Alteraciones hormonales en adolescentes nadadoras deportistas de élite

La realización de entrenamientos de alto rendimiento puede conllevar alteraciones metabólicas y hormonales, pudiendo ser críticas en la pubertad. Este trabajo tiene como objetivo principal determinar la prevalencia de disfunción ovárica en adolescentes nadadoras deportistas de élite. Se realizó un estudio descriptivo, no intervencional. Se incluyeron 31 nadadoras, de 11 a 16 años, en seguimiento por el Centro Regional de Medicina Deportiva, entre noviembre de 2019 y enero de 2020. Las variables principales registradas fueron: antropométricas, clínicas, derivadas de una encuesta nutricional, un test de estrés deportivo (CSAI-2), un test de evaluación de trastornos de la conducta alimentaria (EAT-26) y analíticas hormonales. El grupo presentó una edad de 13,3 años (DE 1,3), realizando 18,0 [15,0- 20,0] horas de entrenamiento/semana. Su IMC era de 19,2 (DE 2,2) kg/m2 . La disponibilidad de energía (DE) calculada fue de 39,4 (DE 23,4) kcal/kg masa libre de grasa (mlg), teniendo un nivel óptimo (>45 kcal/kg/mlg) un 44,4%. Entre las 18 deportistas con edad ginecológica superior a 6 meses, el 61,1% referían irregularidad menstrual, y un 33,3% oligo/amenorrea. No encontramos diferencias significativas al comparar el grupo con vs sin oligo/amenorrea en IMC, horas de entrenamiento, kilocalorías consumidas, test EAT-26 ni en DE. Solo detectamos niveles significativamente menores de ansiedad cognitiva (test CSAI-2) en aquellas con oligo/amenorrea [27,1% (DE 13,6) vs 45,1% (DE 16,6) (p=0,039)]. En nuestras adolescentes deportistas de élite en natación obtuvimos una elevada prevalencia de irregularidades menstruales y más del 50% presentaba una DE subóptima que puede conllevar consecuencias a largo plazo.Grado en Medicin

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients

Tree growth response to drought partially explains regional-scale growth and mortality patterns in Iberian forests

Tree-ring data has been widely used to inform about tree growth responses to drought at the individual scale, but less is known about how tree growth sensitivity to drought scales up driving changes in forest dynamics. Here, we related tree-ring growth chronologies and stand-level forest changes in basal area from two independent data sets to test if tree-ring responses to drought match stand forest dynamics (stand basal area growth, ingrowth, and mortality). We assessed if tree growth and changes in forest basal area covary as a function of spatial scale and tree taxa (gymnosperm or angiosperm). To this end, we compared a tree-ring network with stand data from the Spanish National Forest Inventory. We focused on the cumulative impact of drought on tree growth and demography in the period 1981–2005. Drought years were identified by the Standardized Precipitation Evapotranspiration Index, and their impacts on tree growth by quantifying tree-ring width reductions. We hypothesized that forests with greater drought impacts on tree growth will also show reduced stand basal area growth and ingrowth and enhanced mortality. This is expected to occur in forests dominated by gymnosperms on drought-prone regions. Cumulative growth reductions during dry years were higher in forests dominated by gymnosperms and presented a greater magnitude and spatial autocorrelation than for angiosperms. Cumulative drought-induced tree growth reductions and changes in forest basal area were related, but initial stand density and basal area were the main factors driving changes in basal area. In drought-prone gymnosperm forests, we observed that sites with greater growth reductions had lower stand basal area growth and greater mortality. Consequently, stand basal area, forest growth, and ingrowth in regions with large drought impacts was significantly lower than in regions less impacted by drought. Tree growth sensitivity to drought can be used as a predictor of gymnosperm demographic rates in terms of stand basal area growth and ingrowth at regional scales, but further studies may try to disentangle how initial stand density modulates such relationships. Drought-induced growth reductions and their cumulative impacts have strong potential to be used as early-warning indicators of regional forest vulnerability.This study was financially supported by Xunta de Galicia, Grant/Award Number PGIDIT06PXIB502262PR, GRC GI-1809; INIA, Grant/Award Number RTA2006-00117; CANOPEE, 2014-2020-FEDER funds, Spanish Science Ministry RTI2018-096884-B-C31, RTI2018-096884-B-C33, AGL2017-83828-C2-2R, RTI2018-096884-B-C3,1 and RTI2018-096884-B-C32 projects. Gabriel Sangüesa-Barreda was supported by a “Juan de la Cierva-Formación” grant from MINECO (FJCI 2016-30121). Antonio Gazol and Paloma Ruiz-Benito were supported by a project “2018 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation.” Ana-Maria Hereş was supported by the project PN-III-P1-1.1-TE-2019-1099 financed by the Romanian Ministry of Education and Research through UEFISCDI. Raúl Sánchez-Salguero was supported by VULBOS project (UPO-1263216, FEDER Funds, Andalusia Regional Government, Consejería de Economía, Conocimiento, Empresas y Universidad 2014-2020). Paloma Ruiz-Benito was supported by the Community of Madrid Region under the framework of the multi-year Agreement with the University of Alcalá (Stimulus to Excellence for Permanent University Professors, EPU-INV/2020/010) and the University of Alcalá “Ayudas para la realización de Proyectos para potenciar la Creación y Consolidación de Grupos de Investigación.” Andrea Hevia was supported by PinCaR project (UHU-1266324, FEDER Funds, Andalusia Regional Government, Consejería de Economía, Conocimiento, Empresas y Universidad 2014-2020).Peer reviewe

Alteraciones de splicing en genes supresores de tumores como mecanismo etiopatológico en cáncer de mama y ovario hereditario en pacientes de Castilla y León

Trabajo de investigación ganador de una beca de la VI Convocatoria de Becas de investigación de la Fundación Villalar-Castilla y León.-- Tutores: Cristina Miner Pino y Eladio A. Velasco Sampedro.Becas de investigación de la Fundación Villalar-Castilla y León.Peer Reviewe

Functional classification of BRCA2 DNA variants by splicing assays in a large minigene with 9 exons

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License.Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19 to 27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations.Contract grant sponsors: Instituto de Salud Carlos III (Spanish Ministry of Economy and Competitiveness) grants PI10/2910 and PI13/1749, Consejería de Sanidad (Junta de Castilla y León) grant BIO/VA08/13. AA was supported by the European Social Fund and Consejeria de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007–2013. CHM is supported by Fundación Villalar (Castilla y León, Spain).Peer reviewe

New splicing vector pSAD: Splicing functional analysis of a hybrid "maxi-minigene" with exons 19 to 27 of BRCA2

Resumen del póster presentado a la European Human Genetics Conference celebrada en Paris (Francia) del 9 al 11 de junio de 2013.At least 50% of all mutations identified in BRCA genes in breast/ovarian cancer patients are variants of unknown clinical significance (VUS). Several studies have shown the link between disease and splicing disruptions due to mutations, including predicted nonsense or frameshift mutations. We aimed to investigate the role of splicing aberrations of BRCA2 in breast/ovarian cancer. We followed a simple strategy consisting of bioinformatics analysis with NNSplice and Human Splicing Finder of DNA variants and splicing functional assays of hybrid minigenes. Splicing reporter plasmids allow to perform functional analysis without the need of patient RNA. We designed a new splicing vector, pSAD, which enabled us the creation of a “maxi-minigene” that includes nine exons of BRCA2 (19 to 27, MGBR2_19-27), where exon 27 substituted the second vector exon. The wt minigene produced a mRNA of 2,174 nucleotides. We validated the new MGBR2_19-27 with four splicing variants of the exons 19, 20, 23 and 24, which were tested in a previous report. Thirty-six out of 166 variants reported in these 9 exons were bioinformatically selected and introduced by PCR-mutagenesis in the wt MGBR2_19-27. Fifteen variants (42%) of all types (synonymous, nonsense, frameshift or missense) altered splicing by different mechanisms. Altogether, most variants disrupted the canonical splice sites; one affected a splicing silencer and another the polypyrimidine tract. Aberrant splicing represents a relevant pathogenic mechanism in hereditary breast/ovarian cancer. Splicing functional assays with the new vector pSAD are valuable tools to discriminate between benign and pathogenic DNA variants of any human disease genes.Peer Reviewe

Splicing functional assays of a single minigene with eight exons of the BRCA2 gene

Resumen del póster presentado a la European Human Genetics Conference celebrada en Nuremberg (Alemania) del 23 al 26 de junio de 2012.Splicing disruptions is one key pathogenic mechanism in inherited diseases. We are currently investigating the contribution of aberrant splicing of BRCA1/2 genes to hereditary breast/ovarian cancer. A powerful approach to study the splicing outcomes of DNA variants is a splicing reporter minigene especially when patient RNA is not available. We constructed a single minigene of 8 BRCA2 exons (19 to 26) in a pSPL3-derived plasmid in 5 cloning steps, which is, as far as we know, the largest BRCA2 minigene ever reported. The genomic fragment from exons 19 to 26 is more than 27 kb in length that was reduced to a final insert of 4.7 kb with internal deletions of introns 20, 21, 24 and 25.This construction was transfected in HeLa cells and we observed a main RNA isoform of the expected size of 1.5 Kb that contained the vector constitutive exons and BRCA2 exons 19 to 26. Several splicing variants of each exon were generated in the wild type minigene by PCR mutagenesis and assayed to demonstrate the usefulness and reliability of this large construction. Splicing reporter minigenes are straightforward and robust tools to distinguish between pathogenic mutations and innocuous variants. The use of minigenes with several exons facilitates the analysis of putative splicing variants in a single minigene and emulates the physiological genomic context where the splicing reactions take place.Grants CSI004A10-2 and BIO39/VA27/10 (Junta de Castilla y León) and Grant PI10/2910 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain).Peer Reviewe

Splicing functional assays of a single minigene with eight exons of the BRCA2 gene

Resumen del póster presentado a la European Human Genetics Conference celebrada en Nuremberg (Alemania) del 23 al 26 de junio de 2012.Splicing disruptions is one key pathogenic mechanism in inherited diseases. We are currently investigating the contribution of aberrant splicing of BRCA1/2 genes to hereditary breast/ovarian cancer. A powerful approach to study the splicing outcomes of DNA variants is a splicing reporter minigene especially when patient RNA is not available. We constructed a single minigene of 8 BRCA2 exons (19 to 26) in a pSPL3-derived plasmid in 5 cloning steps, which is, as far as we know, the largest BRCA2 minigene ever reported. The genomic fragment from exons 19 to 26 is more than 27 kb in length that was reduced to a final insert of 4.7 kb with internal deletions of introns 20, 21, 24 and 25.This construction was transfected in HeLa cells and we observed a main RNA isoform of the expected size of 1.5 Kb that contained the vector constitutive exons and BRCA2 exons 19 to 26. Several splicing variants of each exon were generated in the wild type minigene by PCR mutagenesis and assayed to demonstrate the usefulness and reliability of this large construction. Splicing reporter minigenes are straightforward and robust tools to distinguish between pathogenic mutations and innocuous variants. The use of minigenes with several exons facilitates the analysis of putative splicing variants in a single minigene and emulates the physiological genomic context where the splicing reactions take place.Grants CSI004A10-2 and BIO39/VA27/10 (Junta de Castilla y León) and Grant PI10/2910 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain).Peer Reviewe

Splicing functional assays of a BRCA1 minigene with exons 15-19

Resumen del póster presentado a la European Human Genetics Conference celebrada en Paris (Francia) del 9 al 11 de junio de 2013.Deleterious mutations in BRCA1 and BRCA2 increased up to 20-fold the risk of developing breast cancer. Previously published data of our group showed that a third part of pathogenic mutations affected pre-mRNA processing so that splicing could be one of the most relevant etiopathogenic mechanism in hereditary breast/ovarian cancer. We constructed a hybrid minigene with BRCA1 exons 15 to 19 in the new splicing vector pSAD (Spanish patent: P201231427). Bioinformatics studies of DNA variants in exons 16 and 17 of BRCA1 were performed with NNSplice and Human Splicing Finder, to select them according to the following criteria: disruption of canonical splice sites, broken branch points or creation of de novo silencers. Functional assays performed either in lymphocyte RNA from patients or in hybrid minigenes in MCF7/HeLa cells. We investigated the impact on splicing of pre-selected variants listed in the international databases and to correlate these results with those obtained in lymphocyte RNA of patients from Castilla y Leon (Spain). All the RT-PCR products were sequenced to characterize all the splicing outcomes. Splicing functional assays allow a better molecular characterization of DNA variants of unknown clinical significance. The ultimate goal of this research is to improve the quality of life of patients and their families that will benefit from new preventive measures, surveillance and prophylaxis.Peer Reviewe

Body composition symmetry in aircraft pilots

The purpose of this study was to analyze the body composition symmetry in upper and lower body segments of aircrafts pilots. To reach the study aim, body composition in upper and lower body segments of 206 male aircraft pilots of the Spanish Army (23.1 ± 6.87 years) and 105 civilians (24.0 ± 6.29 years) were evaluated by a bioimpedance analyser (InBody 720, Biospace Co. Ltd., Seoul, Korea). Aircraft pilots presented a tendency to dysmetria in upper and lower body segments, showing fitter values in the protagonist side when performing flight functions. Dysmetria could be detrimental during flight manoeuvres and produce injuries in aircraft pilots. It would be recommended to design specific training protocols to improve this imbalance. © 2022 by the authors. Licensee MDPI, Basel, Switzerland