shinyCurves, a shiny web application to analyse multisource qPCR amplification data: a COVID‑19 case study

[EN]Background Quantitative, reverse transcription PCR (qRT-PCR) is currently the gold-standard for SARS-CoV-2 detection and it is also used for detection of other virus. Manual data analysis of a small number of qRT-PCR plates per day is a relatively simple task, but automated, integrative strategies are needed if a laboratory is dealing with hundreds of plates per day, as is being the case in the COVID-19 pandemic. Results Here we present shinyCurves, an online shiny-based, free software to analyze qRT-PCR amplification data from multi-plate and multi-platform formats. Our shiny application does not require any programming experience and is able to call samples Positive, Negative or Undetermined for viral infection according to a number of user-defined settings, apart from providing a complete set of melting and amplification curve plots for the visual inspection of results. Conclusions shinyCurves is a flexible, integrative and user-friendly software that speeds-up the analysis of massive qRT-PCR data from different sources, with the possibility of automatically producing and evaluating melting and amplification curve plots.This project was supported by funding from the UPV/EHU (Accion Especial "Desarrollo e implementacion del test de diagnostico para COVID-19"). The funding body did not play any roles in the study design; nor in the data collection, analysis and interpretation, or in the writing of the paper

La investigación en antropología física. Una mirada al futuro

Desde su creación en 1976 la Sociedad Española de Antropología Física (SEAF) viene celebrando sus congresos bienales de forma ininterrumpida en distintas ciudades españolas. Estos encuentros, destinados a la discusión de los avances y resultados de las investigaciones bioantropológicas, suponen un foro de debate científico en el que participan investigadores de diversas áreas interesados en los estudios de Antropología Física, promoviendo la interacción entre especialistas e investigadores en formación. Asimismo, y dado el interés que esta disciplina despierta en la sociedad en general, la celebración de los Congresos de la SEAF permite mostrar las novedades acontecidas en el ámbito de la ciencia bioantropológica, facilitando la difusión del conocimiento más allá de la esfera académica. En este marco de reuniones científicas, los profesores del área de Antropología Física de la Facultad de Ciencia y Tecnología de la Universidad del País Vasco-Euskal Herriko Unibertsitatea (UPV/EHU) han sido los encargados de organizar el XVIII Congreso de la SEAF, celebrado en Bilbao durante los días 19 a 21 de junio de 2013. Este libro recoge las ponencias y principales comunicaciones presentadas en el XVIII Congreso, seleccionadas para su publicación y agrupadas en diferentes bloques temáticos que recogen algunas de los conocimientos actuales de la investigación bioantropológica: Ecología Humana, Biología Esquelética y Antropología Forense Paleoantropología y Primatología, y Diversidad Genética Humana. Se ha pretendido mostrar algunas de las cuestiones más relevantes actualmente y la proyección futura de la investigación en Antropología biológica, encaminada tanto al cuestionamiento de paradigmas (como la preponderancia de África en la evolución humana o la existencia de una única especie humana a partir del Pleistoceno Superior, entre otros), como a la comprensión de la relación fenotipo-genotipo, entre otras cuestiones de gran importancia para la reconstrucción de la historia evolutiva de nuestra especie. Sirvan las ponencias invitadas como ejemplo del nuevo enfoque de algunas áreas de la investigación en Antropología

BRAF V600E mutational load as a prognosis biomarker in malignant melanoma

Analyzing the mutational load of driver mutations in melanoma could provide valuable information regarding its progression. We aimed at analyzing the heterogeneity of mutational load of BRAF V600E in biopsies of melanoma patients of different stages, and investigating its potential as a prognosis factor. Mutational load of BRAF V600E was analyzed by digital PCR in 78 biopsies of melanoma patients of different stages and 10 nevi. The BRAF V600E load was compared among biopsies of different stages. Results showed a great variability in the load of V600E (0%-81%). Interestingly, we observed a significant difference in the load of V600E between the early and late melanoma stages, in the sense of an inverse correlation between BRAF V600E mutational load and melanoma progression. In addition, a machine learning approach showed that the mutational load of BRAF V600E could be a good predictor of metastasis in stage II patients. Our results suggest that BRAF V600E is a promising biomarker of prognosis in stage II patients.This research was supported by the Basque Government (grants ELKARTEK-KK2016-036 and KK2017-041 to MDB, grant IT1138-16 to SA and predoctoral fellowship PRE_2014_1_419 to AS), and by the University of the Basque Country (UPV/EHU) (grant GIU17/066). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co‑transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.This project was supported by grants from the Basque Government (KK2017-041 and KK2020-00069 to M.D.B.), the UPV/EHU (GIU17/066 to M.D.B.), H2020-ESCEL JTI (15/01 to M.D.B.) and MINECO (PCIN-2015-241 to M.D.B.). CP holds a predoctoral fellowship from the Basque Government. Part of this project is under European patent No. EP3051291 (EP14796149.4): “Method for diagnosis and prognosis of cutaneous melanoma”, Univer- sity of the Basque Country (UPV/EHU). The authors acknowledge the technical support SGIker resources at the UPV/EHU for the computational calculations, which were carried out in the Arina Informatics Cluster. The authors are grateful to the Basque Biobank for providing the biopsy samples and in particular, to María Jesús Fernández and Arantza Perez Dobaran for their technical support with the immunohistochemistry

Computational and Experimental Evaluation of the Immune Response of Neoantigens for Personalized Vaccine Design

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.This work was supported by Basque Government funding (IT456-22; IT1448-22, IT693-22 and IT1524-22; ONKOVAC 2021111042), as well as by the UPV/EHU (GIU20/035; US21/27; US18/21; PIF18/295) and Basque Center of Applied Mathematics (US21/27 and US18/21)

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Genes implicados en el desarrollodel esqueleto. Selección adaptativa en regiones reguladoras no-codificantes

Uno de los rasgos que nos identifica como humanos es nuestro esqueleto. En este trabajo buscamos identificar aquellas zonas reguladoras de genes implicadas en la morfología esquelética y que presentan cambios en su secuencia de ADN que son específicos humanos. Con ello perseguimos identificar aquellos factores implicados en las adaptaciones esqueléticas humanas, como labipedia o morfología craneal.Gure eskeletoa da gizaki bezala identifikatzen gaituen ezaugarrietariko bat. Lan honetan, eskeletoaren morfologian inplikatuta dauden geneen eskualde erregulatzaileak, zeintzuk euren ADN sekuentzian aurkezten dituzten aldaketak gizakiaren berariazkoak diren, identifikatzea bilatzen dugu. Honekin, gizakiaren eskeletoaren moldaketan, hala nola bipedian edo garezurraren egituran, inplikatutako eragileak identifikatu nahi dugu.L'un des traits qui nous identifie en tant qu'humains est notre squelette. Dans ce travail nous cherchons à identifier les zones régulatrices des gènes impliquées dans la morphologie squelettique et qui présentent des changements dans leur séquence d'ADN qui sont des spécifiques humains. De cette façon nous cherchons à identifier les facteurs impliqués dans les adaptations squelettiques humaines, comme la bipédie ou morphologie crânienne.One of the characteristics that identify us as human beings is our skeleton. In this work westrive to identify those regulating areas of genes involved in the skeleton morphology and that present changes in their DNA sequence which are specifically human. With this we try to identifythose factors envolved in human skeleton adaptations, such as bipedia or skull morphology

La Iniciación a la experimentación: una oportunidad para abordar el trabajo fin de grado de manera colaborativa e integradora

En la UPV/EHU, el Trabajo Fin de Grado (TFG) en Biología se ha estructurado en dos etapas secuenciales: el Módulo I, de iniciación a la experimentación; y el Módulo II, correspondiente a un trabajo individual. En esta comunicación se presenta el trabajo que un grupo de profesores de las áreas de Genética, Biología Celular y Antropología Física hemos desarrollado para diseñar, implementar y valorar la calidad, en términos de aprendizaje, de una propuesta formativa transversal y original desarrollada para el Módulo I del Grado en Biología (GBIOL), basada en la realización de un proyecto experimental enmarcado en la especialidad denominada Biología Celular, Molecular y Genética (BIOCELMOLGEN