Análisis de la respuesta de las levaduras a las condiciones de estrés osmótico y ausencia de nitrógeno durante la vinificación

La fermentación alcohólica es un proceso complejo en el que los azúcares presentes en el mosto se convierten mayoritariamente en etanol y CO2 (Fleet and Heard, 1993), gracias a la acción de las levaduras, principalmente Saccharomyces cerevisiae. Durante la vinificación, las levaduras tienen que hacer frente a un conjunto de situaciones de estrés, de las que destacamos el estrés hiperosmótico al inicio del proceso, debido las elevadas concentraciones de azúcares en los mostos (150-250 g/L) (Attfiled, 1997), o el causado por el agotamiento de nutrientes, especialmente la depleción de nitrógeno. Existen diferentes trabajos en los que se estudia la respuesta de las levaduras a diferentes situaciones de estrés. En el llevado a cabo por Gasch et al. (2000) se describen alrededor de 900 genes que ven alterada su expresión cuando se exponen a diferentes tipos de estrés, lo que se denominó respuesta a estrés ambiental (ESR). Existen otros trabajos centrados en el proceso de la fermentación alcohólica, como el de Rossignol et al. (2003) donde se observó que más de 2000 genes variaban sus niveles durante la vinificación. En la primera parte se describe la respuesta de las levaduras al agotamiento de nitrógeno y la adición de diferentes fuentes. Se observa que la adición de estas fuentes a vinificaciones limitantes permite completar el proceso fermentativo de forma similar a la vinificación control, aunque la naturaleza de éstas sí influye, en el perfil organoléptico del producto final (Jiménez-Martí et al., 2007), como también influye en la reprogramación celular tras las adiciones (Jiménez-Martí et al., 2008). Asimismo, se han descrito posibles marcadores moleculares de la limitación de nitrógeno como la actividad arginasa o los niveles de expresión del gen ACA1 (Jiménez-Martí et al., 2007). En la segunda parte se estudia la respuesta de las levaduras al estrés hiperosmótico causado por elevadas concentraciones de glucosa. Se deduce que hay una mayor represión por catabolito de carbono en 20% de glucosa que en 2%. Se analizó también la implicación de algunas de las principales rutas de señalización en respuesta a este tipo particular de estrés; observándose la implicación de la ruta HOG y también una cierta participación de las rutas PKA y TOR. Se identificaró un grupo de genes de función desconocida más expresados en 20% de glucosa que en 2%. Uno de ellos es YHR087W, cuyo mutante de deleción muestra defectos de crecimiento y consumo de glucosa en medios conteniendo elevadas concentraciones de glucosa, además de presentar mayor susceptibilidad a otras condiciones de estrés que su cepa parental, por lo que se considera un gen de respuesta a estrés. Estudios proteómicos comparativos entre dicho mutante y su cepa parental en medios con 20% de glucosa muestran cambios en los niveles de expresión de proteínas pertenecientes a la categoría de plegamiento de proteínas. También se realizó un estudio transcriptómico global comparativo entre una cepa vínica y de laboratorio frente a elevadas concentraciones de azúcares. Los resultados sugieren que la mayor viabilidad de la vínica en estos medios podría estar relacionada con una mayor expresión de los genes implicados en glicolisis, fermentación y procesos biosintéticos. Finalmente, se llevaron a cabo diferentes aproximaciones con la intención de mejorar el comportamiento fermentativo de algunas cepas vínicas. Esto se consiguió, por un lado con la preadaptación de cepas con problemas al inicio de la fermentación en medios de rehidratación con determinadas concentraciones de glucosa. Por otro lado, se realizaron manipulaciones genéticas de los genes HSP26 e YHR087W. Éstas resultan, en general, en un aumento de los niveles de expresión de los genes, y en algunos casos en una mayor resistencia a diferentes condiciones de estrés, así como un menor tiempo para finalizar la fermentación (Jiménez-Martí et al., 2009).Alcoholic fermentation is a complex process in which sugars presents in grape must become ethanol and CO2 through the action of yeasts, especially Saccharomyces cerevisiae. Throughout wine production yeast cells are affected by a plethora of stress conditions, such as nitrogen starvation, or osmotic stress due to high glucose concentrations. In this work we analyzed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion condtions in order to understand yeast metabolic changes. We found that the nature of the nitrogen source added introduces changes to the volatile compounds profile and to the gene expression, and also there is a differential cellular reprogramming after addition depending on the nitrogen source. On the other hand, arginase activity and the expression of ACA1 gene are useful as a molecular markers of nitrogen depletion/addition during vinification. Besides, we present a comprehensive study of the response to high glucose stress which indicates important transcriptomic changes, especially in terms of the genes involved in both stress response and respiration, and the implication of the HOG pathway. We also describe several genes of an unknown function highly expressed under 20% (w/v) glucose than under 2% . In this work we focus on the YHR087W gene, whose relevance for the response to high sugar and other stress conditions and the relationship of the encoded protein with several Hsp proteins suggest its implication in stress response. Indeed, we introduce several genetic manipulations in HSP26 and YHR087W in two different wine yeasts in order to improve their fermentative behavior. Our results indicate that some of these modifications result in strains with higher expression of these genes, better resistance to certain stress conditions, and even improved fermentative behavior. We also compare several laboratory and wine yeast strains in terms of their ability to start growth in must. We propose several clues for yeast strains to adapt to the wine production conditions: the high expression of genes involved in both biosynthetic processes and glycerol biosynthesis and content. Moreover, we demonstrate the usefulness of pre-adaptation of wine yeast with problems at the beginning of the process introducing a certain percentage of glucose in the rehydratation media

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metalions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion

The LEGACy study: a European and Latin American consortium to identify risk factors and molecular phenotypes in gastric cancer to improve prevention strategies and personalized clinical decision making globally

Gastric cancer; Tumor microenvironment; PreventionCàncer gàstric; Microambient tumoral; PrevencióCáncer gástrico; Microambiente tumoral; PrevenciónBackground Gastric Cancer (GC) is the fourth most deadly cancer worldwide. Enhanced understanding of its key epidemiological and molecular drivers is urgently needed to lower the incidence and improve outcomes. Furthermore, tumor biology in European (EU) and Latin American (LATAM) countries is understudied. The LEGACy study is a Horizon 2020 funded multi-institutional research approach to 1) detail the epidemiological features including risk factors of GC in current time and 2) develop cost-effective methods to identify and integrate biological biomarkers needed to guide diagnostic and therapeutic approaches with the aim of filling the knowledge gap on GC in these areas. Methods This observational study has three parts that are conducted in parallel during 2019–2023 across recruiting centers from four EU and four LATAM countries: Part 1) A case-control study (800 cases and 800 controls) using questionnaires on candidate risk factors for GC, which will be correlated with clinical, demographic and epidemiological parameters. Part 2) A case-control tissue sampling study (400 cases and 400 controls) using proteome, genome, microbiome and immune analyses to characterize advanced (stage III and IV) GC. Patients in this part of the study will be followed over time to observe clinical outcomes. The first half of samples will be used as training cohort to identify the most relevant risk factors and biomarkers, which will be selected to propose cost-effective diagnostic and predictive methods that will be validated with the second half of samples. Part 3) An educational study, as part of our prevention strategy (subjects recruited from the general public) to test and disseminate knowledge on GC risk factors and symptoms by a questionnaire and informative video. Patients could be recruited for more than one of the three LEGACy studies. Discussion The LEGACy study aims to generate novel, in-depth knowledge on the tumor biological characteristics through integrating epidemiological, multi-omics and clinical data from GC patients at an EU-LATAM partnership. During the study, cost-effective panels with potential use in clinical decision making will be developed and validated.This work was funded by the European Union’s Horizon 2020 research and innovation program (Grant agreement No GA825832). The European Union will not be involved in the collection, analysis and interpretation of data, in writing future manuscripts or deciding to submit manuscripts for publication

On improving the short-wavelength response and efficiency of photovoltaic modules via luminescent down-shifting of the incident light

This thesis investigates the technology of luminescent down-shifting (LDS) of light for improving the short-wavelength response and efficiency of photovoltaic (PV) modules. A critical literature review of previously published work is presented identifying the opportunity to include the luminescent species in the encapsulation layer of certain PV technologies. A range of luminescent materials and mixtures thereof were tested in ethylene vinyl acetate (EVA) host. They all exhibited very high luminescent efficiencies and did not impair the transmittance of the encapsulant. LDS EVA sheets were used to encapsulate multi-crystalline silicon (mc-Si) and chalcopyrite (CIGS) solar cells. An increase in short-λ external quantum efficiency of up to 25 % was achieved for mc-Si devices. For CIGS, the increase was up to 25 % and 40 % for 50-nm- and 100-nm-thick buffers respectively. The overall efficiency of mc-Si devices was improved by 0.2 % in the best case and gains of up to 0.2 mA / cm2 and 0.6 mA / cm2 were achieved for 50-nm- and 100-nm-thick buffer CIGS devices. LDS offers the additional benefit of device colouration, which can encourage the further uptake of PV in applications where colour is a desirable property

Clinical and radiological characteristics and outcome of wake-up intracerebral hemorrhage

There is little information on the characteristics of patients with wake-up intracerebral hemorrhage (WU-ICH). We aimed to evaluate frequency and relevant differences between WU-ICH and while-awake (WA) ICH patients. This is a retrospective study of a prospective database of consecutive patients with spontaneous ICH, who were classified as WU-ICH, WA-ICH or UO-ICH (unclear onset). We collected demographic, clinical and radiological data, prognostic and therapeutic variables, and outcome [(neurological deterioration, mortality, functional outcome (favorable when modified Rankin scale score 0-2)]. From a total of 466 patients, 98 (25.8%) were classified as UO-ICH according to the type of onset and therefore excluded. We studied 368 patients (mean age 73.9 ± 13.8, 51.4% men), and compared 95 (25.8%) WU-ICH with 273 (74.2%) WA-ICH. Patients from the WU-ICH group were significantly older than WA-ICH (76.9 ± 14.3 vs 72.8 ± 13.6, p = 0.01) but the vascular risk factors were similar. Compared to the WA-ICH group, patients from the WU-ICH group had a lower GCS score or a higher NIHSS score and a higher ICH score, and were less often admitted to a stroke unit or intensive care unit. There were no differences between groups in location, volume, rate of hematoma growth, frequency of intraventricular hemorrhage and outcome. One in five patients with spontaneous ICH are WU-ICH patients. Other than age, there are no relevant differences between WU and WA groups. Although WU-ICH is associated with worse prognostic markers vital and functional outcome is similar to WA-ICH patients

Diagnóstico del Refugio Nacional de Vida Silvestre Caño Negro, Arenal - Huetar Norte, Costa Rica

Se ha elaborado un diagnóstico a partir de un exhaustivo análisis del Refugio Nacional de Vida Silvestre Caño Negro. Esta zona pertenece al Área de Conservación Arenal Huetar Norte, situado al norte de Costa Rica. El refugio forma parte de la cuenca de río Frío y consiste en un sistema de humedales continentales. El diagnóstico contiene la descripción de los aspectos biofísicos, socioeconómicos y administrativo-institucionales del área. La metodología utilizada se basa en un estudio de la bibliografía sobre la zona y posteriormente un análisis detallado de este para observar las problemáticas y oportunidades. Se ha observado que el humedal consta de una gran riqueza ecológica tanto de flora como de fauna; presenta una situación socioeconómica con cifras preocupantes de empleo, salud y educación, por tratarse de una zona aislada. Administrativamente hablando tiene fuertes déficits tanto de presupuesto como de personal. También se trata de un ecosistema con grandes oportunidades como la gran biodiversidad que presenta (por lo que incluso recibió un reconocimiento RAMSAR), su gran potencial eco-turístico, su gran importancia migratoria para la avifauna e incluso su riqueza paisajística. A partir de esta realidad se han planteado unas estrategias de mejora como son la incorporación de nuevas actividades, una mejor gestión de los recursos o incluso un mayor control del Refugio para así mejorar la situación actual.Nota: Aquest projecte està desenvolupat conjuntament amb el titulat "Diagnóstico de la cuenca de río Frío, Arenal - Huetar Norte, Costa Rica"

The H-ATOMIC Criteria for the Etiologic Classification of Patients with Intracerebral Hemorrhage

Background and Purpose There are no generally accepted criteria for the etiologic classification of intracerebral hemorrhage (ICH). For this reason, we have developed a set of etiologic criteria and have applied them to a large number of patients to determine their utility. Methods The H-ATOMIC classification includes 7 etiologic categories: Hypertension, cerebral Amyloid angiopathy, Tumour, Oral anticoagulants, vascular Malformation, Infrequent causes and Cryptogenic. For each category, the etiology is scored with three degrees of certainty: Possible(3), Probable(2) and Definite(1). Our aim was to perform a basic study consisting of neuroimaging, blood tests, and CT-angio when a numerical score (SICH) suggested an underlying structural abnormality. Combinations of >1 etiologic category for an individual patient were acceptable. The criteria were evaluated in a multicenter and prospective study of consecutive patients with spontaneous ICH. Results Our study included 439 patients (age 70.8 ± 14.5 years; 61.3% were men). A definite etiology was achieved in 176 (40.1% of the patients: Hypertension 28.2%, cerebral Amyloid angiopathy 0.2%, Tumour 0.2%, Oral anticoagulants 2.2%, vascular Malformation 4.5%, Infrequent causes 4.5%). A total of 7 patients (1.6%) were cryptogenic. In the remaining 58.3% of the patients, ICH was attributable to a single (n = 56, 12.7%) or the combination of 2 (n = 200, 45.5%) possible/probable etiologies. The most frequent combinations of etiologies involved possible hypertension with possible CAA (H3A3, n = 38) or with probable CAA (H3A2, n = 29), and probable hypertension with probable OA (H2O2, n = 27). The most frequent category with any degree of certainty was hypertension (H1+2+3 = 80.6%) followed by cerebral amyloid angiopathy (A1+2+3 = 30.9%). Conclusions According to our etiologic criteria, only about 40% patients received a definite diagnosis, while in the remaining patients ICH was attributable to a single possible/probable etiology or to more than one possible/probable etiology. The use of these criteria would likely help in the management of patients with ICH.This work was supported by Ministery of Health-Instituto de Salud Carlos III: RETICS (Redes temáticas de Investigación Cooperativa) INVICTUS RD012/0014 (JM-F, PC-R, AM-D, LP-S, RD-M), FEDER (Fondo Europeo de Desarrollo Regional)

Engineered Macroscale Cardiac Constructs Elicit Human Myocardial Tissue-like Functionality

In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term "CardioSlice." PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorde

Altered methylation pattern in EXOC4 is associated with stroke outcome: an epigenome-wide association study

Background and purpose: The neurological course after stroke is highly variable and is determined by demographic, clinical and genetic factors. However, other heritable factors such as epigenetic DNA methylation could play a role in neurological changes after stroke. Methods: We performed a three-stage epigenome-wide association study to evaluate DNA methylation associated with the difference between the National Institutes of Health Stroke Scale (NIHSS) at baseline and at discharge (Delta NIHSS) in ischaemic stroke patients. DNA methylation data in the Discovery (n = 643) and Replication (n = 62) Cohorts were interrogated with the 450 K and EPIC BeadChip. Nominal CpG sites from the Discovery (p value < 10(-06)) were also evaluated in a meta-analysis of the Discovery and Replication cohorts, using a random-fixed effect model. Metabolic pathway enrichment was calculated with methylGSA. We integrated the methylation data with 1305 plasma protein expression levels measured by SOMAscan in 46 subjects and measured RNA expression with RT-PCR in a subgroup of 13 subjects. Specific cell-type methylation was assessed using EpiDISH. Results: The meta-analysis revealed an epigenome-wide significant association in EXOC4 (p value = 8.4 x 10(-08)) and in MERTK (p value = 1.56 x 10(-07)). Only the methylation in EXOC4 was also associated in the Discovery and in the Replication Cohorts (p value = 1.14 x 10(-06) and p value = 1.3 x 10(-02), respectively). EXOC4 methylation negatively correlated with the long-term outcome (coefficient = - 4.91) and showed a tendency towards a decrease in EXOC4 expression (rho = - 0.469, p value = 0.091). Pathway enrichment from the meta-analysis revealed significant associations related to the endocytosis and deubiquitination processes. Seventy-nine plasma proteins were differentially expressed in association with EXOC4 methylation. Pathway analysis of these proteins showed an enrichment in natural killer (NK) cell activation. The cell-type methylation analysis in blood also revealed a differential methylation in NK cells. Conclusions: DNA methylation of EXOC4 is associated with a worse neurological course after stroke. The results indicate a potential modulation of pathways involving endocytosis and NK cells regulation