RNA silencing in the dermatophyte Microsporum canis

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption method

RNA silencing in the dermatophyte Microsporum canis

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption method

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates

Production of lipid-based fuels and chemicals from microalgae: An integrated experimental and model-based optimization study

Abstract Cultivation of microalgae is a promising long-term, sustainable candidate for biomass and oil for the production of fuel, food, nutraceuticals and other added-value products. Attention has been drawn to the use of computational and experimental validation studies aiming at the optimisation and the control of microalgal oil productivity either through the improvement of the growth mechanism or through the application of metabolic engineering methods to microalgae. Optimisation of such a system can be achieved through the evaluation of organic carbon sources, nutrients and water supply, leading to high oil yield. The main objective of this work is to develop a novel integrated experimental and computational approach, utilising a microalgal strain grown at bench-scale, with the aim to systematically identify the conditions that optimise growth and lipid production, in order to ultimately develop a cost-effective process to improve the system economic viability and overall sustainability. To achieve this, a detailed model has been constructed through a multi-parameter quantification methodology taking into account photo-heterotrophic biomass growth. The corresponding growth rate is based on carbon substrate concentration, nitrogen and light availability. The developed model also considers the pH of the medium. Parameter estimation was undertaken using the proposed model in conjunction with an extensive number of experimental data taken at a range of operating conditions. The model was validated and utilised to determine the optimal operating conditions for bench-scale batch lipid oil production

Bourdieusian Reflections on Language: Unavoidable Conditions of the Real Speech Situation

The main purpose of this paper is to shed light on Pierre Bourdieu’s conception of language. Although he has dedicated a significant part of his work to the study of language and even though his analysis of language has been extensively discussed in the literature, almost no attention has been paid to the factthat Bourdieu’s account of language is based on a number of ontological presuppositions, that is, on a set of universal assumptions about the very nature of language. This article aims to fill this gap in the literature by offering a detailed overview of 10 key features which, from a Bourdieusian point of view, can be regarded as inherent in language. On the basis of this enquiry,the study seeks todemonstrate that——contraryto commonbelief——there is not only a Bourdieusian sociology of language but also a Bourdieusian philosophy of language, which provides a useful theoretical framework for examining the unavoidable conditions of the real speech situation. The paper draws to a close by reflecting on the flaws and limitations of Bourdieu’s approach to language

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter-and intraspecies genomic variation. We further predicted 17,903 carbohydrateactive enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.Peer reviewe