Metabolic and molecular adaptation of wine yeasts at low temperature fermentation: strategies for their genetic improvement

La fermentación a baja temperatura incrementa el perfil aromático del vino. Sin embargo presenta algunos inconvenientes: descenso de la tasa de crecimiento, fermentaciones lentas o con paradas. Se comparó el metaboloma de una levadura vínica creciendo a 12 ºC y 28 ºC. Las principales diferencias se observaron en el metabolismo lipídico y en la homeostasis redox. Se comparó el metaboloma de levaduras criotolerantes, Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii, con el de S. cerevisiae creciendo a 12 ºC Las principales diferencias se encontraron en el metabolismo de la fructosa. Se analizó la capacidad fermentativa y de crecimiento de cepas mutantes y sobre-expresantes del metabolismo lipídico. El incremento de la dosis génica de los genes PSD1, LCB3 y OLE1mejoraron el crecimiento y la capacidad fermentativa. Se desarrollaron cepas mejor adaptadas al frío mediante evolución dirigida, se analizaron los cambios moleculares, observándose una inducción de 4 genes de la familia DAN/TIR.Low temperature alcoholic fermentations are becoming more frequent as they enhance wine’s aromatic profile but present some disadvantages: reduced growth rate, long lag phase, sluggish or stuck fermentations. We compared the metabolome of wine yeast growing at 12 ºC and 28 ºC in a synthetic must. The main differences were observed in lipid metabolism and redox homeostasis. We also compared the metabolome of the cryotolerant yeasts, Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii, growing at 12 ºC to the metabolome of S. cerevisiae. The main differences were found for fructose metabolism. We also analyzed the growth and fermentation capacity of lipid mutants and overexpressing strains. The increase in gene-dosage of PSD1, LCB3 and OLE1 genes improved both growth and fermentation activity. Finally, we developed cold adapted wine yeast strains by evolutionary engineering, and deciphered the underlying changes, resulting in new strain with up-regulation of 4 genes belonging to DAN/TIR family

Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature

11 pages, 1 table, 6 figures.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.This work has been financially supported by grants AGL2010-22001-C02-01 and PROMETEOII/2014/042 from the Spanish government and the Generalitat Valenciana, respectively, awarded to JMG. MLM wishes to thank the Spanish government for her FPI grant.Peer reviewe

Estudio descriptivo y comparativo entre jugadores de voleibol con y sin antecedentes de dolor de hombro

El dolor de hombro tiene una alta prevalencia en jugadores de voleibol, al ser una actividad deportiva que implica movimientos repetitivos por encima de la cabeza. Las alteraciones en el rango de movilidad y los desequilibrios de fuerza de la musculatura del hombro, parecen estar relacionados con el riesgo de sufrir dolor o disfunción de hombro. Este estudio, sugiere que los jugadores con antecedentes de dolor de hombro, presentan una mayor movilidad de rotación externa y un mayor umbral de dolor a la presión en el redondo mayor, respecto a los jugadores sin antecedentes de dolor de hombro.<br /

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.Ministerio de Economia, Industria y Competitividad BFU2017-82574-P, BFU2016-75352-

The Application of Reproductive Technologies to Natural Populations of Red Deer

P. 93-102Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer (Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed her

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle

Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM)

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment

The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function

[Background] In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype.[Methods] This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2–3 studying a relationship between FGF23 and vascular dysfunction.[Results] In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2–3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222.[Conclusion] In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.This work was supported by a Spanish government grant from the Programa Nacional I+D+I 2013–2016 and Instituto de Salud Carlos III (ISCIII) grants PI18/0138 and PI21/0654 co-financing from European Funds (FEDER), Consejería de Salud (grants PI-0136 and PI-0169-2020) from the Junta de Andalucía, Framework Programme 7 Syskid UE grant FP7-241544, and EUTOX and REDinREN from the ISCIII. N.V. and J.M.D.-T. were supported by Consejería de Economía, Innovación, Ciencia y Empleo (grant CVI-7925) from the Junta de Andalucía. Y.A. and J.R.M.-C. are senior researchers supported by the Nicolás Monardes Programme, Consejería de Salud-Servicio Andaluz de Salud (Junta de Andalucía).Peer reviewe

Supplemental Material The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function

3 pages. -- Figure S1. Supplemental Material. Effects of anti-miR-221 and miR-222. -- Figure S1. Supplemental Material: A) Anti-miR-221 and B) anti-miR-222 transfection for 48 h decreased not significantly the expression of miR-221 and miR-222 in VSMC. -- Figure S2. Supplemental Material. Recombinant Klotho administration did not modify the expression of contractile markers of VSMC. -- Figure S3. Histological quantifications in thoracic aortas of rats of synthetic markers of VSMC.Peer reviewe