Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

The BRCT domain and the specific loop 1 of human Polμ are targets of Cdk2/cyclin A phosphorylation

Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polμ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polμ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polμ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polμ. We identified Ser¹², Thr²¹ (located in the BRCT domain) and Ser³⁷² (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser³⁷² is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polμ could be regulated in vivo by phosphorylation of the BRCT domain (Ser¹²/Thr²¹) and of Ser³⁷², affecting the function of loop1. Consequently, Polμ’s most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.     

Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(−/−) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(−/−)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(−/−) mice. However, in RIP-Irs-2/IRS-2(−/−) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(−/−) mice. GK and GKRP mRNA levels in liver of IRS-2(−/−) were significantly lower, whereas in RIP-Irs-2/IRS-2(−/−) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(−/−) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(−/−) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(−/−) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.     

Preclinical Demonstration of Synergistic Active Nutrients/Drug (AND) Combination as a Potential Treatment for Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a poor prognosis disease lacking adequate therapy. We have previously shown that ascorbic acid administration is toxic to MPM cells. Here we evaluated a new combined therapy consisting of ascorbate/epigallocatechin-3-gallate/gemcitabine mixture (called AND, for Active Nutrients/Drug). In vitro effects of AND therapy on various MPM cell lines revealed a synergistic cytotoxic mechanism. In vivo experiments on a xenograft mouse model for MPM, obtained by REN cells injection in immunocompromised mice, showed that AND strongly reduced the size of primary tumor as well as the number and size of metastases, and prevented abdominal hemorrhage. Kaplan Meier curves and the log-rank test indicated a marked increase in the survival of AND-treated animals. Histochemical analysis of dissected tumors showed that AND induced a shift from cell proliferation to apoptosis in cancer cells. Lysates of tumors from AND-treated mice, analyzed with an antibody array, revealed decreased TIMP-1 and -2 expressions and no effects on angiogenesis regulating factors. Multiplex analysis for signaling protein phosphorylation exhibited inactivation of cell proliferation pathways. The complex of data showed that the AND treatment is synergistic in vitro on MPM cells, and blocks in vivo tumor progression and metastasization in REN-based xenografts. Hence, the AND combination is proposed as a new treatment for MPM.     

Traditional Nets Interfere with the Uptake of Long-Lasting Insecticidal Nets in the Peruvian Amazon: The Relevance of Net Preference for Achieving High Coverage and Use

Background

While coverage of long-lasting insecticide-treated nets (LLIN) has steadily increased, a growing number of studies report gaps between net ownership and use. We conducted a mixed-methods social science study assessing the importance of net preference and use after Olyset® LLINs were distributed through a mass campaign in rural communities surrounding Iquitos, the capital city of the Amazonian region of Peru.

Methods

The study was conducted in the catchment area of the Paujil and Cahuide Health Centres (San Juan district) between July 2007 and November 2008. During a first qualitative phase, participant observation and in-depth interviews collected information on key determinants for net preference and use. In a second quantitative phase, a survey among recently confirmed malaria patients evaluated the acceptability and use of both LLINs and traditional nets, and a case control study assessed the association between net preference/use and housing structure (open vs. closed houses).

Results

A total of 10 communities were selected for the anthropological fieldwork and 228 households participated in the quantitative studies. In the study area, bed nets are considered part of the housing structure and are therefore required to fulfil specific architectural and social functions, such as providing privacy and shelter, which the newly distributed Olyset® LLINs ultimately did not. The LLINs'' failure to meet these criteria could mainly be attributed to their large mesh size, transparency and perceived ineffectiveness to protect against mosquitoes and other insects, resulting in 63.3% of households not using any of the distributed LLINs. Notably, LLIN usage was significantly lower in houses with no interior or exterior walls (35.2%) than in those with walls (73.8%) (OR = 5.2, 95CI [2.2; 12.3], p<0.001).

Conclusion

Net preference can interfere with optimal LLIN use. In order to improve the number of effective days of LLIN protection per dollar spent, appropriate quantitative and qualitative methods for collecting information on net preference should be developed before any LLIN procurement decision is made.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.     

Disability Affects the 6-Minute Walking Distance in Obese Subjects (BMI>40 kg/m2)

Introduction

In obese subjects, the relative reduction of the skeletal muscle strength, the reduced cardio-pulmonary capacity and tolerance to effort, the higher metabolic costs and, therefore, the increased inefficiency of gait together with the increased prevalence of co-morbid conditions might interfere with walking. Performance tests, such as the six-minute walking test (6MWT), can unveil the limitations in cardio-respiratory and motor functions underlying the obesity-related disability. Therefore the aims of the present study were: to explore the determinants of the 6-minute walking distance (6MWD) and to investigate the predictors of interruption of the walk test in obese subjects.

Methods

Obese patients [body mass index (BMI)>40 kg/m²] were recruited from January 2009 to December 2011. Anthropometry, body composition, specific questionnaire for Obesity-related Disabilities (TSD-OC test), fitness status and 6MWT data were evaluated. The correlation between the 6MWD and the potential independent variables (anthropometric parameters, body composition, muscle strength, flexibility and disability) were analysed. The variables which were singularly correlated with the response variable were included in a multivariated regression model. Finally, the correlation between nutritional and functional parameters and test interruption was investigated.

Results

354 subjects (87 males, mean age 48.5±14 years, 267 females, mean age 49.8±15 years) were enrolled in the study. Age, weight, height, BMI, fat mass and fat free mass indexes, handgrip strength and disability were significantly correlated with the 6MWD and considered in the multivariate analysis. The determination coefficient of the regression analysis ranged from 0.21 to 0.47 for the different models. Body weight, BMI, waist circumference, TSD-OC test score and flexibility were found to be predictors of the 6MWT interruption.

Discussion

The present study demonstrated the impact of disability in obese subjects, together with age, anthropometric data, body composition and strength, on the 6-minute walking distance.     

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin''s tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin''s tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin''s tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.     

Intragroup Behavioral Changes Following Intergroup Conflict in Mountain Gorillas (Gorilla beringei beringei)

International Journal of Primatology - Group-living animals face a number of threats from extragroup conspecifics: from individuals seeking mating opportunities to rival groups attempting to access...