The program includes students taking care of an Infant Simulator; despite growing popularity and an increasing global presence of such programs, there is no published evidence of their long-term impact. The aim of this trial is to evaluate the Virtual Infant Parenting (VIP) program by investigating pre-conceptual health and risk behaviours, teen pregnancy and the resultant birth outcomes, early child health and maternal health.

Methods and Design

Fifty-seven schools (86% of 66 eligible secondary schools) in Perth, Australia were recruited to the clustered (by school) randomised trial, with even randomisation to the intervention and control arms. Between 2003 and 2006, the VIP program was administered to 1,267 participants in the intervention schools, while 1,567 participants in the non-intervention schools received standard curriculum. Participants were all female and aged between 13-15 years upon recruitment. Pre and post-intervention questionnaires measured short-term impact and participants are now being followed through their teenage years via data linkage to hospital medical records, abortion clinics and education records. Participants who have a live birth are interviewed by face-to-face interview. Kaplan-Meier survival analysis and proportional hazards regression will test for differences in pregnancy, birth and abortion rates during the teenage years between the study arms.

Discussion

This protocol paper provides a detailed overview of the trial design as well as initial results in the form of participant flow. The authors describe the intervention and its delivery within the natural school setting and discuss the practical issues in the conduct of the trial, including recruitment. The trial is pragmatic and will directly inform those who provide Infant Simulator based programs in school settings.

Trial registration

ISRCTN24952438     

Thermal clamping of temperature-regulating flowers reveals the precision and limits of the biochemical regulatory mechanism

The flowers of several families of seed plants warm themselves when they bloom. In some species, thermogenesis is regulated, increasing the rate of respiration at lower ambient temperature (T _a) to maintain a somewhat stable floral temperature (T _f). The precision of this regulation is usually measured by plotting T _f over T _a. However, such measurements are influenced by environmental conditions, including wind speed, humidity, radiation, etc. This study eliminates environmental effects by experimentally ‘clamping’ T _f at constant, selected levels and then measuring stabilized respiration rate. Regulating flowers show decreasing respiration with rising T _f (Q ₁₀ < 1). Q ₁₀ therefore becomes a measure of the biochemical ‘precision’ of temperature regulation: lower Q ₁₀ values indicate greater sensitivity of respiration to T _f and a narrower range of regulated temperatures. At the lower end of the regulated range, respiration is maximal, and further decreases in floral temperature cause heat production to diminish. Below a certain tissue temperature (‘switching temperature’), heat loss always exceeds heat production, so thermoregulation becomes impossible. This study compared three species of thermoregulatory flowers with distinct values of precision and switching temperature. Precision was highest in Nelumbo nucifera (Q ₁₀ = 0.16) moderate in Symplocarpus renifolius (Q ₁₀ = 0.48) and low in Dracunculus vulgaris (Q ₁₀ = 0.74). Switching temperatures were approximately 30, 15 and 20°C, respectively. There were no relationships between precision, switching temperature or maximum respiration rate. High precision reveals a powerful inhibitory mechanism that overwhelms the tendency of temperature to increase respiration. Variability in the shape and position of the respiration–temperature curves must be accounted for in any explanation of the control of respiration in thermoregulatory flowers.     

Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic patterns following imbibition

The correlation between changes in seed protein electrophoretic patterns and the hydrolysis of lipid and protein reserves of loblolly pine ( Pinus taeda L.) seed was studied. Seeds were incubated at 30°C for up to 12 days following stratification, then megagametophytes and embryos were assayed for lipid and protein content after each day of imbibition. The megagametophyte of mature seed was found to contain 20% lipid and 12% storage protein on a fresh weight basis. The embryo contained 26% lipid and 15% protein. Both lipid and protein reserves were depleted constantly following imbibition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble and insoluble protein fractions showed a 60 kDa protein that was representative of crystalloid-like proteins. These crystalloid-like proteins comprised 85% of the insoluble protein storage reserves. A small number of insoluble storage proteins, including a 47 kDa protein, were distinct in that they were unaffected by 2-mercaptoethanol treatment. The soluble fractions from both tissues were labelled with [³⁵S]-methionine, and incorporation was visualized by two-dimensional electrophoresis. Proteins were found to belong to one of three categories, those synthesized constitutively (comprising the bulk of newly synthesized proteins), those synthesized during germination or those synthesized after radicle emergence. Accompanying seed reserve hydrolysis were developmental shifts in protein pattern and synthesis, suggesting the possibility that control of hydrolysis is at the level of enzyme accumulation.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.