Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells

Background Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established. Methods D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon+ cells. Results Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon+ cells co-expressed neurofilament middle and some glucagon-like peptide-1+ cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1+ cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon+ cells did not display Ca2+ oscillations typical of pancreatic α-cells. Discussion These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins

High relative expression of two genes of a melon near-isogenic line versus its parental during ripening

[SPA] Con el fin de comparar la expresión génica de una línea casi isogénica (NIL) SC10-2 de melón y su Piel de Sapo (PS) parental durante la maduración y para comprender los mecanismos de diferenciación, se realizó una secuenciación de transcriptoma. Los genes CmTCP15 (Factor de actividad de transcripción) y CmGDSL (actividad de la esterasa y la lipasa) tenían una alta expresión diferencial en el NIL SC10-2 en comparación con el PS debido a la introgresión en LG X. En consecuencia, algunos atributos de calidad de fruto como el aroma, dulzura y, probablemente otros pueden estar afectados por tales genes. [ENG] In order to compare the gene expression of a melon Near-isogenic Line (NIL) SC10-2 and its parental Piel de Sapo (PS) during ripening and to understand the differentiate mechanisms, a transcriptome sequencing was performed. CmTCP15 (Transcription factor activity) and CmGDSL (Esterase and lipase activity) genes were high differentially expressed in the NIL SC10-2 compared with PS due to the introgression in LG X. Consequently, some fruit quality traits such as aroma, sweetness and probably others can be affected by such genes.Financial support: Fundación Séneca de la Región de Murcia (11784/PI/09), MINECO & UE-FEDER funds (AGL2010-20858). Thanks for the technical assistance to P. Varó and his team in CIFEA-Torre Pacheco (Consejería de Agricultura, Región de Murcia), N. Dos-Santos, E. Cuadros, M. García-Gutiérrez, A. Hakmaoui (UPCT), M.J. Roca (SAIT-UPCT), and IRTA-CRAG for the seeds of the NIL

Gene expression and volatile production during melon ripening

[SPA] Se realizó una secuenciación de transcriptoma para analizar los genes implicados en la formación de aromas expresados durante la maduración y para comprender los mecanismos moleculares que diferencian una línea casi isogénica (NIL) SC10-2 de melón y su parental Piel de Sapo (PS). El gen CmLOX18 (similar a la lipoxigenasa 4) se expresó diferencialmente comparando la NIL SC10-2 y PS y se asoció a la producción de hexanal, un compuesto diana e indicador del proceso de maduración no climatérica. La expresión del gen de la CmACO1 (1-aminocyclopropane-1-carboxylate oxidase 1) implicado en la biosíntesis de etileno no manifestó diferencias durante la maduración. La introgresión en LG X estuvo asociada a la diferente producción de hexanal entre la NIL y PS. Se propone un eQTL en el LG X que controla la producción de aromas del gen CmLOX18 localizado en LG I. [ENG] Transcriptome sequencing was performed in order to analyze the genes associated to volatile synthesis expressed during ripening and to understand the molecular mechanisms that differentiate a melon Near-isogenic Line (NIL) SC10-2 and its parental Piel de Sapo (PS). CmLOX18 gene (Similar to Lipoxygenase 18) was differentially expressed in the NIL SC10-2 compared with PS associated with the aroma volatile compound hexanal as a target compound of the non-climacteric ripening. The expression of CmACO1 (1-aminocyclopropane-1-carboxylate oxidase 1) gene associated with ethylene biosynthesis did not change during ripening. The introgression in LG X was associated with the differential hexanal production of the NIL and PS. An eQTL located in LG X is probably controlling the production of aroma volatiles due to CmLOX18 in LG I.Financial support: Fundación Séneca de la Región de Murcia (11784/PI/09), MINECO & UE-FEDER funds (AGL2010-20858). Thanks for the technical assistance to P. Varó and his team in CIFEA-Torre Pacheco (Consejería de Agricultura, Región de Murcia), N. Dos-Santos, E. Cuadros, M. García-Gutiérrez, A. Hakmaoui (UPCT), M.J. Roca (SAIT-UPCT), and IRTA-CRAG for the seeds of the NIL

Lower relative differential expression of two genes is associated with delayed ripening in melon

[SPA] Con el fin de comparar la expresión génica de un melón cerca de la línea isogénica (NIL) SC10-2 y su parental Piel de Sapo (PS) durante la maduración y para comprender los mecanismos de diferenciación, se realizó una secuenciación transcriptoma. Dos genes de CmGGP (GDP-L-galactosa fosforilasa 1) y CmRAP2-11 (factor de transcripción sensible al etileno RAP2-11) mostraron menor expresión relativa en la NIL SC10 -2 versus PS debido a la introgresión en LG X. Sin embargo, no existieron diferencias en expresión de CmAP2-like X1 (factor de transcripción sensible al etileno, similar a AP2 TOE3 isoforma X1). En consecuencia, la expresión de genes que mapearon en el grupo de ligamiento X como un factor de transcripción de respuesta a etileno o del metabolismo del ácido ascórbico estuvieron probablemente asociados con el retraso de maduración. [ENG] The expression of selected genes during ripening was studied considering a melon Near-isogenic Line (NIL) SC10-2 and its parental “Piel de Sapo” (PS). The expression of CmGGP (GDP-L-galactose phosphorylase 1), CmAP2-like X1 (AP2-like ethylene-responsive transcription factor TOE3 isoform X1) and CmRAP2-11 (ethylene-responsive transcription factor RAP2-11) were differentially expressed in the NIL SC10-2 compared with PS. Consequently, expression of genes that mapped in LG X such as one ethylene response transcription factors or ascorbic acid metabolism gene were probably associated with delayed ripening.Financial support: Fundación Séneca de la Región de Murcia (11784/PI/09), MINECO & UE-FEDER funds (AGL2010-20858). Thanks for the technical assistance to P. Varó and his team in CIFEA-Torre Pacheco (Consejería de Agricultura, Región de Murcia) for crop management and IRTA-CRAG for the seeds of the NIL

The atrial natriuretic peptide and guanylyl cyclase-A system modulates pancreatic beta-cell function

Atrial natriuretic peptide (ANP) and its guanylyl cyclase-A (GC-A) receptor are being involved in metabolism, although their role in the endocrine pancreas is still greatly unknown. The aim of this work is to study a possible role for the ANP/GC-A system in modulating pancreatic beta-cell function. The results presented here show a direct effect of the GC-A receptor in regulating glucose-stimulated insulin secretion (GSIS) and beta-cell mass. GC-A activation by its natural ligand, ANP, rapidly blocked ATP-dependent potassium (K(ATP)) channel activity, increased glucose-elicited Ca(2+) signals, and enhanced GSIS in islets of Langerhans. The effect in GSIS was inhibited in islets from GC-A knockout (KO) mice. Pancreatic islets from GC-A KO mice responded to increasing glucose concentrations with enhanced insulin secretion compared with wild type (WT). Remarkably, islets from GC-A KO mice were smaller, presented lower beta-cell mass and decreased insulin content. However, glucose-induced Ca(2+) response was more vigorous in GC-A KO islets, and basal K(ATP) channel activity in GC-A KO beta-cells was greatly diminished compared with WT. When protein levels of the two K(ATP) channel constitutive subunits sulfonylurea receptor 1 and Inward rectifier potassium channel 6.2 were measured, both were diminished in GC-A KO islets. These alterations on beta-cell function were not associated with disruption of glucose tolerance or insulin sensitivity in vivo. Glucose and insulin tolerance tests were similar in WT and GC-A KO mice. Our data suggest that the ANP/GC-A system may have a modulating effect on beta-cell function

Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening

This is the peer reviewed version of the following article: Zarid, M., García-Carpintero, V., Esteras, C., Esteva, J., Bueso, M.C., Cañizares, J., Picó, M.B., Monforte, A.J. and Fernández-Trujillo, J.P. (2021), Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening. J Sci Food Agric, 101: 754-777, which has been published in final form at https://doi.org/10.1002/jsfa.10688. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] BACKGROUND A near-isogenic line (NIL) of melon (SC10-2) with introgression in linkage group X was studied from harvest (at firm-ripe stage of maturity) until day 18 of postharvest storage at 20.5 degrees C together with its parental control ('Piel de Sapo', PS). RESULTS SC10-2 showed higher flesh firmness and whole fruit hardness but lower juiciness than its parental. SC10-2 showed a decrease in respiration rate accompanied by a decrease in ethylene production during ripening, both of which fell to a greater extent than in PS. The introgression affected 11 volatile organic compounds (VOCs), the levels of which during ripening were generally higher in SC10-2 than in PS. Transcriptomic analysis from RNA-Seq revealed differentially expressed genes (DEGs) associated with the effects studied. For example, 909 DEGs were exclusive to the introgression, and only 23 DEGs were exclusive to postharvest ripening time. Major functions of the DEGs associated with introgression or ripening time were identified by cluster analysis. About 37 genes directly and/or indirectly affected the delay in ripening of SC10-2 compared with PS in general and, more particularly, the physiological and quality traits measured and, probably, the differential non-climacteric response. Of the former genes, we studied in more detail at least five that mapped in the introgression in linkage group (LG) X, and 32 outside it. CONCLUSION There is an apparent control of textural changes, VOCs and fruit ripening by an expression quantitative trait locus located in LG X together with a direct control on them due to genes presented in the introgression (CmTrpD,CmNADH1,CmTCP15,CmGDSL esterase/lipase, andCmHK4-like) and CmNAC18.This work was funded by grants 11784/PI/09 (Seneca Foundation, Region of Murcia) and Ministry of Economy and Innovation (AGL2010-20858). M Zarid acknowledges an UE-Erasmus predoctoral fellowship, a program coordinated by the University of Murcia in the framework of CMN. Thanks are due to Semillas Fitó SA (Barcelona, Spain), for providing seeds of PS melons and IRTACRAG for the seeds of SC10-2. We acknowledge the assistance of P Varó and his team in CIFEA-Torre Pacheco for crop management, to N Dos-Santos, M Medina, M García-Gutiérrez, A Hakmaoui, E Cuadros, I Canales and AA Escudero (UPCT) for sampling and technical assistance, to SAIT-UPCT for GC-MS analysis, to AG Sifres (COMAV) for RNA extraction, and to CNAG (Barcelona) for professional assistance in RNA-Seq. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Zarid, M.; García-Carpintero, V.; Esteras Gómez, C.; Esteva, J.; Bueso, MC.; Cañizares Sales, J.; Picó Sirvent, MB.... (2021). Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening. Journal of the Science of Food and Agriculture. 101(2):754-777. https://doi.org/10.1002/jsfa.10688S7547771012Ríos, P., Argyris, J., Vegas, J., Leida, C., Kenigswald, M., Tzuri, G., … Garcia-Mas, J. (2017). 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CPEB alteration and aberrant transcriptome-polyadenylation lead to a treatable SLC19A3 deficiency in Huntington's disease

Huntington’s disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophy–linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine–responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD

Vertidos tóxicos al río Guadiamar: propuestas técnicas para su corrección

Inmediatamente de producirse el vertido tóxico al río Guadiamar, el Grupo T.A.R. se lanzó sin pensarlo dos veces a la búsqueda de soluciones técnicas a un panorama desolador y de efectos desconocidos, todos ellos amenazantes. El ácido “se comía el suelo inundado” por la riada, el agua retenida en Entremuros a pH 3, y con un enorme contenido de metales pesados, ocupaba una extensión de kilómetros. Nos hundimos en el agua hasta el cuello, y cuando nos cubría cogimos la barca, metimos el río a pedazos en nuestro laboratorio, para trabajar todas las hipótesis, ensayar todas las posibilidades. Peleando con la realidad le sacamos datos al Guadiamar, diseñamos actuaciones, poniéndole ingeniería a cuantas hipótesis nos planteaba la situación. En primera fila observamos las mejores actuaciones que nadie diseñó. El propio río, activando sus defensas naturales, mejoró la calidad del agua retenida en el dique de Entremuros subiendo el pH y precipitando los metales pesados. Los mecanismos de entrada de los metales pesados en la cadena trófica parecían ser lentos, dando tiempo a que la retirada de los lodos tóxicos llevada a cabo por la Administración fuera eficaz y diera tiempo a realizar tanto esfuerzo. Aunque el Guadiamar ha trabajado muy duro en su propia recuperación, con su ayuda hemos elaborado una gran cantidad de propuestas técnicas; unas para actuaciones de emergencia, otras a corto, medio y largo plazo. También hemos dado forma a un Plan frente a las previsibles avenidas de este primer otoño después del vertido. Nuestro objetivo ha sido poner a disposición soluciones preparadas para todo tipo de problemas, en primera o en segunda instancia. Prevenir no solo una o dos contingencias, se ha tratado de estar preparado para la mayor cantidad de eventualidades posibles. Por ello algunas serán utilizables, otras estarán en reserva, y muchas irían al cajón de los papeles. Pero ahí están por si acaso. Este libro recoge los trabajos de campo, los ensayos de laboratorio y la ingeniería desarrollada en los primeros cuatro meses. Durante el siguiente preparamos la edición del mismo, mientras, en paralelo, continuábamos en el trabajo experimental y el diseño. Cuando se cumpla el quinto mes, el 25 de Septiembre de 1998, lo presentaremos, ciento cincuenta días después... Con la financiación de la Diputación de Sevilla hemos preparado la primera edición en formato CD Rom e Internet, con muy poco coste para acceder a su contenido. En poco tiempo saldrá la edición en papel, con la misma financiación que la primera. Nos gustaría que este documento fuera entendido como lo que es, en nuestra opinión, una llamada urgente al debate de las ideas. Tratamos de ofrecer la información necesaria y el foro donde recoger las propuestas que seguramente muchos pueden aportar sin saber como transmitir sus experiencias. El Grupo de Tratamiento de Aguas Residuales (T.A.R.) abre con este libro la MESA DE DISCUSIÓN, para buscar un poco de luz, avanzar en las soluciones técnicas a la inmensa tarea de recuperar el río Guadiamar. El libro presenta lagunas, unas por la enorme prisa, otra por falta de datos, muchas por nuestra escasez de conocimientos. Dicen en España que “lo mejor es enemigo de lo bueno”...,y nos gustaría recoger ideas hoy mejor que mañana, que podría ser tarde. Nos comprometemos a seguir trabajando en soluciones técnicas, innovaciones tecnológicas e investigación aplicada a la recuperación del Guadiamar, a conocer lo ocurrido y su remedio. Nos comprometemos a publicar de la misma forma los resultados obtenidos, de manera que la discusión y el debate sigan siempre abiertos. El grupo T.A.R. podría ser un punto de intercambio de conocimientos universal, abierto, respetuoso y tolerante, universitario en definitiva, y por tanto útil en el cumplimiento de sus obligaciones. La primera necesidad de responder urgentemente, está dando paso a unas actuaciones programadas, a medida de los efectos de las correcciones introducidas. Deben instaurarse políticas de prevención y nuevas actuaciones para recuperar el Guadiamar, mejorar urgentemente las condiciones del entorno. Aprender de las soluciones adoptadas y generar mejores prácticas, puede ser una buena conclusión del trabajo realizado por tanta gente. Lo que empezó siendo una carrera de velocidad se nos convierte en un maratón, ya no hay que correr explosivamente, hay que mantener un ritmo en la carrera; hay que persistir en el esfuerzo todos los días durante mucho tiempo. Este nuevo desafío sigue siendo duro y difícil. Podéis contar con el Grupo T.A.R. para recorrer el duro camino de la Recuperación

Insulinotropic Effect of the Non-Steroidal Compound STX in Pancreatic β-Cells

The non-steroidal compound STX modulates the hypothalamic control of core body temperature and energy homeostasis. The aim of this work was to study the potential effects of STX on pancreatic β-cell function. 1–10 nM STX produced an increase in glucose-induced insulin secretion in isolated islets from male mice, whereas it had no effect in islets from female mice. This insulinotropic effect of STX was abolished by the anti-estrogen ICI 182,780. STX increased intracellular calcium entry in both whole islets and isolated β-cells, and closed the KATP channel, suggesting a direct effect on β-cells. When intraperitoneal glucose tolerance test was performed, a single dose of 100 µg/kg body weight STX improved glucose sensitivity in males, yet it had a slight effect on females. In agreement with the effect on isolated islets, 100 µg/kg dose of STX enhanced the plasma insulin increase in response to a glucose load, while it did not in females. Long-term treatment (100 µg/kg, 6 days) of male mice with STX did not alter body weight, fasting glucose, glucose sensitivity or islet insulin content. Ovariectomized females were insensitive to STX (100 µg/kg), after either an acute administration or a 6-day treatment. This long-term treatment was also ineffective in a mouse model of mild diabetes. Therefore, STX appears to have a gender-specific effect on blood glucose homeostasis, which is only manifested after an acute administration. The insulinotropic effect of STX in pancreatic β-cells is mediated by the closure of the KATP channel and the increase in intracellular calcium concentration. The in vivo improvement in glucose tolerance appears to be mostly due to the enhancement of insulin secretion from β-cells

The wide-field, multiplexed, spectroscopic facility WEAVE : survey design, overview, and simulated implementation

Funding for the WEAVE facility has been provided by UKRI STFC, the University of Oxford, NOVA, NWO, Instituto de Astrofísica de Canarias (IAC), the Isaac Newton Group partners (STFC, NWO, and Spain, led by the IAC), INAF, CNRS-INSU, the Observatoire de Paris, Région Île-de-France, CONCYT through INAOE, Konkoly Observatory (CSFK), Max-Planck-Institut für Astronomie (MPIA Heidelberg), Lund University, the Leibniz Institute for Astrophysics Potsdam (AIP), the Swedish Research Council, the European Commission, and the University of Pennsylvania.WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366-959 nm at R ∼ 5000, or two shorter ranges at R ∼ 20,000. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for ∼ 3 million stars and detailed abundances for ∼ 1.5 million brighter field and open-cluster stars; (ii) survey ∼ 0.4 million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey  ∼ 400 neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in z 1 million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at z > 2. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator.PostprintPeer reviewe