Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxida-tive cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications

Mise au point de la quantification d’un marqueur précoce de stress cellulaire chez les larves aquatiques de Cloeon dipterum (Ephemeroptera, Baetidae) collectées en région Méditerranéenne

National audienceThe aim of this study is to calibrate a biomarker of cellular stress, the heat shock protein (HSP70) induction, in aquatic invertebrates collected in a little Mediterranean run-off, as a tool for early ecological risk assessment. Larvae of the Ephemeroptera Cloeon dipterum (L., 1761), present all over the year and in a suitable amount, are chosen as biological model. The development of the biomarker quantification allows choosing the stage 5 and older larvae for this calibration. As the larvae are obtained in absence of natural thermal stress and from a run-off where sediment pollutant concentrations do not exceed the threshold above which adverse effects frequently occurred, the « Consensus-based PEC » (Probable Effect Concentration) (Mac Donald et al., 2000), the measured HSP70 concentrations reflect a basal level.L’objectif de cette étude est de calibrer un biomarqueur de stress cellulaire, l’induction des protéines de choc thermique HSP70, chez des invertébrés aquatiques prélevés dans une roubine méditerranéenne, pour une utilisation comme outil de diagnostic précoce du risque écologique. Les premiers prélèvements permettent de retenir les larves de l’éphéméroptère Cloeon dipterum (L., 1761) comme modèle d’étude car elles sont les seules à y être présentes toute l’année et en quantité suffisante. La mise au point de la quantification du biomarqueur permet de sélectionner les larves de stade 5 et au-delà pour cette calibration. Les taux de HSP70 mesurés correspondent à des concentrations basales car obtenues chez des larves en absence de stress thermique naturel et dans un milieu où les polluants ne dépassent pas le « PEC » (Probable Effect Concentration), seuil au dessus duquel il est probable d’observer des effets toxiques (Mac Donald et al., 2000)

Cytochrome c - silver nanoparticle interactions: Spectroscopy, thermodynamic and enzymatic activity studies

Cytochrome c, an iron containing metalloprotein in the mitochondria of the cells with an oxide/redox property, plays key role in the cell apoptotic pathway. In this study, the interaction of silver nanoparticles (AgNPs) with cytochrome c (Cyt c) was investigated by using a combination of spectroscopic, imaging and thermodynamic techniques, including dynamic light scattering (DLS), ultraviolet-visible (UV-vis) spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). DLS and UV-vis analysis evidenced the formation of surface complexes of Cyt c on AgNPs. The saturation of surface coverage of AgNPs was observed at 4.36 Cyt c molecules per nm 2 of AgNPs. The surface complexation resulted in a promotion of the Ag dissolution overtime. The negative sign of enthalpic (ΔH) contribution suggested that electrostatic forces are indicative forces in the interaction between protein and AgNPs. Moreover, the fluorescence spectra revealed that the conformation of protein was altered around tryptophan (Trp) and tyrosine (Tyr) residues indicating the alteration of the tertiary structure of Cyt c. CD analysis evidenced that the secondary structure of Cyt c was modified under AgNPs-Cyt c interactions and the binding of Cyt c onto AgNPs resulted in remarkable structural perturbation around the active site heme, which in turn alter the protein enzymatic activity. The results of the present study contributed to a deeper insight on the mechanisms of interaction between NPs and biomacromolecules and could help establish the in vivo fate of AgNPs on cellular redox homeostasis

Direct DNA interaction and genotoxic impact of three metals: Cadmium, nickel and aluminum

International audienceThis study simultaneously investigates direct DNA interaction and genotoxic impact of three typical metals: aluminum, cadmium and nickel, which the high concentration in soils and which the industries use, result in a daily significant exposure to humans. The three of them are suspected to be involved in carcinogenesis which implies genomic lesions. We propose to first study their genotoxic impact in vivo on primary normal human dermal fibroblast (NHDF) cells with comet assay at pH 7 to measure DNA breaks occurrence. Then, to characterize the metal/DNA interaction by isothermal titration calorimetry (ITC). Comet assay shows that Cd and Ni are genotoxic, they are responsible for DNA breaks starting from 1.10-4 mol.L-1 and 5.10-2 mol.L-1, respectively whereas Al has no effect (on DNA at pH7) as studied by ITC at pH 7. Cd and Ni present an electrostatic interaction with DNA phosphate groups. At high Cd concentration, a DNA condensation is observed by contrast. Al has no interaction with DNA phosphate groups, but at pH 4 the electrostatic interaction is strong and the same DNA condensation phenomenon is observed. Metal genotoxic effect seems linked to the electrostatic interaction on DNA phosphate groups. Genotoxic power evolves in parallel to DNA phosphate interaction strength as Cd > Ni > Al. If this study shows that metals do not directly break DNA, this binding could be a preferential site for damage due to reactive oxygen species

Contaminant signatures and stable isotope values qualify European conger (Conger conger) as a pertinent bioindicator to identify marine contaminant sources and pathways

International audienceTo evaluate the capacity of the European conger (Conger conger) as a reliable bioindicator to investigate marine contaminant sources and pathways, the muscles of 24 individuals caught in a semi-enclosed industrialized bay (Gulf of Fos, France) were analyzed for numerous organic compounds, metal elements, and stable isotopes. The contaminant levels were elevated to moderate, which relates to the great anthropic pressure from the surrounding industries and maritime traffic. Stable isotope analyses finely discriminated between three geographical areas and between two age groups. Mercury (Hg) and arsenic (As) concentrations (nd-1.35 mg.kg −1 and 5.94-60.1 mg.kg −1 wet weight (ww), respectively), as well as chlorination by-products, were elevated in particular areas, identifying specific industrial sources. Levels of Σ 42 PCB (7.15-28.67 µg.kg −1 ww), Σ 16 PAH (3.64-9.48 µg.kg −1 ww) and Σ 10 pesticides (1.91-18.42 µg.kg −1 ww) in fish muscles, as expected, did not differ among sites

Characterization of atmospheric emission sources in lichen from metal and organic contaminant patterns

International audienceLichen samples from contrasted environments, influenced by various anthropic activities, were investigated focusing on the contaminant signatures according to the atmospheric exposure typologies. Most of the contaminant concentrations measured in the 27 lichen samples, collected around the industrial harbor of Fos-sur-Mer (France), were moderate in rural and urban environments, and reached extreme levels in industrial areas and neighboring cities (Al up to 6567 mg k

NMR and MD investigations of human galectin-1/oligosaccharide complexes.

International audienceThe specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related beta-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1

The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

International audienceUnlabelled: Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. Importance: Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs

Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium

International audienceThere is evidence that exposure to environmental factors is at least partly responsible for changes in semen quality observed over the past decades. The detection of reproductive toxicants under Registration, Evaluation and Authorisation of Chemicals (REACH) will impact animal use for regulatory safety testing. We first validated a model of culture of rat seminiferous tubules for toxicological studies on spermatogenesis. Then, using this model of culture, we assessed the deleterious effects of 1, 10, and 100 μg/l hexavalent chromium [Cr(VI)] on meiotic cells. The prophase I of meiosis was studied in vivo and ex vivo. Bromo-2′-deoxyuridine (BrdU) was used to describe the kinetics of germ cell differentiation. SCP3 labeling allowed to establish the distribution of the stages of the meiotic prophase I and to perform a qualitative study of the pachytene stage in the absence or presence of Cr(VI). The development of the meiotic step of pubertal rats was similar in vivo and ex vivo. The number of total cells appeared not affected by the presence of Cr(VI) irrespective of its concentration. However, the numbers of late spermatocytes and of round spermatids were decreased by Cr(VI) even at the lower concentration. The percentage of synaptonemal complex abnormalities increased slightly with the time of culture and dramatically with Cr(VI) concentrations. This model of culture appears suitable for toxicological studies. This study shows that Cr(VI) is toxic for meiotic cells even at low concentrations, and its toxicity increases in a dose-dependent manner