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

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-Pccbgl1]-C1 Engineered by Multiple \u3b4-Integration of a Fungal \u3b2-Glucosidase Gene

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple d-integration of the b-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the d-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple d-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains

Estimated prevalence of undiagnosed HCV infected individuals in Italy: A mathematical model by route of transmission and fibrosis progression

Background: The universal treatment of diagnosed patients with chronic HCV infection has been widely conducted in Italy since 2017. However, the pool of individuals diagnosed but yet to be treated in Italy has been estimated to end around 2025, leaving a significant proportion of infected individuals undiagnosed/without care. Estimates of this population are currently unknown. Methods: A probabilistic modelling approach was applied to estimate annual historical HCV incident cases by their age-group (0–100 years) distribution from available literature and Italian National database (1952 to October 2019). Viraemic infection rates were modelled on the main infection routes in Italy: people who inject drugs (PWID), tattoos, sexual transmission, glass syringe use, blood transfusion and vertical transmission. Annual liver fibrosis stage transition probabilities were modelled using a Markov model. The number of HCV viraemic asymptomatic (fibrosis stage F0-F3:potentially undiagnosed/unlinked to care) and symptomatic (fibrosis stage F4: potentially linked to care) individuals was estimated. Results: By October 2019, total viraemic HCV individuals in Italy (excluding treated patients since 1992) were estimated to be 410,775 (0.68 % of current population of Italy; 95 % CI: 0.64−0.71%, based on the current Italian population), of which 281,809 (0.47 %; 95 % CI:0.35−0.60%) were fibrosis stage F0-F3. Among different high risk groups in stage F0-F3, the following distribution was estimated: PWID; 52.0 % (95 % CI:37.9–66.6 %), tattoo; 28.8 % (95 % CI:23–32.3 %), sexual transmission; 12.0 % (95 % CI:9.6–13.7 %), glass syringe and transfusion; 6.4 % (95 % CI:2.4–17.8 %), and vertical transmission; 0.7 % (95 % CI:0.4–1.2 %). Conclusion: Under the assumption that most untreated HCV-infected individuals with stage F0-F3 are undiagnosed, more than 280,000 individuals are undiagnosed and/or unlinked to care in Italy. Marked heterogeneity across the major routes of HCV transmission was estimated. This modelling approach may be a useful tool to characterise the HCV epidemic profile also in other countries, based on country specific epidemiology and HCV main transmission routes

Effect of different dietary tannin extracts on lamb growth performances and meat oxidative stability: comparison between mimosa, chestnut and tara.

Little information is available on the effects of different sources of tannins on ruminant product quality. Nowadays several tannin-rich extracts, produced from different plants, are available and contain tannins belonging to different chemical groups, but most of these have not been used so far as feed supplements. The present study aimed at comparing the effects of feeding three tannin extracts (one containing condensed tannins and two containing hydrolysable tannins) to lambs on growth performances and meat oxidative stability. Comisana male lambs were divided into four groups (n=9 each) and were fed for 75 days: a concentrate-based diet (CON), or CON supplemented with 4% tannin extracts from either mimosa (MI; Acacia mearnsii, De Wild; condensed tannins), chestnut (CH; Castanea sativa, Mill; hydrolysable ellagitannins) or tara (TA; Cesalpinia spinosa, (Molina) Kuntze; hydrolysable gallotannins). Only CH reduced growth rate, final weight, carcass weight and feed intake (P0.05). The TA diet increased (P<0.001) the concentration of γ-tocopherol in muscle and tended to increase that of α-tocopherol (P=0.058). Oxidative stability of raw and cooked meat, or of meat homogenates incubated with pro-oxidants, was not affected by the extracts. These results, compared with those reported in the literature, highlight that some effects of tannins cannot be easily generalized, but may strictly depend on their specific characteristics and on conditions inherent to the basal diet and the metabolic status of the animals

High-performance versatile setup for simultaneous Brillouin-Raman micro-spectroscopy

This is the author accepted manuscript. The final version is available from American Physical Society via the DOI in this record.Brillouin and Raman scattering spectroscopy are established techniques for the nondestructive contactless and label-free readout of mechanical, chemical and structural properties of condensed matter. Brillouin-Raman investigations currently require separate measurements and a site-matched approach to obtain complementary information from a sample. Here we demonstrate a new concept of fully scanning multimodal micro-spectroscopy for simultaneous detection of Brillouin and Raman light scattering in an exceptionally wide spectral range, from fractions of GHz to hundreds of THz. It yields an unprecedented 150 dB contrast, which is especially important for the analysis of opaque or turbid media such as biomedical samples, and spatial resolution on a sub-cellular scale. We report the first applications of this new multimodal method to a range of systems, from a single cell to the fast reaction kinetics of a curing process, and the mechano-chemical mapping of highly scattering biological samples.S. Corezzi acknowledges financial support from MIUR-PRIN (Project No. 2012J8X57P). S. Caponi acknowledges support from PAT (Provincia Autonoma di Trento) (GP/PAT/2012) “Grandi Progetti 2012” Project “MaDEleNA.” P. S., A. M., M. P. acknowledge financial support from Centro Nazionale Trapianti (Project: “Studio di cellule per uso clinico umano, con particolare riferimento a modelli cellulari (liposomi) e linee cellulari in interazione con crioconservanti e con materiali biocompatibili”). L. C. and S. Caponi acknowledge financial support from Consiglio Nazionale delle Ricerche-Istituto Officina dei Materiali. F. P. acnowledges support from the UK Engineering and Physical Sciences Research Council (Grant No. EP/M028739/1 (F. P.)). The authors acknowledge Jacopo Scarponi for valuable help in setting up the hardware and software system for simultaneous Raman and BLS measurements

One fungus, which genes?: development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation elongation factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail