Search

The use of homogeneous Brønsted acid cocatalysts (such as benzoic acid) in hydroacylation reactions via imine intermediates has been extensively studied. However, the use of heterogeneous cocatalysts has been limited to montmorillonite K10. Thus, we can use other solid acids to increase the efficiency of the reaction. In this study, we describe the effects of sulfated zirconia, Al-MCM-41 or superacid modified montmorillonite on the hydroacylation of alkenes and alkynes with aldehydes via imine intermediates and in the presence of the Wilkinson complex. Furthermore, we addressed the dual role of montmorillonite, a redox reagent in the presence of TEMPO and an acid solid, allowing the direct use of benzyl alcohols as substrates to generate saturated or α,β-unsaturated ketones., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Nowadays, the wine industry carries out fermentations at low temperatures because this oenological practice clearly improves the aromatic complexity of the final wines. In addition, nitrogen content of the must also influences the quality of the wine. In this study, we carried out a phenotypic and fermentative analysis of two industrial wine Saccharomyces cerevisiae strains (P5 and P24) at 15 and 28 °C and three nitrogen concentrations (60, 140 and 300 mg N/L) in synthetic must. Our results show that both parameters, temperature and nitrogen, are interrelated and clearly determine the competitiveness of the wine strains and their ability to adapt at low temperatures. The best adapted strain at low temperatures decreased its competitiveness at lower nitrogen concentrations. In addition, our results show that it is not only the quantity of nitrogen transported that is important but also the quality of the nitrogen source used for wine yeast adaptation at low temperatures. The presence of some amino acids, such as arginine, branched chain amino acids, and some aromatic amino acids can improve the growth and fermentation activity of wine yeasts at low temperatures. These results allow us to better understand the basis of wine yeast adaptation to fermentation conditions, providing important information for winemakers to help them select the most appropriate yeast strain, thus reducing the economic costs associated with long and sluggish fermentations. The correlation between some amino acids and better yeast fermentation performance could be used in the future to design inactive dry yeast enriched in some of these amino acids, which could be added as a nutritional supplement during low temperature fermentations.

Due to the severity of CMV infection in immunocompromised individuals the development of a vaccine has been declared a priority. However, despite the efforts made there is no yet a vaccine available for clinical use. We designed an approach to identify new CMV antigens able to inducing a broad immune response that could be used in future vaccine formulations. We have used serum samples from 28 kidney transplant recipients, with a previously acquired CMV-specific immune response to identify viral proteins that were recognized by the antibodies present in the patient serum samples by Western blot. A band of approximately 45 kDa, identified as UL44, was detected by most serum samples. UL44 immunogenicity was tested in BALB/c mice that received three doses of the UL44-pcDNA DNA vaccine. UL44 elicited both, a strong antibody response and CMV-specific cellular response. Using bioinformatic analysis we demonstrated that UL44 is a highly conserved protein and contains epitopes that are able to activate CD8 lymphocytes of the most common HLA alleles in the world population. We constructed a UL44 ORF deletion mutant virus that produced no viral progeny, suggesting that UL44 is an essential viral protein. In addition, other authors have demonstrated that UL44 is one of the most abundant viral proteins after infection and have suggested an essential role of UL44 in viral replication. Altogether, our data suggests that UL44 is a potent antigen, and favored by its abundance, it may be a good candidate to include in a vaccine formulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We measured cytomegalovirus (CMV)-specific antibodies that neutralize epithelial cell infection (CMV-AbNEIs) in 101 CMV-seropositive kidney transplant recipients (KTRs) at baseline and posttransplant months 3 and 6. All the patients received antithymocyte globulin and 3-month valganciclovir prophylaxis. There were no significant differences in pretransplant AbNEIs titers between KTRs that developed or did not develop any-level CMV infection or the composite of high-level infection and/or disease. One-year CMV infection-free survival was comparable between KTRs with or without pretransplant CMV-AbNEIs. No differences were observed by months 3 and 6. We observed no protective role for CMV-AbNEIs among CMV-seropositive KTRs undergoing T-cell-depleting induction., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Human Cytomegalovirus (HCMV) is the most frequent congenital infection worldwide causing important sequelae. However, no vaccine or antiviral treatments are currently available, thus interventions are restricted to behavioral measures. The aim of this systematic review was to assess evidence from available intervention studies using hygiene-based measures to prevent HCMV infection during pregnancy., Methods: Studies published from 1972 to 2023 were searched in Medline, PsycInfo, and Clinical Trials (PROSPERO, CRD42022344840) according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Methodological quality was assessed by two authors, using ROBE-2 and MINORS., Results: After reviewing 6 selected articles, the outcome analysis suggested that implementation of hygiene-based interventions during pregnancy prevent, to some extent, the acquisition of congenital HCMV., Conclusions: However, these conclusions are based on limited and low-quality evidence available from few studies using this type of intervention in clinical practice. Thus, it would be necessary to perform effective and homogeneous intervention studies using hygiene-based measures, evaluated in high-quality randomized controlled trials (RCTs)., (© 2024. The Author(s).)

Human cytomegalovirus (HCMV) is an important pathogen worldwide. Although HCMV infection is often asymptomatic in immunocompetent individuals, it can cause severe or even life-threatening symptoms in immunocompromised patients. Due to limitations of antiviral treatments, it is necessary to search for new therapeutic alternatives. Recent studies have highlighted the contribution of antibodies in protecting against HCMV disease, including neutralizing and non-neutralizing antibodies. Given the immunocompromised target population, monoclonal antibodies (mAbs) may represent an alternative to the clinical management of HCMV infection. In this context, we provide a synthesis of recent data revising the literature supporting and arguing about the role of the humoral immunity in controlling HCMV infection. Additionally, we review the state of the art in the development of therapies based on mAbs., Competing Interests: Declaration of interests P.P-R. is founder and shareholder of Vaxdyn, S.L., a biotechnology company developing vaccines., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Summary: A collection of conformers that exist in a dynamical equilibrium defines the native state of a protein. The structural differences between them describe their conformational diversity, a defining characteristic of the protein with an essential role in multiple cellular processes. Since most proteins carry out their functions by assembling into complexes, we have developed CoDNaS-Q, the first online resource to explore conformational diversity in homooligomeric proteins. It features a curated collection of redundant protein structures with known quaternary structure. CoDNaS-Q integrates relevant annotations that allow researchers to identify and explore the extent and possible reasons of conformational diversity in homooligomeric protein complexes., Availability and Implementation: CoDNaS-Q is freely accessible at http://ufq.unq.edu.ar/codnasq/ or https://codnas-q.bioinformatica.org/home. The data can be retrieved from the website. The source code of the database can be downloaded from https://github.com/SfrRonaldo/codnas-q., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Wine yeast have been exposed to harsh conditions for millennia, which have led to adaptive evolutionary strategies. Thus, wine yeasts from Saccharomyces genus are considered an interesting and highly valuable model to study human-drive domestication processes. The rise of whole-genome sequencing technologies together with new long reads platforms has provided new understanding about the population structure and the evolution of wine yeasts. Population genomics studies have indicated domestication fingerprints in wine yeast, including nucleotide variations, chromosomal rearrangements, horizontal gene transfer or hybridization, among others. These genetic changes contribute to genetically and phenotypically distinct strains. This review will summarize and discuss recent research on evolutionary trajectories of wine yeasts, highlighting the domestication hallmarks identified in this group of yeast., Competing Interests: The author declares no conflict of interest.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200-300 nm) compared to fibroblast-derived DB (100-600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions. Transcriptomic analysis of the three strains revealed a huge complexity of cellular mechanisms and responses. Overall, cold exerted a stronger transcriptional response in the three strains comparing with heat conditions, with a higher number of down-regulating genes than of up-regulating genes regardless the strain analyzed. The comparison of the transcriptome at both sub- and supra-optimal temperatures showed the presence of common genes up- or down-regulated in both conditions, but also the presence of common genes up- or down-regulated in the three studied strains. More specifically, we have identified and validated three up-regulated genes at sub-optimal temperature in the three strains, OPI3, EFM6 and YOL014W. Finally, the comparison of the transcriptomic data with a previous proteomic study with the same strains revealed a good correlation between gene activity and protein abundance, mainly at low temperature. Our work provides a global insight into the specific mechanisms involved in temperature adaptation regarding both transcriptome and proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: There is robust evidence indicating that the SARS-CoV-2-specific humoral response is associated with protection against severe disease. However, relatively little data exist regarding how the humoral immune response at the time of hospital admission correlates with disease severity in unimmunized patients. Our goal was toidentify variables of the humoral response that could potentially serve as prognostic markers for COVID-19 progressionin unvaccinated SARS-CoV-2 patients., Methods: A prospective cross-sectional study was carried out in a cohort of 160 unimmunized, adult COVID-19 patients from the Hospital Universitario 12Octubre. Participants were classified into four clinical groups based on disease severity: non-survivors with respiratory failure (RF), RF survivors, patients requiring oxygen therapy and those not receiving oxygen therapy. Serum samples were taken on admission and IgM, IgG, IgG subclass antibody titers were determined by ELISA, and neutralizing antibody titersusing a surrogate neutralization assay. The differences in the antibody titers between groups and the association between the clinical and analytical characteristics of the patients and the antibody titers were analyzed., Results: Patients that developed RF and survived had IgM titers that were 2-fold higher than non-survivors ( p = 0.001), higher levels of total IgG than those who developed RF and succumbed to infection ( p < 0.001), and than patients who required oxygen therapy ( p < 0.05), and had 5-fold higher IgG1 titers than RF non-survivors ( p < 0.001) and those who needed oxygen therapy ( p < 0.001), and 2-fold higher than patients that did not require oxygen therapy during admission ( p < 0.05). In contrast, RF non-survivorshad the lowest neutralizing antibodylevels, which were significantly lower compared those with RF that survived ( p = 0.03). A positive correlation was found between IgM, total IgG, IgG1 and IgG3 titers and neutralizing antibody titers in the total cohort (p ≤ 0.0036)., Conclusions: We demonstrate that patients with RF that survived infection had significantly higher IgM, IgG, IgG1 and neutralizing titers compared to patients with RF that succumb to infection, suggesting that using humoral response variables could be used as a prognostic marker for guiding the clinical management of unimmunized patients admitted to the hospital for SARS-CoV-2 infection., Competing Interests: MJM and PPR are founders and stockholders of the biotech-nology spin-off company Vaxdyn, which develops vaccines for infections caused by MDR bacteria. Vaxdyn had no role in the elaboration of this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tajuelo, Carretero, García-Ríos, López-Siles, Cano, Vázquez, Más, Rodríguez-Goncer, Lalueza, López-Medrano, Juan, Fernández-Ruiz, Aguado, McConnell and Pérez-Romero.)

In order to demonstrate the feasibility of preparing clinical-grade SARS-CoV-2-specific T-cells from convalescent donors and the ability of these cells to neutralize the virus in vitro, we used blood collected from two COVID-19 convalescent donors (before and after vaccination) that was stimulated with specific SARS-CoV-2 peptides followed by automated T-cell isolation using the CliniMacs Prodigy medical device. To determine cytotoxic activity, HEK 293T cells were transfected to express the SARS-CoV-2 M protein, mimicking SARS-CoV-2 infection. We were able to quickly and efficiently isolate SARS-CoV-2-specific T lymphocytes from both donors before and after they received the Pfizer-BioNTech vaccine. Althoughbefore vaccination, the final product contained up to 7.42% and 30.19% of IFN-γ+ CD3+ T-cells from donor 1 and donor 2, respectively, we observed an enrichment of the IFN-γ+ CD3+ T-cells after vaccination, reaching 70.47% and 42.59%, respectively. At pre-vaccination, the isolated SARS-CoV-2-specific T-cells exhibited cytotoxic activity that was significantly higher than that of unstimulated controls (donor 2: 15.41%, p -value 3.27 × 10 -3 ). The cytotoxic activity of the isolated SARS-CoV-2-specific T-cells also significantly increased after vaccination (donor 1: 32.71%, p -value 1.44 × 10 -5 ; donor 2: 33.38%, p -value 3.13 × 10 -6 ). In conclusion, we demonstrated that SARS-CoV-2-specific T-cells can quickly and efficiently be stimulated from the blood of convalescent donors using SARS-CoV-2-specific peptides followed by automated isolation. Vaccinated convalescent donors have a higher percentage of SARS-CoV-2-specific T-cells and may be more suitable as donors. Although further studies are needed to assess the clinical utility of the functional isolated SARS-CoV-2-specific T-cells in patients, previous studies using the same stimulation and isolation methods applied to other pathologies support this idea.

Summary: Conformational changes in RNA native ensembles are central to fulfill many of their biological roles. Systematic knowledge of the extent and possible modulators of this conformational diversity is desirable to better understand the relationship between RNA dynamics and function. We have developed CoDNaS-RNA as the first database of conformational diversity in RNA molecules. Known RNA structures are retrieved and clustered to identify alternative conformers of each molecule. Pairwise structural comparisons between all conformers within each cluster allows to measure the variability of the molecule. Additional annotations about structural features, molecular interactions and biological function are provided. All data in CoDNaS-RNA is free to download and available as a public website that can be of interest for researchers in computational biology and other life science disciplines., Availability and Implementation: The data underlying this article are available at http://ufq.unq.edu.ar/codnasrna or https://codnas-rna.bioinformatica.org/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CMV is a major cause of morbidity and mortality in immunocompromised individuals that will benefit from the availability of a vaccine. Despite the efforts made during the last decade, no CMV vaccine is available. An ideal CMV vaccine should elicit a broad immune response against multiple viral antigens including proteins involved in virus-cell interaction and entry. However, the therapeutic use of neutralizing antibodies targeting glycoproteins involved in viral entry achieved only partial protection against infection. In this scenario, a better understanding of the CMV proteome potentially involved in viral entry may provide novel candidates to include in new potential vaccine design. In this study, we aimed to explore the CMV genome to identify proteins with putative transmembrane domains to identify new potential viral envelope proteins. We have performed in silico analysis using the genome sequences of nine different CMV strains to predict the transmembrane domains of the encoded proteins. We have identified 77 proteins with transmembrane domains, 39 of which were present in all the strains and were highly conserved. Among the core proteins, 17 of them such as UL10, UL139 or US33A have no ascribed function and may be good candidates for further mechanistic studies.

Primary infection and/or reactivation of cytomegalovirus (CMV) in kidney transplant recipients (KTR) favor rejection and mortality. T follicular helper cells (TFH) could contribute to protection against CMV. Circulatory TFH (cTFH) were studied pretransplant and early posttransplant in 90 CMV seropositive KTR not receiving antithymocyte globulin or antiviral prophylaxis, followed-up for 1 year. Patients who presented CMV infection had significantly lower cTFH and activated cTFH pretransplant and early posttransplant. Pretransplant activated cTFH were also lower within patients who developed CMV disease. Pre- and 14 days posttransplant activated cTFH were an independent protective factor for CMV infection (HR 0.41, p = .01; and 0.52, p = .02, respectively). KTR with low cTFH 7 days posttransplant (<11.9%) had lower CMV infection-free survival than patients with high cTFH (28.2% vs. 67.6%, p = .002). cTFH were associated with CMV-specific neutralizing antibodies (Nabs). In addition, IL-21 increased interferon-γ secretion by CMV-specific CD8 + T cells in healthy controls. Thus, we show an association between cTFH and lower incidence of CMV infection, probably through their cooperation in CMV-specific Nab production and IL-21-mediated enhancement of CD8 + T cell activity. Moreover, monitoring cTFH pre- and early posttransplant could improve CMV risk stratification and help select KTR catalogued at low/intermediate risk who could benefit from prophylaxis., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Severe acute respiratory syndrome coronavirus 2-specific cell-mediated immunity (SARS-CoV-2-CMI) elicited by mRNA-based vaccines in solid organ transplant (SOT) recipients and its correlation with antibody responses remain poorly characterized., Methods: We included 44 (28 kidney, 14 liver, and 2 double organ) recipients who received the full series of the mRNA-1273 vaccine. SARS-CoV-2-CMI was evaluated at baseline, before the second dose, and at 2 wk after completion of vaccination by an ELISpot-based interferon-γ FluoroSpot assay using overlapping peptides covering the S1 domain. SARS-CoV-2 immunoglobulin G seroconversion and serum neutralizing activity against the spike protein were assessed at the same points by commercial ELISA and an angiotensin-converting enzyme-2/spike antibody inhibition method, respectively. Postvaccination SARS-CoV-2-CMI was compared with 28 healthcare workers who received the BNT162b2 vaccine., Results: Positive SARS-CoV-2-CMI increased from 6.8% at baseline to 23.3% after the first mRNA-1273 dose and 59.5% after the completion of vaccination ( P < 0.0001). Lower rates were observed for immunoglobulin G seroconversion (2.3%, 18.6%, and 57.1%, respectively) and neutralizing activity (2.3%, 11.6%, and 31.0%). There was a modest correlation between neutralizing titers and the magnitude of SARS-CoV-2-CMI (Spearman's rho: 0.375; P = 0.015). Fifteen recipients (35.7%) mounted SARS-CoV-2-CMI without detectable neutralizing activity, whereas 3 (7.1%) did the opposite, yielding poor categorical agreement (Kappa statistic: 0.201). Rates of positive SARS-CoV-2-CMI among SOT recipients were significantly decreased compared with nontransplant controls (82.1% and 100.0% after the first dose and completion of vaccination, respectively; P < 0.0001). Kidney transplantation, the use of tacrolimus and prednisone, and the number of immunosuppressive agents were associated with lower cell-mediated responses. Results remained unchanged when 3 recipients with prevaccination SARS-CoV-2-CMI were excluded., Conclusions: Two-thirds of SOT recipients mounted SARS-CoV-2-CMI following vaccination with mRNA-1273. Notable discordance was observed between vaccine-induced cell-mediated and neutralizing humoral immunities. Future studies should determine whether these patients with incomplete responses are effectively protected., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

The palladium-catalysed reaction of aryl halides and allylic alcohols is an attractive method for obtaining α,β-unsaturated aldehydes and ketones, which represent key intermediates in organic synthesis. In this context, a 1,2,3-triazol-5-ylidene (aNHC)-based palladium(ii) complex formed in situ has been found to be a selective catalyst for the syntheses of building blocks from the corresponding aryl halides and allylic alcohols, with yields ranging from 50% to 90%. The lack of toxic effects of the ligand precursor (1,2,3-triazolium salt) of the palladium(ii) complex for the harpacticoid copepod Amphiascoides atopus allowed us to contrast the efficiency of the catalytic system with the potential impact of the principal waste chemical in global aquatic ecosystems, which has not been previously addressed., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

During the last decade, many studies have demonstrated the role of CMV specific T-cell immune response on controlling CMV replication and dissemination. In fact, it is well established that transplanted patients lacking CMV-specific T-cell immunity have an increased occurrence of CMV replication episodes and CMV-related complications. In this context, the use of adoptive transfer of CMV-specific T-cells has been widely investigated and applied to Hematopoietic Stem Cell Transplant patients and may be useful as a therapeutic alternative, to reconstitute the CMV specific T-cell response and to control CMV viremia in patients receiving a transplantation. However, only few authors have explored the use of T-cell adoptive transfer in SOT recipients. We propose a novel review in which we provide an overview of the impact of using CMV-specific T-cell adoptive transfer on the control of CMV infection in SOT recipients, the different approaches to stimulate, isolate and expand CMV-specific T-cells developed over the years and a discussion of the possible use of CMV adoptive cellular therapy in this SOT population. Given the timeliness and importance of this topic, we believe that such an analysis will provide important insights into CMV infection and its treatment/prevention., Competing Interests: PP-R is the founder and shareholder of Vaxdyn, S.L., a biotechnology company developing vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 García-Ríos, Nuévalos, Mancebo and Pérez-Romero.)

Single cell oils (SCO) are a promising source of oils that could be exploited in different industrial areas. SCO for biodiesel production circumvents the controversy food vs. fuel, does not require large land areas for culture, and is independent of climate and seasonal variations, among other advantages in comparison to vegetable oils. In this study, a red yeast isolated from a mountain water source, identified as Rhodotorula glutinis T13, showed high potential for lipid production (40% w/w) with suitable growth parameters, yields, and fatty acids profile. Yeast lipids showed a high content of unsaturated fatty acids (56.44%; C18:1, C18:2), and the fuel properties (cetane number, iodine value, density, kinematic viscosity, etc.) of yeast oil analysed were in good agreement with international biodiesel standards. The results show that R. glutinis T13 can be used in the future as a promising microorganism for the commercial production of biodiesel., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cocoa pulp fermentation is a consequence of the succession of indigenous yeasts, lactic acid bacteria and acetic acid bacteria that not only produce a diversity of metabolites, but also cause the production of flavour precursors. However, as such spontaneous fermentations are less reproducible and contribute to produce variability, interest in a microbial starter culture is growing that could be used to inoculate cocoa pulp fermentations. This study aimed to generate robust S. cerevisiae strains by thermo-adaptive evolution that could be used in cocoa fermentation. We evolved a cocoa strain in a sugary defined medium at high temperature to improve both fermentation and growth capacity. Moreover, adaptive evolution at high temperature (40 °C) also enabled us to unveil the molecular basis underlying the improved phenotype by analysing the whole genome sequence of the evolved strain. Adaptation to high-temperature conditions occurred at different genomic levels, and promoted aneuploidies, segmental duplication, and SNVs in the evolved strain. The lipid profile analysis of the evolved strain also evidenced changes in the membrane composition that contribute to maintain an appropriate cell membrane state at high temperature. Our work demonstrates that experimental evolution is an effective approach to generate better-adapted yeast strains at high temperature for industrial processes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Elucidation of temperature tolerance mechanisms in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- and supra-optimal temperatures under chemostat conditions. We employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains in all conditions was performed by sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS), allowing the quantification of 997 proteins, data available via ProteomeXchange (PXD016567). Our analysis demonstrated that temperature responses differ between the strains; however, we also found some common responsive proteins, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome. The proteomic data evidenced that the cold response involves strong repression of translation-related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation while, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects the yeast proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Cytomegalovirus (CMV) infection elicits a potent immune response that includes the stimulation of antibodies with neutralizing activity. Recent studies have focused on elucidating the role of neutralizing antibodies in protecting against CMV infection and disease and characterizing viral antigens against which neutralizing antibodies are directed. Here, we provide a synthesis of recent data regarding the role of neutralizing antibodies in protection against CMV infection/disease. We consider the role of humoral immunity in the context of the global CMV-specific immune response, and the implications that recent findings have for vaccine and antibody-based therapy design., Competing Interests: Disclaimer Statement M.J.M. and P.P.R. are founders and shareholders of Vaxdyn, S.L., a biotechnology company developing vaccines., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A phenotypic screening of 12 industrial yeast strains and the well-studied laboratory strain CEN.PK113-7D at cultivation temperatures between 12 °C and 40 °C revealed significant differences in maximum growth rates and temperature tolerance. From those 12, two strains, one performing best at 12 °C and the other at 40 °C, plus the laboratory strain, were selected for further physiological characterization in well-controlled bioreactors. The strains were grown in anaerobic chemostats, at a fixed specific growth rate of 0.03 h -1 and sequential batch cultures at 12 °C, 30 °C, and 39 °C. We observed significant differences in biomass and ethanol yields on glucose, biomass protein and storage carbohydrate contents, and biomass yields on ATP between strains and cultivation temperatures. Increased temperature tolerance coincided with higher energetic efficiency of cell growth, indicating that temperature intolerance is a result of energy wasting processes, such as increased turnover of cellular components (e.g. proteins) due to temperature induced damage., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to affect the work reported in this paper., (© 2020 Published by Elsevier B.V.)

Sulfite resistance is an important oenological trait for wine yeasts because this compound is used during winemaking as a microbial inhibitor and antioxidant. The molecular mechanisms by which Saccharomyces cerevisiae responds and tolerates SO 2 have been mainly focused on the sulfite efflux pump encoded by SSU1 . Different chromosomal rearrangements in the regulatory region of this gene have been correlated with improved sulfite tolerance. However, other molecular factors must contribute to this trait because the SSU1 gene activity does not always fit with sulfite tolerance. An interesting approach to shed light onto this issue could be found by Lage et al. (2019). These authors have combined transcriptomic and genome-wide analysis to describe how the poorly characterized transcription factor Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO 2 . Additionally, large-scale phenotyping revealed the identification of 50 Com2-targets contributing to the protection against SO 2 . This information is very interesting for gaining knowledge regarding this important industrial trait., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Sulfite-generating compounds are widely used during winemaking as preservatives because of its antimicrobial and antioxidant properties. Thus, wine yeast strains have developed different genetic strategies to increase its sulfite resistance. The most efficient sulfite detoxification mechanism in Saccharomyces cerevisiae uses a plasma membrane protein called Ssu1 to efflux sulfite. In wine yeast strains, two chromosomal translocations (VIIItXVI and XVtXVI) involving the SSU1 promoter region have been shown to upregulate SSU1 expression and, as a result, increase sulfite tolerance. In this study, we have identified a novel chromosomal rearrangement that triggers wine yeast sulfite adaptation. An inversion in chromosome XVI (inv-XVI) probably due to sequence microhomology, which involves SSU1 and GCR1 regulatory regions, increases the expression of SSU1 and the sulfite resistance of a commercial wine yeast strain. A detailed dissection of this chimeric SSU1 promoter indicates that both the removed SSU1 promoter sequence and the relocated GCR1 sequence contribute to SSU1 upregulation and sulfite tolerance. However, no relevant function has been attributed to the SSU1-promoter-binding transcription factor Fzf1. These results unveil a new genomic event that confers an evolutive advantage to wine yeast strains., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Fermentations carried out at low temperatures (10-15°C) enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. Notwithstanding, as Saccharomyces cerevisiae is the main species responsible for alcoholic fermentation, other species of the genus Saccharomyces , such as cryophilic species Saccharomyces eubayanus , Saccharomyces kudriavzevii and Saccharomyces uvarum , are better adapted to low-temperature fermentations during winemaking. In this work, a Saccharomyces cerevisiae × S. uvarum hybrid was constructed to improve the enological features of a wine S. cerevisiae strain at low temperature. Fermentations of white grape musts were performed, and the phenotypic differences between parental and hybrid strains under different temperature conditions were examined. This work demonstrates that hybridization constitutes an effective approach to obtain yeast strains with desirable physiological features, like low-temperature fermentation capacity, which genetically depend on the expression of numerous genes (polygenic character). As this interspecific hybridization approach is not considered a GMO, the genetically improved strains can be quickly transferred to the wine industry.

Cells face genetic and/or environmental changes in order to outlast and proliferate. Characterization of changes after stress at different "omics" levels is crucial to understand the adaptation of yeast to changing conditions. Wine fermentation is a stressful situation which yeast cells have to cope with. Genome-wide analyses extend our cellular physiology knowledge by pointing out the mechanisms that contribute to sense the stress caused by these perturbations (temperature, ethanol, sulfites, nitrogen, etc.) and related signaling pathways. The model organism, Saccharomyces cerevisiae, was studied in response to industrial stresses and changes at different cellular levels (transcriptomic, proteomic, and metabolomics), which were followed statically and/or dynamically in the short and long terms. This chapter focuses on the response of yeast cells to the diverse stress situations that occur during wine fermentations, which induce perturbations, including nutritional changes, ethanol stress, temperature stress, oxidative stress, etc.

Background: Although Saccharomyces cerevisiae is the most frequently isolated species in wine fermentation, and the most studied species, other species and interspecific hybrids have greatly attracted the interest of researchers in this field in the last few years, given their potential to solve new winemaking industry challenges. S. cerevisiae x S. kudriavzevii hybrids exhibit good fermentative capabilities at low temperatures, and produce wines with smaller alcohol quantities and larger glycerol quantities, which can be very useful to solve challenges in the winemaking industry such as the necessity to enhance the aroma profile., Methods: In this study, we performed a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids in low temperature winemaking conditions., Results: The results revealed that the hybrids have acquired both fermentative abilities and cold adaptation abilities, attributed to S. cerevisiae and S. kudriavzevii parental species, respectively, showcasing their industrially relevant characteristics. For several key genes, we also studied the contribution to gene expression of each of the alleles of S. cerevisiae and S. kudriavzevii in the S. cerevisiae x S. kudriavzevii hybrids. From the results, it is not clear how important the differential expression of the specific parental alleles is to the phenotype of the hybrids., Conclusions: This study shows that the fermentative abilities of S. cerevisiae x S. kudriavzevii hybrids at low temperatures do not seem to result from differential expression of specific parental alleles of the key genes involved in this phentoype., Competing Interests: Competing interests: No competing interests were disclosed.

Background: Although Saccharomyces cerevisiae is the most frequently isolated species in wine fermentation, and the most studied species, other species and interspecific hybrids have greatly attracted the interest of researchers in this field in the last few years, given their potential to solve new winemaking industry challenges. S. cerevisiae x S. kudriavzevii hybrids exhibit good fermentative capabilities at low temperatures, and produce wines with smaller alcohol quantities and larger glycerol quantities, which can be very useful to solve challenges in the winemaking industry such as the necessity to enhance the aroma profile., Methods: In this study, we performed a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids in low temperature winemaking conditions., Results: The results revealed that the hybrids have acquired both fermentative abilities and cold adaptation abilities, attributed to S. cerevisiae and S. kudriavzevii parental species, respectively, showcasing their industrially relevant characteristics. For several key genes, we also studied the contribution to gene expression of each of the alleles of S. cerevisiae and S. kudriavzevii in the S. cerevisiae x S. kudriavzevii hybrids. From the results, it is not clear how important the differential expression of the specific parental alleles is to the phenotype of the hybrids., Conclusions: This study shows that the fermentative abilities of S. cerevisiae x S. kudriavzevii hybrids at low temperatures do not seem to result from differential expression of specific parental alleles of the key genes involved in this phenotype., Competing Interests: Competing interests: No competing interests were disclosed.

Background: Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci., Results: In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature., Conclusions: The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Fermentation at low temperatures is one of the most popular current winemaking practices because of its reported positive impact on the aromatic profile of wines. However, low temperature is an additional hurdle to develop Saccharomyces cerevisiae wine yeasts, which are already stressed by high osmotic pressure, low pH and poor availability of nitrogen sources in grape must. Understanding the mechanisms of adaptation of S. cerevisiae to fermentation at low temperature would help to design strategies for process management, and to select and improve wine yeast strains specifically adapted to this winemaking practice. The problem has been addressed by several approaches in recent years, including transcriptomic and other high-throughput strategies. In this work we used a genome-wide screening of S. cerevisiae diploid mutant strain collections to identify genes that potentially contribute to adaptation to low temperature fermentation conditions. Candidate genes, impaired for growth at low temperatures (12°C and 18°C), but not at a permissive temperature (28°C), were deleted in an industrial homozygous genetic background, wine yeast strain FX10, in both heterozygosis and homozygosis. Some candidate genes were required for growth at low temperatures only in the laboratory yeast genetic background, but not in FX10 (namely the genes involved in aromatic amino acid biosynthesis). Other genes related to ribosome biosynthesis (SNU66 and PAP2) were required for low-temperature fermentation of synthetic must (SM) in the industrial genetic background. This result coincides with our previous findings about translation efficiency with the fitness of different wine yeast strains at low temperature., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity., Biological Significance: Temperature is one of the main relevant environmental variables that microorganisms have to cope with and it is also a key factor in some industrial processes that involve microorganisms. However, we are still far from understanding the molecular and physiological mechanisms of adaptation at low temperatures. The results obtained in this study provided a global atlas of the proteome changes triggered by temperature in three different species of the genus Saccharomyces with different degree of cryotolerance. These results would facilitate a better understanding of mechanisms for how yeast could adapt at the low temperature of growth., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Many factors, such as must composition, juice clarification, fermentation temperature, or inoculated yeast strain, strongly affect the alcoholic fermentation and aromatic profile of wine. As fermentation temperature is effectively controlled by the wine industry, low-temperature fermentation (10-15°C) is becoming more prevalent in order to produce white and "rosé" wines with more pronounced aromatic profiles. Elucidating the response to cold in Saccharomyces cerevisiae is of paramount importance for the selection or genetic improvement of wine strains. Previous research has shown the strong implication of oxidative stress response in adaptation to low temperature during the fermentation process. Here we aimed first to quantify the correlation between recovery after shock with different oxidants and cold, and then to detect the key genes involved in cold adaptation that belong to sulfur assimilation, peroxiredoxins, glutathione-glutaredoxins, and thioredoxins pathways. To do so, we analyzed the growth of knockouts from the EUROSCARF collection S. cerevisiae BY4743 strain at low and optimal temperatures. The growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted and validated as key genes in the background of two commercial wine strains with a divergent phenotype in their low-temperature growth. We identified three genes, AHP1, MUP1, and URM1, whose deletion strongly impaired low-temperature growth.

Background: The wine industry needs better-adapted yeasts to grow at low temperature because it is interested in fermenting at low temperature to improve wine aroma. Elucidating the response to cold in Saccharomyces cerevisiae is of paramount importance for the selection or genetic improvement of wine strains., Results: We followed a global approach by comparing transcriptomic, proteomic and genomic changes in two commercial wine strains, which showed clear differences in their growth and fermentation capacity at low temperature. These strains were selected according to the maximum growth rate in a synthetic grape must during miniaturized batch cultures at different temperatures. The fitness differences of the selected strains were corroborated by directly competing during fermentations at optimum and low temperatures. The up-regulation of the genes of the sulfur assimilation pathway and glutathione biosynthesis suggested a crucial role in better performance at low temperature. The presence of some metabolites of these pathways, such as S-Adenosilmethionine (SAM) and glutathione, counteracted the differences in growth rate at low temperature in both strains. Generally, the proteomic and genomic changes observed in both strains also supported the importance of these metabolic pathways in adaptation at low temperature., Conclusions: This work reveals a novel role of the sulfur assimilation pathway in adaptation at low temperature. We propose that a greater activation of this metabolic route enhances the synthesis of key metabolites, such as glutathione, whose protective effects can contribute to improve the fermentation process.

Wine produced by low-temperature fermentation is mostly considered to have improved sensory qualities. However few commercial wine strains available on the market are well-adapted to ferment at low temperature (10 - 15°C). The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. One strategy to modify lipid composition is to alter transcriptional activity by deleting or overexpressing the key genes of lipid metabolism. In a previous study, we identified the genes of the phospholipid, sterol and sphingolipid pathways, which impacted on growth capacity at low temperature. In the present study, we aimed to determine the influence of these genes on fermentation performance and growth during low-temperature wine fermentations. We analyzed the phenotype during fermentation at the low and optimal temperature of the lipid mutant and overexpressing strains in the background of a derivative commercial wine strain. The increase in the gene dosage of some of these lipid genes, e.g., PSD1 , LCB3, DPL1 and OLE1, improved fermentation activity during low-temperature fermentations, thus confirming their positive role during wine yeast adaptation to cold. Genes whose overexpression improved fermentation activity at 12°C were overexpressed by chromosomal integration into commercial wine yeast QA23. Fermentations in synthetic and natural grape must were carried out by this new set of overexpressing strains. The strains overexpressing OLE1 and DPL1 were able to finish fermentation before commercial wine yeast QA23. Only the OLE1 gene overexpression produced a specific aroma profile in the wines produced with natural grape must., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

The effect of the main environmental factors governing wine fermentation on the fitness of industrial yeast strains has barely received attention. In this study, we used the concept of fitness advantage to measure how increasing nitrogen concentrations (0 to 200 mg N/liter), ethanol (0 to 20%), and temperature (4 to 45°C) affects competition among four commercial wine yeast strains (PDM, ARM, RVA, and TTA). We used a mathematical approach to model the hypothetical time needed for the control strain (PDM) to out-compete the other three strains in a theoretical mixed population. The theoretical values obtained were subsequently verified by competitive mixed fermentations in both synthetic and natural musts, which showed a good fit between the theoretical and experimental data. Specifically, the data show that the increase in nitrogen concentration and temperature values improved the fitness advantage of the PDM strain, whereas the presence of ethanol significantly reduced its competitiveness. However, the RVA strain proved to be the most competitive yeast for the three enological parameters assayed. The study of the fitness of these industrial strains is of paramount interest for the wine industry, which uses them as starters of their fermentations. Here, we propose a very simple method to model the fitness advantage, which allows the prediction of the competitiveness of one strain with respect to different abiotic factors.

Tonsil Actisil FF, which is a commercial bentonitic clay, promotes the formation of cycloveratrylene macrocycles and benzyl oligomers from the corresponding benzyl alcohols in good yields under microwave heating and infrared irradiation in the absence of solvent in both cases. The catalytic reaction is sensitive to the type of substituent on the aromatic ring. Thus, when benzyl alcohol was substituted with a methylenedioxy, two methoxy or three methoxy groups, a cyclooligomerisation process was induced. Unsubstituted, methyl and methoxy benzyl alcohols yielded linear oligomers. In addition, computational chemistry calculations were performed to establish a validated mechanistic pathway to explain the growth of the obtained linear oligomers.

Three new diarylbutane lignans, named 9-acetyl-9'-pentadecanoil-dihydroclusin (1), 2,3-demethoxy-secoisolintetralin monoacetate (4) and dihydroclusin monoacetate (5), have been isolated from the resin of Bursera fagaroides, together with two known ones, 2,3-demethoxy-secoisolintetralin diacetate (2) and dihydroclusin diacetate (3). The complete structure assignments were obtained by means of (1)H and (13)C NMR spectra.

A facile and mild macrolactonization reaction of ω-hydroxy acids was developed based on the transesterification of benzotriazole esters. Treatment of ω-hydroxy acids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxy benzotriazole (HOBT) in chloroform provided macrolactones in excellent yields. The reactions were performed under basic, neutral and acidic conditions using N,N-dimethylaminopyridine (DMAP), tetrabutylammonium tetrafluoroborate (TBABF(4)) and BF(3)·Et(2)O, respectively. A calcined hydrotalcite was also used instead of DMAP. Finally, to test the scope of the protocol in the synthesis of biologically relevant macrolactones, the total synthesis of Sansalvamide A was carried out.