Characterization of the Na⁺/H⁺ antiporter from Yersinia pestis

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na/H antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein''s substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein''s structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na/H antiporter''s structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na/H antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.     

Perturbation of Arabidopsis amino acid metabolism causes incompatibility with the adapted biotrophic pathogen Hyaloperonospora arabidopsidis

Reliance of biotrophic pathogens on living plant tissues to propagate implies strong interdependence between host metabolism and nutrient uptake by the pathogen. However, factors determining host suitability and establishment of infection are largely unknown. We describe a loss-of-inhibition allele of ASPARTATE KINASE2 and a loss-of-function allele of DIHYDRODIPICOLINATE SYNTHASE2 identified in a screen for Arabidopsis thaliana mutants with increased resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa). Through different molecular mechanisms, these mutations perturb amino acid homeostasis leading to overaccumulation of the Asp-derived amino acids Met, Thr, and Ile. Although detrimental for the plant, the mutations do not cause defense activation, and both mutants retain full susceptibility to the adapted obligate biotrophic fungus Golovinomyces orontii (Go). Chemical treatments mimicking the mutants' metabolic state identified Thr as the amino acid suppressing Hpa but not Go colonization. We conclude that perturbations in amino acid homeostasis render the mutant plants unsuitable as an infection substrate for Hpa. This may be explained by deployment of the same amino acid biosynthetic pathways by oomycetes and plants. Our data show that the plant host metabolic state can, in specific ways, influence the ability of adapted biotrophic strains to cause disease.     

Improved templated fluorogenic probes enhance the analysis of closely related pathogenic bacteria by microscopy and flow cytometry

Templated fluorescence activation has recently emerged as a promising molecular approach to detect and differentiate nucleic acid sequences in vitro and in cells. Here, we describe the application of a reductive quencher release strategy to the taxonomic analysis of Gram-negative bacteria by targeting a single nucleotide difference in their 16S rRNA in a two-color assay. For this purpose, it was necessary to develop a release linker containing a quencher suitable for red and near-infrared fluorophores, and to improve methods for the delivery of probes into cells. A cyanine-dye labeled oligonucleotide probe containing the new quencher-release linker showed unprecedentedly low background signal and high fluorescence turn-on ratios. The combination of a fluorescein-containing and a near-IR emitting probe discriminated E. coli from S. enterica despite nearly identical ribosomal target sequences. Two-color analysis by microscopy and the first successful discrimination of bacteria by two-color flow cytometry with templated reactive probes are described.     

Peripheral blood gene expression profiles in COPD subjects

To identify non-invasive gene expression markers for chronic obstructive pulmonary disease (COPD), we performed genome-wide expression profiling of peripheral blood samples from 12 subjects with significant airflow obstruction and an equal number of non-obstructed controls. RNA was isolated from Peripheral Blood Mononuclear Cells (PBMCs) and gene expression was assessed using Affymetrix U133 Plus 2.0 arrays.Tests for gene expression changes that discriminate between COPD cases (FEV1< 70% predicted, FEV1/FVC < 0.7) and controls (FEV1> 80% predicted, FEV1/FVC > 0.7) were performed using Significance Analysis of Microarrays (SAM) and Bayesian Analysis of Differential Gene Expression (BADGE). Using either test at high stringency (SAM median FDR = 0 or BADGE p < 0.01) we identified differential expression for 45 known genes. Correlation of gene expression with lung function measurements (FEV1 & FEV1/FVC), using both Pearson and Spearman correlation coefficients (p < 0.05), identified a set of 86 genes. A total of 16 markers showed evidence of significant correlation (p < 0.05) with quantitative traits and differential expression between cases and controls. We further compared our peripheral gene expression markers with those we previously identified from lung tissue of the same cohort. Two genes, RP9and NAPE-PLD, were identified as decreased in COPD cases compared to controls in both lung tissue and blood. These results contribute to our understanding of gene expression changes in the peripheral blood of patients with COPD and may provide insight into potential mechanisms involved in the disease.     

THetA: inferring intra-tumor heterogeneity from high-throughput DNA sequencing data

Tumor samples are typically heterogeneous, containing admixture by normal, non-cancerous cells and one or more subpopulations of cancerous cells. Whole-genome sequencing of a tumor sample yields reads from this mixture, but does not directly reveal the cell of origin for each read. We introduce THetA (Tumor Heterogeneity Analysis), an algorithm that infers the most likely collection of genomes and their proportions in a sample, for the case where copy number aberrations distinguish subpopulations. THetA successfully estimates normal admixture and recovers clonal and subclonal copy number aberrations in real and simulated sequencing data. THetA is available at http://compbio.cs.brown.edu/software/.     

Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

Background

Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients.

Methods and Findings

Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women''s Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10⁻⁷) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10⁻⁵) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10⁻⁴) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers.

Conclusions

Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients. Please see later in the article for the Editors'' Summary     

Mate-Searching Behaviour of Common and Rare Wasps and the Implications for Pollen Movement of the Sexually Deceptive Orchids They Pollinate

Pollinator behaviour directly affects patterns of pollen movement and outcrossing rates in plants. In orchids pollinated by sexual deception of insects, patterns of pollen movement are primarily determined by the mate-searching behaviour of the deceived males. Here, using a capture-mark-recapture study (CMR) and dietary analysis, we compare mate-searching behaviour in relation to local abundance of two pollinator species and explore the implications for pollen movement in sexually deceptive Drakaea (Orchidaceae). Drakaea are pollinated solely by the sexual deception of male thynnine wasps. The rare Drakaea elastica and widespread D. livida occur sympatrically and are pollinated by the rare but locally common Zaspilothynnus gilesi, and the widespread and abundant Z. nigripes, respectively. Local abundance was significantly different with Z. nigripes twice as abundant as Z. gilesi. For the 653 marked wasps, there was no significant difference in median movement distance between Z. gilesi and Z. nigripes. However, the maximum movement distance was twice as high for Z. gilesi (556 m) compared with Z. nigripes (267 m). This is up to three times greater than previously reported for thynnines in CMR studies. Recapture rates were six times higher in Z. gilesi (57%) compared to Z. nigripes (9%). Pollen loads and wasp longevity were similar, suggesting that this difference in recapture rate arises due to differences in the number of males moving at a scale >500 m rather than through diet or mortality. Differences in the frequency of longer movements may arise due to variation in the spatial distribution of the wingless females. We predict that pollen movement will largely be restricted to within populations of Drakaea (<500 m), with few movements between populations (>500 m).     

Analysis of V2 Antibody Responses Induced in Vaccinees in the ALVAC/AIDSVAX HIV-1 Vaccine Efficacy Trial

The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.     

The MUC5B Variant Is Associated with Idiopathic Pulmonary Fibrosis but Not with Systemic Sclerosis Interstitial Lung Disease in the European Caucasian Population

A polymorphism on the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) but not with systemic sclerosis (SSc) with interstitial lung disease (ILD). We genotyped the MUC5B promoter in the first 142 patients of the French national prospective cohort of IPF, in 981 French patients with SSc (346 ILD), 598 Italian patients with SSc (207 ILD), 1383 French controls and 494 Italian controls. A meta-analysis was performed including all American data available. The T risk allele was present in 41.9% of the IPF patients, 10.8% of the controls (P = 2×10^–44), OR 6.3 [4.6–8.7] for heterozygous patients and OR 21.7 [10.4–45.3] for homozygous patients. Prevalence of the T allele was not modified according to age, gender, smoking in IPF patients. However, none of the black patients with IPF presented the T allele. The prevalence of the T risk allele was similar between French (10%) and Italian (12%) cohorts of SSc whatever the presence of an ILD (11.1% and 13.5%, respectively). Meta-analysis confirmed the similarity between French, Italian and American cohorts of IPF or SSc-ILD. This study confirms 1) an association between the T allele risk and IPF, 2) an absence of association with SSc-ILD, suggesting different pathophysiology.     

Impaired Blood Dendritic Cell Numbers and Functions after Aneurysmal Subarachnoid Hemorrhage

Previous Presentation

Portions of this study were presented at the Annual Congress of Société Française d’Anesthésie et de Réanimation in Paris, September 2012.

Background

Toll-like receptor (TLR) agonists are promising therapy for the prevention of nosocomial infections in critical ill patients. We aimed to analyze the TLR-reactivity of circulating dendritic cells (DC) as assessed by cytokine production after an ex vivo challenge with TLR agonists in aneurysmal subarachnoid hemorrhage (SAH) patients.

Methods and Findings

A single-center prospective observational study took place in one intensive care unit of a teaching hospital. Blood samples were harvested on days 2, 5 and 10 in 21 severe SAH patients requiring mechanical ventilation and 17 healthy controls. DC production of cytokines (Tumour Necrosis Factor, TNF-α; Interleukin, IL-12; and Interferon, IFN-α) was assessed by intracellular immunostaining on TLR-3, 4, 7/8 and 9 stimulations. SAH patients had decreased numbers of blood myeloid (mDCs) and plasmacytoid DCs (pDCs) on days 2, 5 and 10. Compared with the healthy controls, the frequency of mDCs producing TNF-α after TLR-3 stimulation was decreased in the SAH patients. The frequency of myeloid DCs producing IL-12 after TLR-3 and 4 stimulations was also decreased in the SAH patients. In contrast, the mDCs response to TLR-7/8 was not impaired in the SAH patients. The frequency of pDCs producing TNF-α⁺ and IFN-α⁺ on TLR-7/8 stimulation were reduced at all of the tested times in the SAH patients, whereas reactivity to TLR-9 was preserved. On day 2, the pDCs from non-survivor patients (n = 8) had a decreased ability to produce IFN-α on TLR-9 stimulation compared with the survivors.

Conclusions

These data suggest functional abnormalities of circulating pDCs and mDCs that could be important for immunomodulation after SAH.