Transapical aortic ‘valve-in-valve' procedure for degenerated stented bioprosthesis

Standard surgical aortic valve replacement with a biological prosthesis remains the treatment of choice for low- and mid-risk elderly patients (traditionally >65 years of age) suffering from severe symptomatic aortic valve stenosis or insufficiency, and for young patients with formal contraindications to long-lasting anticoagulation. Unfortunately, despite the fact that several technical improvements have noticeably improved the resistance of pericardial and bovine bioprostheses to leaflet calcifications and ruptures, the risk of early valve failure with rapid degeneration still exists, especially for patients under haemodialysis and for patients <60 years of age at the time of surgery. Until now, redo open heart surgery under cardiopulmonary bypass and on cardioplegic arrest was the only available therapeutic option in case of bioprosthesis degeneration, but it carried a higher surgical risk when elderly patients with severe concomitant comorbidities were concerned. Since a few years, the advent of new transcatheter aortic valve procedures has opened new horizons in cardiac surgery and, in particular, the possibility of implanting stented valves within the degenerated stented bioprosthesis, the so-called ‘valve-in-valve' (VinV) concept, has become a clinical practice in experienced cardiac centres. The VinV procedure represents a minimally invasive approach dedicated to high-risk redo patients, and published preliminary reports have shown a success rate of 100% with absence of significant valvular leaks, acceptable transvalvular gradients and low complication rate. However, this procedure is not riskless and the most important concerns are about the size mismatch and the right positioning within the degenerated bioprosthesis. In this article, we review the limited available literature about VinV procedures, underline important technical details for the positioning and provide guidelines to prevent valve-prosthesis mismatch comparing the three sizes of the only commercially available transapical device, the Edwards Sapien™, with the inner diameter of three of the most commonly used stented bioprosthese

On-demand assembly of macromolecules used for the design and application of targeted secretion inhibitors

Neurological and endocrine pathologies such as acromegalie, Cushing’s disease, and neuropathic pain display disregulated exocytosis. Silencing specific cell populations would thus be invaluable to correct these debilitating disorders. To achieve this goal, we reengineered the Botulinum neurotoxin (BoT), a highly potent pharmaceutical compound capable of inhibiting exocytosis, and fused to it a protein “stapling” domain [1,2]. These peptide motifs, that form an irreversible tetrahelical coiled-coil, are able to link a variety of targeting domains onto the enzyme and thus redirect it towards normally unaffected cells. The conformational diversity of this assembly process greatly supersedes traditional protein expression since multiple targeting domains (homo- and hetero-) can be linked onto one scaffold, larger yields can be produced separately, it permits the combination of solid-phase peptide synthesis with recombinant protein expression, and it can avoid the necessity of an N- to C- translational fusion. With only a few dozen building “blocks” it is possible to construct thousands of different complexes specifically tailored for each purpose as every individual component can be linked onto any other cognate stapling moieties

Geração procedural de mapas em jogos de estratégia em tempo real

A geração procedural de conteúdo (PCG, do inglês Procedural Content Generation) é uma técnica bastante utilizada para a criação algorítmica de diversos conteúdos de jogo. Este trabalho analisa, a partir da revisão de literatura, a viabilidade de PCG em jogos de Estratégia em Tempo Real (RTS, do inglês Real-Time Strategy). Para isso, foram estabelecidas métricas de qualidade obtidas através de replays do jogo RTS StarCraft 2 e a partir de um algoritmo genético, foi possível gerar novos mapas para um jogo RTS.Procedural Content Generation (PCG) is a well-known data creation technique. This method uses a genetic algorithm to generate game content. This work analyses, through literature review, the viability of using PCG in Real-Time Strategy (RTS) games. For that, quality metrics were obtained by replaying StarCraft 2 matches. Using a genetic algorithm, it was possible to generate new maps for an RTS game

Measuring the Temperature of a Mesoscopic Quantum Electron System by means of Single Electron Statistics

We measure the temperature of a mesoscopic system consisting of an ultra-dilute two dimensional electron gas at the $Si/SiO_2$ interface in a metal-oxide-semiconductor field effect transistor (MOSFET) quantum dot by means of the capture and emission of an electron in a point defect close to the interface. Contrarily to previous reports, we show that the capture and emission by point defects in Si n-MOSFETs can be temperature dependent down to 800 mK. As the finite quantum grand canonical ensemble model applies, the time domain charge fluctuation in the defect is used to determine the temperature of the few electron gas in the channel.Comment: 4 Figures (color

SNARE based peptide linking as an efficient strategy to retarget botulinum neurotoxin’s enzymatic domain to specific neurons using diverse neuropeptides as targeting domains

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that enables the export of the light chain from the endosome to the cytosol, and the receptor binding domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well as finding safer methods of preparation and administration. Recently, our laboratory has developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A substitution of the Rbd with various neuropeptide sequences permits a large throughput combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3 different Synaptobrevin sequences; we also use a small protein staple, and 26 different Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin (SS), vasoactive intestinal peptide, substance P, opioid peptide analogues, Gonadotropin releasing hormone, and Arginine Vasopressin, which natively function through G protein coupled receptors (GPCR) can undergo agonist induced internalisation upon activation. The ability of our new constructs, once endocytosed, to inhibit neurotransmitter release was tested on different neuronal cell lines with immunoblotting of endogenous SNAP25. This cleavage by Lc reflects the ultimate readout of the enzyme’s efficacy, which incorporates the cell surface binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue. Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE peptides, which will fuse together to form highly stable chimeric proteins with an altered targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex, resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’). Fig. 1. SNARE-linked botulinum neurotoxins used for the retargeting of Bont/A. 29

Comparison of measurement methods of the front velocity of small-scale debris flows

Debris flow is a gravity-driven process, which is characterized by a travelling dense surge including large boulders, and it is followed by a more fluid tail. These characteristics make difficult the measurement of the mean flow velocity by means of common hydraulic techniques. Different methods can be used at real scale and small-scale to measure the front velocity but a dedicate comparison between available methods is still lacking. This research aims to compare the front velocity measurements in the transport zone of a miniature debris flow using three devices: i) a common digital video camera (29 frames per second); ii) a high speed thermo camera (60 fps); and iii) a laser photoelectric sensors system. The statistical analysis of data has highlighted no significant differences exist between front velocities obtained by means of the video camera and the thermo camera, whereas photocells data statistically differ from those achieved via the other systems. Some lack of data recorded by photocell was documented, while the thermo camera technique did not show significant loss of information being also helpful to detect the kinematic behaviour of single particles. Finally, the tests confirmed the influence of the solid volumetric concentration in the debris-flow mechanics, which promotes, ceteris paribus, the debris-flow slowing down

A thermo-responsive, self-assembling biointerface for on demand release of surface-immobilised proteins

Dedicated chemistries for on-demand capture and release of biomolecules at the solid-liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80°C; the second disassembles at a substantially lower temperature of about 45°C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40-45°C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli

Cleaved intracellular SNARE peptides are implicated in a novel cytotoxicity mechanism of botulinum serotype C

Recent advances in intracellular protein delivery have enabled more in-depth analyses of cellular functions. A specialized family of SNARE proteases, known as Botulinum Neurotoxins, blocks neurotransmitter exocytosis, which leads to systemic toxicity caused by flaccid paralysis. These pharmaceutically valuable enzymes have also been helpful in the study of SNARE functions. As can be seen in Figure 1A, SNARE bundle formation causes vesicle docking at the presynapse. Although these toxins are systemically toxic, no known cytotoxic effects have been reported with the curious exception of the Botulinum serotype C [1]. This enzyme cleaves intracellular SNAP25, as does serotype A and E, but also, exceptionally, cleaves Syntaxin 1. Using an array of lipid and polymer transfection reagents we were able to deliver different combinations of Botulinum holoenzymes into the normally unaffected, Neuro2A, SH-SY5Y, PC12, and Min6 cells to analyze the individual contribution of each SNARE protein and their cleaved peptide products