Characterization of the active site and calcium binding in cytochromecnitrite reductases

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype‐specific PCR and deep sequencing

The incidence of mixed genotype hepatitis C virus infections in the UK is largely unknown. As the efficacy of direct acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8% however this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (p<0.05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR-next generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the non-quantitative nature of the technique and despite the designation of false positive thresholds based on negative controls

The Interpersonal Style and Complementarity Between Crisis Negotiators and Forensic Inpatients

Previous negotiation research has explored the interaction and communication between crisis negotiators and perpetrators. A crisis negotiator attempts to resolve a critical incident through negotiation with an individual, or group of persons in crisis. The purpose of this study was to establish the interpersonal style of crisis negotiators and complementarity of the interpersonal interaction between them and forensic inpatients. Crisis negotiators, clinical workers and students (n = 90) used the Check List of Interpersonal Transactions-Revised (CLOIT-R) to identify interpersonal style, along with eight vignettes detailing interpersonal styles. Crisis negotiators were most likely to have a friendly interpersonal style compared to the other non-trained groups. Complementarity theory was not exclusively supported as submissive individuals did not show optimistic judgments in working with dominant forensic inpatients and vice versa. Exploratory analysis revealed that dominant crisis negotiators were optimistic in working with forensic inpatients with a dominant interpersonal style. This study provides insight into the area of interpersonal complementarity of crisis negotiators and forensic inpatients. Whilst further research is required, a potential new finding was established, with significant ‘similarity’ found when dominant crisis negotiators are asked to work with dominant forensic inpatients

Light-Driven H2 Evolution and C═C or C═O Bond Hydrogenation by Shewanella oneidensis : A Versatile Strategy for Photocatalysis by Nonphotosynthetic Microorganisms

Photocatalytic chemical synthesis by coupling abiotic photosensitizers to purified enzymes provides an effective way to overcome the low conversion efficiencies of natural photosynthesis while exploiting the high catalytic rates and selectivity of enzymes as renewable, earth-abundant electrocatalysts. However, the selective synthesis of multiple products requires more versatile approaches and should avoid the time-consuming and costly processes of enzyme purification. Here we demonstrate a cell-based strategy supporting light-driven H2 evolution or the hydrogenation of C═C and C═O bonds in a nonphotosynthetic microorganism. Methylviologen shuttles photoenergized electrons from water-soluble photosensitizers to enzymes that catalyze H2 evolution and the reduction of fumarate, pyruvate, and CO2 in Shewanella oneidensis. The predominant reaction is selected by the experimental conditions, and the results allow rational development of cell-based strategies to harness nature’s intrinsic catalytic diversity for selective light-driven synthesis of a wide range of products

GLAST: Understanding the High Energy Gamma-Ray Sky

We discuss the ability of the GLAST Large Area Telescope (LAT) to identify, resolve, and study the high energy gamma-ray sky. Compared to previous instruments the telescope will have greatly improved sensitivity and ability to localize gamma-ray point sources. The ability to resolve the location and identity of EGRET unidentified sources is described. We summarize the current knowledge of the high energy gamma-ray sky and discuss the astrophysics of known and some prospective classes of gamma-ray emitters. In addition, we also describe the potential of GLAST to resolve old puzzles and to discover new classes of sources.Comment: To appear in Cosmic Gamma Ray Sources, Kluwer ASSL Series, Edited by K.S. Cheng and G.E. Romer

A Multi-Center Study to Evaluate the Performance of Phage Amplified Biologically Assay for Detecting TB in Sputum in the Pulmonary TB Patients

Objective: To evaluate the performance of phage amplified biologically assay (PhaB) for detecting tuberculosis (TB) in sputum in the pulmonary tuberculosis (PTB) patients. Methods: Shanghai Tuberculosis Key Laboratory of Shanghai Pulmonary Hospital participated in the project in collaboration with the laboratories of six hospitals and a total of 1660 eligible participants (1351 PTB patients and 309 non-TB patients) were included in the study. The sputum samples from the participants were detected by smear microscopy, PhaB, and Löwenstein-Jensen (L-J) culture method, respectively. Results: The overall sensitivity of PhaB were higher than that of L-J culture and smear microscopy (p,0.05). The sensitivity of PhaB for detecting smear-negative specimens was obviously higher than that of L-J culture (p,0.05). Compared with L-J culture, the overall sensitivity, specificity, PPV, NPV, ACC and Kappa value of PhaB were 98.4 (95 % Cl: 96.9–99.3), 71.6 (95% Cl: 68.4–74.6), 67.7, 98.7, 81.7 % and 0.643, respectively. The detection median time of PhaB only needed 48 hours, which was significantly less than that (31 days) of L-J culture method. Conclusion: PhaB method is a rapid and sensitive method for detecting TB in sputum in PTB patients; especially for th

Multifrequency Strategies for the Identification of Gamma-Ray Sources

More than half the sources in the Third EGRET (3EG) catalog have no firmly established counterparts at other wavelengths and are unidentified. Some of these unidentified sources have remained a mystery since the first surveys of the gamma-ray sky with the COS-B satellite. The unidentified sources generally have large error circles, and finding counterparts has often been a challenging job. A multiwavelength approach, using X-ray, optical, and radio data, is often needed to understand the nature of these sources. This chapter reviews the technique of identification of EGRET sources using multiwavelength studies of the gamma-ray fields.Comment: 35 pages, 22 figures. Chapter prepared for the book "Cosmic Gamma-ray Sources", edited by K.S. Cheng and G.E. Romero, to be published by Kluwer Academic Press, 2004. For complete article and higher resolution figures, go to: http://www.astro.columbia.edu/~muk/mukherjee_multiwave.pd

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti