RAD51D protects against MLH1-dependent cytotoxic responses to O6-methylguanine

S_N1-type methylating agents generate O⁶-methyl guanine (O⁶-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O⁶-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O⁶-meG has been studied, the mechanisms of recombination induced by O⁶-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d^?/?Mlh1^?/? Trp53^?/? MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of γ-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing γ-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O⁶-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O⁶-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O⁶-meG adducts.     

Effect of Size,Quaternary Structure and Translational Error on the Static and Dynamic Heterogeneity of β-Galactosidase and Measurement of Electrophoretic Dynamic Heterogeneity

Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on β-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli β-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.     

MassSieve: Panning MS/MS peptide data for proteins

We present MassSieve, a Java‐based platform for visualization and parsimony analysis of single and comparative LC‐MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC‐MS/MS‐based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments.     

Fine root biomass and turnover in southern taiga estimated by root inclusion nets

Fine roots play an important part in forest carbon, nutrient and water cycles. The turnover of fine roots constitutes a major carbon input to soils. Estimation of fine root turnover is difficult, labour intensive and is often compounded by artefacts created by soil disturbance. In this work, an alternative approach of using inclusion nets installed in an undisturbed soil profile was used to measure fine root production and was compared to the in-growth core method. There was no difference between fine root production estimated by the two methods in three southern taiga sites with contrasting soil conditions and tree species compositions in the Central Forest State Biosphere Reserve, Russia. Expressed as annual production over standing biomass, Norway spruce fine root turnover was in the region of 0.10 to 0.24 y^-1. The inclusion net technique is suitable for field based assessment of fine root production. There are several advantages over the in-growth core method, due to non-disturbance of the soil profile and its potential for very high rate of replication.     

A fish out of water: gill and skin remodeling promotes osmo- and ionoregulation in the mangrove killifish Kryptolebias marmoratus

The euryhaline, amphibious mangrove killifish Kryptolebias marmoratus is known to survive weeks out of water in moist environments. We tested the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus. We predicted that under terrestrial conditions, gill and skin remodeling would result in an enhanced role for skin and a diminished role for the gills in osmo- and ionoregulation. Fish were exposed to water-either freshwater (FW, 1‰) or hypersaline water (saltwater [SW], 45‰)-or air over a moist surface of FW or SW for 9 d and then recovered in water. When fish were emersed for 9 d, (22)Na and (3)H-H(2)O were exchanged across the cutaneous surface. Homeostasis of whole-body Cl(-) and water levels but not of Na(+) levels was maintained over 9 d in air. In air-exposed fish, there was a significant increase in the size of skin ionocytes (in SW), a decrease in the number of skin mucous cells (in SW), and an increase in the gill interlamellar cell mass relative to those of fish in water. Gill ionocytes were mostly embedded away from the external surface in air-exposed fish, but the number and size of ionocytes increased (in FW). Interestingly, skin ionocytes formed distinct clusters of 20-30 cells. The estimated number of ionocytes over the whole skin surface was comparable to that in the gills. Overall, the findings support the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus in aquatic and terrestrial environments. Reversible cellular and morphological changes to the skin and gills during air exposure probably enhanced the cutaneous contribution to ion and water balance.     

A longitudinal study of medicaid coverage for tobacco dependence treatments in Massachusetts and associated decreases in hospitalizations for cardiovascular disease

Background

Insurance coverage of tobacco cessation medications increases their use and reduces smoking prevalence in a population. However, uncertainty about the impact of this coverage on health care utilization and costs is a barrier to the broader adoption of this policy, especially by publicly funded state Medicaid insurance programs. Whether a publicly funded tobacco cessation benefit leads to decreased medical claims for tobacco-related diseases has not been studied. We examined the experience of Massachusetts, whose Medicaid program adopted comprehensive coverage of tobacco cessation medications in July 2006. Over 75,000 Medicaid subscribers used the benefit in the first 2.5 years. On the basis of earlier secondary survey work, it was estimated that smoking prevalence declined among subscribers by 10% during this period.

Methods and Findings

Using claims data, we compared the probability of hospitalization prior to use of the tobacco cessation pharmacotherapy benefit with the probability of hospitalization after benefit use among Massachusetts Medicaid beneficiaries, adjusting for demographics, comorbidities, seasonality, influenza cases, and the implementation of the statewide smoke-free air law using generalized estimating equations. Statistically significant annualized declines of 46% (95% confidence interval 2%–70%) and 49% (95% confidence interval 6%–72%) were observed in hospital admissions for acute myocardial infarction and other acute coronary heart disease diagnoses, respectively. There were no significant decreases in hospitalizations rates for respiratory diagnoses or seven other diagnostic groups evaluated.

Conclusions

Among Massachusetts Medicaid subscribers, use of a comprehensive tobacco cessation pharmacotherapy benefit was associated with a significant decrease in claims for hospitalizations for acute myocardial infarction and acute coronary heart disease, but no significant change in hospital claims for other diagnoses. For low-income smokers, removing the barriers to the use of smoking cessation pharmacotherapy has the potential to decrease short-term utilization of hospital services. Please see later in the article for the Editors'' Summary