Regulación Transcripcional de la respuesta al estrés en la levadura Saccharomyces cerevisiae. Papel de los factores transcripcionales Msn2p y Msn4p.

RESUMEN Los genes MSN2 y MSN4 de la levadura Saccharomyces cerevisiae fueron aislados como supresores en multicopia de mutaciones termosensibles en el gen de la proteína quinasa Snf1p, necesaria para la desrepresión de genes reprimidos por catabolito. Las proteínas Msn2p y Msn4p están funcionalmente relacionadas. Ambas contienen dos dedos de cinc del tipo Cys2-His2, que guardan una gran similitud con los encontrados en las proteínas de Respuesta Temprana al Crecimiento de mamíferos, la proteína del tumor de Wilms, y el represor de levadura Mig1p. Msn2p y Msn4p son capaces de actuar como activadores transcripcionales, pero cuando se inició este trabajo se desconocía su función fisiológica. Durante la caracterización fenotípica del doble mutante msn2 msn4 se observó que esta cepa era sensible a la ausencia de fuente de carbono. Al extender el estudio a otras condiciones de estrés severo, como el estrés térmico, el estrés osmótico o el estrés oxidativo, se observó en todos los casos una mayor sensibilidad de la cepa mutante respecto a la cepa silvestre. Por el contrario, la sobre-expresión de los genes MSN2 y MSN4 produce un aumento de la resistencia celular a las condiciones de estrés. En la cepa msn2 msn4 se encontraron defectos en la expresión de genes de respuesta a estrés, como HSP12, HSP26, CTT1 y DDR2. Estos genes tienen en común la presencia en sus promotores de varios Elementos de Respuesta General a Estrés (STRE). En Saccharomyces cerevisiae, el elemento STRE (consenso: CCCCT ó AGGGG) puede mediar la activación transcripcional en respuesta a múltiples estreses, entre ellos el choque térmico, el estrés osmótico, el estrés oxidativo, el pH ácido, la presencia de ácidos débiles, o el ayuno de fuente de carbono o nitrógeno. Al analizar la expresión regulada por STRE, mediante una fusión STRE-LEU2-lacZ, en el mutante msn2 msn4, se pudo comprobar que en ausencia de las proteínas Msn2p y Msn4p los niveles de expresión basal vía STRE están muy reducidos, y la activación transcripcional en diversas condiciones de estrés se encuentra prácticamente abolida. Por otra parte, la sobre-expresión de los genes MSN2 y MSN4 produce activación de la expresión dependiente de STRE incluso en condiciones de ausencia de estrés. Msn2p y Msn4p se requieren para una correcta regulación de la respuesta mediada a través de este elemento. Se demostró que estas proteínas se unen directamente al elemento STRE mediante la técnica de retardo en gel: las proteínas Msn2p y Msn4p, purificadas o presentes en extractos totales de proteína, se unen in vitro, de forma específica, a elementos STRE presentes en oligonucleótidos sintéticos. Además, mediante análisis de footprinting in vivo con DMS se demostró que existe unión in vivo de estas proteínas a los elementos STRE presentes en diversos promotores de Saccharomyces cerevisiae. Por ello proponemos que Msn2p y Msn4p son los Factores de Unión a STRE (STRF), que no habían sido identificados con anterioridad. En el segundo capítulo de este trabajo analizamos las causas de un hecho observado en la cepa silvestre W303-1A. La transferencia brusca de células silvestres en fase exponencial de crecimiento en glucosa a un medio con rafinosa produce una larga parada del crecimiento, durante el cual las células no son capaces de sintetizar suficiente cantidad de invertasa. Se demostró que durante las primeras horas de cambio de medio, aunque el gen que codifica la invertasa, SUC2, se desreprime normalmente, la privación brusca de glucosa produce en las células de levadura un bloqueo de la traducción, el cual impide que se exprese el enzima. En este proceso no parecen estar implicadas la proteína quinasa dependiente de AMP cíclico ni la proteína quinasa Gcn2p. __________________________________________________________________________________________________The Saccharomyces cerevisiae genes MSN2 and MSN4 encode homologous and functionally redundant Cys2Hys2 zinc finger proteins. A disruption of both MSN2 and MSN4 genes results in a higher sensitivity of yeast cells to different stresses, including carbon source starvation, heat shock and severe osmotic and oxidative stresses. In this work we show that Msn2p and Msn4p are required for the activation of several yeast genes such as CTT1, HSP104, DDR2 and HSP12, whose induction is mediated through stress-response elements (STREs). Msn2p and Msn4p are important factors for the stress-induced activation of STRE dependent promoters and bind specifically to STRE-containing oligonucleotides. Moreover, by in vivo footprinting we demonstrate that Msn2p and Msn4p bind in vivo to the STREs present in several stress gene promoters. We propose that Msn2p and Msn4p are the previously unknown STRE binding factors, or STRFs. In the second chapter of this work we analyse the causes of an experimentally observed fact: expression of invertase in the yeast Saccharomyces cerevisiae is greatly delayed when derepression occurs in a medium that lacks a usable carbon source. The delay is not a consequence of defects in the transcription of the SUC2 gene but is due to the impossibility of translating the normal levels of mRNA generating under derepressing conditions. In the absence of glucose a complete inhibition of translation occurs, and this causes the lag time before the new carbon source can be used, even if the mRNAs of glucose-repressible genes are transcripted early

Iron regulatory mechanisms in Saccharomyces cerevisiae

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in many cellular processes. However, excess iron can damage cells since it promotes the generation of reactive oxygen species. The budding yeast Saccharomyces cerevisiae has been used as a model organism to study the adaptation of eukaryotic cells to changes in iron availability. Upon iron deficiency, yeast utilizes two transcription factors, Aft1 and Aft2, to activate the expression of a set of genes known as the iron regulon, which are implicated in iron uptake, recycling and mobilization. Moreover, Aft1 and Aft2 activate the expression of Cth2, an mRNA-binding protein that limits the expression of genes encoding for iron-containing proteins or that participate in iron-using processes. Cth2 contributes to prioritize iron utilization in particular pathways over other highly iron-consuming and non-essential processes including mitochondrial respiration. Recent studies have revealed that iron deficiency also alters many other metabolic routes including amino acid and lipid synthesis, the mitochondrial retrograde response, transcription, translation and deoxyribonucleotide synthesis; and activates the DNA damage and general stress responses. At high iron levels, the yeast Yap5, Msn2, and Msn4 transcription factors activate the expression of a vacuolar iron importer called Ccc1, which is the most important high-iron protecting factor devoted to detoxify excess cytosolic iron that is stored into the vacuole for its mobilization upon scarcity. The complete sequencing and annotation of many yeast genomes is starting to unveil the diversity and evolution of the iron homeostasis network in this species

Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts

P. 449–459Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.S

Physical and antioxidant properties of chitosan and methylcellulose based films containing resveratrol

[EN] New trends in edible films focus on the improvement of their functionality through the incorporation of active compounds, such as antimicrobial or antioxidant agents. Resveratrol is a natural antioxidant found in a variety of plant species, such as grapes, and could be used for minimizing or preventing lipid oxidation in food products, retarding the formation of oxidation products, maintaining nutritional quality and prolonging the food shelf life. The aim of this work was to develop and characterize two different polymeric composite films (made with chitosan (CH) and methylcellulose (MC)) containing different amounts of resveratrol. This compound could be incorporated efficiently into both films, but provoke structural changes in the matrices, which became less stretchable (65-70% reduction of deformation at break at the greatest resveratrol content) and resistant to fracture (26 and 54% reduction of tensile at break for MC and CH, respectively, at the greatest resveratrol content) more opaque (significant reduction of the internal transmittance) and less glossy (about 60-65% reduction of gloss at the greatest resveratrol content). Film barrier properties were hardly improved by the presence of resveratrol; water vapour and oxygen permeability tend to slightly decrease when resveratrol was incorporated into both polymers. Composite films showed antioxidant activity, which was proportional to the resveratrol concentration in the film. None of the films showed antimicrobial activity against Penicillium italicum and Botrytis cinerea. Thus, these films could be applied to food products which are sensitive to oxidative processes to prolong their shelf life. (C) 2012 Elsevier Ltd. All rights reserved.The authors acknowledge the financial support from the Spanish Ministerio de Ciencia e Innovación throughout the project AGL2010-20694.Pastor Navarro, C.; Sánchez González, L.; Chiralt, A.; Cháfer Nácher, MT.; González Martínez, MC. (2013). Physical and antioxidant properties of chitosan and methylcellulose based films containing resveratrol. Food Hydrocolloids. 30(1):272-280. https://doi.org/10.1016/j.foodhyd.2012.05.026S27228030

Yeast translation elongation factor eIF5A expression is regulated by nutrient availability through different signalling pathways

Translation elongation factor eIF5A binds to ribosomes to promote peptide bonds between problematic amino acids for the reaction like prolines. eIF5A is highly conserved and essential in eukaryotes, which usually contain two similar but differentially expressed paralogue genes. The human eIF5A-1 isoform is abundant and implicated in some cancer types; the eIF5A-2 isoform is absent in most cells but becomes overexpressed in many metastatic cancers. Several reports have connected eIF5A and mitochondria because it co-purifies with the organelle or its inhibition reduces respiration and mitochondrial enzyme levels. However, the mechanisms of eIF5A mitochondrial function, and whether eIF5A expression is regulated by the mitochondrial metabolism, are unknown. We analysed the expression of yeast eIF5A isoforms Tif51A and Tif51B under several metabolic conditions and in mutants. The depletion of Tif51A, but not Tif51B, compromised yeast growth under respiration and reduced oxygen consumption. Tif51A expression followed dual positive regulation: by high glucose through TORC1 signalling, like other translation factors, to promote growth and by low glucose or non-fermentative carbon sources through Snf1 and heme-dependent transcription factor Hap1 to promote respiration. Upon iron depletion, Tif51A was down-regulated and Tif51B up-regulated. Both were Hap1-dependent. Our results demonstrate eIF5A expression regulation by cellular metabolic status

A genome-wide transcriptional study reveals that iron deficiency inhibits the yeast TORC1 pathway

Iron is an essential micronutrient that participates as a cofactor in a broad range of metabolic processes including mitochondrial respiration, DNA replication, protein translation and lipid biosynthesis. Adaptation to iron deficiency requires the global reorganization of cellular metabolism directed to optimize iron utilization. The budding yeast Saccharomyces cerevisiae has been widely used to characterize the responses of eukaryotic microorganisms to iron depletion. In this report, we used a genomic approach to investigate the contribution of transcription rates to the modulation of mRNA levels during adaptation of yeast cells to iron starvation. We reveal that a decrease in the activity of all RNA polymerases contributes to the down-regulation of many mRNAs, tRNAs and rRNAs. Opposite to the general expression pattern, many genes including components of the iron deficiency response, the mitochondrial retrograde pathway and the general stress response display a remarkable increase in both transcription rates and mRNA levels upon iron limitation, whereas genes encoding ribosomal proteins or implicated in ribosome biogenesis exhibit a pronounced fall. This expression profile is consistent with an activation of the environmental stress response. The phosphorylation stage of multiple regulatory factors strongly suggests that the conserved nutrient signaling pathway TORC1 is inhibited during the progress of iron deficiency. These results suggest an intricate crosstalk between iron metabolism and the TORC1 pathway that should be considered in many disorders.This work was supported by predoctoral contracts from the Spanish Ministry of Science, Innovation and Universities (MICINN) to AMR and LRA; a fellowship from the “Generalitat de Catalunya” (Spain) to SMM; European Union Funds (FEDER) and MICINN grants BIO2017-87828-C2-1-P to SP, BIO2017-87828-C2-2-P to MATR, BFU2016-77728-C3-3-P to JEPO, and BFU2015-71978-REDT to SP and JEPO; and Regional Government of Valencia PROMETEOII 2015/006 grant to JEPO

Characterization of the Germplasm Bank for the Spanish Autochthonous Bull Breed “Asturiana de la Montaña”

[EN] Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm’s viability. This study reports an investigation of the “Asturiana de la Montaña” breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4–6 mL, 5–10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26–43%, progressive 14–28%), with higher values for viability (47–62%). Insemination results showed a good performance for this breed (NRR: 47–56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing resultsSIThis study was funded by MINECO (Spain) grant numbers RZP 2013-006-00-00, RZP 2017-00008-00-00, and AGL2016-81890-REDT (PIVEP research network)

Physical Education Program for university students with intellectual disabilities from service-learning methodology

La experiencia de aprendizaje-servicio que se presenta a continuación se ha desarrollado on el alumnado del Grado de Ciencias de la Actividad Física y el Deporte (9 materias) y el Grado de Magisterio en Educación Primaria con Mención en Educación Física (1 asignatura) de la Universidad Autónoma de Madrid11. La finalidad principal que se persigue es desarrollar las competencias profesionales a través del aprendizaje experiencial, ofreciendo un servicio comprometido y responsable al alumnado universitario con discapacidad intelectual que participa en el Programa Promentor. En concreto, el servicio prestado persigue desarrollar los aprendizajes motrices del alumnado, así como una actitud positiva para la ocupación activa del ocio y la adquisición de hábitos de práctica saludable. Esta metodología requiere llevar a cabo unos protocolos para orientar los procesos de intervención del alumnado del Grado, a través de una serie de acciones formativas sistematizadas y supervisadas por el profesorado universitario (asambleas, diseño e implementación de sesiones, etc.). La evaluación de la experiencia desarrollada se realiza a partir de la información recopilada de los diferentes agentes implicados en el proceso (profesorado y alumnado de Grado y de Promentor)A service-learning experience has been developed with the students of Sport Sciences and Physical Activity Degree (9 subjects) and Primary Education with Physical Education Specialty Degree (1 subject) at Autonomous University of Madrid. The main aim followed is to develop their professional competences through experiential learning offering a committed and responsible service to university students with intellectual disabilities who participate in the Promentor Program. Specifically, the service provided aims to develop student motor skills, as well as a positive attitude for the active occupation of leisure and the acquisition of healthy practice habits. This methology requires carried out some protocols to guide the interventions of Degree students, through some systematized and supervised formative actions by university teachers (conferences, design and implementation of sessions, etc). The experience assessment is carried out with the information gathered from the different agents involved in the process (teachers and Degree and Promentor students

Laccase/TEMPO-mediated bacterial cellulose functionalization: production of paper-silver nanoparticles composite with antimicrobial activity

“This is a post-peer-review, pre-copyedit version of an article published in Cellulose. The final authenticated version is available online at: https://doi.org/10.1007/s10570-019-02678-5Bacterial cellulose (BC) was functionalized applying the Laccase/TEMPO oxidative treatment, leading to a five-fold increase of the concentration of carboxyl groups. Paper produced with this cellulose showed improved mechanical properties while maintaining barrier function against water and greases as compared to paper produced with non-oxidized BC. Also, the negative charge provided by the carboxyl groups on functionalized BC was used to generate silver nanoparticles (AgNPs), obtaining a BC paper and Ag composite. The presence of AgNPs in the composites was validated by SEM, EDS and ICP analysis, showing spherical, uniformly sized particles stabilized in the BC nanofibers matrix. Additionally, antimicrobial property of composites containing AgNPs was tested. The results showed the strong antimicrobial activity of the composites against Gram-positive and Gram-negative bacteria and fungi. The generation of Ag nanoparticles in a matrix that combine the physical characteristics of the BC nanofibers with the stiffness and the mechanical properties of paper produced composites that may have applicability in technological and biomedical usesPeer ReviewedPostprint (author's final draft

Indicators of Good Practices of Service-Learning University

The proliferation of innovative experiences in university education has been confirmed in different forums, as well as the evidence of their impact in research. However, the available tools are limited when trying to address the quality assessment of University Service-Learning (USL) projects. We present a tool for the evaluation of your SL projects. The goal of the Matrix is to specify the essential and secondary indicators needed to develop good practices in USL: IM_USL. A protocol is established to carry out its assessment. Dimensions and phases indicators of a project are specied, as well as the agents or people involved in it. This tool tries to guide the development of USL projects and to specify different points to improve. The application of the IM_USL will allow you to explore, exhaustively, each of the dimensions and indicators that make up a USL project, regarding both the AGENTS involved and the PHASES that make up a programme. In order to gather information on the reality of the projects, the phases and agents involved in a USL project will be reviewed in particular, determining the level of achievement of the indicators that compose it and of the dimensions that specify the particularities of each indicator. Finally, a series of questions are proposed to guide the process of analysis and reflection on the quality of the evaluated project. As a result, we will be able to obtain an X-ray of the quality of the project and the possible aspects for improvement.Funding: Spanish Ministry of Science and Innovation. Grant: I+D 2019. REFERENCIA DEL PROYECTO/AEI/10.13039/50110001103