Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Magnetic and in vitro heating properties of implants formed in situ from injectable formulations and containing superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microparticles for magnetically induced local hyperthermia

The biological and therapeutic responses to hyperthermia, when it is envisaged as an anti-tumor treatment modality, are complex and variable. Heat delivery plays a critical role and is counteracted by more or less efficient body cooling, which is largely mediated by blood flow. In the case of magnetically mediated modality, the delivery of the magnetic particles, most often superparamagnetic iron oxide nanoparticles (SPIONs), is also critically involved. We focus here on the magnetic characterization of two injectable formulations able to gel in situ and entrap silica microparticles embedding SPIONs. These formulations have previously shown suitable syringeability and intratumoral distribution in vivo. The first formulation is based on alginate, and the second on a poly(ethylene-co-vinyl alcohol) (EVAL). Here we investigated the magnetic properties and heating capacities in an alternating magnetic field (141 kHz, 12 mT) for implants with increasing concentrations of magnetic microparticles. We found that the magnetic properties of the magnetic microparticles were preserved using the formulation and in the wet implant at 37 degrees C, as in vivo. Using two orthogonal methods, a common SLP (20 Wg(-1)) was found after weighting by magnetic microparticle fraction, suggesting that both formulations are able to properly carry the magnetic microparticles in situ while preserving their magnetic properties and heating capacities. (C) 2010 Elsevier B.V. All rights reserved.

Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome.

Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide.

Altered structural connectivity in patients with medial temporal lobe epilepsy: A Diffusion Spectrum Imaging and Graph Analysis study

In this study we investigated the effect of medial temporal lobe epilepsy (MTLE) on the global characteristics of brain connectivity estimated by topological measures. We used DSI (Diffusion Spectrum Imaging) to construct a connectivity matrix where the nodes represents the anatomical ROIs and the edges are the connections between any pair of ROIs weighted by the mean GFA/FA values. A significant difference was found between the patient group vs control group in characteristic path length, clustering coefficient and small-worldness. This suggests that the MTLE network is less efficient compared to the network of the control group.

Induction of innate immune responses through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants such as asbestos or silica is linked to inflammation of the lung, fibrosis and lung cancer. How the presence of pathogenic dust is recognised, and how chronic inflammatory diseases are triggered are poorly understood. We will se show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to IL-1b secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. In a model of asbestos inhalation, Nalp3_/_ mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory ''danger" receptor.

A Structural Analysis of the Health Expenditures and Portfolio Choices of Retired Agents

Richer and healthier agents tend to hold riskier portfolios and spend proportionally less on health expenditures. Potential explanations include health and wealth effects on preferences, expected longevity or disposable total wealth. Using HRS data, we perform a structural estimation of a dynamic model of consumption, portfolio and health expenditure choices with recursive utility, as well as health-dependent income and mortality risk. Our estimates of the deep parameters highlight the importance of health capital, mortality risk control, convex health and mortality adjustment costs and binding liquidity constraints to rationalize the stylized facts. They also provide new perspectives on expected longevity and on the values of life and health.

Repression and Protest Structural Models and Strategic Interactions

The recent wave of upheavals and revolts in Northern Africa and the Middle East goes back to an old question often raised by theories of collective action: does repression act as a negative or positive incentive for further mobilization? Through a review of the vast literature devoted to this question, this article aims to go beyond theoretical and methodological dead-ends. The article moves on to non-Western settings in order to better understand, via a macro-sociological and dynamic approach, the causal effects between mobilizations and repression. It pleads for a meso- and micro-level approach to this issue: an approach that puts analytical emphasis both on protest organizations and on individual activists' careers.

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor

Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

In-vivo magnetic resonance imaging of the structural core of the Papez circuit in humans.

Papez circuit is one of the major pathways of the limbic system, and it is involved in the control of memory and emotion. Structural and functional alterations have been reported in psychiatric, neurodegenerative, and epileptic diseases. Despite the clinical interest, however, in-vivo imaging of the entire circuit remains a technological challenge. We used magnetic resonance diffusion spectrum imaging to comprehensively picture the Papez circuit in healthy humans: (i) the hippocampus-mammillary body pathway, (ii) the connections between the lateral subiculum and the cingulate cortex, and (iii) the mammillo-thalamic tract. The diagnostic and therapeutic implications of these results are discussed in the context of recent findings reporting the involvement of the Papez circuit in neurological and psychiatric diseases.

Interactions of Adeno-associated virus Rep78 protein with the host cell

Abstract : Adeno-associated virus (AAV) is a small DNA virus belonging to the familiy of Parvoviridae. Its genome contains two genes : the rep gene encoding four non structural proteins (Rep78, 68, 52 and 40) implicated in transcription, replication and site-specific integration of the viral DNA and the cap gene encoding three capsid proteins. AAV does not cause any disease, but is studied in view of its potential use to treat several diseases. An interesting property of AAV is its antiproliferative effect. Two elements of AAV can inhibit cell growth. Firstly, the single stranded viral DNA is recognized in cells as damaged DNA leading to either a G2 block or cell death depending on p53 status. Secondly, the two larger Rep proteins (Rep78 and 68) also arrest the cell cycle when they are expressed at high levels. Rep78 in particular induces a complete cell cycle arrest in all the phases, including S phase. Such a strong S phase arrest is rarely seen in other conditions. It was thus interesting to determine how Rep78 could induce it. We found that this strong block is the consequence of Rep78's effects on at least two pathways. Rep78 induces a DNA damage response by producing nicks in the cellular chromatin. Furthermore, Rep78 can bind to the cellular phosphatase Cdc25A and prevent its binding to its substrates CDK2 and CDK1, thus inhibiting its activity. A mutational analysis of Rep78 protein determined that its endonuclease activity is responsible for the DNA damage response and its zinc finger domain for Cdc25A inhibition. The combined expression of two mutants each defective for one of these activities, or these two activities obtained independently of Rep78, could restore the complete cell cycle block, indicating that these two effects of Rep78 are likely to explain completely the cell cycle block it induces. Secondly, the lack of pathogenicity of AAV, its broad range of infection and its ability to integrate site-specifically in human chromosome 19 make it an interesting potential vector for gene therapy. However site-specific integration is only possible in the presence of Rep78/68 whose gene is removed in recombinant AAV vectors. In this part of the study, we tried to introduce Rep protein separately from recombinant AAV vectors to promote their site-specific integration. For that purpose, a fusion protein, TAT-Rep, comprising Rep78/68 joined to the human immunodeficiency virus Tat protein was produced. It had the ability to enter cells and remain active there for a short period. Its activity was sufficient to mediate transcription from the p5 promoter, second-strand synthesis of a recombinant AAV and probably site-specific integration. Résumé : Le virus associé à l'adénovirus (AAV) est un petit virus à ADN qui fait partie de la famille des Parvoviridae. Son génome contient deux gènes : le gène rep code pour quatre protéines (Rep78, 68, 52 et 40) qui participent à la transcription, la réplication et l'intégration du virus et le gène cap code pour les trois protéines de capside. AAV ne produit pas de maladie, mais pourrait au contraire être utilisé pour en soigner. Sa bénignité, sa capacité à infecter différents types de cellules et son intégration spécifique en font un vecteur potentiel pour la thérapie génique. Pour qu'il puisse s'intégrer spécifiquement, il a besoin de la protéine Rep78 ou 68, mais ce gène doit être enlevé des vecteurs pour la thérapie génique. Le but de la première partie de cette étude était d'introduire Rep78 ou 68 dans des cellules en même temps qu'un AAV recombinant, mais indépendamment afin de permettre une intégration spécifique. La stratégie utilisée était de produire une protéine de fusion (TAT-Rep) qui peut entrer dans des cellules si elle est présente dans leur milieu. Cette protéine entrait bien dans les cellules et y était active favorisant ainsi l'intégration spécifique. Une deuxième propriété d'AAV, son effet anti-prolifératif, est intéressante dans le cadre de certaines maladies comme le cancer. Deux éléments d'AAV en sont responsables. D'abord, son ADN simple brin active une réponse cellulaire à l'ADN endommagé et arrête les cellules en G2 ou provoque leur mort. De plus, la protéine Rep78 d'AAV peut fortement bloquer le cycle cellulaire à toutes les phases, même en phase S, ce qui est rare. C'est pourquoi nous avons essayé de comprendre cet effet. Nous avons remarqué que Rep78 doit agir sur deux fronts pour obtenir ce fort bloc. D'un côté, Rep78 introduit des coupures simple brin sur l'ADN de la cellule ce qui active une réponse cellulaire à l'ADN endommagé qui passe par ATM. D'un autre côté, Rep78 lie une phosphatase cellulaire, Cdc25A, et l'empêche ainsi de lier ses substrats CDK2 et CDK1 et donc d'être active. Finalement, à l'aide de mutants de Rep78, nous avons déterminé que l'activité endonuclease de Rep78 était nécessaire pour induire une réponse cellulaire via ATM et que le domaine C-terminal appelé «zinc finger » était responsable de la liaison avec Cdc25A. En co-exprimant deux mutants, qui n'ont chacun qu'un des effets de Rep78, ou en obtenant les deux effets de Rep78 indépendamment d'elle, nous avons obtenu un bloc complet du cycle cellulaire similaire à celui obtenu avec Rep78. Il est donc probable que ces deux effets de Rep78 sont suffisants pour expliquer comment elle arrive à arrêter le cycle cellulaire si efficacement.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

Mapping the structural core of human cerebral cortex.

Structurally segregated and functionally specialized regions of the human cerebral cortex are interconnected by a dense network of cortico-cortical axonal pathways. By using diffusion spectrum imaging, we noninvasively mapped these pathways within and across cortical hemispheres in individual human participants. An analysis of the resulting large-scale structural brain networks reveals a structural core within posterior medial and parietal cerebral cortex, as well as several distinct temporal and frontal modules. Brain regions within the structural core share high degree, strength, and betweenness centrality, and they constitute connector hubs that link all major structural modules. The structural core contains brain regions that form the posterior components of the human default network. Looking both within and outside of core regions, we observed a substantial correspondence between structural connectivity and resting-state functional connectivity measured in the same participants. The spatial and topological centrality of the core within cortex suggests an important role in functional integration.