Genome sequencing and oenologically relevant traits of the Uruguayan native yeast Issatchenkia terricola

Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from Uruguayan vineyards; it stands out for its ability to produce high levels of β-glucosidase activity, which contributes to the aromatic complexity of wines. To delve into the potential oenological applications of this strain, its high-quality genome was obtained and explored, allowing the main central carbon and nitrogen metabolic pathways to be reconstructed. I. terricola is able to utilise glycerol as the sole carbon source in a way that has not previously been described for yeasts. The genes of the fermentome and those involved in stress resistance during winemaking were also identified, and differences were found when compared to S. cerevisiae, which may explain why I. terricola is unable to complete fermentation. The pathways responsible for natural aroma synthesis were also reconstructed, and the production of aromatic acids, alcohols, esters, acetates and lactones was verified experimentally. Finally, sequences encoding for β-glucosidases, in addition to the previously characterised one, were identified in the genome. The work presented here lays the groundwork for experimental research focused on the dissection of the metabolism of a native non-Saccharomyces strain and its application for oenological and biotechnological purposes.ANII: FMV_1_2017_1_13657

One-year monitoring SARS-CoV-2 RNA surface contamination in hospitals reveals no correlation with organic material and negative pressure as a limiting factor for contamination

Understanding transmission routes of SARS-CoV-2 is crucial to establish effective interventions in healthcare institutions. Although the role of surface contamination in SARS-CoV-2 transmission has been controversial, fomites have been proposed as a contributing factor. Longitudinal studies about SARS-CoV-2 surface contamination in hospitals with different infrastructure (presence or absence of negative pressure systems) are needed to improve our understanding of their effectiveness on patient healthcare and to advance our knowledge about the viral spread. We performed a one-year longitudinal study to evaluate surface contamination with SARS-CoV-2 RNA in reference hospitals. These hospitals have to admit all COVID-19 patients from public health services that require hospitalization. Surfaces samples were molecular tested for SARS-CoV-2 RNA presence considering three factors: the dirtiness by measuring organic material, the circulation of a high transmissibility variant, and the presence or absence of negative pressure systems in hospitalized patients' rooms. Our results show that: (i) There is no correlation between the amount of organic material dirtiness and SARS-CoV-2 RNA detected on surfaces; (ii) SARS-CoV-2 high transmissible Gamma variant introduction significantly increased surface contamination; (iii) the hospital with negative pressure systems was associated with lower levels of SARS-CoV-2 surface contamination and, iv) most environmental samples recovered from contaminated surfaces were assigned as non-infectious. This study provides data gathered for one year about the surface contamination with SARS-CoV-2 RNA sampling hospital settings. Our results suggest that spatial dynamics of SARS-CoV-2 RNA contamination varies according with the type of SARS-CoV-2 genetic variant and the presence of negative pressure systems. In addition, we showed that there is no correlation between the amount of organic material dirtiness and the quantity of viral RNA detected in hospital settings. Our findings suggest that SARS CoV-2 RNA surface contamination monitoring might be useful for the understanding of SARS-CoV-2 dissemination with impact on hospital management and public health policies. This is of special relevance for the Latin-American region where ICU rooms with negative pressure are insufficient.Agencia Nacional de Investigación e Innovació

Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since most of these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low- and middle-income countries. In order to develop an inexpensive SARS-CoV-2 detection protocol using available resources we evaluated the SYBR Green based detection of SARS-CoV-2 to establish a suitable assay. To do so, we adapted one of the WHO recommended TaqMan-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results indicate that SYBR-Green detection of ORF1b-nsp14 target represents a reliable cost-effective alternative to increase the testing capacity.ANII: FCE_1_2019_1_156157ANII: FCE_1_2019_1_15593

Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit of human mitochondrial trifunctional protein, a key enzyme of lipid metabolism

During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial β-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or β-oxidation activity

Early transcontinental import of SARS-CoV-2 variant of concern 202012/01 (B.1.1.7) from Europe to Uruguay

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 causes a more transmissible and apparently more severe disease. We report its early introduction from Europe to South America from a traveler who arrived in Uruguay from the United Kingdom, even before B.1.1.7 was recognized as a variant of concern. This highlights the risk of introduction of SARS-CoV-2 variants despite strict contingency protocols and underscores the need of improving real-time surveillance worldwide.ANII: POS_NAC_2018_1_15149

Informe final del proyecto: Obtención de una cepa de Saccharomyces cerevisiae productora de una beta-glucosidasa de Issatchenkia terricola y explotación del genoma de esta levadura nativa para la identificación de nuevas enzimas con potencial aplicación en enología

El desarrollo del aroma del vino depende en gran parte de la existencia durante la elaboración, de enzimas (glicosidasas) capaces de actuar eficientemente sobre los sustratos glicosídicos existentes, generando compuestos volátiles. Estudios previos de nuestro grupo con enzimas aisladas de la microbiota de viñedos uruguayos, demostraron que una beta-glucosidasa de la cepa Issatchenkia terrícola presenta propiedades muy promisorias en condiciones enológicas y se destaca por impartir características aromáticas propias a los vinos locales. Sin embargo, los bajos niveles producidos por la cepa autóctona constituyen una limitante para la manipulación y posible aplicación biotecnológica de dicha glucosidasa. Con el objetivo de clonarla y expresarla en Saccharomyces cerevsiae con mayor rendimiento, nos encontramos actualmente focalizados en obtener la secuencia de dicha glucosidasa mediante el diseño de cebadores degenerados, dado que no disponemos aún del genoma de I. terrícola. La cepa generada será utilizada en ensayos de microvinificaciones y análisis químico y sensorial de aromas de los vinos obtenidos. Complementariamente, se propone avanzar en la caracterización molecular mediante secuenciación masiva del genoma de I. terrícola. Esto permitirá identificar la presencia de otros genes codificantes para beta-glucosidasas así como otras glicosidasas. Asimismo, la interpretación del genoma permitirá identificar otras actividades enzimáticas con potencial interés biotecnológico. El proyecto implica el diseño y uso racional del potencial existente en la microbiota nativa enológica, integrando conocimientos desde un enfoque multidisciplinario desde las áreas de bioquímica, biología molecular, genómica y química de aromas. Los resultados podrían generar productos potencialmente transferibles a la industria enológica.Agencia Nacional de Investigación e Innovació

Emergence and spread of a B.1.1.28-derived P.6 lineage with Q675H and Q677H spike mutations in Uruguay

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020–February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay’s capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest

Real-Time genomic surveillance for SARS-CoV-2 variants of concern, Uruguay

We developed a genomic surveillance program for realtime monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARSCoV- 2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay

Superfamilia SCP/TAPS de Mesocestoides corti : contribución a la dilucidación del rol de estas proteínas durante el desarrollo estrobilar

Tribunal: Dra. Cecilia Fernández, Dra. Adriana Esteves, Dra. Verónica Fernánde

Bases per a l'acció socioeducativa amb la infància, setembre 2010

Material docent de la Universitat Oberta de Catalunya.Material docente de la "Universitat Oberta de Catalunya".Learning material of the "Universitat Oberta de Catalunya