Self-assembly Controls Self-cleavage of HHR from ASBVd (−): a Combined SANS and Modeling Study

International audienceIn the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (−) RNAs thus generated are processed by cleavage to unit-length where ASBVd (−) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (−) and its derived 79-nt HHR (−). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (−) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (−) though with a remoter region from an extension of the HI domain

Impact of Imperfect Disease Detection on the Identification of Risk Factors in Veterinary Epidemiology

Risk factors are key epidemiological concepts that are used to explain disease distributions. Identifying disease risk factors is generally done by comparing the characteristics of diseased and non-diseased populations. However, imperfect disease detectability generates disease observations that do not necessarily represent accurately the true disease situation. In this study, we conducted an extensive simulation exercise to emphasize the impact of imperfect disease detection on the outcomes of logistic models when case reports are aggregated at a larger scale (e.g., diseased animals aggregated at farm level). We used a probabilistic framework to simulate both the disease distribution in herds and imperfect detectability of the infected animals in these herds. These simulations show that, under logistic models, true herd-level risk factors are generally correctly identified but their associated odds ratio are heavily underestimated as soon as the sensitivity of the detection is less than one. If the detectability of infected animals is not only imperfect but also heterogeneous between herds, the variables associated with the detection heterogeneity are likely to be incorrectly identified as risk factors. This probability of type I error increases with increasing heterogeneity of the detectability, and with decreasing sensitivity. Finally, the simulations highlighted that, when count data is available (e.g., number of infected animals in herds), they should not be reduced to a presence/absence dataset at the herd level (e.g., presence or not of at least one infected animal) but rather modeled directly using zero-inflated count models which are shown to be much less sensitive to imperfect detectability issues. In light of these simulations, we revisited the analysis of the French bovine abortion surveillance data, which has already been shown to be characterized by imperfect and heterogeneous abortion detectability. As expected, we found substantial differences between the quantitative outputs of the logistic model and those of the zero-inflated Poisson model. We conclude by strongly recommending that efforts should be made to account for, or at the very least discuss, imperfect disease detectability when assessing associations between putative risk factors and observed disease distributions, and advocate the use of zero-inflated count models if count data is available

Biocatalytic reductive amination by native Amine Dehydrogenases to access short chiral alkyl amines and amino alcohols

Small optically active molecules, and more particularly short-chain chiral amines, are key 20 compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-21 biocatalytic production on a commercial scale is already established, mainly through lipase-22 catalyzed resolutions leading to ChiProsTM products among others. Nevertheless, their 23 biocatalytic synthesis still remains challenging for very short-chain C4 to C5 amines due to low 24 enantiomeric excess. To complement the possibilities recently offered by transaminases, this 25 work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without 26 any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, 27 MsmeAmDH, MicroAmDH and MATOUAmDH2) were shown to be efficient for the synthesis 28 of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were 29 reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-30 1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol 31 (99.4%) and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale 32 up experiments were successfully performed at 150 mM substrate concentrations for the 33 synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-34 MOIPA”. Modelling studies provided some preliminary results explaining the basis for the 35 challenging discrimination between similarly sized substituents in the active sites of these 36 enzymes

Seismological evidence for crustal-scale thrusting in the Zagros mountain belt (Iran)

International audienceCrustal receiver functions computed from the records of 45 temporary seismological stations installed on a 620-km long profile across central Zagros provide the first direct evidence for crustal thickening in this mountain belt. Due to a rather short 14-km average station spacing, the migrated section computed from radial receiver functions displays the Moho depth variations across the belt with good spatial resolution. From the coast of the Persian Gulf to 25 km southwest of the Main Zagros Thrust (MZT), the Moho is almost horizontal with slight depth variations around 45 km. Crustal thickness then increases abruptly to a maximum of ~70 km beneath the Sanandaj-Sirjan metamorphic zone, between 50 and 90 km northeast of the surface exposure of the MZT. Further northeast, the Moho depth decreases to ~42 km beneath the Urumieh-Dokhtar magmatic assemblage and the southern part of the Central Iranian micro-continent. The region of thickest crust is located ~75 km to the northeast of the Bouguer anomaly low at –220 mgals. Gravity modelling shows that the measured Moho depth variations can be reconciled with gravity observations by assuming that the crust of Zagros underthrusts the crust of central Iran along the MZT considered as a crustal-scale structure. This hypothesis is compatible with shortening estimates by balanced cross-sections of the Zagros folded belt, as well as with structural and petrological studies of the metamorphic Sanandaj-Sirjan zone

Native amine dehydrogenases can catalyze the direct reduction of carbonyl compounds to alcohols in the absence of ammonia

Native amine dehydrogenases (nat-AmDHs) catalyze the (S)-stereoselective reductive amination of various ketones and aldehydes in the presence of high concentrations of ammonia. Based on the structure of CfusAmDH from Cystobacter fuscus complexed with NADP+ and cyclohexylamine, we previously hypothesized a mechanism involving the attack at the electrophilic carbon of the carbonyl by ammonia followed by delivery of the hydride from the reduced nicotinamide cofactor on the re-face of the prochiral ketone. The direct reduction of carbonyl substrates into the corresponding alcohols requires a similar active site architecture and was previously reported as a minor side reaction of some nat-AmDHs and variants. Here we describe the ketoreductase (KRED) activity of a set of nat-AmDHs and variants, which proved to be significant in the absence of ammonia in the reaction medium but negligible in its presence. Conducting this study on a large set of substrates revealed the heterogeneity of this secondary KRED activity, which was dependent upon the enzyme/substrate pairs considered. In silico docking experiments permitted the identification of some relationships between KRED activity and the structural features of the enzymes. Kinetic studies of MsmeAmDH highlighted the superior performance of this nat-AmD

Native amine dehydrogenases can catalyze the direct reduction of carbonyl compounds to alcohols in the absence of ammonia

Native amine dehydrogenases (nat-AmDHs) catalyze the (S)-stereoselective reductive amination of various ketones and aldehydes in the presence of high concentrations of ammonia. Based on the structure of CfusAmDH from Cystobacter fuscus complexed with Nicotinamide adenine dinucleotide phosphate (NADP+) and cyclohexylamine, we previously hypothesized a mechanism involving the attack at the electrophilic carbon of the carbonyl by ammonia followed by delivery of the hydride from the reduced nicotinamide cofactor on the re-face of the prochiral ketone. The direct reduction of carbonyl substrates into the corresponding alcohols requires a similar active site architecture and was previously reported as a minor side reaction of some native amine dehydrogenases and variants. Here we describe the ketoreductase (KRED) activity of a set of native amine dehydrogenases and variants, which proved to be significant in the absence of ammonia in the reaction medium but negligible in its presence. Conducting this study on a large set of substrates revealed the heterogeneity of this secondary ketoreductase activity, which was dependent upon the enzyme/substrate pairs considered. In silico docking experiments permitted the identification of some relationships between ketoreductase activity and the structural features of the enzymes. Kinetic studies of MsmeAmDH highlighted the superior performance of this native amine dehydrogenases as a ketoreductase but also its very low activity towards the reverse reaction of alcohol oxidation

Transmission of highly pathogenic avian influenza in the nomadic free-grazing duck production system in Viet Nam.

The presence of free-grazing ducks (FGD) has consistently been shown to be associated with highly pathogenic avian influenza virus (HPAIV) H5N1 outbreaks in South-East Asia. However, the lack of knowledge about the transmission pathways limits the effectiveness of control efforts. To address this gap, we developed a probabilistic transmission model of HPAIV H5N1 in the nomadic FGD production system in Viet Nam, assuming different scenarios to address parameter uncertainty. Results suggested that HPAIV H5N1 could spread within the nomadic FGD production system, with an estimated flock-level effective reproduction number (re) ranging from 2.16 (95% confidence interval (CI): 1.39-3.49) to 6.10 (95%CI: 3.93-9.85) depending on the scenario. Indirect transmission via boats and trucks was shown to be the main transmission route in all scenarios. Results suggest that re could be reduced below one with 95% confidence if 86% of FGD flocks were vaccinated in the best-case scenario or 95% in the worst-case scenario. If vaccination was combined with cleaning and disinfection of transport vehicles twice a week, vaccination coverage could be lowered to 60% in the best-case scenario. These findings are of particular relevance for prioritising interventions for effective control of HPAIV in nomadic free-grazing duck production systems

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome

BACKGROUND: Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia. METHODOLOGY/PRINCIPAL FINDINGS: We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation. CONCLUSIONS: We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms