Optimization of production, biochemical characterization and In Vitro evaluation of the therapeutic potential of fibrinolytic enzymes from a new Bacillus Amyloliquefaciens

The capacity of fibrinolytic enzymes to degrade blood clots makes them of high relevance in medicine and in the pharmaceutical industry. In this work, forty-three microorganisms of the genus Bacillus were evaluated for their potential to produce fibrinolytic proteases. Thirty bacteria were confirmed as producers of fibrinolytic enzymes, the best results obtained for the strain Bacillus amyloliquefaciens UFPEDA 485. The optimization of the enzyme production conditions was done by a central composite design (CCD) star 23 that allowed to define the optimal conditions for soybean flour and glucose concentrations and agitation rate. The highest fibrinolytic activity (FA) of 813 U mL-1 and a degradation of blood clot in vitro of 62% were obtained in a medium with 2% (w/v) of soybean flour and 1% (w/v) glucose at 200 rpm after 48 h of cultivation, at pH 7.2 and 37 °C. The obtained fibrinolytic enzyme was characterized biochemically. Fibrinolytic activity was inhibited by PMSF (fluoride methylphenylsulfonyl - C7H7FO2S) 91.52% and EDTA (ethylenediaminetetraacetic acid - C10H16N2O8) 89.4%, confirming to be a serine- metallo protease. The optimum pH and temperature were 7.0 and 37 oC, respectively, and the enzyme was stable for 12 h. The fibrinolytic activity at physiological conditions of this enzyme produced by Bacillus amyloliquefaciens UFPEDA 485, as well as its long term stability, demonstrate that it has suitable characteristics for human and veterinary applications, and promises to be a powerful drug for the treatment of vascular diseases.We express our thanks to Coordination for the Improvement of Higher Level Education Personnel (CAPES) - Doctoral Sandwich Program (PDSE) Nº 0259/ 12-8 and National Council for Scientific and Technological Development (CNPq) - Nº 202026/2011-6 for the financial support

The Making of the NEAM Tsunami Hazard Model 2018 (NEAMTHM18)

The NEAM Tsunami Hazard Model 2018 (NEAMTHM18) is a probabilistic hazard model for tsunamis generated by earthquakes. It covers the coastlines of the North-eastern Atlantic, the Mediterranean, and connected seas (NEAM). NEAMTHM18 was designed as a three-phase project. The first two phases were dedicated to the model development and hazard calculations, following a formalized decision-making process based on a multiple-expert protocol. The third phase was dedicated to documentation and dissemination. The hazard assessment workflow was structured in Steps and Levels. There are four Steps: Step-1) probabilistic earthquake model; Step-2) tsunami generation and modeling in deep water; Step-3) shoaling and inundation; Step-4) hazard aggregation and uncertainty quantification. Each Step includes a different number of Levels. Level-0 always describes the input data; the other Levels describe the intermediate results needed to proceed from one Step to another. Alternative datasets and models were considered in the implementation. The epistemic hazard uncertainty was quantified through an ensemble modeling technique accounting for alternative models’ weights and yielding a distribution of hazard curves represented by the mean and various percentiles. Hazard curves were calculated at 2,343 Points of Interest (POI) distributed at an average spacing of ∼20 km. Precalculated probability maps for five maximum inundation heights (MIH) and hazard intensity maps for five average return periods (ARP) were produced from hazard curves. In the entire NEAM Region, MIHs of several meters are rare but not impossible. Considering a 2% probability of exceedance in 50 years (ARP≈2,475 years), the POIs with MIH >5 m are fewer than 1% and are all in the Mediterranean on Libya, Egypt, Cyprus, and Greece coasts. In the North-East Atlantic, POIs with MIH >3 m are on the coasts of Mauritania and Gulf of Cadiz. Overall, 30% of the POIs have MIH >1 m. NEAMTHM18 results and documentation are available through the TSUMAPS-NEAM project website (http://www.tsumaps-neam.eu/), featuring an interactive web mapper. Although the NEAMTHM18 cannot substitute in-depth analyses at local scales, it represents the first action to start local and more detailed hazard and risk assessments and contributes to designing evacuation maps for tsunami early warning.publishedVersio

Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena

Quantification of 2-acetyl-1-pyrroline in rice by stable isotope dilution assay through headspace solid-phase microextraction coupled to gas chromatography-tandem mass spectrometry

A new and convenient synthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor compound in rice, and its ring-deuterated analog, 2-acetyl-1-d2-pyrroline (2AP-d2), was reported. A stable isotope dilution assay (SIDA), involving headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-positive chemical ionization-ion trap-tandem mass spectrometry (GC-PCI-IT-MS-MS), was developed for 2AP quantification. A divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was used for HS-SPME procedure and parameters affecting analytes recovery, such as extraction time and temperature, pH and salt, were studied. The repeatability of the method (n=10) expressed as relative standard deviation (RSD) was 11.6%. A good linearity was observed from 5.9 to 779ng of 2AP (r2=0.9989). Limits of detection (LOD) and quantification (LOQ) for 2AP were 0.1 and 0.4ngg-1 of rice, respectively. The recovery of spiked 2AP from rice matrix was almost complete. The developed method was applied to the quantification of 2AP in aerial parts and grains of scented and non-scented rice cultivars. © 2010 Elsevier B.V.This research was funded by the Office National Interprofessionnel des Grandes Cultures (ONIGC) as a part of the “Plan de Relance de la Filiere Riz Camarguaise” project. We thank the Centre Français du Riz (CFR, Arles, France) for providing the rice samples. We also thank Languedoc Roussillon region and Cirad for supporting the doctoral fellowship of the author I. Maraval

Mapping of High Modulus Materials at the Nanoscale: Comparative Study between by Atomic Force Microscopy and Nanoindentation

peer reviewe

Quantitative mapping of high modulus materials at the nanoscale: comparative study between atomic force microscopy and nanoindentation

International audienceLocal mechanical properties of submicron features are of particular interest due to their influence on macroscopic material performance and behaviour. This study is focused on local nanomechanical measurements, based on the latest Atomic Force Microscopy (AFM) mode, where the peak force set point is finely controlled at each pixel. After probe calibration, we evaluate the impact of spring constant of two AFM hand-crafted natural full diamond tips with steel cantilevers, used for mapping. Based on the fast capture of the cantilever deflection at each pixel and real time force curve analysis in the elastic region, AFM local measured contact moduli mappings of the silica beads ('50 GPa) incorporated in an epoxy resin matrix, are compared with those determined using classical instrumented nanoindentation tests. Our analyses show that with the two AFM probes, without local residual deformation, the high moduli of the silica beads measured with this advanced AFM mode are within the standard deviation of the values determined by classical nanoindentation. Lay Description: The knowledge of material properties at the nanometer scale is a key parameter for well understanding and determining the behavior of material at macroscopic scale. In this paper, we compare two methods (an advanced mode and a classical one) based on the analysis of probes in interaction with the surface of studied material. We focus on a latest developed mode for determining local mechanical properties with a very high spatial resolution. For the advanced mode, we also consider two different hand-crafted probes. Our analyses show that with the high spatial resolution advanced mode, local mechanical properties are well determined. We also highlight the impact of the properties of the used probes for this advanced mode. In a final step, the power of the presented investigation lies in the fact that it does not modify the topography of the surface

Integrated optimal design of permanent magnet synchronous generator for smart wind turbine using genetic algorithm

In recent years, the investment in the wind energy sector has increased in the context of producing green electricity and saving the environment. The installation of small wind turbines (SWTs) represents an actual strategy for meeting energy needs for off-grid systems and certain specialized applications. SWTs are more expensive per kilowatt installed as compared to large-scale wind turbines. Therefore, the main objective of this study is to produce an economical technology for the wind power market offering low-cost SWTs. The idea consists of considering a simple structure of the wind turbine using direct-drive permanent magnet synchronous generator (DDPMSG). DDPMSGs are the most useful machines in the wind energy field thanks to several advantages, such as elimination of noise and maintenance cost due to suppression of the gearbox and absence of the rotor circuit excitation barriers by the presence of the permanent magnets (PMs). Their major downside is the high cost of active materials, especially the PMs. Thus, the improvement of the generator design is treated as being the main component of the considered chain to assure active materials’ mass and cost reduction. The methodology studied aims to explain the approach of the design integrated by optimization of the considered system. It is based on the elaboration of analytical models to find a feasible structure for the system, taking into account the multi-disciplinary analysis. The relevance of these models is validated by the finite element method using 2D MATLAB-FEMM simulation. The models are integrated to elaborate the optimization problem based on a genetic algorithm to improve the cost of the proposed generator by minimizing the mass of its active constructive materials. As an outcome, an optimal solution is offered for the wind generators market, providing a 16% cost reduction

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway

International audienceUnderstanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A-and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways