Looking for specialized ribosomes in plants. Characterization of the riboprotein family L24.

Translation and its regulation play an important role in plant adaptation. Ribosomes have traditionally been considered passive molecular players regarding which RNA to translate. However, this view is changing due to studies showing that specific and heterogeneous ribosomes can have an active role regulating the translation of different RNA subpools in mammals and bacteria (Genuth & Barna, 2019). In plants, the possibilities for specialization are much higher, as each ribosomal family is encoded by two to seven paralogs and there are several hints in the literature pointing towards differential paralog roles. However, whether this heterogeneity provides selective translation of specific mRNAs under particular cell conditions has yet to be demonstrated. To address this question, we are characterizing two ribosomal families, RPL10 and RPL24, which contain three and two paralogs, respectively, and that are ubiquitously expressed in Arabidopsis. Specific functions have been described for at least one paralog of each family and paralog mutants show different phenotypes as well (Falcone Ferreyra et al., 2020; Zhou et al., 2010) We will provide evidence of phenotypic variance between paralog mutants in families RPL10 and RPL24 under control and abiotic stress conditions. To determine if these phenotypes are due to different RNA populations being translationally affected in each mutant, we have performed RNA-seq from total and polysomal RNA from WT and mutant plants. In addition, we are studying mutant complementation by Recombineering (Brumos et al, 2020), leveraging the system to exchange exons between paralogs maintaining each other ́s regulatory elements therefore shedding light on whether they are functionally equivalent. We will present our progress regarding these objectives.Ministerio de Ciencia e Innovación, Ministerio de Economia, Industria y Competitividad y Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Translational regulation of plant developmental and environmental responses

Translational regulation has long been recognized as a vital process in the cell responses to the environment and in the execution of developmental programs, yet still little is known about the selective translation of specific mRNAs and its regulation (1). Our research tries to understand the role of translational regulation in developmental and environmental responses, and to that, we are using different perspectives: 1) we are studying ribosome heterogeneity in Arabidopsis and trying to determine whether it is involved in the selective translation of specific mRNAs under different cell conditions, and we have been able to show that there is phenotypic variance within paralog mutants in each family, in both control and abiotic stress conditions; 2) we are trying to identify the translational machinery and RNA-binding proteins involved in the translational regulation of hormonal, nutritional and defence signalling pathways using well-known translationally regulated genes (2–4) ; and 3) we are trying to determine de translational landscape of the plant-virus interaction, for which we are using both virus-resistant and susceptible plants (5). To carry out these tasks, we are employing phenotypical analyses, polysome fractionation, RNA-seq, RNA immunoprecipitation, TRAP-seq and Ribo-seq. Our progress regarding these objectives will be presented.Plan Propio de investigación de la UMA. Universidad de Málaga. Campus de Excelencia internacional Andalucía Tech and Grants BIO2017-82720-P and RYC-2017-22323 from the Ministerio de Economía, Industria y Competitividad, P18-RT-1218 from Junta de Andalucía, and UMA20-FEDERJA-100 from FEDER funds to C.M PRE2018-083348 predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades to JADC Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Looking for specialized ribosomes in plants. Characterization of the riboprotein families L10 and L24

Translation and its regulation play an important role in plant adaptation. Ribosomes have traditionally been considered passive molecular players regarding which RNA to translate. However, this view is changing due to studies showing that specific and heterogeneous ribosomes can have an active role regulating the translation of different RNA subpools in mammals and bacteria (Genuth & Barna, 2019). In plants, the possibilities for specialization are much higher, as each ribosomal family is encoded by two to seven paralogs and there are several hints in the literature pointing towards differential paralog roles. However, whether this heterogeneity provides selective translation of specific mRNAs under particular cell conditions has yet to be demonstrated. To address this question, we are characterizing two ribosomal families, RPL10 and RPL24, which contain three and two paralogs, respectively, and that are ubiquitously expressed in Arabidopsis. Specific functions have been described for at least one paralog of each family and paralog mutants show different phenotypes as well (Falcone Ferreyra et al., 2020; Zhou et al., 2010) We will provide evidence of phenotypic variance between paralog mutants in families RPL10 and RPL24 under control and abiotic stress conditions. To determine if these phenotypes are due to different RNA populations being translationally affected in each mutant, we have performed RNAseq from total and polysomal RNA from WT and mutant plants. In addition, we are studying mutant complementation by Recombineering (Brumos et al, 2020), leveraging the system to exchange exons between paralogs maintaining each other´s regulatory elements therefore shedding light on whether they are functionally equivalent. We will present our progress regarding these objectives.This work is funded by Grants BIO2017-82720-P and RYC-2017-22323 from the Ministerio de Economía, Industria y Competitividad to C.M., a fellowship PRE2018-083348 from Ministerio de Ciencia, Innovación y Universidades to JADC and Plan Propio de investigación from Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Towards understanding the role of heterogeneous ribosomes in Arabidopsis. Characterization of the ribosomal protein family eL24.

Translation and its regulation play an important role in plant adaptation. Despite being the key molecular machines that synthesize proteins, ribosomes have traditionally been considered passive molecular players in determining which mRNA to translate. This view is changing due to studies showing that ribosomes can have an active role in regulating the translation of different mRNA subpools in mammals and bacteria. In plants, the potential specialization is significantly greater, as each ribosomal family is encoded by two to seven paralogs. Moreover, several indications in the literature point towards differential roles among these paralogs. However, whether this heterogeneity provides selective translation of specific mRNAs under particular cell conditions has yet to be demonstrated. To address this question, we are characterizing the ribosomal family eL24, composed of two paralogs, eL24z and eL24y. Both are ubiquitously expressed in Arabidopsis at a very similar level. It was described that the el24y displayed important growth retardation and auxin-defective phenotypes, while little was known about the eL24z paralog. By characterizing mutants in both paralogs, we have provided evidence that both eL24y and eL24z are are involved in the assembly of the 80S ribosomes, are constituents of actively translating ribosomes, and exert common functions in translation. However, our sequencing studies also indicate a greater impact on the translational machinery in el24y. Since we also show evidence that overall translation is unaffected in any of the mutants, the phenotypic differences between them may be due to the specific function of the paralog y in translation reinitiation, a process in which paralog z seems to be less important according to our results. Our ongoing experiments are designed toward definitively answering the question of whether the two paralogs within this family play different functions in translation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cuprizone-Induced Neurotoxicity in Human Neural Cell Lines Is Mediated by a Reversible Mitochondrial Dysfunction: Relevance for Demyelination Models

Suitable in vivo and in vitro models are instrumental for the development of new drugs aimed at improving symptoms or progression of multiple sclerosis (MS). The cuprizone (CPZ)-induced murine model has gained momentum in recent decades, aiming to address the demyelination component of the disease. This work aims at assessing the differential cytotoxicity of CPZ in cells of different types and from different species: human oligodendroglial (HOG), human neuroblastoma (SH-SY5Y), human glioblastoma (T-98), and mouse microglial (N-9) cell lines. Moreover, the effect of CPZ was investigated in primary rat brain cells. Cell viability was assayed by oxygen rate consumption and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based (MTT) method. Our results demonstrated that CPZ did not cause death in any of the assayed cell models but affected mitochondrial function and aerobic cell respiration, thus compromising cell metabolism in neural cells and neuron-glia co-cultures. In this sense, we found differential vulnerability between glial cells and neurons as is the case of the CPZ-induced mouse model of MS. In addition, our findings demonstrated that reduced viability was spontaneous reverted in a time-dependent manner by treatment discontinuation. This reversible cell-based model may help to further investigate the role of mitochondria in the disease, and study the molecular intricacies underlying the pathophysiology of the MS and other demyelinating diseases. Keywords: neurodegenerative diseases, copper chelator, pathophysiology, cell metabolism, gli

Electrochemical oxidation of lignin for the simultaneous production of bioadhesive precursors and value-added chemicals

Electrochemical oxidation of lignin has been widely regarded as a clean and reliable alternative to obtain value-added products from lignin, such as vanillin or guaiacol. This work aims to go one step beyond the production of low molecular weight molecules and explore the possibility of using lignin residues from electrochemical treatments in the context of biorefinery. To this end, a two-way valorization of lignin by electrochemical oxidation is proposed, in order to obtain a liquid phase enriched in low molecular weight organic oligomers and a solid phase of modified lignin to be used as bioadhesive precursor. Hydroxylation of lignin by electrochemical oxidation using boron-doped diamond (BDD) anodes was observed according to the FTIR and MALDI-TOF results, concluding that an applied current density of 10 mA cm−2 leads to promising modifications for the formulation of bioadhesives. Furthermore, NIPU bioadhesives with electrochemically modified lignin were successfully prepared and tested for use in particleboard panels, showing satisfactory mechanical properties, and thus paving the way for more environmentally friendly lignin modification procedures for the wood industryJJC acknowledges financial support from Galician Government though a postdoctoral fellowship (ED481B-2021/015). SG-R and GE predoctoral and postdoctoral fellowships (BES-2017-081677 and RYC-2018-024846-I, respectively) were funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. JJC, SG-R, TAL-C, GE and MTM belong to the Galician Competitive Research Group (GRC) ED431C-2021/37. LERMAB is financed by the French Agence Nationale de la Recherche (ANR) as part of the laboratory of excellence (LABEX) ARBRE. The authors would also like to thank the use of the analytical facilities of IR-Raman Spectroscopy Unit and Mass Spectrometry Unit from RIAIDT-USC.S

Translational regulation of hormone synthesis and signaling mRNAs

Translational regulation has long been recognized as a vital process in the cell responses to the environment and in the execution of developmental programs, yet still little is known about the selective translation of specific mRNAs and its regulation (1). Our research tries to understand the role of translational regulation in developmental and environmental responses and to that, we are using different perspectives: 1) we are studying ribosome heterogeneity in Arabidopsis and trying to determine whether it is involved in the selective translation of specific mRNAs under different cell conditions, and 2) we are trying to identify the translational machinery and RNA-binding proteins involved in the translational regulation of hormonal pathways using well-known translationally regulated hormone- biogenesis and signaling genes (2-4). We have been able to show that certain ribosomal protein paralogs are required for the specific translation of uORF-containing hormone-related mRNAs, and are now trying to understand the molecular mechanisms that control this regulation. Our progress regarding this objective will be presented.This work has been funded by grants BIO2017-82720-P, RYC2017-22323, UMA20-FEDERJA-100, and PID2021-123240NB-100 to CM; a PRE2018-083348 fellowhip to JADC; and Plan Propio (UMA): Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Cuprizone-Induced Neurotoxicity in Human Neural Cell Lines Is Mediated by a Reversible Mitochondrial Dysfunction: Relevance for Demyelination Models

Suitable in vivo and in vitro models are instrumental for the development of new drugs aimed at improving symptoms or progression of multiple sclerosis (MS). The cuprizone (CPZ)-induced murine model has gained momentum in recent decades, aiming to address the demyelination component of the disease. This work aims at assessing the differential cytotoxicity of CPZ in cells of different types and from different species: human oligodendroglial (HOG), human neuroblastoma (SH-SY5Y), human glioblastoma (T-98), and mouse microglial (N-9) cell lines. Moreover, the effect of CPZ was investigated in primary rat brain cells. Cell viability was assayed by oxygen rate consumption and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based (MTT) method. Our results demonstrated that CPZ did not cause death in any of the assayed cell models but affected mitochondrial function and aerobic cell respiration, thus compromising cell metabolism in neural cells and neuron-glia co-cultures. In this sense, we found differential vulnerability between glial cells and neurons as is the case of the CPZ-induced mouse model of MS. In addition, our findings demonstrated that reduced viability was spontaneous reverted in a time-dependent manner by treatment discontinuation. This reversible cell-based model may help to further investigate the role of mitochondria in the disease, and study the molecular intricacies underlying the pathophysiology of the MS and other demyelinating diseases.Los modelos in vivo e in vitro adecuados son fundamentales para el desarrollo de nuevos fármacos destinados a mejorar los síntomas o la progresión de la esclerosis múltiple (EM). El modelo murino inducido por cuprizona (CPZ) ha ganado impulso en las últimas décadas, con el objetivo de abordar el componente de desmielinización de la enfermedad. Este trabajo tiene como objetivo evaluar la citotoxicidad diferencial de CPZ en células de diferentes tipos y de diferentes especies: oligodendroglial humano (HOG), neuroblastoma humano (SH-SY5Y), glioblastoma humano (T-98) y microglial de ratón (N-9). líneas celulares. Además, se investigó el efecto de CPZ en células cerebrales primarias de rata. La viabilidad celular se ensayó mediante el consumo de oxígeno y mediante el método basado en bromuro de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT). Nuestros resultados demostraron que CPZ no causó la muerte en ninguno de los modelos celulares ensayados, pero afectó la función mitocondrial y la respiración celular aeróbica, comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neuronas y glías. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neurona y glía. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. comprometiendo así el metabolismo celular en las células neurales y los cocultivos de neurona y glía. En este sentido, encontramos una vulnerabilidad diferencial entre las células gliales y las neuronas, como es el caso del modelo de EM en ratón inducido por CPZ. Además, nuestros hallazgos demostraron que la reducción de la viabilidad se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. nuestros hallazgos demostraron que la viabilidad reducida se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes. nuestros hallazgos demostraron que la viabilidad reducida se revirtió espontáneamente de manera dependiente del tiempo al suspender el tratamiento. Este modelo reversible basado en células puede ayudar a investigar más a fondo el papel de las mitocondrias en la enfermedad y estudiar las complejidades moleculares que subyacen a la fisiopatología de la EM y otras enfermedades desmielinizantes

Comparación en el tiempo de la adquisición de competencias en soporte vital básico (SVB) en alumnos de 2º curso del Grado de Medicina

Memoria ID2022-178 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023