Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily

The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond

HAZARD ANALYSIS SOFTWARE

Washington Safety Management Solutions, LLC developed web-based software to improve the efficiency and consistency of hazard identification and analysis, control selection and classification, and to standardize analysis reporting at Savannah River Site. In the new nuclear age, information technology provides methods to improve the efficiency of the documented safety analysis development process which includes hazard analysis activities. This software provides a web interface that interacts with a relational database to support analysis, record data, and to ensure reporting consistency. A team of subject matter experts participated in a series of meetings to review the associated processes and procedures for requirements and standard practices. Through these meetings, a set of software requirements were developed and compiled into a requirements traceability matrix from which software could be developed. The software was tested to ensure compliance with the requirements. Training was provided to the hazard analysis leads. Hazard analysis teams using the software have verified its operability. The software has been classified as NQA-1, Level D, as it supports the analysis team but does not perform the analysis. The software can be transported to other sites with alternate risk schemes. The software is being used to support the development of 14 hazard analyses. User responses have been positive with a number of suggestions for improvement which are being incorporated as time permits. The software has enforced a uniform implementation of the site procedures. The software has significantly improved the efficiency and standardization of the hazard analysis process

The impact of environmental and climatic variation on the spatiotemporal trends of hospitalized pediatric diarrhea in Ho Chi Minh City, Vietnam.

It is predicted that the integration of climate-based early warning systems into existing action plans will facilitate the timely provision of interventions to diarrheal disease epidemics in resource-poor settings. Diarrhea remains a considerable public health problem in Ho Chi Minh City (HCMC), Vietnam and we aimed to quantify variation in the impact of environmental conditions on diarrheal disease risk across the city. Using all inpatient diarrheal admissions data from three large hospitals within HCMC, we developed a mixed effects regression model to differentiate district-level variation in risk due to environmental conditions from the overarching seasonality of diarrheal disease hospitalization in HCMC. We identified considerable spatial heterogeneity in the risk of all-cause diarrhea across districts of HCMC with low elevation and differential responses to flooding, air temperature, and humidity driving further spatial heterogeneity in diarrheal disease risk. The incorporation of these results into predictive forecasting algorithms will provide a powerful resource to aid diarrheal disease prevention and control practices in HCMC and other similar settings

Long-Term Weekly Iron-Folic Acid and De-Worming Is Associated with Stabilised Haemoglobin and Increasing Iron Stores in Non-Pregnant Women in Vietnam

BACKGROUND: The prevalence of anaemia and iron deficiency in women remains high worldwide. WHO recommends weekly iron-folic acid supplementation where anaemia rates in non-pregnant women of reproductive age are higher than 20%. In 2006, a demonstration project consisting of weekly iron-folic acid supplementation and regular de-worming was set up in two districts in a northern province in Vietnam where anaemia and hookworm rates were 38% and 76% respectively. In 2008 the project was expanded to all districts in the province, targeting some 250,000 women. The objectives of this study were to: 1) examine changes in haemoglobin, iron stores and soil transmitted helminth infection prevalence over three years and 2) assess women's access to and compliance with the intervention. METHODS AND FINDINGS: The study was a semi-cross-sectional, semi-longitudinal panel design with a baseline survey, three impact surveys at three-, twelve- and thirty months after commencement of the intervention, and three compliance surveys after ten weeks, eighteen and thirty six months. RESULTS: After thirty months, mean haemoglobin stabilised at 130.3 g/L, an increase of 8.2 g/L from baseline, and mean serum ferritin rose from 23.9 µg/L to 52 µg/L. Hookworm prevalence fell from 76% to 22% over the same period. After thirty six months, 81% of the target population were receiving supplements and 87% were taking 75% or more of the supplements they received. CONCLUSIONS: Weekly iron-folic acid supplementation and regular de-worming was effective in significantly and sustainably reducing the prevalence of anaemia and soil transmitted helminth infections and high compliance rates were maintained over three years

Risk factors for the development of severe typhoid fever in Vietnam

Background Typhoid fever is a systemic infection caused by the bacterium Salmonella enterica serovar Typhi. Age, sex, prolonged duration of illness, and infection with an antimicrobial resistant organism have been proposed risk factors for the development of severe disease or fatality in typhoid fever. Methods We analysed clinical data from 581 patients consecutively admitted with culture confirmed typhoid fever to two hospitals in Vietnam during two periods in 1993–1995 and 1997–1999. These periods spanned a change in the antimicrobial resistance phenotypes of the infecting organisms i.e. fully susceptible to standard antimicrobials, resistance to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole (multidrug resistant, MDR), and intermediate susceptibility to ciprofloxacin (nalidixic acid resistant). Age, sex, duration of illness prior to admission, hospital location and the presence of MDR or intermediate ciprofloxacin susceptibility in the infecting organism were examined by logistic regression analysis to identify factors independently associated with severe typhoid at the time of hospital admission. Results The prevalence of severe typhoid was 15.5% (90/581) and included: gastrointestinal bleeding (43; 7.4%); hepatitis (29; 5.0%); encephalopathy (16; 2.8%); myocarditis (12; 2.1%); intestinal perforation (6; 1.0%); haemodynamic shock (5; 0.9%), and death (3; 0.5%). Severe disease was more common with increasing age, in those with a longer duration of illness and in patients infected with an organism exhibiting intermediate susceptibility to ciprofloxacin. Notably an MDR phenotype was not associated with severe disease. Severe disease was independently associated with infection with an organism with an intermediate susceptibility to ciprofloxacin (AOR 1.90; 95% CI 1.18-3.07; p = 0.009) and male sex (AOR 1.61 (1.00-2.57; p = 0.035). Conclusions In this group of patients hospitalised with typhoid fever infection with an organism with intermediate susceptibility to ciprofloxacin was independently associated with disease severity. During this period many patients were being treated with fluoroquinolones prior to hospital admission. Ciprofloxacin and ofloxacin should be used with caution in patients infected with S. Typhi that have intermediate susceptibility to ciprofloxacin

Viral Etiology of Encephalitis in Children in Southern Vietnam: Results of a One-Year Prospective Descriptive Study

Viral encephalitis is associated with high morbidity and mortality in Vietnam. However little is known about the causes of the disease due to a lack of diagnostic facilities in this relatively resource-poor setting. Knowledge about the etiologies and clinical outcome of viral encephalitis is necessary for future design of intervention studies targeted at improvement of clinical management, treatment and prevention of the disease. We report the viral agents, clinical outcome and prognostic factors of mortality of encephalitis in children admitted to a referral hospital for children in southern Vietnam. We show that about one third of the enrolled patients die acutely, and that mortality is independently associated with patient age and Glasgow Coma Scale on admission. Japanese encephalitis, dengue virus and enterovirus (including enterovirus 71) are the major viruses detected in our patients. However, more than half of the patients remain undiagnosed, while mortality in this group is as high as in the diagnosed group. This study will benefit clinicians and public health in terms of clinical management and prevention of childhood encephalitis in Vietnam

High Pro-Inflammatory Cytokine Secretion and Loss of High Avidity Cross-Reactive Cytotoxic T-Cells during the Course of Secondary Dengue Virus Infection

BACKGROUND: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. METHODS AND FINDINGS: Virus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis. CONCLUSION: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease

An Outbreak of Severe Infections with Community-Acquired MRSA Carrying the Panton-Valentine Leukocidin Following Vaccination

Background: Infections with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging worldwide. We investigated an outbreak of severe CA-MRSA infections in children following out-patient vaccination. Methods and Findings: We carried out a field investigation after adverse events following immunization (AEFI) were reported. We reviewed the clinical data from all cases. S. aureus recovered from skin infections and from nasal and throat swabs were analyzed by pulse-field gel electrophoresis, multi locus sequence typing, PCR and microarray. In May 2006, nine children presented with AEFI, ranging from fatal toxic shock syndrome, necrotizing soft tissue infection, purulent abscesses, to fever with rash. All had received a vaccination injection in different health centres in one District of Ho Chi Minh City. Eight children had been vaccinated by the same health care worker (HCW). Deficiencies in vaccine quality, storage practices, or preparation and delivery were not found. Infection control practices were insufficient. CA-MRSA was cultured in four children and from nasal and throat swabs from the HCW. Strains from children and HCW were indistinguishable. All carried the Panton-Valentine leukocidine (PVL), the staphylococcal enterotoxin B gene, the gene complex for staphylococcal-cassette-chromosome mec type V, and were sequence type 59. Strain HCM3A is epidemiologically unrelated to a strain of ST59 prevalent in the USA, althoughthey belong to the same lineage. Conclusions. We describe an outbreak of infections with CA-MRSA in children, transmitted by an asymptomatic colonized HCW during immunization injection. Consistent adherence to injection practice guidelines is needed to prevent CA-MRSA transmission in both in- and outpatient settings