Therapeutic Potential of EWSR1-FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma.

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1-FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1-FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1-FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1-FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1-FLI1 at the cell level should be considered a therapeutic approach to develop in the future.This research was funded by the Instituto de Salud Carlos III, grant numbers PI20CIII/00020, DTS18CIII/00005, PI16CIII/00026; Asociación Pablo Ugarte, grant numbers TRPV205/18, TPI-M 1149/13; Asociación Candela Riera, Asociación Todos Somos Iván & Fundación Sonrisa de Alex, grant numbers TVP333-19, TVP-1324/15; ASION, grant number TVP141/17, and by the Spanish Center for Biomedical Network Research on Rare Diseases (CIBERER, ER19P5AC728/2021, grant to M.M.), and by the Regional Government of Madrid (CAM, B2017/BMD3721, grant to M.A.M.-P.). R.M.M-F.d.M. was supported by a grant from the Spanish Center for Biomedical Network Research on Rare Diseases (CIBERER).S

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494

Dinosaur bonebed amber from an original swamp forest soil

[EN] Dinosaur bonebeds with amber content, yet scarce, offer a superior wealth and quality of data on ancient terrestrial ecosystems. However, the preserved palaeodiversity and/or taphonomic characteristics of these exceptional localities had hitherto limited their palaeobiological potential. Here, we describe the amber from the Lower Cretaceous dinosaur bonebed of Ariño (Teruel, Spain) using a multidisciplinary approach. Amber is found in both a root layer with amber strictly in situ and a litter layer mainly composed of aerial pieces unusually rich in bioinclusions, encompassing 11 insect orders, arachnids, and a few plant and vertebrate remains, including a feather. Additional palaeontological data-charophytes, palynomorphs, ostracods- are provided. Ariño arguably represents the most prolific and palaeobiologically diverse locality in which fossiliferous amber and a dinosaur bonebed have been found in association, and the only one known where the vast majority of the palaeontological assemblage suffered no or low-grade pre-burial transport. This has unlocked unprecedentedly complete and reliable palaeoecological data out of two complementary windows of preservation-the bonebed and the amber-from the same site.Funding Ministerio de Ciencia, Innovación y Universidades: CGL2017-84419 (Eduardo Barrón, Xavier Delclòs); Ministerio de Ciencia, Innovación y Universidades: PGC2018-094034-B-C22 (Luis Alcalá) ; Ministerio de Economía y Competitividad: CGL2015-69805-P (Carles Martín-Closas) ; Generalitat de Catalunya: 2017SGR-824 (Carles Martín-Closas, Xavier Delclòs) ; Generalitat de Catalunya: 2020FI_B1 00002 (Sergio Álvarez-Parra) ; Oxford University Museum: Research Fellowship (Ricardo Pérez-de la Fuente) ; Ministerio de Ciencia, Innovación y Universidades: BES-2016-076469 (Jordi Pérez-Cano) ; Austrian Academy of Sciences: Project 661 (Khaled Trabelsi) ; Université de Tunis: LR18 ES07 (Khaled Trabelsi) ; Generalitat Valenciana: APOSTD2019 (Alba Sánchez-García) ; European Regional Development Fund: IGME13-4E-1518 (Rafael P Lozano)Peer reviewe

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

Desarrollo de una web de análisis de datos NGS para la detección de mosaicismo genético en enfermedades raras y experimentos de edición genómica (CRISPR-CAS9)

Trabajo de fin de máster en Bioinformática y Biología ComputacionalEste proyecto responde a la creciente necesidad de analizar y visualizar datos de secuenciación de nueva generación (NGS) producidos a través de experimentos de edición genética. Particularmente para aquellos utilizando tecnología de CRISPR-CAS9 [1]. Actualmente existen múltiples herramientas online que llevan a cabo cierto nivel de análisis de estos datos [2, 3], sin embargo proporcionan información aglutinada en forma de gráficos de in-dels (inserciones y deleciones) o gráficos de tarta sobresimplificados. No proporcionan una caracterización específica de los efectos de mosaicismo que pueden surgir a raíz de este tipo de experimentos de edición, efectos que pueden resultar determinantes para concluir que un experimento se ha llevado a cabo con éxito o no [4, 5, 6, 7, 8, 9]. Por ello el objetivo de esta aplicación es el de analizar y visualizar estos datos de secuenciación masiva de una manera sencilla y comprensible incluso para aquellos sin gran conocimiento bioinformático. Para llevar a cabo el proyecto se hizo uso del popular entorno de R [10] en un sistema operativo Linux. R tiene disponible una potente herramienta de desarrollo de páginas web, ShinyR [11]. Mediante su uso uno puede combinar las increíbles capacidades de procesado de datos de R con el desarrollo de una interfaz intuitiva y adaptable. El desarrollo se subdividió en dos fases principales: el análisis y el agrupamiento de los datos; y el procesado y visualización de estos. El análisis de los datos no es distinto de el procesado llevado a cabo para la identificación de variantes [12]. Comprobaciones de calidad, trimming de los adaptadores y primers, filtrado por calidad y tamaño y joining de secuencias en caso de estar trabajando con datos en paired-end [13]. Tras este procesamiento comienza el agrupamiento de los datos. En un proceso de identificación de variantes ahora precederíamos con un mapeo y usaríamos un llamador de variantes [14], sin embargo en este caso simplemente comparamos cada una de las secuencias entre ellas y las agrupamos según su similitud. Por cada grupo se determina una secuencia representativa o consenso y esta se la alinea frente a una secuencia de referencia. Donde la secuencia de referencia es la secuencia mayoritaria de la muestra sin editar. De esta manera si parte de las secuencias han sido mutadas mediante CRISPR-CAS9 , el alineamiento las identificaría al detectar in-dels (inserciones-deleciones) o cambios de base con respecto a la secuencia representativa. Ya que también somos capaces de asignar a cada grupo de secuencias un valor de Abundancia podemos determinar qué porcentaje de las secuencias han sido correctamente editadas frente a las que no. El proceso por el que CRISPR-CAS9 introduce mutaciones discretas es también susceptible de producir mutaciones aleatorias en posiciones aleatorias [15]. Estas mutaciones aleatorias pueden ser más o menos abundantes, y pueden tener un efecto patogénico o no, motivo por el cual su visualización a nivel de secuencia es necesaria. La aplicación proporciona muchas opciones a la hora de visualizar los resultados provenientes del análisis. Si el usuario proporciona un secuencia Target la aplicación llevará a cabo una búsqueda a través de todos los grupos buscandola e indicará al usuario qué grupo es el más parecido a ella. También facilita el análisis de las secuencias mediante BLASTn [16], la descarga de archivos de alineamiento y los archivos FASTA de todas o algunas de las secuencias representativas,múltiples gráficos e informe customizable que permite al usuario seleccionar la información que él o ella estime relevante

Flint and other raw materials as evidence of contacts between hunter-gatherer groups during the Upper Palaeolithic of Cantabrian Spain: Synthesis of available information

Numero de páginas:46, 7 Figuras[EN] Flint was the most widely used lithic raw material in Europe in Prehistory and, more specifically, was a fundamental resource in the economic and social networks of hunter-gatherer groups in the Cantabrian Spain during the Upper Palaeolithic. The undeniable preference for it compared with other resources was due to a series of factors, such as its easy availability because of its abundance and wide distribution of outcrops, and its excellent qualities for knapping. This summary of the available information about flint and other raw materials used by hunter-gatherers is framed in the context of Upper Palaeolithic occupations in Cantabrian Spain. First, it presents the studies focusing on the provenance of the different types of flint that are found in those occupations; their quantitative representation at each of the sites; their preference, if that is the case, over other raw materials; and the model of their diffusion across the territory. Then other resources are considered, such as quartzite, ochre and different metamorphic, sedimentary and igneous rocks, as well as some materials of biological origin, such as amber, jet/lignite and fossils of animal origin. However, the information available about the use of the latter raw materials in Cantabrian Spain during the Upper Palaeolithic is quite limited and studies of their characterisation are very recent.[FR] Le silex est la principale matière première lithique utilisée dans la Préhistoire du continent européen et c’est une ressource fondamentale dans les circuits économiques et sociaux des groupes de chasseurs-cueilleurs pendant le Paléolithique supérieur de la région cantabrique (nord de l’Espagne). Sa préférence incontestable par rapport aux autres ressources repose sur une série de qualités telles qu’une disponibilité facile du fait de sa grande abondance, de la grande dispersion des affleurements, ainsi que les excellentes qualités pour sa taille. Dans cet article, nous présentons une synthèse des informations disponibles à partir des études sur le silex et d’autres matières premières utilisées par les groupes de chasseurs-cueilleurs. Cette révision a été abordée dans le cadre des occupations archéologiques de la région cantabrique datées du Paléolithique supérieur. D’une part, il s’agit des études relatives à la provenance des différents types de silex trouvés, leur représentation quantitative dans chacun des gisements révisées, préférence, si c’est le cas, par rapport aux autres matières premières documentées, ainsi que leur modèle de diffusion sur le territoire. D’autre part, d’autres ressources sont traitées, comme le quartzite, l’ocre et différentes roches métamorphiques, sédimentaires et ignées, ainsi que quelques matériaux d’origine biologique, comme l’ambre, le jais/lignite et les fossiles d’origine animale. Les informations disponibles concernant l’utilisation de ces matières premières dans le contexte du Paléolithique supérieur cantabrique sont assez rares et leurs études de caractérisation sont récentes.S.M.-J. est chercheur pre´ doctoral pour la Junta de Castilla y Leo´n et le Fonds social europe´ en. Il participe aux projets PID2020-114462GB-100 (E.A.-F.) et PID2020-118359GB-I00 (A.T.), tous deux finance´ s par le programme d’E´ tat pour la promotion de la ge´ne´ ration de connaissances et Renforcement Scientifique et Technologique, du Ministe` re de la Science et de l’Innovation (Espagne). A.P. est chercheur postdoctoral dans le programme Marı´a Zambrano de l’Universite´ du Pays Basque (UPV/EHU), MAZAM 21/22, finance´ par des fonds Next Generation EU, et participe aux projets PID2021-126937NB-I00, finance´ s par CIN/AEI/10.13039/501100011033 et FEDER, Una manera de hacer Europa, ainsi que PID2019-103987GB-C33. Ce travail est une contribution aux projets CRE CGL2017-84419 AEI/FEDER, UE du Ministe` re de la Science, de l’Innovation et des Universite´ s (Espagne), et celui promu par EL SOPLAO S.L. (accord de recherche #20963 avec l’Universite´ de Barcelone et contrat de recherche Ref. VAPC 20225428 avec IGME-CSIC, tous deux pour la pe´ riode 2022–2025), qui comprend l’e´tude de l’ambre du Cre´ tace´ en EspagnePeer reviewe

CRISPR/Cas9-mediated allele-specific disruption of a dominant COL6A1 pathogenic variant improves collagen VI network in patient fibroblasts

Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1

Amber and the Cretaceous Resinous Interval

Amber is fossilized resin that preserves biological remains in exceptional detail, study of which has revolutionized understanding of past terrestrial organisms and habitats from the Early Cretaceous to the present day. Cretaceous amber outcrops are more abundant in the Northern Hemisphere and during an interval of about 54 million years, from the Barremian to the Campanian. The extensive resin production that generated this remarkable amber record may be attributed to the biology of coniferous resin producers, the growth of resiniferous forests in proximity to transitional sedimentary environments, and the dynamics of climate during the Cretaceous. Here we discuss the set of interrelated abiotic and biotic factors potentially involved in resin production during that time. We name this period of mass resin production by conifers during the late Mesozoic, fundamental as an archive of terrestrial life, the 'Cretaceous Resinous Interval' (CREI)