High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription

Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp

Fungi are important for biomass degradation processes in mangrove forests. Given the presence of sea water in these ecosystems, mangrove fungi are adapted to high salinity. Here we isolate Pestalotiopsis sp. NCi6, a halotolerant and lignocellulolytic mangrove fungus of the order Xylariales. We study its lignocellulolytic enzymes and analyse the effects of salinity on its secretomes. De novo transcriptome sequencing and assembly indicate that this fungus possesses of over 400 putative lignocellulolytic enzymes, including a large fraction involved in lignin degradation. Proteomic analyses of the secretomes suggest that the presence of salt modifies lignocellulolytic enzyme composition, with an increase in the secretion of xylanases and cellulases and a decrease in the production of oxidases. As a result, cellulose and hemicellulose hydrolysis is enhanced but lignin breakdown is reduced. This study highlights the adaptation to salt of mangrove fungi and their potential for biotechnological applications

Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study

<p>Abstract</p> <p>Background</p> <p>The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, <it>Trichoderma reesei </it>is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. <it>T. reesei </it>has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from <it>T. reesei</it>, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.</p> <p>Results</p> <p>After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from <it>T. reesei</it>.</p> <p>Conclusions</p> <p>Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus <it>T. reesei </it>gives new insights into the physicochemical parameters and biodiversity in this family.</p

Tracking the connection between evolutionary and functional shifts using the fungal lipase/feruloyl esterase A family

BACKGROUND: There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/feruloyl esterase A family, whose functional diversification makes it a very promising candidate. RESULTS: The results suggested functional shift following a duplication event where neofunctionalisation of feruloyl esterase A had occurred with conservation of the ancestral lipase function. Evolutionary shift was detected using the branch-site model for testing positive selection on individual codons along specific lineages. Positively selected amino acids were detected. Furthermore, biological data obtained from site-directed mutagenesis experiments clearly demonstrated that certain amino acids under positive selection were involved in the functional shift. We reassessed evolutionary history in terms of environmental response, and hypothesized that environmental changes such as colonisation by terrestrial plants might have driven adaptation by functional diversification in Euascomycetes (Aspergilli), thus conferring a selective advantage on this group. CONCLUSION: The results reported here illustrate a rare example of connection between fundamental events in molecular evolution. We demonstrated an unequivocal connection between evolutionary and functional shifts, which led us to conclude that these events were probably linked to environmental change

Oxidation of model lipids representative for main paper pulp lipophilic extractives by the laccase-mediator system.

Several model lipids representative for main paper pulp lipophilic extractives - including alkanes, fatty alcohols, fatty acids, resin acids, free and esterified sterols, and triglycerides – were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator. The reaction products were analyzed by GC and GC-MS. The laccase alone decreased the amount of some unsaturated lipids, however, the most rapid and extensive lipid modification was obtained with the laccase-mediator system. Most unsaturated lipids were largely oxidized and the dominant oxidation products detected were epoxy and hydroxy-fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. In contrast, saturated lipids were not modified, although some of them were oxidized when the enzymatic reactions were carried out in the presence of unsaturated lipids. The results obtained are discussed in the context of enzymatic control of pitch deposits, to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.This study was funded by the BIORENEW EU-project (NMP2-CT-2006-026456) and the Spanish MEC (BIO2007-28719-E). Beldem (Andenne, Belgium) is acknowledged for laccase supply. S.M. thanks the Spanish CSIC and CELESA for an I3P contract and J.R thanks the CSIC for an I3P fellowship.Peer reviewe

ecocomDP: A flexible data design pattern for ecological community survey data

The idea of harmonizing data is not new. Decades of amassing data in databases according to community standards - both locally and globally - have been more successful for some research domains than others. It is particularly difficult to harmonize data across studies where sampling protocols vary greatly and complex environmental conditions need to be understood to apply analytical methods correctly. However, a body of longterm ecological community observations is increasingly becoming publicly available and has been used in important studies. Here, we discuss an approach to preparing harmonized community survey data by an environmental data repository, in collaboration with a national observatory. The workflow framework and repository infrastructure are used to create a decentralized, asynchronous model to reformat data without altering original data through cleaning or aggregation, while retaining metadata about sampling methods and provenance, and enabling programmatic data access. This approach does not create another data ‘silo’ but will allow the repository to contribute subsets of available data to a variety of different analysis-ready data preparation efforts. With certain limitations (e.g., changes to the sampling protocol over time), data updates and downstream processing may be completely automated. In addition to supporting reuse of community observation data by synthesis science, a goal for this harmonization and workflow effort is to contribute these datasets to the Global Biodiversity Information Facility (GBIF) to increase the data’s discovery and use

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina

International audienceBackground: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and beta-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries

Standardized Neon Organismal Data for Biodiversity Research

Understanding patterns and drivers of species distribution and abundance, and thus biodiversity, is a core goal of ecology. Despite advances in recent decades, research into these patterns and processes is currently limited by a lack of standardized, high-quality, empirical data that span large spatial scales and long time periods. The NEON fills this gap by providing freely available observational data that are generated during robust and consistent organismal sampling of several sentinel taxonomic groups within 81 sites distributed across the United States and will be collected for at least 30 years. The breadth and scope of these data provide a unique resource for advancing biodiversity research. To maximize the potential of this opportunity, however, it is critical that NEON data be maximally accessible and easily integrated into investigators\u27 workflows and analyses. To facilitate its use for biodiversity research and synthesis, we created a workflow to process and format NEON organismal data into the ecocomDP (ecological community data design pattern) format that were available through the ecocomDP R package; we then provided the standardized data as an R data package (neonDivData). We briefly summarize sampling designs and data wrangling decisions for the major taxonomic groups included in this effort. Our workflows are open-source so the biodiversity community may: add additional taxonomic groups; modify the workflow to produce datasets appropriate for their own analytical needs; and regularly update the data packages as more observations become available. Finally, we provide two simple examples of how the standardized data may be used for biodiversity research. By providing a standardized data package, we hope to enhance the utility of NEON organismal data in advancing biodiversity research and encourage the use of the harmonized ecocomDP data design pattern for community ecology data from other ecological observatory networks

The REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) statement.

Routinely collected health data, obtained for administrative and clinical purposes without specific a priori research goals, are increasingly used for research. The rapid evolution and availability of these data have revealed issues not addressed by existing reporting guidelines, such as Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The REporting of studies Conducted using Observational Routinely collected health Data (RECORD) statement was created to fill these gaps. RECORD was created as an extension to the STROBE statement to address reporting items specific to observational studies using routinely collected health data. RECORD consists of a checklist of 13 items related to the title, abstract, introduction, methods, results, and discussion section of articles, and other information required for inclusion in such research reports. This document contains the checklist and explanatory and elaboration information to enhance the use of the checklist. Examples of good reporting for each RECORD checklist item are also included herein. This document, as well as the accompanying website and message board (http://www.record-statement.org), will enhance the implementation and understanding of RECORD. Through implementation of RECORD, authors, journals editors, and peer reviewers can encourage transparency of research reporting

Main Recent Contributions to SHS from France

International audienceBoth in situ TRXRD and IR thermography, on the one hand, and different levels of modeling, on the other, have generated a strong progress in the knowledge and control of numerous SHS reactions. The SHS of simple binary materials, such as intermetallics (FeAl, MoSi2, NbAl3, etc.), oxides (e.g. ZrO2), carbides (e.g. SiC) or nitrides, more complex materials, such as mullite, SiAlONs, MAX phases, composites (SiO2-Al2O3, NiAl-ZrO2 ), powders in their more complicated states, such as well controlled microstructures, fully densified intermetallics, smart composites, and hard coatings carried out by GFA researchers greatly contributed to the worldwide competition to harness the potential of SHS. On the occasion of the 40th anniversary of SHS, this paper is giving an overview of the main results obtained by GFA over the last ten years