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Language for specific purposes
\"Language for Specific Purposes provides a concise history of the roots of LSP, and an analysis of the current trends in globalization, technology and applied linguistics that directly affect the learning, teaching and researching of LSP. The book provides insights into language course planning, design and implementation, assessment and evaluation, as well as issues in pedagogy, classroom management and professional development of particular interest to LSP practitioners. Another important focus of the book is research: the nature of research in LSP, typical research methods in the field, and ideas for researchable projects. Numerous case studies and lists of useful resources for further study and research make this book a rich resource, not only for graduate students planning to teach LSP, but also for professionals looking to update or extend their knowledge of the field\"-- Provided by publisher.
Book
The ClusPro web server for protein–protein docking
ClusPro is a web server that performs rigid-body docking of two proteins by sampling billions of conformations. Low-energy docked structures are clustered, and centers of the largest clusters are used as likely models of the complex.
The ClusPro server (
https://cluspro.org
) is a widely used tool for protein–protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format. However, ClusPro also offers a number of advanced options to modify the search; these include the removal of unstructured protein regions, application of attraction or repulsion, accounting for pairwise distance restraints, construction of homo-multimers, consideration of small-angle X-ray scattering (SAXS) data, and location of heparin-binding sites. Six different energy functions can be used, depending on the type of protein. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low-energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in <4 h.
Journal Article
The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins
This protocol describes the FTMap family of web servers for determining and characterizing ligand-binding hot spots of macromolecules, including FTSite for predicting ligand-binding sites, FTFlex for accounting for side chain flexibility, FTMap/param for parameterizing additional probes, and FTDyn for mapping ensembles of protein structures.
FTMap is a computational mapping server that identifies binding hot spots of macromolecules—i.e., regions of the surface with major contributions to the ligand-binding free energy. To use FTMap, users submit a protein, DNA or RNA structure in PDB (Protein Data Bank) format. FTMap samples billions of positions of small organic molecules used as probes, and it scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots in good agreement with experimental data. FTMap serves as the basis for other servers, namely FTSite, which is used to predict ligand-binding sites, FTFlex, which is used to account for side chain flexibility, FTMap/param, used to parameterize additional probes and FTDyn, for mapping ensembles of protein structures. Applications include determining the druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures and providing input for fragment-based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and it is much faster than the more-recent approaches to protein mapping based on mixed molecular dynamics. By using 16 probe molecules, the FTMap server finds the hot spots of an average-size protein in <1 h. As FTFlex performs mapping for all low-energy conformers of side chains in the binding site, its completion time is proportionately longer.
Journal Article
Exploring the structural origins of cryptic sites on proteins
Molecular dynamics (MD) simulations of proteins reveal the existence of many transient surface pockets; however, the factors determining what small subset of these represent druggable or functionally relevant ligand binding sites, called “cryptic sites,” are not understood. Here, we examine multiple X-ray structures for a set of proteins with validated cryptic sites, using the computational hot spot identification tool FTMap. The results show that cryptic sites in ligand-free structures generally have a strong binding energy hot spot very close by. As expected, regions around cryptic sites exhibit above-average flexibility, and close to 50% of the proteins studied here have unbound structures that could accommodate the ligand without clashes. Nevertheless, the strong hot spot neighboring each cryptic site is almost always exploited by the bound ligand, suggesting that binding may frequently involve an induced fit component. We additionally evaluated the structural basis for cryptic site formation, by comparing unbound to bound structures. Cryptic sites are most frequently occluded in the unbound structure by intrusion of loops (22.5%), side chains (19.4%), or in some cases entire helices (5.4%), but motions that create sites that are too open can also eliminate pockets (19.4%). The flexibility of cryptic sites frequently leads to missing side chains or loops (12%) that are particularly evident in low resolution crystal structures. An interesting observation is that cryptic sites formed solely by the movement of side chains, or of backbone segments with fewer than five residues, result only in low affinity binding sites with limited use for drug discovery.
Journal Article
Early postpartum HbA1c after hyperglycemia first detected in pregnancy—Imperfect but not without value
South African women of childbearing age are disproportionally affected by obesity and at significant risk of Type 2 Diabetes Mellitus (T2DM). Unless pregnant, they do not readily undergo screening for T2DM. With a local focus on improved antenatal care, hyperglycemia is often first detected in pregnancy (HFDP). This may erroneously be attributed to Gestational Diabetes Mellitus (GDM) in all without considering T2DM. Glucose evaluation following pregnancy is essential for early detection and management of women with T2DM in whom persistent hyperglycemia is to be expected. Conventional testing with an oral glucose tolerance test (OGTT) is cumbersome, prompting investigation for alternate solutions.
To compare the diagnostic performance of HbA1c to the current gold standard OGTT in women with HFDP 4-12 weeks post-delivery.
Glucose homeostasis was assessed with OGTT and HbA1c in 167 women with HFDP, 4-12 weeks after delivery. Glucose status was based on American Diabetes Association criteria.
Glucose homeostasis was assessed at 10 weeks (IQR 7-12) after delivery. Of the 167 participants, 52 (31%) had hyperglycemia, which was comprised of 34 (20%) prediabetes and 18 (11%) T2DM. Twelve women in the prediabetes subgroup had diagnostic fasting plasma glucose (FPG) and 2-hour plasma glucose (2hPG), but in two-thirds of the patients (22/34) only one time point proved diagnostic. The FPGs and the 2hPGs of six women with HbA1c-based T2DM were both within the prediabetes diagnostic range. According to the HbA1c measurements, 85% of 52 participants with gold standard OGTT defined hyperglycemia (prediabetes and T2DM) as well as 15 of 18 women with postpartum persistent T2DM were correctly classified. According to FPG, 15 women with persistent hyperglycemia would have been missed (11 with prediabetes and four with T2DM; 29%). When compared to an OGTT, a single HbA1c of 6.5% (48mmol/mol) postpartum demonstrated a sensitivity of 83% and specificity of 97% for the identification of T2DM.
HbA1c may improve access to postpartum testing in overburdened clinical settings where the required standards of OGTT cannot be guaranteed. HbA1c is a valuable test to detect women who will benefit most from early intervention but cannot unequivocally replace OGTT.
Journal Article
Prostaglandin E2 Signaling Mediates Oenocytoid Immune Cell Function and Lysis, Limiting Bacteria and Plasmodium Oocyst Survival in Anopheles gambiae
Lipid-derived signaling molecules known as eicosanoids have integral roles in mediating immune and inflammatory processes across metazoans. This includes the function of prostaglandins and their cognate G protein-coupled receptors (GPCRs) to employ their immunological actions. In insects, prostaglandins have been implicated in the regulation of both cellular and humoral immune responses, yet in arthropods of medical importance, studies have been limited. Here, we describe a prostaglandin E2 receptor ( Ag PGE2R) in the mosquito Anopheles gambiae and demonstrate that its expression is most abundant in oenocytoid immune cell populations. Through the administration of prostaglandin E2 (PGE2) and AgPGE2R- silencing, we demonstrate that prostaglandin E2 signaling regulates a subset of prophenoloxidases (PPOs) and antimicrobial peptides (AMPs) that are strongly expressed in populations of oenocytoids. We demonstrate that PGE2 signaling via the Ag PGE2R significantly limits both bacterial replication and Plasmodium oocyst survival. Additional experiments establish that PGE2 treatment increases phenoloxidase (PO) activity through the increased expression of PPO1 and PPO3 , genes essential to anti- Plasmodium immune responses that promote oocyst killing. We also provide evidence that the mechanisms of PGE2 signaling are concentration-dependent, where high concentrations of PGE2 promote oenocytoid lysis, negating the protective effects of lower concentrations of PGE2 on anti- Plasmodium immunity. Taken together, our results provide new insights into the role of PGE2 signaling on immune cell function and its contributions to mosquito innate immunity that promote pathogen killing.
Journal Article
A genome-wide CRISPR screen in Anopheles mosquito cells identifies fitness and immune cell function-related genes
Anopheles
mosquitoes are the sole vector of malaria, the most burdensome vector-borne disease worldwide. At present, strategies for reducing mosquito populations or limiting their ability to transmit disease show the most promise for disease control. Therefore, improving our understanding of mosquito biology and immune function may aid new approaches to limit malaria transmission. Here, we perform genome-wide CRISPR screens in
Anopheles
mosquito cells to identify genes required for fitness and that confer resistance to clodronate liposomes, which are used to ablate immune cells. The cellular fitness screen identifies 1280 fitness-related genes (393 at highest confidence) that are highly enriched for roles in fundamental cell processes. The clodronate screen identifies resistance factors that impair clodronate liposome function. For the latter, we confirm roles in liposome uptake and processing through in vivo validation in
Anopheles gambiae
that provide new mechanistic detail of phagolysosome formation and clodronate liposome processing. Altogether, we present a genome-wide CRISPR knockout platform in a major malaria vector and identify genes important for fitness and immune-related processes.
Mosquitoes are major vectors for the transmission of many serious pathogens. This study uses genome-wide CRISPR screens in the mosquito, Anopheles gambiae, to reveal new insights into mosquito fitness and the function of clodronate-liposome mediated immune cell ablation.
Journal Article
Structural conservation of druggable hot spots in protein–protein interfaces
Despite the growing number of examples of small-molecule inhibitors that disrupt protein–protein interactions (PPIs), the origin of druggability of such targets is poorly understood. To identify druggable sites in protein–protein interfaces we combine computational solvent mapping, which explores the protein surface using a variety of small \"probe\" molecules, with a conformer generator to account for side-chain flexibility. Applications to unliganded structures of 15 PPI target proteins show that the druggable sites comprise a cluster of binding hot spots, distinguishable from other regions of the protein due to their concave topology combined with a pattern of hydrophobic and polar functionality. This combination of properties confers on the hot spots a tendency to bind organic species possessing some polar groups decorating largely hydrophobic scaffolds. Thus, druggable sites at PPI are not simply sites that are complementary to particular organic functionality, but rather possess a general tendency to bind organic compounds with a variety of structures, including key side chains of the partner protein. Results also highlight the importance of conformational adaptivity at the binding site to allow the hot spots to expand to accommodate a ligand of drug-like dimensions. The critical components of this adaptivity are largely local, involving primarily low energy side-chain motions within 6 Å of a hot spot. The structural and physicochemical signature of druggable sites at PPI interfaces is sufficiently robust to be detectable from the structure of the unliganded protein, even when substantial conformational adaptation is required for optimal ligand binding.
Journal Article
Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection
The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called ‘FLip’ mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.
Many emerging SARS-CoV-2 variants partially escape the humoral immune response. Here, Liu et al. characterize 28 antibodies from BA.4/5 breakthrough infections and find attrition of neutralization and complete loss of function for variants with Spike mutations at positions 455 and 456.
Journal Article
Mosquito immune cells enhance dengue and Zika virus infection in Aedes aegypti
Mosquito-borne arboviruses cause more than 400 million annual infections, yet despite their public health importance, the mechanisms by which arboviruses infect and disseminate in the mosquito host are not well understood. Here, we provide evidence that dengue virus and Zika virus actively infect
Aedes aegypti
hemocytes and demonstrate, through phagocyte depletion, that hemocytes facilitate virus infection to peripheral tissues including the ovaries and salivary glands. Adoptive transfer experiments further reveal that virus-infected hemocytes efficiently confer virus infection to naïve recipient mosquitoes. Together, these data support a model of arbovirus dissemination where infected hemocytes enhance virus infection of mosquito tissues required for transmission, which parallels vertebrate systems where immune cell populations promote virus dissemination. This study significantly advances our understanding of virus infection dynamics in the mosquito host and highlights potential conserved roles of immune cells in arbovirus infection across vertebrate and invertebrate systems.
Infection of mosquito immune cells by dengue and Zika virus enhances the spread of virus infection to mosquito tissues, such as the salivary glands, to promote virus transmission and highlights conserved roles of immune cells in virus dissemination.
Journal Article