Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin‐dependent trafficking

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti‐HCV action ofsilymarin‐derived compounds at the molecular level. By using live‐cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV‐infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin‐coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin‐coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well‐known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin‐mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad‐spectrum antiviral therapy.     

Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.     

Evolution of Influenza B Virus in Kuala Lumpur,Malaysia, between 1995 and 2008

Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.     

What Do Eye Gaze Metrics Tell Us about Motor Imagery?

Many of the brain structures involved in performing real movements also have increased activity during imagined movements or during motor observation, and this could be the neural substrate underlying the effects of motor imagery in motor learning or motor rehabilitation. In the absence of any objective physiological method of measurement, it is currently impossible to be sure that the patient is indeed performing the task as instructed. Eye gaze recording during a motor imagery task could be a possible way to “spy” on the activity an individual is really engaged in. The aim of the present study was to compare the pattern of eye movement metrics during motor observation, visual and kinesthetic motor imagery (VI, KI), target fixation, and mental calculation. Twenty-two healthy subjects (16 females and 6 males), were required to perform tests in five conditions using imagery in the Box and Block Test tasks following the procedure described by Liepert et al. Eye movements were analysed by a non-invasive oculometric measure (SMI RED250 system). Two parameters describing gaze pattern were calculated: the index of ocular mobility (saccade duration over saccade + fixation duration) and the number of midline crossings (i.e. the number of times the subjects gaze crossed the midline of the screen when performing the different tasks). Both parameters were significantly different between visual imagery and kinesthesic imagery, visual imagery and mental calculation, and visual imagery and target fixation. For the first time we were able to show that eye movement patterns are different during VI and KI tasks. Our results suggest gaze metric parameters could be used as an objective unobtrusive approach to assess engagement in a motor imagery task. Further studies should define how oculomotor parameters could be used as an indicator of the rehabilitation task a patient is engaged in.     

Construction of Chromosome Segment Substitution Lines in Peanut (Arachis hypogaea L.) Using a Wild Synthetic and QTL Mapping for Plant Morphology

Chromosome segment substitution lines (CSSLs) are powerful QTL mapping populations that have been used to elucidate the molecular basis of interesting traits of wild species. Cultivated peanut is an allotetraploid with limited genetic diversity. Capturing the genetic diversity from peanut wild relatives is an important objective in many peanut breeding programs. In this study, we used a marker-assisted backcrossing strategy to produce a population of 122 CSSLs from the cross between the wild synthetic allotetraploid (A. ipaënsis×A. duranensis)^4x and the cultivated Fleur11 variety. The 122 CSSLs offered a broad coverage of the peanut genome, with target wild chromosome segments averaging 39.2 cM in length. As a demonstration of the utility of these lines, four traits were evaluated in a subset of 80 CSSLs. A total of 28 lines showed significant differences from Fleur11. The line×trait significant associations were assigned to 42 QTLs: 14 for plant growth habit, 15 for height of the main stem, 12 for plant spread and one for flower color. Among the 42 QTLs, 37 were assigned to genomic regions and three QTL positions were considered putative. One important finding arising from this QTL analysis is that peanut growth habit is a complex trait that is governed by several QTLs with different effects. The CSSL population developed in this study has proved efficient for deciphering the molecular basis of trait variations and will be useful to the peanut scientific community for future QTL mapping studies.     

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.     

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecular basis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct pharmacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation.     

Protein-Protein Interactions in a Crowded Environment: An Analysis via Cross-Docking Simulations and Evolutionary Information

Large-scale analyses of protein-protein interactions based on coarse-grain molecular docking simulations and binding site predictions resulting from evolutionary sequence analysis, are possible and realizable on hundreds of proteins with variate structures and interfaces. We demonstrated this on the 168 proteins of the Mintseris Benchmark 2.0. On the one hand, we evaluated the quality of the interaction signal and the contribution of docking information compared to evolutionary information showing that the combination of the two improves partner identification. On the other hand, since protein interactions usually occur in crowded environments with several competing partners, we realized a thorough analysis of the interactions of proteins with true partners but also with non-partners to evaluate whether proteins in the environment, competing with the true partner, affect its identification. We found three populations of proteins: strongly competing, never competing, and interacting with different levels of strength. Populations and levels of strength are numerically characterized and provide a signature for the behavior of a protein in the crowded environment. We showed that partner identification, to some extent, does not depend on the competing partners present in the environment, that certain biochemical classes of proteins are intrinsically easier to analyze than others, and that small proteins are not more promiscuous than large ones. Our approach brings to light that the knowledge of the binding site can be used to reduce the high computational cost of docking simulations with no consequence in the quality of the results, demonstrating the possibility to apply coarse-grain docking to datasets made of thousands of proteins. Comparison with all available large-scale analyses aimed to partner predictions is realized. We release the complete decoys set issued by coarse-grain docking simulations of both true and false interacting partners, and their evolutionary sequence analysis leading to binding site predictions. Download site: http://www.lgm.upmc.fr/CCDMintseris/