Biomaterials

Cell-assembled extracellular matrix (CAM) has been used to produce vascular grafts. While these completely biological vascular grafts performed well in clinical trials, the in vivo remodeling and inflammatory response of this truly "bio" material has not yet been investigated. In this study, human CAM yarns were implanted subcutaneously in nude rats to investigate the innate immune response to this matrix. The impact of processing steps relevant to yarn manufacturing was evaluated (devitalization, decellularization, gamma sterilization, and twisting). We observed that yarns were still present after six months, and were integrated into a non-inflamed loose connective tissue. The CAM was repopulated by fibroblastic cells and blood vessels. While other yarns caused minor peripheral inflammation at an early stage (two weeks of implantation), gamma sterilization triggered a more intense host response dominated by the presence of M1 macrophages. The inflammatory response was resolved at six months. Yarn mechanical strength was decreased two weeks after implantation except for the more compact "twisted" yarn. While the strength of other yarns was stable after initial remodeling, the gamma-sterilized yarn continued to lose mechanical strength over time and was weaker than devitalized (control) yarns at six months. This is the first study to formally demonstrate that devitalized human CAM is very long-lived in vivo and does not trigger a degradative response, but rather is very slowly remodeled. This data supports a strategy to produce human textiles from CAM yarn for regenerative medicine applications where a scaffold with low inflammation and long-term mechanical properties are critical

Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses

Étanchéité des différents types de connexions implantaires : conséquences biologiques et mécaniques

Most dental implant systems consist of two main parts: the abutment and the implant body. The implant-abutment connection plays an essential role in implant stability and is often cited as responsible for peri-implant bone loss. This work is a review of the literature on the different types of implant connections, their biological and mechanical behavior and their consequences. The first part will present an overview of the existing types of implants and,in particular,the different types of connections available.In the second part, the effect of micro-gap on bacterial sealing capability and its consequences will be presented. The existing types of implant-abutment connections will be reviewed to establish their strengths and weaknesses. The third part will focus on the connections’ biomechanical aspects and their consequences on sealing and longevity. The different type of implant-abutment connections will be reviewed as well.La plupart des systèmes implantaires sont composés de deux parties : l’implant et le pilier. La connexion entre ces deux parties joue un rôle essentiel dans la stabilité implantaire et est souvent citée comme un des facteurs impliqués dans perte osseuse peri-implantaire. Ce travail propose donc d’effectuer un état des lieux de la littérature portant sur les différents types de connexions implantaires, de leur comportement biologiques et mécaniques et de leurs conséquences afin de les comparer. Dans une première partie les différents types de systèmes implantaires et de connexions existantes seront présentés.Dans une seconde partie l’influence du micro-gap sur l’étanchéité bactérienne et ses conséquences sera présentée. Les différents types de connexions implantaires actuellement disponibles seront passésen revue afin de déterminer leurs points forts et leurs points faibles. La troisième partie se penchera sur les aspects biomécaniques du omportement des connexions et sur leurs conséquences sur l’étanchéité et la pérennité. Une étude de la littérature sera également effectuée afin de comparer les différents types de connexions

Stratégies de formation de la structure coiffe chez les virus à ARN

La plupart des virus utilisent la machinerie de traduction dépendante de la coiffe pour assurer l’expression de leurs ARN messagers (ARNm). L’addition d’une structure coiffe à l’extrémité 5’ des ARNm viraux est par conséquent une étape essentielle pour la réplication de nombreux virus. En effet, la coiffe protège les ARNm de la dégradation par les nucléases cellulaires et neutralise la détection des ARNm viraux par les mécanismes de l’immunité innée. L’acquisition de la coiffe des ARN viraux se fait soit en utilisant les enzymes cellulaires de formation de la coiffe de la cellule infectée ou en subtilisant la coiffe des ARNm de la cellule infectée, soit par des machineries enzymatiques virales dédiées. De nombreuses enzymes virales impliquées dans la synthèse de la coiffe ont récemment été caractérisées du point de vue structural et fonctionnel. Ces études ont révélé des mécanismes de synthèse originaux qui ouvrent la voie pour le développement d’inhibiteurs spécifiques à potentiel antiviral

J Tissue Eng

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts . A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures

Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex.

International audienceTo date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

International audienceCoronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold