Characterization of the Arsenate Respiratory Reductase from Shewanella sp. Strain ANA-3

Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of ~131 kDa, composed of one ArrA subunit (~95 kDa) and one ArrB subunit (~27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41°C, a Km of 5 µM, and a Vmax of 11,111 µmol of As(V) reduced min–1 mg of protein–1 and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family

Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis

Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

The prevalence and causes of vision loss in Indigenous Australians: the National Indigenous Eye Health Survey

Aim: To determine the prevalence and causes of vision loss in Indigenous Australians. Design, setting and participants: A national, stratified, random cluster sample was drawn from 30 communities across Australia that each included about 300 Indigenous people of all ages. A sample of non-Indigenous adults aged 40 years was also tested at several remote sites for comparison. Participants were examined using a standardised protocol that included a questionnaire (self-administered or completed with the help of field staff), visual acuity (VA) testing on presentation and after correction, visual field testing, trachoma grading, and fundus and lens photography. The data were collected in 2008. Main outcome measures: VA; prevalence of low vision and blindness; causes of vision loss; rates of vision loss in Indigenous compared with non-Indigenous adults. Results: 1694Indigenouschildrenand1189Indigenousadultswereexamined, representing recruitment rates of 84% for children aged 5–15 years and 72% for adults aged 40 years. Rates of low vision (VA \u3c 6/12 to 6/60) were 1.5% (95% CI, 0.9%–2.1%) in children and 9.4% (95% CI, 7.8%–11.1%) in adults. Rates of blindness (VA \u3c 6/60) were 0.2% (95% CI, 0.04%–0.5%) in children and 1.9% (95% CI, 1.1%–2.6%) in adults. The principal cause of low vision in both adults and children was refractive error. The principal causes of blindness in adults were cataract, refractive error and optic atrophy. Relative risks (RRs) of vision loss and blindness in Indigenous adults compared with adults in the mainstream Australian population were 2.8 and 6.2, respectively. By contrast, RRs of vision loss and blindness in Indigenous children compared with mainstream children were 0.2 and 0.6, respectively. Conclusion: Many causes of vision loss in our sample were readily avoidable. Better allocation of services and resources is required to give all Australians equal access to eye health services

Design and characterization of structured protein linkers with differing flexibilities

Engineered fusion proteins containing two or more functional polypeptides joined by a peptide or protein linker are important for many fields of biological research. The separation distance between functional units can impact epitope access and the ability to bind with avidity; thus the availability of a variety of linkers with different lengths and degrees of rigidity would be valuable for protein design efforts. Here, we report a series of designed structured protein linkers incorporating naturally occurring protein domains and compare their properties to commonly used Gly_4Ser repeat linkers. When incorporated into the hinge region of an immunoglobulin G (IgG) molecule, flexible Gly_4Ser repeats did not result in detectable extensions of the IgG antigen-binding domains, in contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein and tetratricopeptide repeats. This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the construction of antibodies with enhanced binding properties or other fusion proteins

Are Musculoskeletal Conditions Neglected in National Health Surveys?

Concise report[Abstract] Objective: To describe the proportion of national health surveys that contain questions on the prevalence and consequences of musculoskeletal conditions. Methods: We used a comprehensive search strategy to obtain national health surveys from the 218 countries listed by the World Bank. Two authors independently extracted information from each national health survey. Outcomes were the proportion of surveys that contained questions on the prevalence of musculoskeletal conditions using the Global Burden of Disease categorization of RA, OA, low back pain, neck pain, gout and other and contained condition-specific questions about activity limitation, severity of pain and work absence. We also measured how frequently the prevalence of low back pain was measured using a consensus-based standard definition for low back pain prevalence studies. Results: We identified national health surveys from 170 countries. Sixty-two (36.4%), the majority from high-income countries (n = 43), measured the prevalence of at least one musculoskeletal condition. OA [53 (85.4%)], low back pain [39 (62.9%)] and neck pain [37 (59.7%)] were most commonly measured, while RA and gout prevalence were only measured in 10 (5.9%) and 3 (1.8%) surveys, respectively. A minority of surveys assessed condition-specific activity limitations [6 (3.6%)], pain severity [5 (2.9%)] and work absence [1 (0.6%)]. Only one survey used the consensus-based standard definition for low back pain. Conclusion: Musculoskeletal conditions are neglected in the majority of national health surveys. Monitoring musculoskeletal conditions through ongoing surveys is crucial for the development and evaluation of health policies to reduce their burden

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV

Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires