YwdL in Bacillus cereus: Its Role in Germination and Exosporium Structure

In members of the Bacillus cereus group the outermost layer of the spore is the exosporium, which interacts with hosts and the environment. Efforts have been made to identify proteins of the exosporium but only a few have so far been characterised and their role in determining spore architecture and spore function is still poorly understood. We have characterised the exosporium protein, YwdL. ΔywdL spores have a more fragile exosporium, subject to damage on repeated freeze-thawing, although there is no evidence of altered resistance properties, and coats appear intact. Immunogold labelling and Western blotting with anti-YwdL antibodies identified YwdL to be located exclusively on the inner surface of the exosporium of B. cereus and B. thuringiensis. We conclude that YwdL is important for formation of a robust exosporium but is not required to maintain the crystalline assembly within the basal layer or for attachment of the hairy nap structure. ΔywdL spores are unable to germinate in response to CaDPA, and have altered germination properties, a phenotype that confirms the expected defect in localization of the cortex lytic enzyme CwlJ in the coat

Sex in the shadow of HIV:A systematic review of prevalence, risk factors, and interventions to reduce sexual risk-taking among HIVpositive adolescents and youth in sub-Saharan Africa

Background Evidence on sexual risk-taking among HIV-positive adolescents and youth in sub-Saharan Africa is urgently needed. This systematic review synthesizes the extant research on prevalence, factors associated with, and interventions to reduce sexual risk-taking among HIV-positive adolescents and youth in sub-Saharan Africa. Methods Studies were located through electronic databases, grey literature, reference harvesting, and contact with researchers. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Quantitative studies that reported on HIV-positive participants (10-24 year olds), included data on at least one of eight outcomes (early sexual debut, inconsistent condom use, older partner, transactional sex, multiple sexual partners, sex while intoxicated, sexually transmitted infections, and pregnancy), and were conducted in sub-Saharan Africa were included. Two authors piloted all processes, screened studies, extracted data independently, and resolved any discrepancies. Due to variance in reported rates and factors associated with sexual risk-taking, meta-analyses were not conducted. Results 610 potentially relevant titles/abstracts resulted in the full text review of 251 records. Forty-two records (n=35 studies) reported one or multiple sexual practices for 13,536 HIV-positive adolescents/youth from 13 sub-Saharan African countries. Seventeen cross-sectional studies reported on individual, relationship, family, structural, and HIV-related factors associated with sexual risk-taking. However, the majority of the findings were inconsistent across studies, and most studies scored Conclusions Sexual risk-taking among HIV-positive adolescents and youth is high, with inconclusive evidence on potential determinants. Few known studies test secondary HIV-prevention interventions for HIV-positive youth. Effective and feasible low-cost interventions to reduce risk are urgently needed for this group.</p

Determination of an optimal potential window for catalysis by E. coli dimethyl sulfoxide reductase and hypothesis on the role of Mo(V) in the reaction pathway.

Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential. The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)). At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0). Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance. The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state

Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.

The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli

Detection and interpretation of redox potential optima in the catalytic activity of enzymes.

It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation

“I did not want to give birth to a child who has HIV”: Experiences using PrEP during pregnancy among HIV-uninfected Kenyan women in HIV-serodiscordant couples

Objectives: The perceptions, motivations, and beliefs of HIV-uninfected women about pre-exposure prophylaxis (PrEP) use during pregnancy can influence its uptake and adherence. This study elicited the views of HIV-uninfected women with personal experience taking PrEP during pregnancy. Design: Qualitative interviews were conducted with HIV-uninfected women who had personal experience taking PrEP while pregnant. Methods: Semistructured interviews were conducted with 21 HIV-uninfected Kenyan women in HIV-serodiscordant couples enrolled in an open-label PrEP demonstration project who became pregnant while using PrEP and continued PrEP through their pregnancy. Interviews were audio-recorded and transcribed into English. A qualitative descriptive analysis was performed, using a constant comparison approach to identify key themes related to PrEP use in pregnancy. Results: Desire to remain HIV uninfected and have an HIV-free infant were strong motivators influencing continued use of PrEP during pregnancy. Supporting HIV-infected partners and childbearing within an HIV-serodiscordant relationship were also motivators. Women had challenges distinguishing normal pregnancy symptoms from PrEP side effects and were concerned that observed side effects could be signs of danger for the infant related to PrEP exposure. Health care providers were important conduits of knowledge about PrEP, and continuity of PrEP providers throughout pregnancy facilitated adherence. Conclusions: HIV-uninfected women in HIV-serodiscordant couples were motivated to use PrEP during pregnancy to remain HIV uninfected and to have an HIV-free child but had concerns about side effects. Health care providers will be important for PrEP messaging and adherence support in this unique population

HIV-uninfected Kenyan adolescent and young women share perspectives on using pre-exposure prophylaxis during pregnancy

To optimize scale-up of pre-exposure prophylaxis (PrEP) for pregnant women at risk of HIV in high HIV burden settings, implementation strategies must be developed that account for perceptions of PrEP in this unique population. Semistructured focus group discussions were conducted with 68 HIV-uninfected Kenyan pregnant and postpartum women without prior PrEP knowledge or experience. A qualitative descriptive analysis was performed, using a constant comparison approach, to identify key themes related to the values and rationale impacting potential PrEP use in pregnancy. Median age was 19.5 years and participants were either pregnant or had 1–2 children. Almost all (96%) were married or had a steady partner. Women felt pregnancy was a time of high HIV risk because they desired sex less frequently, which may lead their partners to have outside partnerships. This made PrEP an attractive HIV prevention option for themselves and their infants. Although women believed male partner behaviors influenced their HIV risk, many women perceived that male partners would react negatively, including becoming physically violent, if they discovered that women used PrEP. Clinicians were identified as potential facilitators of PrEP use who could explain PrEP to male partners on behalf of pregnant women. Women said that community-level stigma against HIV and potential for conflating PrEP with antiretroviral therapy (ART) would necessitate that PrEP use be discreet. Our results indicate the importance of addressing risk perception of women, concerns of male partners, HIV stigma, and benefits of PrEP for HIV prevention as programs are developed for pregnant women