Enhanced lipid extraction from unbroken microalgal cells using enzymes

The marine microalga Nannochloropsis sp. was chosen as a model organism to investigate the feasibility of using cell wall-degrading enzymes to enhance the recovery of intracellular lipids. An enzyme cocktail containing galactomannanase, 1,4-β-cellobiosidase and β-glucosidase as main components was prepared from commercial enzyme preparations. The effects of pretreatment time (P), enzyme dosage (D), pH and temperature (T) on the amount of extracted lipids were investigated using response surface methodology. Under the best conditions (P = 90 min, D = 1.3 mg g–1, pH = 5, T = 36°C) over 70% of the lipids present in the microalga were recovered. SEM and TEM characterization of enzyme-treated microalgae showed extensive cell damage with significant disruption of the cell wall and release of algal material. Overall, the results of this study strongly support the use of commercial enzyme preparations to improve lipid recovery from microalgae and provide useful information on the influence of process conditions on the treatment efficiency

seismic response of a deep continental basin including velocity inversion the sulmona intramontane basin central apennines italy

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Newly identified active faults in the Pollino seismic gap, southern Italy, and their seismotectonic significance

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Multiple Lines of Evidence for a Potentially Seismogenic Fault Along the Central-Apennine (Italy) Active Extensional Belt–An Unexpected Outcome of the MW6.5 Norcia 2016 Earthquake

The Apenninic chain, in central Italy, has been recently struck by the Norcia 2016 seismic sequence. Three mainshocks, in 2016, occurred on August 24 (MW6.0), October 26 (MW 5.9) and October 30 (MW6.5) along well-known late Quaternary active WSW-dipping normal faults. Coseismic fractures and hypocentral seismicity distribution are mostly associated with failure along the Mt Vettore-Mt Bove (VBF) fault. Nevertheless, following the October 26 shock, the aftershock spatial distribution suggests the activation of a source not previously mapped beyond the northern tip of the VBF system. In this area, a remarkable seismicity rate was observed also during 2017 and 2018, the most energetic event being the April 10, 2018 (MW4.6) normal fault earthquake. In this paper, we advance the hypothesis that the Norcia seismic sequence activated a previously unknown seismogenic source. We constrain its geometry and seismogenic behavior by exploiting: 1) morphometric analysis of high-resolution topographic data; 2) field geologic- and morphotectonic evidence within the context of long-term deformation constraints; 3) 3D seismological validation of fault activity, and 4) Coulomb stress transfer modeling. Our results support the existence of distributed and subtle deformation along normal fault segments related to an immature structure, the Pievebovigliana fault (PBF). The fault strikes in NNW-SSE direction, dips to SW and is in right-lateral en echelon setting with the VBF system. Its activation has been highlighted by most of the seismicity observed in the sector. The geometry and location are compatible with volumes of enhanced stress identified by Coulomb stress-transfer computations. Its reconstructed length (at least 13 km) is compatible with the occurrence of MW≥6.0 earthquakes in a sector heretofore characterized by low seismic activity. The evidence for PBF is a new observation associated with the Norcia 2016 seismic sequence and is consistent with the overall tectonic setting of the area. Its existence implies a northward extent of the intra-Apennine extensional domain and should be considered to address seismic hazard assessments in central Italy

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5?9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 ?M. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists

Les fiches d’indexation minimale du conseil de l’Europe pour l’inventaire normalisé du patrimoine culturel mondial

IntroductionFigure 1Tunisie. Sbeitla (Sufetula). Vue du Capitolium© Photo Memar, 1998Désormais l’inventaire du patrimoine culturel mondial a pris l’aspect définitif d’un véritable « babélisme de normes standards ». La situation est préoccupante, mais non pas sans espoir, puisqu’une forte veine commune parcourt les informations françaises et italiennes, informations qui constituent à elles seules, selon les estimations de l’étude, la plus grande partie des fiches informatisées qui existent au ..

Approches intégratives visant à décoder le rôle de co-traduction de la peptide déformylase du phage Vp16 et comment il compromet la viabilité de l'hôte

L'excision de la méthionine N-terminale (NME) est la première modification se produisant au N-terminal des protéines (NPM). Les peptides déformylases (PDF) sont les enzymes impliquées dans ce processus co-traductionnel essentiel et conservé. Les PDFs suppriment le groupe formyle lié à la méthionine initiatrice (iMet) présente au début de toutes les chaînes procaryotes naissantes. Les PDFs agissent au niveau du tunnel de sortie des ribosomes, plaque tournante de nombreux facteurs de biogénèse des protéines associées aux ribosomes (PRB), impliqués non seulement sur les MNP, mais également dans le repliement et la translocation des protéines. La déformylation N-terminale implique 95% du protéome bactérien et contribue directement à la stabilité des protéines. Le récent séquençage à haut débit de milliers de génomes a révolutionné notre perception de la distribution des PDFs dans les différents règnes, révélant des PDFs putatives dans tous les organismes, y compris les virus. En particulier, les études concernant les virus présents dans les échantillons microbiens océaniques ont permis d’identifier des gènes inhabituels de PDF chez les phages, constituant la famille la plus abondante de protéines auxiliaires conservées de ces génomes. La comparaison des séquences identifiées révèle que les PDF virales présentent une forte conservation des trois motifs constituant le site catalytique. Cependant, ces PDFs virales ne présentent pas d'extension C-terminale, région réputée importante des PDFs des autres organismes. Sachant que cette extension est impliquée dans la liaison de la PDF d’E. coli au ribosome et est requise pour son activité déformylase in vivo, il était incertain que les PDFs de phage découvertes avaient une activité déformylase classique. Ainsi, la découverte de ces PDFs virales soulève un certain nombre de questions parmi lesquelles: a) Ces PDFs virales présentent-elles une activité déformylase classique? b) Ces PDFs sont-elles capables de se lier aux ribosomes ? c) Pourquoi autant de virus portent-ils une ou plusieurs déformylases ? Dans ce contexte, l’objectif de ma thèse a été d’entreprendre la caractérisation de ces PDFs de phages marins et en particulier la PDF de Vp16 provenant de bactériophages isolés à l’origine de la souche 16 de Vibrio parahaemolyticus. Nos études révèlent que ces PDFs de phages présentent une activité déformylase à la fois in vitro et in vivo, avec une spécificité de substrat similaire à celle des autres PDFs bactériennes. D'autre part, nous avons montré par des études biochimiques et structurales, combinées à des analyses par mutagenèse dirigée, que les propriétés de la PDF de Vp16 diffèrent significativement de celle des autres PDFs caractérisées précédemment. Il faut aussi noter que l'expression de la PDF Vp16 dans les souches d'E. Coli, même à de faibles concentrations, montre un effet bactéricide marqué à une température inférieure à 37 °C. L’effet bactéricide de la PDF Vp16 est indépendant de la présence de la PDF endogène bactérienne et repose strictement sur son activité déformylase. La caractérisation de ce phénotype a révélé que la létalité induite par Vp16 PDF montrait un lien génétique fort avec des gènes codants pour des facteurs cellulaires impliqués dans le ciblage et le repliement précoce des protéines (Trigger Factor et Sec). Contrairement à ce qui a été montré pour les PDFs bactériennes, Vp16 PDF a une forte affinité pour le ribosome bactérien d’E. coli en cours de traduction, interagissant avec une région ribosomale chevauchant celles des facteurs impliqués dans le transit des protéines vers les voies de sécrétion. Une compétition au niveau du ribosome entre Vp16 PDF et ces RPBs pourrait contribuer à la lyse cellulaire de l’hôte. Mon travail suggère un nouveau mécanisme utilisé par les bactériophages permettant de contrôler la viabilité de l'hôte.N-terminal Methionine Excision (NME) is the first occurring N-terminal Protein Modification (NPMs). Peptide deformylases (PDFs) are the enzymes involved in this essential and conserved co-translational process. PDFs remove the formyl group bound to the iMet present at the beginning of all prokaryotic nascent chains. PDFs act on the nascent chain at the level of the ribosome exit tunnel, a central hub for a number of Ribosome-associated Protein Biogenesis factors (RPBs) involved not only on NPMs but also in protein folding and translocation. Deformylation involves 95% of bacterial proteome and it is suggested to directly contribute to protein stability. Recent high-throughput sequencing of thousands of genomes has strongly contributed to revolutionizing our perception of the distribution of PDFs among kingdoms, revealing putative PDFs in all organisms, including viruses. In particular, studies of viruses within oceanic microbial samples retrieved unusual PDFs genes as the most abundant family in most of phage genomes. Sequence comparisons reveal that viral PDFs show high conservation in the three motifs that build the catalytic site; however, viral PDFs do not display a C-terminal extension when compared to the different active PDFs from other organisms. Since this C-terminal extension was shown to be important for PDF-ribosome binding and is required for the in vivo deformylase activity of E. coli PDF, it was unclear whether the discovered phage PDFs might support a classical deformylase activity. Thus, the discovery of these viral PDFs raises a number of questions among which: a) Have these viral PDFs a classical deformylase activity? b) Are these PDFs able to still bind to the ribosomes? c) Why so many viruses carry a peptide deformylase? In this context, the objective of my thesis was to undertake the characterization of these marine phage PDFs and particularly Vp16 PDF derived from the bacteriophages originally isolated from Vibrio Parahaemolyticus strain 16. Our studies reveal that phage PDFs display deformylase activity both in vitro and in vivo with a substrate specificity similar to that of other bacterial PDFs. On the other hand, we showed by biochemical and structural data, combined with site-directed mutagenesis analyses, that Vp16 PDF significantly differs from previously characterized PDFs in terms of their properties, which can be related to its few uncommon peculiarities. Interestingly, expression of Vp16 PDF in E. coli strains, even at low concentrations, exhibited a severe bactericidal effect at temperature lower than 37 °C. This bactericidal effect of Vp16 PDF was independent of the presence of the bacterial endogenous PDF and strictly relied on its PDF activity. Characterization of this phenotype revealed that Vp16 PDF-induced lethality showed a strong genetic link with genes encoding cellular factors involved in nascent pre-secretory protein targeting and folding (Trigger Factor and Sec). Differently from bacterial PDF, I could show that Vp16 PDF has strong affinity for ribosomes with a specific nascent chain, interacting with a ribosomal region overlapping that of factors involved in pre-secretory protein targeting. A competition between Vp16 PDF and these RPBs at the level of the ribosome may contribute to the host lysis, revealing a possible new unrecognized mechanism developed by viruses to control host viability