Biochemistry and physiology of rehydration and adaptation of active dry wine yeast for winemaking.

Alcoholic fermentation is a process mainly characterised by several factors that induce yeast stress response (low pH, high osmotic pressure, low nitrogen content, etc). Thus, stress response is essential to yeast in order to assure its survival, adaptation and growth, in both natural habitats and industrial conditions such as winemaking. One of the main features to optimise and improve alcoholic fermentation is the establishment of metabolic and physiological changes by the yeast just after must inoculation. In this early phase, yeast must quickly change its metabolism to achieve maximum advantage in the new growth medium. Oenological industry uses selected active dry wine yeast (ADWY) to inoculate the musts in order to get a better control over the fermentation process. Those dry yeasts have to be rehydrated before must inoculation. Therefore, yeast rehydration is the first step to ensure healthy cells and a good fermentation performance. The knowledge of the early yeast metabolic responses, from a biochemical and physiological point of view, were the main objectives of this thesis. The studied objectives were: Analysis of trehalose and glycogen metabolism (reserve carbohydrates) in a commercial wine yeast strain, considering these carbohydrates markers of stress response. Study of their metabolism in winemaking conditions, taking into account the fermentation temperature (low 13ºC and control 25ºC), nitrogen content (low 60 mg/l, control 300 mg/l and high 1200 mg/l) and the ADWY rehydration and pre-adaptation. Determination of early transcriptional responses of yeast just after must inoculation. It was considered the presence of glucose and fructose, the osmotic shock and the yeast metabolic activation, including the presence of the drug Cycloheximide, which is an inhibitor of protein synthesis. Characterisation of the standard protocol of ADWY rehydration in an oenological context, which is rehydrate in warm water (37ºC) during 30 minutes.Low fermentation temperatures affected fermentation kinetics, but not induced any additional stress response. In fact, high levels of trehalose were accumulated at control temperature than at 13ºC. In industrial conditions, after a period of pre-adaptation before inoculation, carbohydrates were completely depleted once yeasts were inoculated into the must. Their synthesis started simultaneously with a change in the phase of growth, from exponential to stationary, coinciding with nitrogen depletion. We also studied rehydration and characterised the early response to synthetic must but also to relevant conditions that ADWY must cope after inoculation (osmotic shock, presence of carbon sources). Glycogen content did not presented any change. However, trehalose was early mobilised in those media capable to ensure yeast growth. The effect of nitrogen availability upon trehalose metabolism was also studied. Neither glucose nor nitrogen exhaustion were involved in the regulation of trehalose metabolism. TPS1 (gene which codifies for trehalose-6-phosphate synthase) was induced just before trehalose accumulation, simultaneously with growth arrest. Microarray technique was used to determine whole gene expression after rehydration and in the same conditions previously studied. We can conclude that 30 minutes of rehydration in water is enough for the yeast to fully recover and that longer times in this medium are detrimental. The yeast transcriptional switch is the presence of fermentable sugars, and this is mostly related to ribosome and protein synthesis, glycolysis and ethanol synthesis.La fermentació alcohòlica és un procés bàsicament caracteritzat per una sèrie de factors que indueixen estrès al llevat, el microorganisme responsable de la seva consecució. La resposta a estrès per part del llevat és essencial per a la seva supervivència, adaptació i creixement, tant en medis naturals com en processos industrials (enologia). Una de les principals característiques per a l'optimització de la fermentació alcohòlica és l'establiment de canvis metabòlics i fisiològics del llevat just després de la inoculació en el most. És en aquesta fase inicial quan el llevat ha de canviar ràpidament el seu metabolisme per tal d'adquirir el màxim d'avantatge en el nou medi de creixement. La indústria enològica disposa de llevats secs actius (LSA) seleccionats que s'inoculen al most per tal d'assolir un millor control sobre el procés fermentatiu. Aquests llevats secs requereixen d'una rehidratació prèvia a la inoculació en el most. La rehidratació és el primer pas per tal d'assegurar el bon estat cel.lular i una bona fermentació alcohòlica. El coneixement de les respostes metabòliques inicials del llevat des d'un punt de vista bioquímic i fisiològic foren els objectius fonamentals d'aquest treball. Els objectius van ésser:- Anàlisi del metabolisme de trehalosa i glicogen (carbohidrats de reserva) en una soca de llevat comercial, com a indicadors de resposta a estrès. Estudi del metabolisme en condicions víniques, considerant com a variables la temperatura de fermentació (baixa, 13ºC i control, 25ºC), contingut en nitrogen (baix 60 mg/l, control 300 mg i elevat 1200 mg/l) i la rehidratació i preadaptació del LSA.- Determinació de les respostes transcripcionals inicials del llevat immediatament després de la seva inoculació en el most. Es va considerar la presència de glucosa i fructosa, el shock osmòtic i l'activació metabòlica del llevat, incloent la presència de cicloheximida, un inhibidor de la síntesi proteica.- Caracterització del protocol estàndard de rehidratació del llevat dins el context enològic, que és la utilització d'aigua a 37-40ºC durant 30 minuts. Les baixes temperatures de fermentació van incidir sobre la cinètica fermentativa, però no sobre una resposta a estrès. De fet, es va trobar major acumulació de trehalosa a temperatura control que a 13ºC. En condicions industrials, després d'un període d'adaptació previ a la inoculació (peu de cuba), els nivells de carbohidrats van ser nuls. La seva síntesi es va produir al canvi de fase exponencial a estacionària, coincidint amb l'esgotament de nitrogen. Es va estudiar la rehidratació i permanença en diferents medis (aigua, glucosa/fructosa, most sintètic i sorbitol). La concentració intracel.lular en glicogen no presentà cap modificació. Però la trehalosa es va metabolitzar en les fases inicials, en aquells medis favorables per al creixement del llevat.Es va estudiar l'efecte de la disponibilitat de nitrogen sobre el metabolisme de la trehalosa. Independentment del contingut nitrogenat, el seu metabolisme no es va veure modificat. TPS1 (gen que codifica trehalosa-6-fosfat sintasa) es va induir just abans de la acumulació de trehalosa, coincidint amb l'aturada del creixement cel.lular. Mitjançant la tècnica dels "microarrays" es va determinar l'expressió gènica global del llevat després de rehidratar i romandre en els medis anteriors. Podem concloure que el temps de rehidratació utilitzat en enologia assegura la total recuperació de LSA. Temps més llargs afectaren negativament la fisiologia del llevat, reduint significativament la seva vitalitat. Les modificacions transcripcionals al most foren degudes bàsicament a la presència de glucosa i fonts nitrogenades, induint la síntesi proteica, glucòlisi i producció d'etanol

Alfabetització biotecnològica: molt més que una història de gens

La biotecnologia moderna (la genòmica i les seves tecnologies associades) s’està desenvolupant a una velocitat vertiginosa, tant en la recerca bàsica com en les seves aplicacions tecnològiques (de la medicina a l’agricultura). A la nostra societat, la biotecnologia cada cop exercirà un major impacte, i es requeriran ciutadans informats i crítics capaços de prendre decisions sobre aspectes biotecnològics. Aquest article reflexiona al voltant de la necessitat d’alfabetitzar biotecnològicament en les etapes formatives obligatòries.Modern biotechnology (the genome and its associated technologies) is developing at breakneck speed, both in terms of basic research and its technological applications (from medicine to agriculture). In modern society, biotechnology exerts an increasingly important impact which will require critically informed citizens capable of taking decisions regarding biotechnological issues. This article reflects on the need to increase biotechnological literacy during compulsory education

Control of ATP homeostasis during the respiro-fermentative transition in yeast

Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition

Entrando por la otra puerta: la indagación para promover el razonamiento científico en educación infantil

La percepción negativa hacia la enseñanza de las ciencias agrava otras que incrementan la dimensión excluyente del aprendizaje de las ciencias (diversidad de género, étnica, socio-económica, cultural, etc.). Por ello, quisimos valorar las percepciones sobre la ciencia y su enseñanza en docentes y estudiantes (5-6 años) de Educación Infantil, a través de entrevistas, tras la implementación en el aula de una secuencia de actividades basadas en indagación. Observamos que los maestros cambian su percepción sobre la ciencia al ver una herramienta didáctica que presenta la ciencia como algo que se construye participativamente y por la que muestran gran interés sus estudiantes. La desafección previa se debe a su escaso dominio del contenido y del conocimiento didáctico del contenido adquirido durante su formación. Al finalizar, se interesan por implementar actividades similares con materiales preparados. No se detectan barreras que limiten la percepción positiva de la ciencia entre los estudiantes. Por tanto, parece que la enseñanza de las ciencias basada en la indagación es una estrategia didáctica que mejora la percepción de las ciencias en EI, eliminando barreras de exclusión, convirtiendo a los maestros en pieza clave de la dimensión transformadora para la enseñanza de las ciencias en etapas tempranas

Virtual Water and Water Footprint: A Case Study from Spain

The World Water Week in Stockholm is the annual meeting place for the planet’s most urgent water-related issues. Organised by the Stockholm International Water Institute (SIWI), it brings together experts, practitioners, decision makers and leader

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations

Family History and Breast Cancer Hormone Receptor Status in a Spanish Cohort

Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women.A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles.Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER−&PR−. Women with a family history of breast cancer were more likely to have ER−&PR− tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91–2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34–5.81).An increased proportion of ER−&PR− breast cancer was observed among younger Spanish women with a family history of the disease

Effects of intubation timing in patients with COVID-19 throughout the four waves of the pandemic : a matched analysis

The primary aim of our study was to investigate the association between intubation timing and hospital mortality in critically ill patients with COVID-19-associated respiratory failure. We also analysed both the impact of such timing throughout the first four pandemic waves and the influence of prior non-invasive respiratory support on outcomes. This is a secondary analysis of a multicentre, observational and prospective cohort study that included all consecutive patients undergoing invasive mechanical ventilation due to COVID-19 from across 58 Spanish intensive care units (ICU) participating in the CIBERESUCICOVID project. The study period was between 29 February 2020 and 31 August 2021. Early intubation was defined as that occurring within the first 24 h of intensive care unit (ICU) admission. Propensity score (PS) matching was used to achieve balance across baseline variables between the early intubation cohort and those patients who were intubated after the first 24 h of ICU admission. Differences in outcomes between early and delayed intubation were also assessed. We performed sensitivity analyses to consider a different timepoint (48 h from ICU admission) for early and delayed intubation. Of the 2725 patients who received invasive mechanical ventilation, a total of 614 matched patients were included in the analysis (307 for each group). In the unmatched population, there were no differences in mortality between the early and delayed groups. After PS matching, patients with delayed intubation presented higher hospital mortality (27.3% versus 37.1%, p =0.01), ICU mortality (25.7% versus 36.1%, p=0.007) and 90-day mortality (30.9% versus 40.2%, p=0.02) when compared to the early intubation group. Very similar findings were observed when we used a 48-hour timepoint for early or delayed intubation. The use of early intubation decreased after the first wave of the pandemic (72%, 49%, 46% and 45% in the first, second, third and fourth wave, respectively; first versus second, third and fourth waves p<0.001). In both the main and sensitivity analyses, hospital mortality was lower in patients receiving high-flow nasal cannula (n=294) who were intubated earlier. The subgroup of patients undergoing NIV (n=214) before intubation showed higher mortality when delayed intubation was set as that occurring after 48 h from ICU admission, but not when after 24 h. In patients with COVID-19 requiring invasive mechanical ventilation, delayed intubation was associated with a higher risk of hospital mortality. The use of early intubation significantly decreased throughout the course of the pandemic. Benefits of such an approach occurred more notably in patients who had received high-flow nasal cannul