23 research outputs found

    AMPK phosphorylation by Ssp1 is required for proper sexual differentiation in fission yeast

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    The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, which, in response to a fall in intracellular ATP levels, activates energy-producing pathways and inhibits energy-consuming processes. Here, we report that fission yeast cells lacking AMPK activity are unable to advance entry into mitosis in response to nitrogen starvation and cannot undergo proper G1 arrest and cell differentiation. We also show that AMPK is important in the promotion of the nuclear localization and accumulation of the Ste11 transcription factor. As in animal cells, the fission yeast CaMKK ortholog (Ssp1) phosphorylates and activates the catalytic subunit of AMPK (Ssp2) in its activation loop (Thr189) when cells are starved for nitrogen or glucose. Interestingly, we found that the phosphorylation of Ssp2 on Thr189 is required for nuclear accumulation of AMPK. Our data demonstrate the existence of a signal transduction pathway activated by nutrient starvation that triggers Ssp2 phosphorylation and AMPK redistribution from the cytoplasm to the nucleus. This pathway is important to advance fission cells into mitosis and to establish a timely pre-Start G1 cell cycle arrest for mating.N.V. is supported by a postdoctoral grant from the Carlos III Institute, Ministerio de Sanidad. Our group is supported by the Ministerio de Economía y Competitividad [grant numbers BFU2008-01808, BFU2011-28274, Consolider CSD2007-00015]; and the Junta de Castilla y León Grupo de Excelencia GR 265.Peer Reviewe

    The Vam6 and Gtr1-Gtr2 pathway activates TORC1 in response to amino acids in fission yeast

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    The Rag family of GTPases has been implicated in the TORC1 activation in Drosophila and in mammalian cells in response to amino acids. We have investigated the role of the Rag GTPases Gtr1 and Gtr2 in TORC1 regulation in Schizosaccharomyces pombe. Fission yeast Gtr1 and Gtr2 are non-essential proteins that enhance cell growth in the presence of amino acids in the medium. The function of Gtr1 and Gtr2 in nutrient signaling is further supported by the observation that even in rich medium the deletion of either gene results in the promotion of mating, meiosis and sporulation, consistent with the downregulation of TORC1. We show that Gtr1 and Gtr2 colocalize with TORC1 in vacuoles, where TORC1 is presumably activated. Epistasis analyses indicated that Gtr1 and Gtr2 function downstream of Vam6 and upstream of TORC1 in response to amino acid signals. Our data demonstrate the existence of an evolutionarily conserved pathway with the Vam6 and Gtr1-Gtr2 pathway activating TORC1, which in turns stimulates cell growth and inhibits sexual differentiation. © 2012.This work is supported by grants from Ministerio de Economía y Competitividad [grant numbers BFU2008-01808, BFU2011-28274, Consolider CSD2007-00015] and Junta de Castilla y León Grupo de Excelencia [grant number GR 265] to S.M. N.V. is supported by a postdoctoral grant from the Carlos III Institute, Ministerio de Sanidad.Peer Reviewe

    Affinity purification of SARS-COV-2 spike protein receptor binding domain produced in a C1 fungal expression system

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    The Receptor Binding Domain (RBD)of the spike protein of SARS-CoV-2 has shown promise for diagnosis, treatment, and development of vaccines for COVID-19. However, two problems persist with large scale production of RBD: 1) lack of high productivity upstream cell culture, 2) absence of a commercial, highly selective affinity resin. In an effort to overcome these limitations, we evaluated two novel technologies for the production and purification of RBD. Briefly, RBD was expressed using C1, an engineered fungal strain of Thermothelomyces heterothallica (DyadicInternational1). The C1 platform expresses glycosylated antigens with high productivity, stability, and purity. RBD was purified using a novel affinity resin2 known to produce yields of 90% to 95% purity in one chromatography step. Affinity purification did not affect protein quality, as demonstrated by ACE-2 binding of RBD. The novel affinity resin showed excellent base stability, consistent product quality, and similar ACE-2 binding activity over 40 cycles. Please click Download on the upper right corner to see the full abstract

    Development of the filamentous fungus Thermothelomyces heterothallica C1 into a next-generation production platform for human and animal vaccines

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    engineering. The thermophilic fungus Thermothelomyces heterothallica is a robust and versatile fungal expression system for the rapid production of proteins at very high levels. In the last 6 years, the C1 protein production platform has been further improved to become a safe and efficient expression system with the prime objective of speeding up the development and production of commercial scale human and animal vaccines, monoclonal antibodies, biosimilars, as well as other therapeutic proteins at larger quantities and lower cost. C1 is a very efficient platform to produce antigens, even to generate multicomponent vaccines. The production levels of engineered C1 strains are similar in terms of yield and purity, reaching in some cases more than 2.5 g/L (in 4-5 days). In contrast to other vaccine platforms, C1 has a higher safety profile, and production can be scaled up in a more cost-effective manner using standard microbial E. coli fermenters. Stable cell lines have been developed to produce different antigens as influenza, neuraminidase, west Nile, rabies, rift valley fever..etc. Please click Download on the upper right corner to see the full abstract

    Estimation of the measurement uncertainty and practical suggestion for the description of the metrological traceability in clinical laboratories

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    Clinicians request a large part of measurements of biological quantities that clinical laboratories perform for diagnostic, prognostic or diseases monitoring purposes. Thus, laboratories need to provide patient's results as reliable as possible. Metrological concepts like measurement uncertainty and metrological traceability allow to know the accuracy of these results and guarantee their comparability over time and space. Such is the importance of these two parameters that the estimation of measurement uncertainty and the knowledge of metrological traceability is required for clinical laboratories accredited by ISO 15189:2012. Despite there are many publications or guidelines to estimate the measurement uncertainty in clinical laboratories, it is not entirely clear what information and which formulae they should use to calculate it. On the other hand, unfortunately, there are a small number of clinical laboratories that know and describe the metrological traceability of their results, even though they are aware of the lack of comparability that currently exists for patient's results. Thus, to try to facilitate the task of clinical laboratories, this review aims to provide a proposal to estimate the measurement uncertainty. Also, different suggestions are shown to describe the metrological traceability. Measurement uncertainty estimation is partially based on the ISO/TS 20914:2019 guideline, and the metrological traceability described using the ISO 17511:2020. Different biological quantities routinely measured in clinical laboratories are used to exemplify the proposal and suggestions

    Educate to transform: An innovative experience for faculty training.

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    Learning-focussed educational models require the development of pedagogical, methodological, assessment and technological competences among the faculty community. The COVID-19 pandemic has accentuated the need for this training. This study evaluates the impact of the training project “Educate to Transform” on teacher attitudes, knowledge and on the implementation of innovative teaching methodologies. There were 695 faculty participants in the program conducted at the Universidad Francisco de Vitoria (UFV). Participants included full-time professors (FULLPROF) and part-time professors (PART-PROF). The measurement instrument was validated using the entire sample and a subsample of 357 participants was used to analyse the impact of the program (pre and post measurement). Professor attitudes and knowledge of or familiarity with innovative methodologies and their application in the classroom were evaluated. The findings show that the program improved the attitudes of teachers towards innovation, raised the level of awareness and number of methodologies implemented in the classroom. The methodology towards more experiential and collaborative learning is effective in transforming teaching practice. Furthermore, the implementation of the program through the CANVAS platform, making teachers live the experience as learners, seems to have contributed to improve the teachers’ attitude towards the LMS. The only difference found among the participants was a worse attitude towards innovation on the part of medical teachers, with a clearly differentiated profile of teachers and students, as well as a greater implementation of active methodologies by teachers with a lower teaching load. Overall, it may be concluded that the program achieved its proposed objectives.post-print830 K

    TOR and PKA Pathways Synergize at the Level of the Ste11 Transcription Factor to Prevent Mating and Meiosis in Fission Yeast

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    [Background]: In the fission yeast Schizosaccharomyces pombe, the TOR (target of rapamycin) and PKA (protein kinase A) signaling transduction pathways regulate the expression of genes required for cell growth and sexual differentiation in response to the nutritional environment. Inhibition of Tor2 signaling results in the induction of genes involved in sexual differentiation, and the cells undergo mating and meiosis, even under good nutritional conditions. The same phenotype is observed in mutants in which the PKA pathway is inactive. By contrast, Tor2 overexpression or mutations that hyperactivate PKA signaling impair sexual differentiation, even under poor nutritional conditions. Accordingly, a very important question is to understand the molecular mechanism by which these two pathways coordinately regulate gene expression in response to nutrients. [Methodology/Principal Findings]: Here we demonstrate that TOR and PKA pathways operate coordinately to negatively regulate sexual differentiation by inhibiting the nuclear accumulation of the Ste11 transcription factor. However, the Tor2 pathway is unable to block the nuclear localization of Ste11 under good nutritional conditions when the PKA pathway is inactive. Using microarray analyses, we found that both pathways inhibit sexual differentiation by blocking ste11-dependent gene expression. [Conclusions/Significance]: We conclude that both the PKA and the TOR pathways inhibit Ste11 nuclear accumulation to repress Ste11-dependent gene expression. However, the PKA pathway plays a quantitatively more important role than the TOR pathway in this process.N.V. is supported by a postdoctoral grant from the Carlos III Institute, Ministerio de Sanidad. Our group is supported by grants from la Junta de Castilla y Leon (Grupo de Excelencia grant GR265) and the Spanish Ministry of Science and Innovation (BFU2008-01808 and Consolider Ingenio CSD2007-00015).Peer reviewe

    Fuerza de ventas determinante de la competitividad empresarial

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    El objetivo de este artículo es describir como la fuerza de ventas influye dentro y fuera de la organización empresarial para alcanzar la competitividad. Para ello se vincula la labor de la fuerza de ventas en la cadena de valor, y se estudia este importante eslabón de la cadena. La metodología utilizada es documental o bibliográfica. Se parte del supuesto de que la fuerza de ventas es la encargada de interrelacionar el entorno interno y externo de la organización, produciendo el ingreso necesario para el inicio y fin del ciclo productivo, a través de la realización de diversas actividades. Se concluye que para alcanzar los objetivos empresariales y la competitividad, es vital contar con una excelente fuerza de ventas; representativa de un factor de cambio y diferenciación de las organizaciones hacia el logro y permanencia de una empresa competitiva

    Fuerza de ventas determinante de la competitividad empresarial

    No full text
    El objetivo de este artículo es describir como la fuerza de ventas influye dentro y fuera de la organización empresarial para alcanzar la competitividad. Para ello se vincula la labor de la fuerza de ventas en la cadena de valor, y se estudia este importante eslabón de la cadena. La metodología utilizada es documental o bibliográfica. Se parte del supuesto de que la fuerza de ventas es la encargada de interrelacionar el entorno interno y externo de la organización, produciendo el ingreso necesario para el inicio y fin del ciclo productivo, a través de la realización de diversas actividades. Se concluye que para alcanzar los objetivos empresariales y la competitividad, es vital contar con una excelente fuerza de ventas; representativa de un factor de cambio y diferenciación de las organizaciones hacia el logro y permanencia de una empresa competitiva
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