Enhancement of synchronized vortex lattice motion in hybrid magnetic/amorphous superconducting nanostructures

et al.Superconducting a-Mo3Si and Nb films have been grown on arrays of Ni nanodots. We have studied the vortex lattice dynamics close to critical temperatures. Different vortex lattice configurations are obtained with the same array unit cell. These different vortex lattices occur at matching conditions between the vortex lattice and the array unit cell. The interplay between the random intrinsic pinning of the superconducting films and the periodic pinning of the array govern the vortex lattice configurations. Different vortex lattice configurations and enhancement of synchronized vortex lattice motion are obtained by increasing the periodic pinning strength and decreasing the random pinning strength.This work was supported by the Spanish Ministerio Ciencia e Innovacion, Grant Nos. NAN2004-09087 and FIS2005-07392, Consolider Grant Nos. CSD2007-00010 and FIS2008-06249 (Grupo Consolidado), CAM Grant No. S-0505/ESP/0337, and Fondo Social Europeo and Junta de Comunidades de Castilla-La Mancha Grant No. PAI08-0067-2673. A.A. wants to thank PCTI BP06-109.Peer reviewe

The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.Consejería de Educación, Juventud y Deportes–Comunidad de Madrid,Grant/Award Number: B2017/BMD‐36involving contributions from the EuropeanFunds for Regional Development (EFRD) andthe Social European Fund (SEF); ConsejoSuperior de Investigaciones Científicas, Grant/Award Number: COOPA20053;Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: SAF2014‐52048‐R; Agencia Española de Cooperación Internacional para el Desarrollo, Grant/Award Numbers: A/019018/08,A/5444/06, A/8197/0

RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis

Cancer; Cell biologyCàncer; Biologia cel·lularCáncer; Biología CelularRho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear β-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.This work was supported by the IRBLleida Immunohistochemistry and Histology Core Facility. This study was partially funded by grants of Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union (PI19/00993, PI22/00773), European Regional Development Funds (AC19/00095-EuroNanoMed III, AC20/00022-European Joint Program for Rare Diseases), and the Spanish Association Against Cancer (AECC GCA15152966ARAN) to D.A. and grants of Instituto de Salud Carlos III (ISCIII), the European Union (PI23/01062), and Diputació de Lleida (PIRS21/03) to A.M.-B

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression

This research received no external funding. K.G.-H is supported by The European Social Fund (ESF) through a Río Ortega Grant for Health Research Projects by the Carlos III Health Institute (ISCIII) (CM20/00098).B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan–Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Mechanisms of inactivation of the tumour suppressor gene RHOA in colorectal cancer

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF- β /SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms

Regulación de los receptores ErbB y de Src por la calmodulina

[ES]: La calmodulina es una proteína receptora de calcio que interviene en multitud de procesos celulares regulando una gran diversidad de proteínas, entre ellas diversas tirosina quinasas. Aunque el aumento de calcio citosólico permite la activación de la calmodulina; también es capaz de regular distintas funciones celulares en forma de apocalmoculina (calmodulina no unida a calcio) y tras sufrir modificaciones postraduccionales como la fosforilación. Se ha demostrado in vivo que, en presencia de ligando, el complejo Ca2+/calmodulina se une al dominio de unión de la calmodulina del receptor del factor de crecimiento epidérmico (EGFR), que se encuentra en la región citosólica y uxtamembranal, activando la tirosina quinasa del receptor. En cambio, se ha observado in vitro que la calmodulina inhibe esta actividad tirosina quinasa cuando el receptor se activa por su ligando. Además, es sabido que diferentes especies de fosfo-calmodulina realizan distintas funciones celulares. En este trabajo, hemos querido profundizar acerca de la regulación que ejerce la calmodulina sobre las proteína tirosina quinasas. Hemos estudiado, en células vivas, el papel regulador de la misma sobre receptores ErbB y sobre la tirosina quinasa no receptora Src. Asimismo, se ha realizado un ensayo in vitro para determinar el papel de la calmodulina y de un mutante fosfo-mimético de la misma sobre la fosforilación del EGFR.[EN]: Calmodulin is a calcium-binding protein which is involved in the regulation of many cellular processes and a wide variety of proteins, including several protein tyrosine kinases. The increase of free cytosolic calcium allows calmodulin activation; but its function is not only due to the presence of calcium because this protein is also capable to regulate various cell functions as apocalmoculin (calcium-free calmodulin) and after undergoing post-translational modifications (such as phosphorylation). It has been shown in vivo that the Ca2+/ calmodulin complex binds to the calmodulin-binding domain of the EGFR, which is located in its citosolic juxtamembrane region in the region of the EGFR. This region activates the tyrosine kinase activity of the EGFR. In contrast, it has been demostrated in vitro that calmodulin inhibits the ligand-dependent activity of the EGFR. Furthermore, it is known that different phospho-calmodulin species perform different cellular functions. Therefore, in this work, for understanding more about the regulation that calmodulin exerts on protein tyrosine kinases, we have studied in living cells the role of calmodulin on ErbB receptors and the non-receptor tyrosine kinase Src. Likewise, it has been performed a study in vitro to determine the role of wild type calmodulin and a phospho-mimetic calmodulin mutant on EGFR phosphorylation.Parte de este trabajo ha dado lugar a la presentación de una comunicación al congreso de la SEBBM 2014 en Granada.Peer Reviewe

The Small GTPase RHOA in cancer

RhoA és una proteïna GTPasa amb un paper rellevant en importants processos cel·lulars. Els nivells d'expressió de RhoA i/o la seva activitat es troben alterats en molts tipus de càncer, però curiosament s'han trobat mutacions recurrents i específiques de tumor en càncer gàstric de tipus difús (CGD), carcinoma de cèl·lules escamoses de cap i coll (CCECC), limfoma de Burkitt, leucèmia/limfoma T de l'adult i limfoma T angioimmunoblàstic, suggerint un paper diferent y depenent de tumor per cada proteïna mutant. En aquest treball hem caracteritzat a nivell funcional set de les mutacions recurrents en RHOA. Els nostres resultats indiquen que, alguns dels mutants exhibeixen una disminució en l'estabilitat de la proteïna i una localització predominant al nucli en comparació amb la proteïna RhoA nativa. A més, l'avaluació dels efectes de la sobreexpressió de les formes de RhoA mutades en l'organització del citoesquelet va mostrar una reducció general en la formació de filaments d'actina i una major capacitat d'adhesió a substrat. Finalment, la majoria dels mutants estudiats mostraren una senyalització depenent de les vies NFkB i del factor de resposta sèrica reduïda. Per conèixer els mecanismes moleculars desregulats per les mutacions de RhoA, s'avaluà l'interactoma. La capacitat d'unió de les diferents proteïnes mutants de RhoA a Rhotekina resultà ser depenent de la seva estabilitat proteica. A més, l'avaluació de la interacció entre les diferents proteïnes RhoA i els seus efectors més comuns mitjançant un sistema de doble híbrid en llevat, demostrà la incapacitat de tots els mutants de DGC estudiants d'unir PKN1. Tanmateix, el mutant de CCECC RhoA E40Q resultà deficient en la unió a NET1 y kinectina. El CCECC és un grup molt heterogeni de càncers. Les mancances en la millora de la supervivència i en els tractaments personalitzats han promogut la investigació activa dels mecanismes moleculars associats al seu desenvolupament. Encara que RHOA està mutat en només l'1,5% dels pacients de CCECC, més del 60% d'aquestes mutacions succeeixen a E40Q, el que suggereix un paper determinant en el procés de carcinogènesi. L'avaluació immunohistoquímica dels nivells de RhoA en mostres de teixit de CCECC va indicar que l'expressió de RhoA s'associa amb una pitjor supervivència en pacients amb tumors de laringe específicament. Per estudiar el paper de RhoA nativa a CCECC, es van generar línies cel·lulars isogèniques deplecionades de RHOA mitjançant ARN d'interferència (RHOA KD) i inactivació genètica mitjançant la tecnologia CRISPR/Cas9 (RHOA KO). Els resultats van mostrar un alentiment del creixement cel·lular in vitro i in vivo, així com una disminució del potencial de migració in vitro en línies cel·lulars tumorals derivades de llengua i laringe. Aquests resultats confirmen que RHOA actua com un oncogèn a CCECC. Seguidament, es va avaluar el paper de la mutació E40Q en sistemes cel·lulars de sobreexpressió de RhoA de manera induïble amb doxiciclina. Sorprenentment, la sobreexpressió de RhoA nativa o E40Q en cèl·lules de faringe va reduir el creixement cel·lular in vitro. Per tant, vam decidir reintroduir RhoA nativa o E40Q en els sistemes cel·lulars de llengua i laringe RHOA KD i RHOA KO generats prèviament. Curiosament, la reintroducció de RhoA nativa i E40Q no va afectar el creixement cel·lular ni les capacitats de migració de les cèl·lules. En conjunt, la complexitat del patró mutacional de RHOA sense precedents, específic del tipus de tumor, i els nostres resultats, mostren que els diferents mutants de RhoA presenten diferents papers oncogènics. A més, demostrem que RhoA a CCECC proporciona un avantatge de creixement per a les cèl·lules tumorals que donen suport al possible paper oncogènic en aquest tipus tumoral.RhoA es una proteína GTPasa con un papel relevante en importantes procesos celulares. Los niveles de expresión de RhoA y/o su actividad se encuentran alterados en muchos tipos de cáncer, pero curiosamente, se han encontrado mutaciones recurrentes y específicas de tumor en cáncer gástrico de tipo difuso (CGD), carcinoma de células escamosas de cabeza y cuello (CCECC), linfoma de Burkitt, leucemia/linfoma T del adulto y linfoma T angioinmunoblástico, sugiriendo un papel distinto y dependiente de tumor para cada proteína mutante. En este trabajo hemos caracterizado a nivel funcional siete de las mutaciones recurrentes en RHOA. Nuestros resultados indican que, algunos de los mutantes exhiben una disminución en la estabilidad de la proteína y una localización predominante en el núcleo en comparación con la proteína RhoA nativa. Además, la evaluación de los efectos de la sobreexpresión de las formas de RhoA mutadas en la organización del citoesqueleto mostró una reducción general en la formación de filamentos de actina, y una mayor capacidad de adhesión a sustrato. Finalmente, la mayoría de los mutantes estudiados mostraron una señalización dependiente de las vías NFkB y del factor de respuesta sérica reducida. Para conocer los mecanismos moleculares desregulados por las mutaciones de RhoA, se evaluó su interactoma. La capacidad de unión de las diferentes proteínas mutantes de RhoA a Rhotekina resultó ser dependiente de su estabilidad proteica. Además, la evaluación de a interacción entre las diferentes proteína RhoA y sus efectores más comunes mediante un sistema de doble híbrido en levadura, demostró la incapacidad de todos los mutantes de DGC estudiados de unir a PKN1. Sin embargo, el mutante de CCECC RhoA E40Q resultó deficiente en la unión a NET1 y kinectina. El CCECC es un grupo muy heterogéneo de cánceres. Las carencias en la mejora de la supervivencia y en los tratamientos personalizados han promovido la investigación activa de los mecanismos moleculares asociados su desarrollo. Aunque RHOA está mutado sólo en el 1,5% de los pacientes de CCECC, más del 60% de estas mutaciones ocurren en E40Q, lo que sugiere un papel determinante en el proceso de carcinogénesis. La evaluación inmunohistoquímica de los niveles de RhoA en muestras de tejido de CCECC indicó que la expresión de RhoA se asocia con una peor supervivencia en pacientes con tumores de laringe específicamente. Para estudiar el papel de RhoA nativa en CCECC, se generaron líneas celulares isogénicas deplecionadas de RHOA mediante ARN de interferencia (RHOA KD) e inactivación genética mediante la tecnología CRISPR/Cas9 (RHOA KO). Los resultados mostraron una ralentización del crecimiento celular in vitro e in vivo, así como una disminución del potencial de migración in vitro en líneas celulares tumorales derivadas de lengua y laringe. Estos resultados confirman que RHOA actúa como un oncogén en CCECC. Seguidamente, se evaluó el papel de la mutación E40Q en sistemas celulares de sobreexpresión de RhoA de manera inducible con doxiciclina. Sorprendentemente, la sobreexpresión de RhoA nativa o E40Q en células de faringe redujo el crecimiento celular in vitro. Por consiguiente, decidimos reintroducir RhoA nativa o E40Q en los sistemas celulares de lengua y laringe RHOA KD y RHOA KO generados previamente. Curiosamente, la reintroducción de RhoA nativa y E40Q no afectó el crecimiento celular ni las capacidades de migración de las células. En conjunto, la complejidad del patrón mutacional de RHOA sin precedentes, específico del tipo de tumor, y nuestros resultados muestran que los distintos mutantes de RhoA presentan diferentes papeles oncogénicos. Además, demostramos que RhoA en CCECC proporciona una ventaja de crecimiento para las células tumorales que apoyan el posible papel oncogénico en este tipo tumoral.RhoA is a small GTPase with an important role in key cellular processes. RhoA expression levels and/or activity are altered in multiple cancers, but interestingly, recurrent and tissue-specific hotspot mutations have been recently identified in diffuse gastric cancer (DGC), head and neck squamous cell carcinoma (HNSCC), Burkitt lymphoma, adult T-cell leukemia/lymphoma and angioimmunoblastic T-cell lymphoma, suggesting a distinctive and tumor-dependent role of each mutant protein. In this work, seven recurrent RHOA mutations have been functionally characterized. Our results indicate that some RhoA mutants display lower protein stability and a predominant nuclear localization compared to the wild-type (wt) RhoA. Moreover, the evaluation of the effects of mutant RhoA overexpression on the cytoskeleton organization showed a general reduction in the formation of actin filaments, and a higher cell adhesion capacity. Finally, we observed a reduced NFkB and serum response factor signaling in most of the hotspot RhoA mutants studied. To dissect the molecular mechanisms deregulated due to RhoA mutations, we investigated the protein interactome. Binding of the different RhoA mutants to Rhotekin effector protein trough a pull-down assay was dependent on RhoA protein levels. A yeast-two-hybrid system approach between RhoA forms and the most common RhoA effector proteins, evidenced that all the studied DGC mutants were unable to bind to PKN1; whereas, the HNSCC hotspot mutant RhoA E40Q failed to bind to NET1 and kinectin. HNSCC is a very heterogeneous group of cancers. The lack of survival improvement and personalized treatments has promoted an active research into the molecular mechanisms of HNSCC. RHOA is mutated only in around of 1.5% of the HNSCC cases, but interestingly, more than 60% of these mutations occur in E40Q, suggesting a possible active role in the tumorigenic process. Immunohistochemical analysis of RhoA expression in HNSCC tissue microarray indicated that RHOA expression is associated with shorter survival outcomes in patients with larynx tumors. To study the role of wt RhoA in HNSCC, we engineered isogenic cell line systems downregulated for RHOA through the directed targeting of RHOA by shRNA (RHOA KD) and by CRISPR/Cas9 technology (RHOA KO). Results showed an impairment of cell growth in vitro and in vivo, as well as a decrease of the migration potential in vitro, both in tongue and larynx-derived tumor cell lines. These results confirm that RHOA acts as an oncogene in HNSCC. Next, the role of E40Q hotspot mutation was evaluated in doxycycline-inducible RhoA overexpressing cell systems. Surprisingly, RhoA wt or E40Q overexpression in pharynx cells reduced cell growth in vitro. Hence, we decided to re-introduce RhoA wt or E40Q in tongue and larynx cell lines RHOA KD and RHOA KO cell systems. Intriguingly, the reintroduction of RhoA wt and E40Q did not affect cell growth and migration capabilities of the cells. Altogether, the unprecedented tumor-type-dependent complexity in the mutational landscape of RhoA and our results indicate that the different RhoA hotspot mutations exhibit different oncogenic roles. Furthermore, specifically in HNSCC we demonstrate that RhoA provides a growth advantage for the cancer cells supporting a possible oncogenic role of RhoA in this tumor type

Ca2+ signaling and Src-kinases-controlled cellular functions

Calcium-mediated signaling and the functionality of Src-family tyrosine kinases (SFKs) are two interconnected processes. Activation of these kinases, which are coupled to a series of receptors, mediates Ca2+ mobilization by regulating Ca2+ channels, and the generated Ca2+ signal in turn exerts control on the kinase activity via calmodulin. In this review, we shall cover the regulation of selected processes where crosstalk between the functionality of SFKs and the Ca2+ signal occurs during the lifespan of the cell, when subjected to different extracellular or intracellular stimuli. These events result in the modulation of many physiological processes, which are essential to maintain organismal homeostasis. We discuss the importance of these mechanisms, and the implicated signaling pathways on essential cellular processes, comprising proliferation, differentiation, cell adhesion, migration, cytoskeletal remodeling, oocyte fertilization, apoptosis and autophagy. Thereafter, we discuss the role that Ca2+ and SFKs exert in the control of selected physiological functions, including hormones/neurotransmitters release, striated and smooth muscle contraction, glomerular filtration, stress response, and the cellular response to viral and bacterial infections.The work in the authors laboratory was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación grant SAF2014-52048-R, and the Consejería de Educación, Juventud y Deportes – Comunidad de Madrid grant B2017/BMD-36 - MULTI-TARGET&VIEW-CM (to AV).Peer reviewe

Src-family tyrosine kinases and the Ca2 + signal

In this review, we shall describe the rich crosstalk between non-receptor Src-family kinases (SFKs) and the Ca2+ transient generated in activated cells by a variety of extracellular and intracellular stimuli, resulting in diverse signaling events. The exchange of information between SFKs and Ca2+ is reciprocal, as it flows in both directions. These kinases are main actors in pathways leading to the generation of the Ca2+ signal, and reciprocally, the Ca2+ signal modulates SFKs activity and functions. We will cover how SFKs participate in the generation of the cytosolic Ca2+ rise upon activation of a series of receptors and the mechanism of clearance of this Ca2+ signal. The role of SFKs modulating Ca2+-translocating channels participating in these events will be amply discussed. Finally, the role of the Ca2+ sensor protein calmodulin on the activity of c-Src, and potentially on other SFKs, will be outlined as well. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.The work in the authors laboratory was funded by grants (to AV) from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2014-52048-R) and the CSIC program i-COOP + 2014 (COOPA20053).Peer reviewe