Targeting VEGF signalling via the neuropilin co-receptor.

The blockade of tumour vascularisation and angiogenesis continues to be a focus for drug development in oncology and other pathologies. Historically, targeting vascular endothelial growth factor (VEGF) activity and its association with VEGF receptors (VEGFRs) has represented the most promising line of attack. More recently, the recognition that VEGFR co-receptors, neuropilin-1 and -2 (NRP1 and NRP2), are also engaged by specific VEGF isoforms in tandem with the VEGFRs has expanded the landscape for the development of modulators of VEGF-dependent signalling. Here, we review the recent structural characterisation of VEGF interactions with NRP subdomains and the impact this has had on drug development activity in this area

Crystal structures of PI3K-C2 alpha PX domain indicate conformational change associated with ligand binding

Background: PX domains have specialized protein structures involved in binding of phosphoinositides ( PIs). Through binding to the various PIs PX domains provide site- specific membrane signals to modulate the intracellular localisation and biological activity of effector proteins. Several crystal structures of these domains are now available from a variety of proteins. All PX domains contain a canonical core structure with main differences exhibited within the loop regions forming the phosphoinositide binding pockets. It is within these areas that the molecular basis for ligand specificity originates.Results: We now report two new structures of PI3K-C2 alpha PX domain that crystallised in a P3(1)21 space group. The two structures, refined to 2.1 angstrom and 2.5 angstrom, exhibit significantly different conformations of the phosphoinositide-binding loops. Unexpectedly, in one of the structures, we have detected a putative-ligand trapped in the binding site during the process of protein purification and crystallisation.Conclusion: The two structures reported here provide a more complete description of the phosphoinositide binding region compared to the previously reported 2.6 angstrom crystal structure of human PI3K-C2 alpha PX where this region was highly disordered. The structures enabled us to further analyse PI specificity and to postulate that the observed conformational change could be related to ligand-binding

Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family

Dynamic Allostery in PLC gamma 1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation

Spatial patterns of mercury in biota of Adirondack, New York lakes

We studied the spatial distribution patterns of mercury (Hg) in lake water, littoral sediments, zooplankton, crayfish, fish, and common loons in 44 lakes of the Adirondacks of New York State, USA, a region that has been characterized as a “biological Hg hotspot”. Our study confirmed this pattern, finding that a substantial fraction of the lakes studied had fish and loon samples exceeding established criteria for human and wildlife health. Factors accounting for the spatial variability of Hg in lake water and biota were lake chemistry (pH, acid neutralizing capacity (ANC), percent carbon in sediments), biology (taxa presence, trophic status) and landscape characteristics (land cover class, lake elevation). Hg concentrations in zooplankton, fish and common loons were negatively associated with the lake water acid-base status (pH, ANC). Bioaccumulation factors (BAF) for methyl Hg (MeHg) increased from crayfish (mean log10 BAF = 5.7), to zooplankton (5.9), to prey fish (6.2), to larger fish (6.3), to common loons (7.2). MeHg BAF values in zooplankton, crayfish, and fish (yellow perch equivalent) all increased with increasing lake elevation. Our findings support the hypothesis that bioaccumulation of MeHg at the base of the food chain is an important controller of Hg concentrations in taxa at higher trophic levels. The characteristics of Adirondack lake-watersheds (sensitivity to acidic deposition; significant forest and wetland land cover; and low nutrient inputs) contribute to elevated Hg concentrations in aquatic biota

Supervised learning for the automated transcription of spacer classification from spoligotype films

<p>Abstract</p> <p>Background</p> <p>Molecular genotyping of bacteria has revolutionized the study of tuberculosis epidemiology, yet these established laboratory techniques typically require subjective and laborious interpretation by trained professionals. In the context of a Tuberculosis Case Contact study in The Gambia we used a reverse hybridization laboratory assay called spoligotype analysis. To facilitate processing of spoligotype images we have developed tools and algorithms to automate the classification and transcription of these data directly to a database while allowing for manual editing.</p> <p>Results</p> <p>Features extracted from each of the 1849 spots on a spoligo film were classified using two supervised learning algorithms. A graphical user interface allows manual editing of the classification, before export to a database. The application was tested on ten films of differing quality and the results of the best classifier were compared to expert manual classification, giving a median correct classification rate of 98.1% (inter quartile range: 97.1% to 99.2%), with an automated processing time of less than 1 minute per film.</p> <p>Conclusion</p> <p>The software implementation offers considerable time savings over manual processing whilst allowing expert editing of the automated classification. The automatic upload of the classification to a database reduces the chances of transcription errors.</p

Male reproductive health and environmental xenoestrogens

EHP is a publication of the U.S. government. Publication of EHP lies in the public domain and is therefore without copyright. Research articles from EHP may be used freely; however, articles from the News section of EHP may contain photographs or figures copyrighted by other commercial organizations and individuals that may not be used without obtaining prior approval from both the EHP editors and the holder of the copyright. Use of any materials published in EHP should be acknowledged (for example, "Reproduced with permission from Environmental Health Perspectives") and a reference provided for the article from which the material was reproduced.Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood. Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.Supported by EU Contract BMH4-CT96-0314

Integrating organizational, social, and individual perspectives in Web 2.0-based workplace e-learning

From the issue entitled 'Special Issue: Emerging Social and Legal Aspect'E-learning is emerging as a popular approach of education in the workplace by virtue of its flexibility to access, just-in-time delivery, and cost-effectiveness. To improve social interaction and knowledge sharing in e-learning, Web 2.0 is increasingly utilized and integrated with e-learning applications. However, existing social learning systems fail to align learning with organizational goals and individual needs in a systemic way. The dominance of technology-oriented approaches makes elearning applications less goal-effective and poor in quality and design. To solve the problem, we address the requirement of integrating organizational, social, and individual perspectives in the development of Web 2.0 elearning systems. To fulfill the requirement, a key performance indicator (KPI)-oriented approach is presented in this study. By integrating a KPI model with Web 2.0 technologies, our approach is able to: 1) set up organizational goals and link the goals with expertise required for individuals; 2) build a knowledge network by linking learning resources to a set of competences to be developed and a group of people who learn and contribute to the knowledge network through knowledge creation, sharing, and peer evaluation; and 3) improve social networking and knowledge sharing by identifying each individual's work context, expertise, learning need, performance, and contribution. The mechanism of the approach is explored and elaborated with conceptual frameworks and implementation technologies. A prototype system for Web 2.0 e-learning has been developed to demonstrate the effectiveness of the approach. © Springer Science + Business Media, LLC 2009.postprin

Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of β2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis.

IgG aPL against domain I of β2-glycoprotein I (β2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS

Overexpression of cathepsin K during silica-induced lung fibrosis and control by TGF-β

BACKGROUND: Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s) (Cat) are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis. METHODS: Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B) was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts. RESULTS: In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-β expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-β1. CONCLUSION: Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-β appears to limit its overexpression in response to silica particles