Single strain high-depth ngs reveals high rdna (Its-lsu) variability in the four prevalent pathogenic species of the genus candida

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra-and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput highdepth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology

Extensive pollen flow in a natural fragmented population of Patagonian cypress Austrocedrus chilensis

Habitat fragmentation might significantly affect mating and pollen dispersal patterns in plant populations, contributing to the decline of remnant populations. However, wind-pollinated species are able to disperse pollen at longer distances after opening of the canopy. Our objectives were to characterize the mating system parameters and to estimate the average distance of effective pollen dispersal in the wind-pollinated conifer Austrocedrus chilensis. We sampled 19 “mother trees,” 200 progeny, and 81 additional adults (both male and female), in a fragmented population at the Argentinean Patagonian steppe. We registered the spatial positions of individuals and genotyped all samples with five microsatellite markers. We found a high genetic diversity, a moderated rate of biparental inbreeding (tm  − ts = 0.105), and a complete absence of correlated paternity (rp = −0.015). The effective number of pollen donors contributing to a single mother (Nep) was 13.9. Applying TWOGENER, we estimated a low but significant differentiation among the inferred pollen pools (ΦFT = 0.036, p = 0.001) and a very large average pollen dispersal distance (d = 1,032.3 m). The leptokurtic distribution (b = 0.18) presumes a potential for even larger dispersal distances. The high genetic diversity, the mating patterns, and the extensive pollen dispersal presume that habitat fragmentation did not have a negative impact on pollen movement in this population of A. chilensis. Genetic connectivity among fragmented populations scattered in the Patagonian region is possible, and we stress the need of management policies at the landscape level.Fil: Colabella, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Gallo, L. A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Moreno, Angela Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Marchelli, Paula. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

From Georgia and Canada with FTIR: metabolomic of vine and wine related strains

Concern is recently growing among wine producers about the importance of introducing high quality wines to the market that exhibit geographical characteristics and complexity (Harvey et al., 2014). Terroir has been defined as the concept that links the sensory features of wine to the environmental conditions of vineyards. Moreover, these elements may condition what has been defined as the microbial biogeography of grapes (Bokulich et al., 2014), as unique microbial strains have been associated with specific geographical locations (Tofalo et al., 2013). Thus, the description of this microbial diversity can be the first step of the selection of a consortium of native yeast microbiota emulating spontaneous fermentation that could be used for the production of wines with a characteristic footprint. In this framework, we investigated the biodiversity of Canadian and Georgian spontaneous vines, maple trees and wineries to investigate the relationship between the ecological characteristics and the metabolomic profile of this indigenous microbiota. These two different regions of isolation represents two different oenological environments, with recent and almost naive (Canada) and ancient (Georgia) oenological history that influenced significantly the related microbial diversity. In this work, we analyzed 207 yeasts strains. One hundred forty four were isolated from different Canadian sites while sixty three from Georgian cellars. All strains were characterized with molecular analysis using ITS and LSU D1/D2 regions for the taxonomic assignment. FTIR (Fourier Transform Infrared Spectroscopy) fingerprint was employed to elucidate if any significant variation exists between strains of different origin and if it is possible to correlate their metabolic profile with the ecological traits. Result showed that the substrate of isolation, and therefore the type of selection, is particularly important in triggering the evolution of the metabolomic characters, as identified by FTIR metabolomic fingerprint

Making the Hypar Up pavilion: (in)efficiencies of upcycling surplus timber products

This paper illustrates the design and fabrication processes of the Hypar Up pavilion, which served as a proof-of-concept to demonstrate the viability of a design-to-fabrication workflow for complex yet modular architectural geometries that utilise small and planar timber offcuts geometries discretised as Planar Quadrilateral (PQ) meshes. By integrating computational design and optimisation with efficient manufacturing processes, this research highlights the technical challenges of repurposing materials with unknown characteristics, notably detailing solutions, and evaluates the efficiency of design-to-manufacturing workflows with surplus timber products, using a quantitative cost analysis of the fabrication and assembly phases. While exploring the potential of repurposing scrap wood into hypar-shaped modular construction components, this work expands on existing research on segmented shells and investigates methods and means to move beyond the use of shell structures as monolithic and static artefacts. The pavilion is intended as a 1:1 modular prototype that can be resized to accommodate different dimensions of the timber panel offcuts and potential applications to be tested in future applications, such as load-bearing walls and facade retrofitting

Exploring and FTIR-ing yeast diversity towards the development of superior strains for lignocellulosic ethanol

Robust yeast strains with high inhibitors tolerance remain a critical requirement for the production of lignocellulosic bioethanol. These stress factors are known to severely hinder yeast growth and fermentation performance (Jönsson et al., 2013). This study aims at the design of industrial yeast suitable for the lignocellulose-to-bioethanol route. Grape marc was selected as extreme environment to search for innately tolerant yeast because of its limited nutrients, exposure to solar radiation, weak acid and ethanol content (Favaro et al., 2013 and 2014). One hundred and twenty Saccharomyces cerevisiae strains were recently described to have inhibitors, temperature and osmotic tolerance greater than those exhibited by industrial strains and showed remarkable potential for bioethanol production. With the aim of further investigating their industrial fitness, a Fourier Transform InfraRed Spectroscopy (FTIR)-based bioassay was employed to explore the yeast metabolomic and viability responses to inhibitors. Three different strains of S. cerevisiae (Fm17, Fm84 and DSM70049) were chosen as representative for the top, medium and low inhibitors tolerance. The yeast were exposed to acetic acid, formic acid, furfural and 5-hydroxymethyl-2- furaldehyde (HMF) and analysed by a FTIR-based assay to detect the extent of the metabolomic stress, the type of response and the induced mortality. To deeper investigate the effect of the co-presence of inhibitors on yeast metabolism, FTIR analysis was also conducted on yeast cells challenged with mixtures composed by increasing dosages of each single inhibitory compound

FTIR Metabolomic Fingerprint Reveals Different Modes of Action Exerted by Structural Variants of N-Alkyltropinium Bromide Surfactants on Escherichia coli and Listeria innocua Cells

Surfactants are extremely important agents to clean and sanitize various environments. Their biocidal activity is a key factor determined by the interactions between amphiphile structure and the target microbial cells. The object of this study was to analyze the interactions between four structural variants of N-alkyltropinium bromide surfactants with the Gram negative Escherichia coli and the Gram positive Listeria innocua bacteria. Microbiological and conductometric methods with a previously described FTIR bioassay were used to assess the metabolomic damage exerted by these compounds. All surfactants tested showed more biocidal activity in L. innocua than in E. coli. N-tetradecyltropinium bromide was the most effective compound against both species, while all the other variants had a reduced efficacy as biocides, mainly against E. coli cells. In general, the most prominent metabolomic response was observed for the constituents of the cell envelope in the fatty acids (W1) and amides (W2) regions and at the wavenumbers referred to peptidoglycan (W2 and W3 regions). This response was particularly strong and negative in L. innocua, when cells were challenged by N-tetradecyltropinium bromide, and by the variant with a smaller head and a 12C tail (N-dodecylquinuclidinium bromide). Tail length was critical for microbial inhibition especially when acting against E. coli, maybe due the complex nature of Gram negative cell envelope. Statistical analysis allowed us to correlate the induced mortality with the metabolomic cell response, highlighting two different modes of action. In general, gaining insights in the interactions between fine structural properties of surfactants and the microbial diversity can allow tailoring these compounds for the various operative conditions

Merging FT-IR and NGS for simultaneous phenotypic and genotypic identification of pathogenic Candida species

The rapid and accurate identification of pathogen yeast species is crucial for clinical diagnosis due to the high level of mortality and morbidity induced, even after antifungal therapy. For this purpose, new rapid, high-throughput and reliable identification methods are required. In this work we described a combined approach based on two high-throughput techniques in order to improve the identification of pathogenic yeast strains. Next Generation Sequencing (NGS) of ITS and D1/D2 LSU marker regions together with FTIR spectroscopy were applied to identify 256 strains belonging to Candida genus isolated in nosocomial environments. Multivariate data analysis (MVA) was carried out on NGS and FT-IR datasets, separately. Strains of Candida albicans, C. parapsilosis, C. glabrata and C. tropicalis, were identified with high-throughput NGS sequencing of ITS and LSU markers and then with FTIR. Inter-and intra-species variability was investigated by consensus principal component analysis (CPCA) which combines high-dimensional data of the two complementary analytical approaches in concatenated PCA blocks normalized to the same weight. The total percentage of correct identification reached around 97.4% for C. albicans and 74% for C. parapsilosis while the other two species showed lower identification rates. Results suggested that the identification success increases with the increasing number of strains actually used in the PLS analysis. The absence of reliable FT-IR libraries in the current scenario is the major limitation in FTIR-based identification of strains, although this metabolomics fingerprint represents a valid and affordable aid to rapid and high-throughput to clinical diagnosis. According to our data, FT-IR libraries should include some tens of certified strains per species, possibly over 50, deriving from diverse sources and collected over an extensive time period. This implies a multidisciplinary effort of specialists working in strain isolation and maintenance, molecular taxonomy, FT-IR technique and chemo-metrics, data management and data basing