Functional Relevance of PP2A-B55 Phosphatases in the Mammalian Cell Cycle

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 04-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 04-03-2019La fosforilación reversible de proteínas es un mecanismo esencial en la regulación del ciclo celular. Mientras que el papel llevado a cabo en mitosis por las proteínas quinasa ha sido profundamente caracterizado, la identidad y la función específica de las fosfatasas en la mitosis de mamíferos está aún por determinar. La proteína fosfatasa 2A (PP2A) es una fosfatasa de residuos serina y treonina de gran importancia en células eucarióticas. Existen evidencias de que los complejos formados por PP2A con subunidades reguladoras de la familia B55 juegan un importante papel en la desfosforilación de sustratos de quinasas dependiente de ciclina (CDKs) durante mitosis en diferentes organismos. Este hecho sugiere su posible relevancia también en la mitosis de mamíferos. Con el objetivo de dilucidar la función de estos complejos PP2A-B55 en mitosis en mamíferos, hemos generado modelos de ratón deficientes para los genes Ppp2r2a (B55α) y Ppp2r2d (B55δ) que codifican dos de las cuatro isoformas de esta familia de subunidades reguladoras; en particular, aquellas consideradas ubicuas y previamente relacionadas con el ciclo celular. El estudio de estos modelos ha revelado que B55α, pero no B55δ, es necesaria durante el desarrollo embrionario y resulta esencial para la supervivencia del animal. Además, a nivel celular, ambas isoformas tienen funciones específicas y redundantes. Las células deficientes para los complejos PP2A-B55 presentan alteraciones en la duración de mitosis y en la segregación cromosómica que ocurre en esta etapa del ciclo celular, dando lugar a defectos de proliferación. El análisis detallado de la progresión del ciclo celular en células deficientes para B55 ha revelado un nuevo papel de estos complejos en la agrupación cromosómica durante mitosis, mediada al menos parcialmente por la proteína pericromosómica Ki-67. El tratamiento con compuestos que afectan la polimerización de microtúbulos, en las células deficientes para B55, provoca dispersión cromosómica durante mitosis, una acumulación de la proteína Ki-67 y, en algunos casos, muerte celular. Estos datos ponen de manifiesto la importancia de estos complejos en la regulación de mitosis en mamíferos y en la respuesta a los fármacos que afectan la polimerización de microtúbulos, los cuales se utilizan frecuentemente en tratamientos contra el cáncer.Reversible protein phosphorylation is an essential mechanism of cell cycle regulation. Whereas the role of mitotic kinases has been deeply characterized, the identity and specific functions of mitotic phosphatases in mammalian cells has not been fully resolved yet. Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase in eukaryotic cells and there is evidence that PP2A complexes containing the B55 family of regulatory subunits play a key role in dephosphorylating CDK substrates during mitosis in different organisms. This fact makes these phosphatase complexes might be important for mitosis in mammals. To address the functional relevance of those PP2A-B55 complexes in mammalian cell cycle, we have generated loss-of-function mouse models for Ppp2r2a (B55α) and Ppp2r2d (B55δ), which encode the ubiquitous and cell cycle-related isoforms out of the four existing ones in mammals (B55α, β, γ, δ). Using these models we have found that B55α, but not B55δ, is required during late embryonic development and therefore essential for mouse survival. Moreover, at the cellular level, both isoforms have specific and overlapping roles in cell cycle regulation. PP2A-B55-null cells display defects in timing and chromosome segregation during mitosis resulting in impaired proliferation. Interestingly, analysis of cell cycle progression in B55-null cells has also revealed a new role for PP2A/B55 complexes in chromosome clustering during mitosis, which is mediated through the perichromosomal protein Ki-67. Treatment of B55 deficient cells with microtubule depolymerizing drugs leads to massive chromosome scattering in mitosis, excessive Ki67 accumulation and eventually mitotic cell death. These data highlight the importance of these phosphatase complexes in regulating mammalian mitosis and the response to microtubule poisons, a common chemotherapeutic reagent used for cancer treatmen

Sydowia polyspora dominates fungal communities carried by two Tomicus species in pine plantations threatened by Fusarium circinatum

Producción CientíficaBark beetles (Coleoptera, Scolytinae) carry a diverse filamentous fungal community sometimes acting as vectors or carriers of phytopathogens. In this study, mycobiota carried by two Tomicus species (Tomicus piniperda and Tomicus destruens) were investigated through (i) morphological and molecular identification of taxa; (ii) taxonomic richness, diversity, evenness, dominance and phoresy indices; (iii) ecological network analysis and (iv) statistical co-occurrence analysis. The studied mycobiota were formed by eleven taxa and showed a moderate fungal diversity with low evenness. The fungus Sydowia polyspora was significantly abundant and dominated the community. All the fungal taxa were randomly associated. Both insect species (T. piniperda and T. destruens) were collected from plantations of Pinus radiata infected by Fusarium circinatum. The ecological factors that could drive community ecology and phoretic links between fungi and bark beetles are discussed.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R)Ministerio de Economía, Industria y Competitividad (project AGL2012-39912)Junta de Castilla y León - Fondo Europeo de Desarrollo Regional (grant ORDEN EDU/1083/2013

Combination of BeGraft and Solaris stent grafts for the covered endovascular reconstruction of aortic bifurcation—BS-CERAB technique

Producción CientíficaBackground: This study examines the impact of the use of the combination of BeGraft and Solaris stent grafts on the outcomes during the covered endovascular reconstruction of aortic bifurcation (BS-CERAB) technique and extension to the iliac arteries. Methods: Consecutive patients with aortoiliac occlusive disease who underwent endovascular treatment using BS-CERAB between January 2020 and December 2023 were included. Patient demographics, symptoms, lesion characteristics, and procedural and follow-up details were collected and analyzed. Perioperative complications and reinterventions were also identified. Results: A total of 42 patients met the inclusion criteria (32 men, 76.2%, median age 72 years, range 59–85). Indications for treatment were intermittent claudication (42.9%) and critical limb ischemia (57.1%). Procedure success was achieved in all cases. The median patient follow-up time was 14 months (1–36). One patient died at a 10-month follow-up due to lung cancer. The mean pre-operative ABI increased from 0.37 ± 0.19 before intervention to 0.71 ± 1.23 post-operatively at 12 months (p = 0.037). The estimated primary patency rates at 3, 6, and 12 months were 90.5%, 85.7%, and 81.0% and primary assisted patency rates were 90.5%, 90.5%, and 85.7%, respectively. Secondary patency was 95.2% at 3 and 6 months and 90.5% at a 12-month follow-up. Active cancer (p = 0.023, OR 2.12 95%CI 1.14–3.25) was a risk factor for restenosis. Conclusions: This mid-term experience shows that the CERAB technique using the combination of BeGraft and Solaris stents grafts, for the endovascular treatment of severe aortoiliac atherosclerotic disease, may allow an effective reconstruction of the aortic bifurcation and iliac arteries related to high-patency and lower-reintervention rates.Junta de Castilla y León, Fundación Instituto de Estudios de Ciencias de la Salud de Castilla y León (IESCYL) - (grant PIP0277) (project VA171P20

Estudio comparativo de un test sensorial de nutrición enteral con adultos sanos y enfermos hematológicos

Se trata en un estudio en el que se realizó un test sensorial con tres productos de nutrición enteral con adultos sanos y enfermos hematológicos. El objetivo del estudio fue comparar si existían diferencias entre las valoraciones que hacen los pacientes de los productos respecto a la que hacen los controles sanos. Además, se realizó un tratamiento de prueba consistente en que los pacientes tomasen los productos durante un día, simulando un tratamiento real. Tras esto, realizamos de nuevo del test sensorial y comparamos los resultados obtenidos en el primer test sensorial en los pacientes frente a los obtenidos tras este tratamiento de prueba.<br /

Increased p53 gene dosage reduces neointimal thickening induced by mechanical injury but has no effect on native atherosclerosis

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Cardiovascular Research following peer review. The definitive publisher-authenticated version Cardiovasc Res. 75 (4):803-12. is available online at: http://cardiovascres.oxfordjournals.org/cgi/content/full/75/4/803OBJECTIVE: The tumor suppressor p53 regulates cell proliferation and apoptosis, two key processes in the pathogenesis of occlusive vascular disease. Here, we examined the consequences of heightening p53 function on neointimal lesion formation in the setting of atherosclerosis and mechanical injury. METHODS: (1) Immunohistopathological characterization of neointimal lesions in atherosclerosis-prone apolipoprotein E-null mice with normal p53 gene dosage (apoEKO) and carrying a p53 transgene (Super-p53/apoE-KO); (2) molecular studies in macrophages and smooth muscle cells (SMCs) obtained from these mice. RESULTS: The p53 transgene conferred p53 gain-of-function in cultured cells and mice. In vitro, survival of irradiated Super-p53 macrophages and femoral SMCs was reduced, but only Super-p53 SMCs exhibited attenuated proliferation. In vivo, whereas the size of spontaneously formed and diet-induced aortic atheromas was undistinguishable in apoE-KO and Super-p53/apoE-KO mice, the latter exhibited attenuated neointimal thickening in mechanically-injured femoral artery. In both models, neither apoptosis nor cell proliferation were affected by additional p53 gene dosage when examined in established neointimal lesions. However, at 2 days after mechanical injury when neointimal lesions were not formed yet, cell proliferation was significantly attenuated within medial SMCs of Super-p53/apoEKO mice. CONCLUSION: Heightening p53 function has differential effects on in vitro proliferation of macrophages (unaffected) versus SMCs (reduced), and on native atherosclerosis (unaffected) versus mechanically-induced neointimal thickening (reduced) in apoE-KO mice. The protective effect of p53 in mechanically-injured femoral artery coincided with limited medial SMC proliferation at early time points preceding neointima formation, but neither medial nor neointimal cell proliferation was affected in vessels with established occlusive lesions. These findings corroborate p53 gain-of-function as a promising therapeutic strategy to limit post-angioplasty restenosis but not native atherosclerosis.Work financed by grants from Ministerio de Sanidad y Consumo/Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares, RECAVA), from the Regional Government of Valencia (GV04B-288) and from Ministerio de Educación y Ciencia and the European Regional Development Fund (SAF2004-03057). S.M.S.-G. and J.M.G received salary support from Instituto de Salud Carlos III, and J.J.F. from CSIC-I3P predoctoral fellowship program cosponsored by the European Social Fund.Peer reviewe

Gαq activation modulates autophagy by promoting mTORC1 signaling.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.We thank Paula Ramos, Susana Rojo-Berciano, and Laura López for helpful technicalassistance. Dr. Marta Cruces (Universidad Autónoma de Madrid, Spain) for herinvaluable help regarding the liver explants experiments, Dr. Badford Berk (University ofRochester, NY, USA) for providing the GFP-Flag-PB1-p62 plasmid, Drs. Stefan Offer-manns and Nina Wettschureck (Max-Planck-Institute for Heart and Lung Research,Germany) for providing Tie2-CreERT2; Gnaq f/f; Gna11−/−[EC-q/11-KO) mice, andDr. Guzmán Sánchez for scientific advice. We thank also Ricardo Ramos from theGenomic facility of Fundación Parque Científico de Madrid (Universidad Autónoma deMadrid, Spain) and Gemma Rodríguez-Tarduchy from the Genomic facility of theInstituto de Investigaciones Biomédicas“Alberto Sols”for their help with cell linesauthentication. The help from CBMSO Animal Care, Flow Cytometry, Electron andOptical and Confocal Microscopy facilities is also acknowledged. This work was sup-ported by Ministerio de Economía; Industria y Competitividad (MINECO) of Spain(grant SAF2017-84125-R to F.M.), (grant BFU2017-83379-R to A.M.A.), Instituto deSalud Carlos III (PI18/01662 to CR, co-funded with European FEDER contribution),CIBERCV-Instituto de Salud Carlos III, Spain (grant CB16/11/00278 to F.M., co-fundedwith European FEDER contribution), Fundación Ramón Areces (to C.R. and F.M.) andPrograma de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to F.M. and NIH grants AG021904 and AG038072 to A.M.C. Wealso acknowledge the support of a Contrato para la Formación del Profesorado Uni-versitario (FPU13/04341) and (FPU14/06670), an EMBO short-term fellowship (ASTF600-2016). We also acknowledge institutional support to the CBMSO from FundaciónRamón Areces.S

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Incidence and risk factors of pharyngocutaneus fistula formation after total laryngectomy. Review

Introduction and objective: The pharyngocutaneous fistula is a troublesome complication after total laryngectomy, increasing morbidity and mortality. We aimed to determine the incidence of pharyngocutaneus fistula after total laryngectomy and to define the possible predictors for pharyngocutaneus fistula formation. Method: We conducted a review of 31 articles with a total of 1100 patients, to evaluate the incidence of fistula in patients with total laryngectomy and risks factors involved. Results: The overall incidence of pharyngocutaneus fistula is 22,3%, and ranges from 3 to 65%. The review revealed that prior radiation treatment was the most common antecedent, following this associated comorbidity, hypoalbuminemia, anemia, and history of tracheotomy prior to laryngectomy. Discussion: Among the series studied, there is significant heterogeneity in the results, because although irradiated patients have a greater number of pharyngostomas, in some studies no relationship was found, which could explain the association with other risk factors. Conclusions: The incidence of pharyngocutaneus fistula is very variable and there are a large number of risk factors involved, the most frequent is radiotherapy associated or not with chemotherapy.Introducción y objetivo: La fístula faringocutánea tras la laringectomía total es una complicación que conlleva un incremento de la morbilidad y mortalidad. Realizamos una revisión con el objetivo de identificar los factores que implican un aumento de su incidencia. Método: Se realizó una revisión de 31 artículos con un total 1100 pacientes con esta complicación, recogiéndose su incidencia del total de laringectomías totales realizadas, así como los factores de riesgo asociados a su aparición. Resultados: La incidencia de esta complicación se estima en un 22,3% con un rango que oscila de un 3 al 65% entre todas las series incluidas en la revisión. De la totalidad de factores de riesgo implicados, el que aparece con mayor frecuencia es la radioterapia preoperatoria, seguida de la comorbilidad asociada, hipoalbuminemia, anemia y antecedentes de traqueotomía previa a la laringectomía. Discusión: Los pacientes irradiados tienen mayor número de faringostomas. Aun así, según las series estudiadas, hay una heterogeneidad en los resultados pues en algunas no se encuentra relación, lo que podría explicarse por la asociación con otros factores de riesgo. Conclusiones: La incidencia en la formación de fístulas faringocutáneas es muy variable y existe un gran número de factores de riesgo implicados siendo el más frecuente la radioterapia asociada o no a quimioterapia

Dry needling of the flexor digitorum brevis muscle reduces postural control in standing: A pre-post stabilometric study

[Abstract] There are studies that show the better balance after dry needling in lumbar pain. However, the postural control effects after foot dry needling are unknown. Our objective was to check if dry needling reduces postural control. Eighteen subjects with flexor digitorum brevis (FDB) muscle Myofascial trigger point were evaluated pre-and post-deep dry needling. We measured stabilometric variables in a pre-post study. We have found significant differences in three stabilometric variables: surface with eyes closed (29.36–53.21 mm2) (p = 0.000), medium speed of the laterolateral displacement with eyes closed (1.42–1.64 mm/s) (p = 0.004), and medium speed of the anteroposterior displacement with eyes closed (1.30–1.53 mm/s) (p = 0.025). Dry needling therapy application in FDB muscle reduces standing postural control with eyes closed