Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).     

Incidence of HIV in Windhoek, Namibia: demographic and socio-economic associations

Objective

To estimate HIV incidence and prevalence in Windhoek, Namibia and to analyze socio-economic factors related to HIV infection.

Method

In 2006/7, baseline surveys were performed with 1,753 private households living in the greater Windhoek area; follow-up visits took place in 2008 and 2009. Face-to-face socio-economic questionnaires were administrated by trained interviewers; biomedical markers were collected by nurses; GPS codes of household residences were recorded.

Results

The HIV prevalence in the population (aged>12 years) was 11.8% in 2006/7 and 14.6% in 2009. HIV incidence between 2007 and 2009 was 2.4 per 100 person year (95%CI = 1.9–2.9). HIV incidence and prevalence were higher in female populations. HIV incidence appeared non-associated with any socioeconomic factor, indicating universal risk for the population. For women a positive trend was found between low per-capita consumption and HIV acquisition. A HIV knowledge score was strongly associated with HIV incidence for both men and women. High HIV prevalence and incidence was concentrated in the north-western part of the city, an area with lower HIV knowledge, higher HIV risk perception and lower per-capita consumption.

Discussion

The HIV incidence and prevalence figures do not suggest a declining epidemic in Windhoek. Higher vulnerability of women is recorded, most likely related to economic dependency and increasing transactional sex in Namibia. The lack of relation between HIV incidence and socio-economic factors confirms HIV risks for the overall urban community. Appropriate knowledge is strongly associated to lower HIV incidence and prevalence, underscoring the importance of continuous information and education activities for prevention of infection. Geographical areas were identified that would require prioritized HIV campaigning.     

Psychophysiological responses to stress after stress management training in patients with rheumatoid arthritis

Background

Stress management interventions may prove useful in preventing the detrimental effects of stress on health. This study assessed the effects of a stress management intervention on the psychophysiological response to stress in patients with rheumatoid arthritis (RA).

Methods

Seventy-four patients with RA, who were randomly assigned to either a control group or a group that received short-term stress management training, performed a standardized psychosocial stress task (Trier Social Stress Test; TSST) 1 week after the stress management training and at a 9-week follow-up. Psychological and physical functioning, and the acute psychophysiological response to the stress test were assessed.

Results

Patients in the intervention group showed significantly lower psychological distress levels of anxiety after the training than did the controls. While there were no between-group differences in stress-induced tension levels, and autonomic (α-amylase) or endocrine (cortisol) responses to the stress test 1 week after the intervention, levels of stress-induced tension and cortisol were significantly lower in the intervention group at the 9-week follow-up. Overall, the response to the intervention was particularly evident in a subgroup of patients with a psychological risk profile.

Conclusion

A relatively short stress management intervention can improve psychological functioning and influences the psychophysiological response to stress in patients with RA, particularly those psychologically at risk. These findings might help understand how stress can affect health and the role of individual differences in stress responsiveness.

Trial Registration

TrialRegister.nl NTR1193     

Cultivation of high-rate sulfate reducing sludge by pH-based electron donor dosage

A novel self-regulating bioreactor concept for sulfate reduction is proposed aiming for high biomass concentrations and treatment capacities. The system consists of a cell suspension of sulfate reducing bacteria in a continuous stirred tank reactor (30 degrees C) fed with a mixture of both electron donor and electron acceptor (formic acid and sulfuric acid, respectively), nutrients and phosphate buffer via a pH controller. The pH rise due to sulfate reduction is balanced with dosage of the sulfate reducing substrates as acids. The reactor concept was shown to be capable of full sulfate reduction without competition for the electron donor by methanogens and acetogens. Activity assays revealed that hardly any methanogenic activity on formate was left in the suspension by the end of the continuous run (130 days). In addition, the sulfidogenic activity with formate and H2/CO2 had increased, respectively, 3.9 and 11.6 times at the end of the experimental run. The evolution of the particle size distribution of the cell suspension over time indicated that newly grown cells have the tendency to attach together in flocs or to the existing agglomerates.     

Genetic and physiological architecture of early vigor in Aegilops tauschii, the D-genome donor of hexaploid wheat. A quantitative trait loci analysis

Plant growth can be studied at different organizational levels, varying from cell, leaf, and shoot to the whole plant. The early growth of seedlings is important for the plant's establishment and its eventual success. Wheat (Triticum aestivum, genome AABBDD) seedlings exhibit a low early growth rate or early vigor. The germplasm of wheat is limited. Wild relatives constitute a source of genetic variation. We explored the physiological and genetic relationships among a range of early vigor traits in Aegilops tauschii, the D-genome donor. A genetic map was constructed with amplified fragment-length polymorphism and simple sequence repeat markers, and quantitative trait loci (QTL) analysis was performed on the F(4) population of recombinant inbred lines derived from a cross between contrasting accessions. The genetic map consisted of 10 linkage groups, which were assigned to the seven chromosomes and covered 68% of the D genome. QTL analysis revealed 87 mapped QTLs (log of the odds >2.65) in clusters, 3.1 QTLs per trait, explaining 32% of the phenotypic variance. Chromosomes 1D, 4D, and 7D harbored QTLs for relative growth rate, biomass allocation, specific leaf area, leaf area ratio, and unit leaf rate. Chromosome 2D covered QTLs for rate and duration of leaf elongation, cell production rate, and cell length. Chromosome 5D harbored QTLs for the total leaf mass and area and growth rate of the number of leaves and tillers. The results show that several physiological correlations between growth traits have a genetic basis. Genetic links between traits are not absolute, opening perspectives for identification of favorable alleles in A. tauschii to improve early vigor in wheat.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.     

Cytokine production of stimulated whole blood cultures in rheumatoid arthritis patients receiving short-term infliximab therapy

Patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) strategies have an increased susceptibility to infections, especially those caused by intracellular pathogens. In this study we assessed the cytokine production capacity in patients with RA and we further investigated whether anti-TNF therapy modulates the production of pro-inflammatory cytokines involved in the resistance against infections. Whole blood cultures from 10 RA patients and 10 healthy controls were stimulated with heat-killed Candida albicans, Salmonella typhimurium, Staphyloccocus aureus, Aspergillus fumigatus or Mycobacterium tuberculosis and production of interleukin (IL)-1beta, IL-6, IL-10, interferon (IFN)-gamma and TNF-alpha was measured. Before anti-TNF therapy, whole blood cultures from RA patients released significantly less IFN-gamma than healthy controls after stimulation with all tested microorganisms. Short-term anti-TNF therapy did not have an inhibitory effect on the release of the cytokines tested. We conclude that cells of patients with RA have a strongly reduced production capacity of IFN-gamma after bacterial challenge. Although short-term therapy with anti-TNF agents did not further decrease the release of other proinflammatory cytokines, the combination of defective IFN-gamma production in basal conditions and TNF neutralization during anti-TNF therapy is likely to be responsible for the higher susceptibility to infections in patients with RA.     

Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio)

The environmental fate and ecotoxicological effect of pharmaceuticals are poorly understood, and standardized tests to detect and evaluate their potential effects in the environment are not available. We developed a zebrafish brain-specific microarray containing 682 neurologically relevant cDNA-fragments. To investigate the applicability of this microarray for studying neurotoxic modes-of-action and impact assessment of neuro-active pharmaceuticals in zebrafish, chlorpromazine was used as a model compound. After exposure to chlorpromazine (75 μg/L) for 2, 4, 14 and 28 days or control treatment RNA was extracted from brains of males and females. Fluorescently labeled cDNA was prepared and hybridized to the custom microarray. In total, 56 genes were differentially expressed in brains of male and/or female zebrafish, of which most genes were down-regulated. A clear difference in response to chlorpromazine exposure between males and females was observed with exposure time as well as in functional classes of affected genes. The presented study is one of the first reports on molecular effects of human neuro-active pharmaceuticals in aquatic non-target organisms. This new genomic tool successfully detected gene expression effects of exposure to chlorpromazine in the brain of zebrafish. Reported gene expression effects are found to be consistent with literature data for other laboratory animals.     

An experimental evaluation of a loop versus a reference design for two-channel microarrays

MOTIVATION: Despite theoretical arguments that so-called 'loop designs' for two-channel DNA microarray experiments are more efficient, biologists continue to use 'reference designs'. We describe two sets of microarray experiments with RNA from two different biological systems (TPA-stimulated mammalian cells and Streptomyces coelicolor). In each case, both a loop and a reference design were used with the same RNA preparations with the aim of studying their relative efficiency. RESULTS: The results of these experiments show that (1) the loop design attains a much higher precision than the reference design, (2) multiplicative spot effects are a large source of variability, and if they are not accounted for in the mathematical model, for example, by taking log-ratios or including spot effects, then the model will perform poorly. The first result is reinforced by a simulation study. Practical recommendations are given on how simple loop designs can be extended to more realistic experimental designs and how standard statistical methods allow the experimentalist to use and interpret the results from loop designs in practice. AVAILABILITY: The data and R code are available at http://exgen.ma.umist.ac.uk CONTACT: veronica.vinciotti@brunel.ac.uk.