Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.

TRAPs: the 'elevator-with-an-operator' mechanism.

Tripartite ATP-independent periplasmic (TRAP) transporters are nutrient-uptake systems found in bacteria and archaea. These evolutionary divergent transporter systems couple a substrate-binding protein (SBP) to an elevator-type secondary transporter, which is a first-of-its-kind mechanism of transport. Here, we highlight breakthrough TRAP transporter structures and recent functional data that probe the mechanism of transport. Furthermore, we discuss recent structural and biophysical studies of the ion transporter superfamily (ITS) members and highlight mechanistic principles that are relevant for further exploration of the TRAP transporter system.

Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.

Structure of Reelin repeat 8 and the adjacent C-terminal region.

Neuronal development and function are dependent in part on the several roles of the secreted glycoprotein Reelin. Endogenous proteases process this 400 kDa, modular protein, yielding N-terminal, central, and C-terminal fragments that each have distinct roles in Reelin's function and regulation. The C-terminal fragment comprises Reelin repeat (RR) domains seven and eight, as well as a basic stretch of 32 amino acid residues termed the C-terminal region (CTR), influences Reelin signaling intensity, and has been reported to bind to Neuropilin-1, which serves as a co-receptor in the canonical Reelin signaling pathway. Here, we present a crystal structure of RR8 at 3.0 Å resolution. Analytical ultracentrifugation and small-angle x-ray scattering confirmed that RR8 is monomeric and enabled us to identify the CTR as a flexible, yet compact subdomain. We conducted structurally informed protein engineering to design a chimeric RR8 construct guided by the structural similarities with RR6. Experimental results support a mode of Reelin-receptor interaction reliant on the multiple interfaces coordinating the binding event. Structurally, RR8 resembles other individual RRs, but its structure does show discrete differences that may account for Reelin receptor specificity toward RR6.

Synthesis of N-acetylmannosamine-6-phosphate derivatives to investigate the mechanism of N-acetylmannosamine-6-phosphate 2-epimerase.

The synthesis of analogues of natural enzyme substrates can be used to help deduce enzymatic mechanisms. N-Acetylmannosamine-6-phosphate 2-epimerase is an enzyme in the bacterial sialic acid catabolic pathway. To investigate whether the mechanism of this enzyme involves a re-protonation mechanism by the same neighbouring lysine that performed the deprotonation or a unique substrate-assisted proton displacement mechanism involving the substrate C5 hydroxyl, the syntheses of two analogues of the natural substrate, N-acetylmannosamine-6-phosphate, are described. In these novel analogues, the C5 hydroxyl has been replaced with a proton and a methyl ether respectively. As recently reported, Staphylococcus aureus N-acetylmannosamine-6-phosphate 2-epimerase was co-crystallized with these two compounds. The 5-deoxy variant bound to the enzyme active site in a different orientation to the natural substrate, while the 5-methoxy variant did not bind, adding to the evidence that this enzyme uses a substrate-assisted proton displacement mechanism. This mechanistic information may help in the design of potential antibacterial drug candidates.

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization.

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.

Selective Nutrient Transport in Bacteria: Multicomponent Transporter Systems Reign Supreme.

Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems.

The structure-function relationship of a signaling-competent, dimeric Reelin fragment.

Reelin operates through canonical and non-canonical pathways that mediate several aspects of brain development and function. Reelin's dimeric central fragment (CF), generated through proteolytic cleavage, is required for the lipoprotein-receptor-dependent canonical pathway activation. Here, we analyze the signaling properties of a variety of Reelin fragments and measure the differential binding affinities of monomeric and dimeric CF fragments to lipoprotein receptors to investigate the mode of canonical signal activation. We also present the cryoelectron tomography-solved dimeric structure of Reelin CF and support it using several other biophysical techniques. Our findings suggest that Reelin CF forms a covalent parallel dimer with some degree of flexibility between the two protein chains. As a result of this conformation, Reelin binds to lipoprotein receptors in a manner inaccessible to its monomeric form and is capable of stimulating canonical pathway signaling.

Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.

While humans lack the biosynthetic pathways for meso-diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum (VsDapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso-diaminopimelate. Kinetic studies show that VsDapL follows a Michaelis-Menten mechanism, with a KMapp of 4.0 mM toward its substrate l,l-diaminopimelate. The kcat (0.46 s-1) and the kcat/KM (115 s-1 M-1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that VsDapL exists in a monomer-dimer self-association, with a KD2-1 of 7.4 µM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in VsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

OpenWorm: overview and recent advances in integrative biological simulation of Caenorhabditis elegans.

The adoption of powerful software tools and computational methods from the software industry by the scientific research community has resulted in a renewed interest in integrative, large-scale biological simulations. These typically involve the development of computational platforms to combine diverse, process-specific models into a coherent whole. The OpenWorm Foundation is an independent research organization working towards an integrative simulation of the nematode Caenorhabditis elegans, with the aim of providing a powerful new tool to understand how the organism's behaviour arises from its fundamental biology. In this perspective, we give an overview of the history and philosophy of OpenWorm, descriptions of the constituent sub-projects and corresponding open-science management practices, and discuss current achievements of the project and future directions.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

Crystal structures and kinetic analyses of N-acetylmannosamine-6-phosphate 2-epimerases from Fusobacterium nucleatum and Vibrio cholerae.

Sialic acids are nine-carbon sugars that are found abundantly on the cell surfaces of mammals as glycoprotein or glycolipid complexes. Several Gram-negative and Gram-positive bacteria have the ability to scavenge and catabolize sialic acids to use as a carbon source. This gives them an advantage in colonizing sialic acid-rich environments. The genes of the sialic acid catabolic pathway are generally present as the operon nanAKE. The third gene in the operon encodes the enzyme N-acetylmannosamine-6-phosphate 2-epimerase (NanE), which catalyzes the conversion of N-acetylmannosamine 6-phosphate to N-acetylglucosamine 6-phosphate, thus committing it to enter glycolysis. The NanE enzyme belongs to the isomerase class of enzymes possessing the triose phosphate isomerase (TIM) barrel fold. Here, comparative structural and functional characterizations of the NanE epimerases from two pathogenic Gram-negative bacteria, Fusobacterium nucleatum (Fn) and Vibrio cholerae (Vc), have been carried out. Structures of NanE from Vc (VcNanE) with and without ligand bound have been determined to 1.7 and 2.7 Šresolution, respectively. The structure of NanE from Fn (FnNanE) has been determined to 2.2 Šresolution. The enzymes show kinetic parameters that are consistent with those of Clostridium perfringens NanE. These studies allowed an evaluation of whether NanE may be a good drug target against these pathogenic bacteria.

Multi-institutional comparison of simulated treatment delivery errors in ssIMRT, manually planned VMAT and autoplan-VMAT plans for nasopharyngeal radiotherapy.

PURPOSE: To quantify the impact of simulated errors for nasopharynx radiotherapy across multiple institutions and planning techniques (auto-plan generated Volumetric Modulated Arc Therapy (ap-VMAT), manually planned VMAT (mp-VMAT) and manually planned step and shoot Intensity Modulated Radiation Therapy (mp-ssIMRT)). METHODS: Ten patients were retrospectively planned with VMAT according to three institution's protocols. Within one institution two further treatment plans were generated using differing treatment planning techniques. This resulted in mp-ssIMRT, mp-VMAT, and ap-VMAT plans. Introduced treatment errors included Multi Leaf Collimator (MLC) shifts, MLC field size (MLCfs), gantry and collimator errors. A change of more than 5% in most selected dose metrics was considered to have potential clinical impact. The original patient plan total Monitor Units (MUs) were correlated to the total number of dose metrics exceeded. RESULTS: The impact of different errors was consistent, with ap-VMAT plans (two institutions) showing larger dose deviations than mp-VMAT created plans (one institution). Across all institutions' VMAT plans the significant errors included; ±5° for the collimator angle, ±5mm for the MLC shift and +1, ±2 and ±5mm for the MLC field size. The total number of dose metrics exceeding tolerance was positively correlated to the VMAT total plan MUs (r=0.51, p<0.001), across all institutions and techniques. CONCLUSIONS: Differences in VMAT robustness to simulated errors across institutions occurred due to planning method differences. Whilst ap-VMAT was most sensitive to MLC errors, it also produced the best quality treatment plans. Mp-ssIMRT was most robust to errors. Higher VMAT treatment plan complexity led to less robust plans.

OpenWorm: an open-science approach to modeling Caenorhabditis elegans.

OpenWorm is an international collaboration with the aim of understanding how the behavior of Caenorhabditis elegans (C. elegans) emerges from its underlying physiological processes. The project has developed a modular simulation engine to create computational models of the worm. The modularity of the engine makes it possible to easily modify the model, incorporate new experimental data and test hypotheses. The modeling framework incorporates both biophysical neuronal simulations and a novel fluid-dynamics-based soft-tissue simulation for physical environment-body interactions. The project's open-science approach is aimed at overcoming the difficulties of integrative modeling within a traditional academic environment. In this article the rationale is presented for creating the OpenWorm collaboration, the tools and resources developed thus far are outlined and the unique challenges associated with the project are discussed.

Doing Anger Differently: two controlled trials of percussion group psychotherapy for adolescent reactive aggression.

This study evaluates efficacy and effectiveness of 'Doing Anger Differently' (DAD), a group treatment for reactively aggressive 12-15 year old males. DAD uses percussion exercises to aid treatment. Study 1 compared a ten-week treatment with a waitlist control at pre, post and 6 month (treatment group only) follow-up. Study 2 replicated Study 1, but also followed up controls at 6 months. In study 1 (N = 54) the treatment resulted in lowered trait anger (Cohen's d = -1.3), aggression-reports (d = -1.0) and depression (d = -0.6), and increased self-esteem (d = 0.6), all maintained at six months. In study 2 (N = 65), aggression-reports fell to one fifth of pre-treatment levels at nine months follow-up (d = -1.2), with lowered trait anger (d = -0.4) and anger expression (d = -0.3) post-treatment.

High-efficiency organic solar concentrators for photovoltaics.

The cost of photovoltaic power can be reduced with organic solar concentrators. These are planar waveguides with a thin-film organic coating on the face and inorganic solar cells attached to the edges. Light is absorbed by the coating and reemitted into waveguide modes for collection by the solar cells. We report single- and tandem-waveguide organic solar concentrators with quantum efficiencies exceeding 50% and projected power conversion efficiencies as high as 6.8%. The exploitation of near-field energy transfer, solid-state solvation, and phosphorescence enables 10-fold increases in the power obtained from photovoltaic cells, without the need for solar tracking.

Surface dose extrapolation measurements with radiographic film.

Assessment of surface dose delivered from radiotherapy x-ray beams for optimal results should be performed both inside and outside the prescribed treatment fields. An extrapolation technique can be used with radiographic film to perform surface dose assessment for open field high energy x-ray beams. This can produce an accurate two-dimensional map of surface dose if required. Results have shown that the surface percentage dose can be estimated within +/-3% of parallel plate ionization chamber results with radiographic film using a series of film layers to produce an extrapolated result. Extrapolated percentage dose assessment for 10 cm, 20 cm and 30 cm square fields was estimated to be 15% +/- 2%, 29% +/- 3% and 38% +/- 3% at the central axis and relatively uniform across the treatment field. The corresponding parallel plate ionization chamber measurements are 16%, 27% and 37%, respectively. Surface doses are also measured outside the treatment field which are mainly due to scattered electron contamination. To achieve this result, film calibration curves must be irradiated to similar x-ray field sizes as the experimental film to minimize quantitative variations in film optical density caused by varying x-ray spectrum with field size.

Doing anger differently: a group percussion therapy for angry adolescent boys.

This paper describes Doing Anger Differently (DAD), a group treatment for young adolescent boys with high levels of anger. The approach is school-based, 10 weeks long, and utilizes music in the form of percussion to engage this difficult to treat population into treatment and to represent the experience of anger. A tri-level intervention is described: the experience of anger and its influence on action; the formation of meaning and identity resulting from anger and aggression; and the emphasis on group work and the interpersonal basis of anger. Techniques used throughout the group are discussed and illustrative case vignettes are provided.