Improving the biotreatment of hydrocarbons-contaminated soils by addition of activated sludge taken from the wastewater treatment facilities of an oil refinery

Addition of activated sludge taken from the wastewater treatment facilities ofan oil refinery to a soil contaminated with oily sludge stimulated hydrocarbonbiodegradation in microcosms, bioreactors and biopile. Microcosms containing50 g of soil to which 0.07 % (w/w) of activated sludge was added presented ahigher degradation of alkanes (80 % vs 24 %) and polycyclic aromatic hydrocarbons(PAHs) (77 % vs 49 %) as compared to the one receiving only water, after 30days of incubation at room temperature. Addition of ammonium nitrate or sterilesludge filtrate instead of activated sludge resulted in a similar removal of PAHsbut not of alkanes suggesting that the nitrogen contained in the activated sludgeplays a major role in the degradation of PAHs while microorganisms of thesludge are active against alkanes. Addition of sludge also stimulated hydrocarbonbiodegradation in 10-kg bioreactors operated during 60 days and in a 50-m³ biopile operated during 126 days. This biopile treatment allowed the use of the soil for industrial purpose based on provincial regulation (``C' criteria). In contrast, the soil of the control biopile that received only water still exceeded C criteria for C₁₀–C₅₀ hydrocarbons, total PAHs, chrysene and benzo[a]anthracene.The stimulation effect of sludge was stronger on the 4-rings than on 2-rings PAHs.The soil of the biopile that received sludge was 4–5 times less toxic than the control. These results suggest that this particular type of activated sludge could be used to increase the efficiency of the treatment of hydrocarbon-contaminated soils in a biopile.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.     

Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one "consensus" (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T(m)) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/l) of chitosan, respectively. T(m) were increased by 11.6 degrees C (N174-CONS), 13.8 degrees C (CSN-A104L), 15.6 degrees C (N174-K164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T(m) in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.     

Energy Sources,Self-organization,and the Origin of Life

The emergence and early developments of life are considered from the point of view that contingent events that inevitably marked evolution were accompanied by deterministic driving forces governing the selection between different alternatives. Accordingly, potential energy sources are considered for their propensity to induce self-organization within the scope of the chemical approach to the origin of life. Requirements in terms of quality of energy locate thermal or photochemical activation in the atmosphere as highly likely processes for the formation of activated low-molecular weight organic compounds prone to induce biomolecular self-organization through their ability to deliver quanta of energy matching the needs of early biochemical pathways or the reproduction of self-replicating entities. These lines of reasoning suggest the existence of a direct connection between the free energy content of intermediates of early pathways and the quanta of energy delivered by available sources of energy.     

Group Choice as a Function of Group Size Differences and Assessment Time in Fish: The Influence of Species Vulnerability to Predation

The shoal-choice behaviour of two species of fish that differ in their vulnerability to predation was compared. Individuals of threespine stickleback, Gasterosteus aculeatus, and creek chub, Semotilus atromaculatus, were presented with a simultaneous choice of two equidistant stimulus shoals of conspecifics that differed in membership size (5 vs. 6 fish, 5 vs. 7, 5 vs. 8, 5 vs. 9 and 5 vs. 10). Test fish were allowed to view the stimulus shoals from a standard distance for either 10–20 or 120–150s before being frightened with a stimulus from an overhead light and released to join either shoal. We observed which shoal (the smaller or the larger one) the test fish approached. Preference for the larger stimulus shoal generally increased with increasing shoal size difference and with the duration of the assessment period, and was more pronounced in chub (the more vulnerable of the two species). For the short assessment period, chub showed a significantly stronger preference for the larger stimulus shoal than sticklebacks, whereas there was no significant difference between species for the long assessment period. Furthermore, chub responded more readily to small differences in shoal size (of 1–3 fish) than sticklebacks, for both short and long assessment periods. The above results are consistent with the hypothesis that chub, as the more vulnerable of the two species (in terms of predation), should be able to identify the larger of two shoals more quickly and should be more sensitive to small differences in shoal size than sticklebacks.     

Discovery and process development of a novel TACE inhibitor for the topical treatment of psoriasis

Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this pathway, TACE stands out as a druggable target and has been the focus of in-house research programs. In this article, we present the discovery of clinical candidate 26a. Starting from hits plagued with poor solubility or genotoxicity, 26a was identified through thorough multiparameter optimisation. Showing robust in vivo activity in an oxazolone-mediated inflammation model, the compound was selected for development. Following a polymorph screen, the hydrochloride salt was selected and the synthesis was efficiently developed to yield the API in 47% overall yield.     

Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.