Dichotomous colorectal cancer behaviour.

Colorectal cancer (CRC) is the third most common malignant tumor and one of the deadliest cancers. At molecular level, CRC is a heterogeneous disease that could be divided in four Consensus Molecular Subtypes. Given the differences in the disease due to its anatomical location (proximal and distal colon), another classification should be considered. Here, we review the current knowledge on CRC dichotomic´s behaviour based on two different entities; right and left-sided tumors, their impact on clinical trial data, microbiota spatial composition and the interaction with the nervous system. We discuss recent advances in understanding how the spatial tumor heterogeneity influences the tumor growth, progression, and responses to current therapiespost-print3748 K

LAMP2 deficiency attenuates the neurodegeneration markers induced by HSV-1 infection

Mounting evidence suggests a major role of infectious agents in the pathogenesis of sporadic Alzheimer's disease (AD). Among them, herpes simplex virus type 1 (HSV-1) infection has emerged as a major factor in the etiology of AD. HSV-1 is able to induce some of the main alterations of the disease such as hyperphosphorylation of tau protein and accumulation of amyloid-β peptide. Functional genomic analysis of a cell model of HSV-1 infection and oxidative stress developed in our laboratory revealed lysosomal system to be the main pathway altered, and the lysosome-associated membrane protein 2 (LAMP2) gene one of the most strongly modulated genes. The aim of this work is to study LAMP2 as an AD candidate gene and to investigate its role in the neurodegeneration induced by HSV-1 using a LAMP2 knockdown cell model. LAMP2 deficiency led to a significant reduction of viral DNA replication and formation of infectious particles. In addition, tau hyperphosphorylation and inhibition of Aβ secretion induced by the virus were attenuated by the absence of LAMP2. Finally, genetic association studies revealed LAMP2 genetic variants to be associated with AD risk. In summary, our data indicate that LAMP2 could be a suitable candidate to mediate the AD-like phenotype caused by HSV-1.This work was supported by the Spanish Ministerio de Ciencia e Innovación (SAF 2017-85747-R); and the Ramon Areces Foundatio

Sprouty1 controls genitourinary development via its N-terminal tyrosine

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) is a group of diseases that include a broad spectrum of developmental defects of the genitourinary system. Mouse models indicate that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of CAKUT. Sprouty1 is an intracellular Ret inhibitor whose mutation results in supernumerary kidneys, megaureters, and hydronephrosis in mice. Both the molecular mechanisms and the structural domains critical for Sprouty function are a matter of controversy, partly because studies pursuing this objective rely on ectopic overexpression in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for their function in vitro. Methods: We have generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We have characterized development of the genitourinary systems of these mice via different methods, including the use of reporter mice expressing EGFP form the Ret locus, and whole mount cytokeratin staining. Results: Mice lacking this tyrosine grow ectopic ureteric buds that ultimately will form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. Conclusions: Tyrosine 53 is absolutely necessary to convey Sprouty1 function during genitourinary development.This work was supported by grants BFU2010-47175-P and BFU2017-83646-P (AEI/FEDER, UE) from MINECO to ME. MV was supported by a predoctoral fellowship from AGAUR. CA was supported by a predoctoral fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the EU. We are grateful to Dr. Sanjay Jain (Washington University, St Louis) for sharing RetEGFP mice, and to Dr. Tung-Tien Sun (New York University) for Uroplakin antibody. We thank Anna Macià (IRB Lleida) for her contribution to the initial development of this manuscript, as well as Marta Hereu, Maria Santacana, Mónica Domingo and Maria Carrele for their excellent technical assistance

A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants BFU2017-83646-P (MINECO) and PID2020-114947 GB-I00 (MCIU) (both supported by funds from AEI/FEDER, UE) to ME. MV was supported by a predoctoral fellowship from AGAUR. GA and CA and GA are supported by a fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the E

Studies of the Protein-protein interaction between the AtKAT1 potassium channel and its putative regulators

[EN] Potassium is one of the most important cationic nutrients for all living organisms. Among its various roles, it takes part in plant stomatal aperture as it influences osmotic potential of the cells surrounding the stomata (guard cells). This direct link between [K+ ] and plant transpiration rate, and therefore plant water loss, makes K+ transport extremely interesting, especially for the development of drought resistant plants. The KAT1 inward rectifying potassium channel has been proposed to be the major pathway for potassium influx in guard cells. This channel belongs to the Shaker family, which are selective voltage gated channels conserved throughout all Kingdoms. They are essential for stomatal responses to fluctuating environment or internal signalling. Therefore, understanding KAT1 regulation has become one of the main goals of research in the field. Screening of a cDNA library of Arabidopsis thaliana genes with the Split-Ubiquitin proteinprotein interaction assay produced 18 proteins as putative KAT1 interactors. Fourteen of these possible candidates were further confirmed in terms of their specificity for KAT1. For this reason, the purpose of this project is to employ the GoldenBraid cloning system, a standardized assembly system based on type IIS restriction enzymes, to study the interaction between the KAT1 potassium channel and one of the candidate proteins in plants. This system allows multigenic assemblies of reusable modules, which are not only useful in this study, but also for the testing of the other candidate interactors. Using the GoldenBraid technology, a series of constructs will be generated in order to carry out Bimolecular Fluorescence Complementation assay (BiFC) in planta (by means of Agrobacterium-mediated transient transformation of Nicotinana benthamiana). In the BiFC assay, the yellow fluorescent protein (YFP) is divided into 2 non-fluorescent segments and fused to the interacting proteins. Upon protein-protein interaction, both fragments of YFP are able to associate, re-fold and generate fluorescence which can be visualized using fluorescence microscopy.[ES] El potasio es uno de los nutrientes catiónicos más importantes para todos los organismos vivos. Entre sus muchas funciones participa en la apertura de estomas de las plantas ya que tiene influencia sobre el potencial osmótico de las células que se encuentran alrededor de los estomas (las células guarda). Un vínculo tan directo entre la [K+ ] y la transpiración en plantas, y por consecuencia con la pérdida de agua, convierte el transporte de potasio en un punto de especial interés en el desarrollo de especies más resistentes a la sequía. El canal rectificador de entrada de potasio KAT1 se ha propuesto como la mayor ruta de flujo de potasio hacia el interior de las células guarda. Este canal forma parte de la familia de canales Shaker, una familia de canales selectivos dependientes de voltaje que están conservados en todos los reinos. Son esenciales para las respuestas de los estomas al ambiente variable o a los diferentes estímulos internos. Por ello, la comprensión de la regulación de KAT1 se ha convertido en un objetivo prioritario para la investigación en este campo. El rastreo de una biblioteca de cDNA de genes de Arabidopsis thaliana por el sistema de estudio de interacción proteína - proteína Split-Ubiquitina devolvió 18 proteínas putativas de interactuar con KAT1. De catorce de ellos se reafirmó la especificidad de la interacción con la proteína KAT1. Por ello, el objetivo de este proyecto es el empleo del sistema de clonaje GoldenBraid, un sistema de ensamblaje estandarizado basado en enzimas de restricción tipo IIS, al estudio de la interacción del canal de potasio KAT1 con una de las proteínas candidatas a interactuar con él. Este sistema permite ensamblajes multigénicos de módulos reutilizables, siendo de esta forma no solo útil en este estudio sino también para el estudio de las interacciones de KAT1 con otras posibles proteínas reguladoras. Empleando la tecnología GoldenBraid se producirán una serie de construcciones para llevar a cabo un ensayo bimolecular de complementación de fluorescencia (Bimolecular Fluorescence Complementation - BiFC), directamente sobre las células de la planta (mediante la transformación transitoria de Nicotiana benthamiana mediada por Agrobacterium). En los ensayos BiFC la proteína fluorescente amarilla se divide en dos segmentos no fluorescentes y se fusiona a las proteínas a estudiar. Si se da la interacción proteína-proteína los fragmentos de YFP se asocian, reconstituyendo la proteína en su estructura original y se genera fluorescencia, que puede visualizarse con un microscopio de fluorescencia.Aljama López, S. (2017). Studies of the Protein-protein interaction between the AtKAT1 potassium channel and its putative regulators. http://hdl.handle.net/10251/86686.TFG