Characterization of a Conserved Interaction between DNA Glycosylase and ParA in Mycobacterium smegmatis and M. tuberculosis

The chromosome partitioning proteins, ParAB, ensure accurate segregation of genetic materials into daughter cells and most bacterial species contain their homologs. However, little is known about the regulation of ParAB proteins. In this study, we found that 3-methyladenine DNA glycosylase I MsTAG(Ms5082) regulates bacterial growth and cell morphology by directly interacting with MsParA (Ms6939) and inhibiting its ATPase activity in Mycobacterium smegmatis. Using bacterial two-hybrid and pull-down techniques in combination with co-immunoprecipitation assays, we show that MsTAG physically interacts with MsParA both in vitro and in vivo. Expression of MsTAG under conditions of DNA damage induction exhibited similar inhibition of growth as the deletion of the parA gene in M. smegmatis. Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA. Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction. Based on protein co-expression and fluorescent co-localization assays, MsParA and MsTAG were further found to co-localize in mycobacterial cells. In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis. Our findings provide important new insights into the regulatory mechanism of cell growth and division in mycobacteria

VapC Toxins from Mycobacterium tuberculosis Are Ribonucleases that Differentially Inhibit Growth and Are Neutralized by Cognate VapB Antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism

Mapping and simulating systematics due to spatially-varying observing conditions in DES Science Verification data

Spatially-varying depth and characteristics of observing conditions, such as seeing, airmass, or sky background, are major sources of systematic uncertainties in modern galaxy survey analyses, in particular in deep multi-epoch surveys. We present a framework to extract and project these sources of systematics onto the sky, and apply it to the Dark Energy Survey (DES) to map the observing conditions of the Science Verification (SV) data. The resulting distributions and maps of sources of systematics are used in several analyses of DES SV to perform detailed null tests with the data, and also to incorporate systematics in survey simulations. We illustrate the complementarity of these two approaches by comparing the SV data with the BCC-UFig, a synthetic sky catalogue generated by forward-modelling of the DES SV images. We analyse the BCC-UFig simulation to construct galaxy samples mimicking those used in SV galaxy clustering studies. We show that the spatially-varying survey depth imprinted in the observed galaxy densities and the redshift distributions of the SV data are successfully reproduced by the simulation and well-captured by the maps of observing conditions. The combined use of the maps, the SV data and the BCC-UFig simulation allows us to quantify the impact of spatial systematics on $N(z)$, the redshift distributions inferred using photometric redshifts. We conclude that spatial systematics in the SV data are mainly due to seeing fluctuations and are under control in current clustering and weak lensing analyses. The framework presented here is relevant to all multi-epoch surveys, and will be essential for exploiting future surveys such as the Large Synoptic Survey Telescope (LSST), which will require detailed null-tests and realistic end-to-end image simulations to correctly interpret the deep, high-cadence observations of the sky

In vivo and in silico determination of essential genes of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>In the United Kingdom, the thermophilic <it>Campylobacter </it>species <it>C. jejuni </it>and <it>C. coli </it>are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate <it>C. jejuni </it>and <it>C. coli </it>from the food chain.</p> <p>Results</p> <p>A metabolic model of <it>C. jejuni </it>was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium <it>Helicobacter pylori</it>, and extensive literature mining. Using this model, we have used <it>in silico </it>Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this <it>in silico </it>approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published <it>Campylobacter </it>protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.</p> <p>Conclusions</p> <p>We have constructed the first curated metabolic model for the food-borne pathogen <it>Campylobacter jejuni </it>and have presented the resulting metabolic insights. We have shown that the combination of <it>in silico </it>and <it>in vivo </it>approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to <it>C. jejuni</it>, which are all potential novel <it>Campylobacter </it>intervention targets.</p

Effects of Hydrographic Variability on the Spatial, Seasonal and Diel Diving Patterns of Southern Elephant Seals in the Eastern Weddell Sea

Weddell Sea hydrography and circulation is driven by influx of Circumpolar Deep Water (CDW) from the Antarctic Circumpolar Current (ACC) at its eastern margin. Entrainment and upwelling of this high-nutrient, oxygen-depleted water mass within the Weddell Gyre also supports the mesopelagic ecosystem within the gyre and the rich benthic community along the Antarctic shelf. We used Conductivity-Temperature-Depth Satellite Relay Data Loggers (CTD-SRDLs) to examine the importance of hydrographic variability, ice cover and season on the movements and diving behavior of southern elephant seals in the eastern Weddell Sea region during their overwinter feeding trips from Bouvetøya. We developed a model describing diving depth as a function of local time of day to account for diel variation in diving behavior. Seals feeding in pelagic ice-free waters during the summer months displayed clear diel variation, with daytime dives reaching 500-1500 m and night-time targeting of the subsurface temperature and salinity maxima characteristic of CDW around 150–300 meters. This pattern was especially clear in the Weddell Cold and Warm Regimes within the gyre, occurred in the ACC, but was absent at the Dronning Maud Land shelf region where seals fed benthically. Diel variation was almost absent in pelagic feeding areas covered by winter sea ice, where seals targeted deep layers around 500–700 meters. Thus, elephant seals appear to switch between feeding strategies when moving between oceanic regimes or in response to seasonal environmental conditions. While they are on the shelf, they exploit the locally-rich benthic ecosystem, while diel patterns in pelagic waters in summer are probably a response to strong vertical migration patterns within the copepod-based pelagic food web. Behavioral flexibility that permits such switching between different feeding strategies may have important consequences regarding the potential for southern elephant seals to adapt to variability or systematic changes in their environment resulting from climate change

The Influence of Number and Timing of Pregnancies on Breast Cancer Risk for Women With BRCA1 or BRCA2 Mutations

International audienceBACKGROUND:Full-term pregnancy (FTP) is associated with a reduced breast cancer (BC) risk over time, but women are at increased BC risk in the immediate years following an FTP. No large prospective studies, however, have examined whether the number and timing of pregnancies are associated with BC risk for BRCA1 and BRCA2 mutation carriers.METHODS:Using weighted and time-varying Cox proportional hazards models, we investigated whether reproductive events are associated with BC risk for mutation carriers using a retrospective cohort (5707 BRCA1 and 3525 BRCA2 mutation carriers) and a prospective cohort (2276 BRCA1 and 1610 BRCA2 mutation carriers), separately for each cohort and the combined prospective and retrospective cohort.RESULTS:For BRCA1 mutation carriers, there was no overall association with parity compared with nulliparity (combined hazard ratio [HRc] = 0.99, 95% confidence interval [CI] = 0.83 to 1.18). Relative to being uniparous, an increased number of FTPs was associated with decreased BC risk (HRc = 0.79, 95% CI = 0.69 to 0.91; HRc = 0.70, 95% CI = 0.59 to 0.82; HRc = 0.50, 95% CI = 0.40 to 0.63, for 2, 3, and ≥4 FTPs, respectively, P trend < .0001) and increasing duration of breastfeeding was associated with decreased BC risk (combined cohort P trend = .0003). Relative to being nulliparous, uniparous BRCA1 mutation carriers were at increased BC risk in the prospective analysis (prospective hazard ration [HRp] = 1.69, 95% CI = 1.09 to 2.62). For BRCA2 mutation carriers, being parous was associated with a 30% increase in BC risk (HRc = 1.33, 95% CI = 1.05 to 1.69), and there was no apparent decrease in risk associated with multiparity except for having at least 4 FTPs vs. 1 FTP (HRc = 0.72, 95% CI = 0.54 to 0.98).CONCLUSIONS:These findings suggest differential associations with parity between BRCA1 and BRCA2 mutation carriers with higher risk for uniparous BRCA1 carriers and parous BRCA2 carriers

Association of genomic domains in BRCA1 and BRCA2 with prostate cancer risk and aggressiveness

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. Weevaluated whether PSVs inBRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 30 region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25-2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63-5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71-4.68; P = 0.00004) and elevated risk of Gleason 8+prostate cancer (HR = 4.95; 95% CI, 2.12-11.54; P = 0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. Significance: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.Peer reviewe

Commercial Nucleic-Acid Amplification Tests for Diagnosis of Pulmonary Tuberculosis in Respiratory Specimens: Meta-Analysis and Meta-Regression

BACKGROUND: Hundreds of studies have evaluated the diagnostic accuracy of nucleic-acid amplification tests (NAATs) for tuberculosis (TB). Commercial tests have been shown to give more consistent results than in-house assays. Previous meta-analyses have found high specificity but low and highly variable estimates of sensitivity. However, reasons for variability in study results have not been adequately explored. We performed a meta-analysis on the accuracy of commercial NAATs to diagnose pulmonary TB and meta-regression to identify factors that are associated with higher accuracy. METHODOLOGY/PRINCIPAL FINDINGS: We identified 2948 citations from searching the literature. We found 402 articles that met our eligibility criteria. In the final analysis, 125 separate studies from 105 articles that reported NAAT results from respiratory specimens were included. The pooled sensitivity was 0.85 (range 0.36-1.00) and the pooled specificity was 0.97 (range 0.54-1.00). However, both measures were significantly heterogeneous (p<.001). We performed subgroup and meta-regression analyses to identify sources of heterogeneity. Even after stratifying by type of commercial test, we could not account for the variability. In the meta-regression, the threshold effect was significant (p = .01) and the use of other respiratory specimens besides sputum was associated with higher accuracy. CONCLUSIONS/SIGNIFICANCE: The sensitivity and specificity estimates for commercial NAATs in respiratory specimens were highly variable, with sensitivity lower and more inconsistent than specificity. Thus, summary measures of diagnostic accuracy are not clinically meaningful. The use of different cut-off values and the use of specimens other than sputum could explain some of the observed heterogeneity. Based on these observations, commercial NAATs alone cannot be recommended to replace conventional tests for diagnosing pulmonary TB. Improvements in diagnostic accuracy, particularly sensitivity, need to be made in order for this expensive technology to be worthwhile and beneficial in low-resource countries