African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ∼5nm, connected to the membrane by a 6nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER. © 2015 John WileyDeutsche Forschungsgemeinschaft personal grant KR2173 to Jacomine Krijnse Locker. German Andres is supported by the ‘Amarouto Program for senior scientists from the Comunidad Autónoma de Madrid’ and by grants BFU2009-08085 and AGL2013-48998-C2- 2-R from the Spanish Ministerio de Economía y CompetitividadPeer Reviewe

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.Fil: Bussi, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Peralta Ramos, Javier María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Arroyo, Daniela Soledad. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Ronchi, Paolo. European Molecular Biology Laboratory; AlemaniaFil: Kolovou, Androniki. European Molecular Biology Laboratory; AlemaniaFil: Wang, Ji M.. National Cancer Institute at Frederick; Estados UnidosFil: Florey, Oliver. Babraham Institute; Reino UnidoFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Schwab, Yannick. European Molecular Biology Laboratory; AlemaniaFil: Ktistakis, Nicholas. Babraham Institute; Reino UnidoFil: Iribarren, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; Argentin

Membrane acquisition of African swine fever virus uses open intermediates, similar to mimi- and Vaccinia virus

Trabajo presentado en la XX International Poxvirus, Asfarvirus & Iridovirus Conference, celebrada en Victoria (Canadá) del 26 al 30 de septiembre de 2014

Application of electron tomography to the study of the africanswinw fever virus morphogenesis

Trabajo presentado en la XIX international poxvirus, asfarvirus & iridovirus conference, celebrada en Salamanca (España) del 24 al 28 de junio de 2012

Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model

Nanoparticles which surface adsorb proteins in an uncontrolled and non-reproducible manner will have limited uses as nanomedicinal products. A promising approach to avoid nanoparticle non-specific interactions with proteins is to design bio-hybrids by purposely pre-forming a protein corona around the inorganic cores. Here, we investigate, in vitro and in vivo, the newly acquired bio-identity of superparamagnetic iron oxide nanoparticles (SPIONs) upon their functionalization with a pre-formed and well-defined bovine serum albumin (BSA) corona. Cellular uptake, intracellular particle distribution and cytotoxicity were studied in two cell lines: adherent and non-adherent cells. BSA decreases nanoparticle internalization in both cell lines and protects the iron core once they have been internalized. The physiological response to the nanoparticles is then in vivo evaluated by oral administration to Caenorhabditis elegans, which was selected as a model of a functional intestinal barrier. Nanoparticle biodistribution, at single particle resolution, is studied by transmission electron microscopy. The analysis reveals that the acidic intestinal environment partially digests uncoated SPIONs but does not affect BSA-coated ones. It also discloses that some particles could enter the nematode's enterocytes, likely by endocytosis which is a different pathway than the one described for the worm nutrients. Statement of Significance Unravelling meaningful relationships between the physiological impact of engineered nanoparticles and their synthetic and biological identity is of vital importance when considering nanoparticles biomedical uses and when establishing their nanotoxicological profile. This study contributes to better comprehend the inorganic nanoparticles’ behavior in real biological milieus. We synthesized a controlled pre-formed BSA protein corona on SPIONs to lower unspecific cell uptake and decrease nanoparticle fouling with other proteins. Such findings may be of relevance considering clinical translation and regulatory issues of inorganic nanoparticles. Moreover, we have advanced in the validation of C. elegans as a simple animal model for assessing biological responses of engineering nanomaterials. The physiological response of BSA coated SPIONs was evaluated in vivo after their oral administration to C. elegans. Analyzing ultra-thin cross-sections of the worms by TEM with single-particle precision, we could track NP biodistribution along the digestive tract and determine unambiguously their translocation through biological barriers and cell membranes.This research was partially funded by the Spanish Ministry of Economy (MAT2015-64442-R, Ramon y Cajal program (AL, RyC-2010-06082), FPU program (LGM, FPU12/05549), Severo Ochoa Program (SEV-2015-0496) co-funded by European Social Funds), the Generalitat de Catalunya (2014SGR213), People Program of the European Commission (grant agreement no. 303630, co-funded by the European Social Fund), a Chinese Scholarship Council fellowship (SMY, 201206150053), the Christian Boulin fellowship 2015 from EMBL (LGM), the COST Actions HINT (Action No. MP1202), and GENIE (Action No. BM1408-A). We thank Lorenzo Albertazzi (IBEC, Spain) for the critical reading of the manuscript.Peer Reviewe

Effect of Moderate Wine Consumption on Oxidative Stress Markers in Coronary Heart Disease Patients

Evidence from research studies reports that wine consumption is associated with lower cardiovascular disease risk, partly through the amelioration of oxidative stress. The aim of the present study was to examine the effect of regular light to moderate wine consumption from coronary heart disease (CHD) patients compared to the effect induced by alcohol intake without the presence of wine microconstituents, on oxidation-induced macromolecular damage as well as on endogenous antioxidant enzyme activity. A randomized, single-blind, controlled, three-arm parallel intervention was carried out, in which 64 CHD patients were allocated to three intervention groups. Group A consumed no alcohol, and Group B (wine) and Group C (ethanol) consumed 27 g of alcohol/day for 8 weeks. Blood and urine samples were collected at baseline and at 4 and 8 weeks. Urine oxidized guanine species levels, protein carbonyls, thiobarbituric acid substances (TBARS) levels, as well as superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, were measured. Oxidized guanine species and protein carbonyl levels were significantly increased in the ethanol group during the intervention and were significantly decreased in the wine group. These results support the idea that wine’s bioactive compounds may exert antioxidant actions that counteract the macromolecular oxidative damage induced by alcohol in CHD patients

Data from: AFRICAN SWINE FEVER VIRUS ASSEMBLES A SINGLE MEMBRANE DERIVED FROM RUPTURE OF THE ENDOPLASMIC RETICULUM

<p>Collective evidence argues that two members of the Nucleo-cytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus (ASFV). By electron tomography (ET) the virion assembles a single bilayer derived open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal ER proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ~5nm, connected to the membrane by a 6nm wide structure displaying thin striations, as assessed by several complementary EM imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.</p