Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key <i>Klebsiella pneumoniae</i> Capsular Exopolysaccharides.

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections

Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections

Search for R-Parity Violating Decays of Supersymmetric Particles in e+e−e^{+}e^{-} Collisions at Centre-of-Mass Energies near 183 GeV

Searches for pair-production of supersymmetric particles under the assumption that R-parity is violated via a single dominant $LL{\bar E}$, $LQ{\bar D}$ or ${\bar U} {\bar D} {\bar D}$ coupling are performed using the data collected by the \ALEPH\ collaboration at centre-of-mass energies of 181--184~\gev. The observed candidate events in the data are in agreement with the Standard Model expectations. Upper limits on the production cross-sections and lower limits on the masses of charginos, sleptons, squarks and sneutrinos are de rived

Simulating rewetting events in intermittent rivers and ephemeral streams: A global analysis of leached nutrients and organic matter

Climate change and human pressures are changing the global distribution and the ex‐ tent of intermittent rivers and ephemeral streams (IRES), which comprise half of the global river network area. IRES are characterized by periods of flow cessation, during which channel substrates accumulate and undergo physico‐chemical changes (precon‐ ditioning), and periods of flow resumption, when these substrates are rewetted and release pulses of dissolved nutrients and organic matter (OM). However, there are no estimates of the amounts and quality of leached substances, nor is there information on the underlying environmental constraints operating at the global scale. We experi‐ mentally simulated, under standard laboratory conditions, rewetting of leaves, river‐ bed sediments, and epilithic biofilms collected during the dry phase across 205 IRES from five major climate zones. We determined the amounts and qualitative character‐ istics of the leached nutrients and OM, and estimated their areal fluxes from riverbeds. In addition, we evaluated the variance in leachate characteristics in relation to selected environmental variables and substrate characteristics. We found that sediments, due to their large quantities within riverbeds, contribute most to the overall flux of dis‐ solved substances during rewetting events (56%–98%), and that flux rates distinctly differ among climate zones. Dissolved organic carbon, phenolics, and nitrate contrib‐ uted most to the areal fluxes. The largest amounts of leached substances were found in the continental climate zone, coinciding with the lowest potential bioavailability of the leached OM. The opposite pattern was found in the arid zone. Environmental vari‐ ables expected to be modified under climate change (i.e. potential evapotranspiration, aridity, dry period duration, land use) were correlated with the amount of leached sub‐ stances, with the strongest relationship found for sediments. These results show that the role of IRES should be accounted for in global biogeochemical cycles, especially because prevalence of IRES will increase due to increasing severity of drying event

Tumor-Infiltrating T Cells Correlate with NY-ESO-1-Specific Autoantibodies in Ovarian Cancer

BACKGROUND: Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens. METHODOLOGY AND PRINCIPAL FINDINGS: We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-gamma ELISPOT and MHC class I pentamer staining. CONCLUSION AND SIGNIFICANCE: We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe