Virulence and adaptation of Pseudomonas savastanoi to woody hosts

Pseudomonas savastanoi pv. savastanoi (Psv) is a tumour-inducing pathogen of woody hosts, causing olive (Olea europaea) knot disease. During epiphytic colonization, Psv can multiply on the surface of healthy olive leaves, and these large epiphytic populations can spread by rain, wind, insects and cultural practices, entering the plant through wounds. During the last few years, our work focused on the molecular mechanisms governing the transition from the epiphytic to the endopathogenic lifestyle in Psv. Besides confirmation of several virulence factors previously associated to P. savastanoi infections, such as the phytohormones indole-3-acetic acid and cytokinins and the type-III secretion system, we have identified several novel virulence genes and metabolic pathways required for full fitness of Psv in olive knots. Here, we will present our recent results concerning i) the characterization of a genomic region of the Psv chromosome that is absent in all sequenced P. syringae and P. savastanoi strains infecting herbaceous plants (non-lignified), but it is shared with other pathovars infecting woody hosts (lignified) and, ii) the role in virulence of two enzymes involved in the metabolism of cyclic diguanylate-GMP (c-di-GMP), which are also encoded by other Pseudomonas spp.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy

Background Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. Methods NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. Results We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. Conclusions These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.This work was funded by Instituto de Salud Carlos III (grants PI19/01533, CP19/00029 to S.G.-P.), Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (grant P29/22/02 to S.G.-P.), by MCIN/AE (grant RTI2018.101309B-C22 and PID2022-140151OB-C22 funded by MCIN/AEI https://doi.org/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR to J.A.M.), the Chair “Doctors Galera-Requena in cancer stem cell research” (CMC-CTS963 to J.A.M.), the European Regional Development Fund (European Union), Fundación Científica Asociación Española Contra el Cáncer, Junta Provincial de Jaén (AECC) (grant PRDJA19001BLAY to J.L.B.-C.,), Proyectos Intramurales ibs.GRANADA (grant INTRAIBS-2021-09 to C.G.-L.), Junta de Andalucía, Plan Andaluz de Investigación, Desarrollo e Innovación (grant POSTDOC_21_638 to C.G.-L.), Ministerio de Ciencia, Innovación y Universidades (grant FPU19/04450 to A.L.-T.), Junta de Andalucía, Consejería de Transformación Económica, Industria, Conocimiento y Universidades (grant DOC_01686 to J.C.).Peer reviewe

Role of c-di-GMP metabolism in the virulence of Pseudomonas spp.

El diguanilato ciclico (c-di-GMP) es un segundo mensajero que participa en el control de numerosas funciones celulares tales como la biosíntesis y secreción de adhesinas y exopolisacáridos, la motilidad bacteriana, y la virulencia de bacterias patógenas de animales y plantas (Jenal and Malone, 2006; Tamayo et al., 2007; Romling, 2012; Romling et al., 2013). El c-di-GMP se sintetiza a partir de dos nucleótidos de GTP gracias a la acción de diguanilato ciclasas (DGC) y se hidroliza por fosfodiesterasas específicas (PDE). La actividad DGC está asociada con el dominio GGDEF (Camilli y Bassler, 2006; Chang et al., 2001) mientras que la actividad PDE se encuentra asociada con dominios EAL o HD-GYP (Christen et al., 2005; Ryan et al., 2006). Un trabajo previo llevado a cabo por nuestro grupo de investigación para la identificación de nuevos factores de virulencia en Pseudomonas savastanoi pv. savastanoi NCPPB 3335, agente causal de la tuberculosis del olivo, permitió la identificación de dos mutantes en genes relacionados con el metabolismo del c-di-GMP (Matas et al., 2012). La caracterización de los mismos (PSA3335_4049 y PSA3335_000620), fue el objetivo inicial de esta Tesis Doctoral. Posteriormente, este objetivo se expandió a otra cepa del complejo Pseudomonas syringae como es el patógeno de tomate y Arabidopsis, P. syringae pv. tomato DC3000, así como al patógeno oportunista de humanos Pseudomonas aeruginosa. Para llevar a cabo este objetivo general, se plantearon los siguientes objetivos específicos: 1) Analizar la respuesta de P. savastanoi pv. savastanoi NCPPB 3335 a niveles elevados de c-di-GMP causados por la sobreexpresión de la DGC de Caulobacter crescentus PleD*; 2) analizar el papel del gen bifA en fenotipos relacionados con la virulencia de P. savastanoi pv. savastanoi NCPPB 3335 y P. syringae pv. tomato DC3000 y; 3) estudiar el papel de la hipotética DGC codificada por el gen PAS3335_0620 (dgcP) en fenotipos relacionados con la virulencia de P. savastanoi pv. savastanoi y P. aeruginosa. Las conclusiones más relevantes obtenidas en este trabajo son las que se presentan a continuación: i) el aumento de los niveles intracelulares de c-di-GMP en P. savastanoi pv. savastanoi NCPPB 3335 debido a la sobreexpresión de la DGC PleD*, causa una reducción de la movilidad tipo swimming y un incremento en la producción de exopolisacáridos y en la formación de biofilms, así como induce el desarrollo de tumores de mayor tamaño en plantas de olivo, los cuales muestran una necrosis reducida; ii) la proteína BifA regula positivamente la movilidad en P. savastanoi pv. savastanoi NCPPB 3335 y P. syringae pv. tomato DC3000, así como afecta negativamente a la producción de exopolisácaridos y la formación de biofilms en NCPPB 3335; iii) la deleción del gen bifA provoca una disminución de la virulencia de P. savastanoi pv. savastanoi NCPPB 3335 en olivo y de P. syringae pv. tomato DC3000 en tomate; iv) la proteína PDE BifA de Pseudomonas aeruginosa PAO1 complementa todos los fenotipos relacionados con la virulencia que se encuentran alterados en los mutantes bifA de P. savastanoi pv. savastanoi NCPPB 3335 y P. syringae pv. tomato DC3000; v) el gen dgcP de P. savastanoi pv. savastanoi NCPPB 3335 y P. aeruginosa PAK se co-transcribe en forma de operón junto con el gen dsbA y otro gen codificante de una endonucleasa/ exonucleasa/ fosfatasa (EEPP), este operón está conservado en el género Pseudomonas; vi) la proteína DgcP de P. savastanoi pv. savastanoi NCPPB335 es una DGC cuya actividad es dependiente del dominio GGDEF; vii) la proteína DgcP regula positivamente la movilidad y negativamente la formación de biofilms tanto en P. savastanoi pv. savastanoi NCPPB 3335 como en P. aeruginosa PAK; viii) la sobreexpresión del gen dgcP en P. aeruginosa PAK inhibe el sistema de secreción tipo III e induce el sistema de secreción tipo IV; ix) el gen dgcP es esencial para la virulencia completa de P. savastanoi pv. savastanoi NCPPB 3335 en plantas de olivo y de P. aeruginosa PAK en ratones. REFERENCIAS BIBLIOGRÁFICAS Christen, M., Christen, B., Folcher, M., Schauerte, A., and Jenal, U. 2005. Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J Biol Chem 280:30829-30837. Jenal, U., and Malone, J. 2006. Mechanisms of cyclic-di-GMP signaling in bacteria. Annu Rev Genet 40:385-407. Matas, I.M., Lambertsen, L., Rodriguez-Moreno, L., and Ramos, C. 2012. Identification of novel virulence genes and metabolic pathways required for full fitness of Pseudomonas savastanoi pv. savastanoi in olive (Olea europaea) knots. New Phytol 196:1182-1196. Romling, U. 2012. Cyclic di-GMP, an established secondary messenger still speeding up. Environ Microbiol 14:1817-1829. Romling, U., Galperin, M.Y., and Gomelsky, M. 2013. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1-52. Ryan, R.P., Fouhy, Y., Lucey, J.F., Crossman, L.C., Spiro, S., He, Y.W., Zhang, L.H., Heeb, S., Camara, M., Williams, P., and Dow, J.M. 2006. Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci U S A 103:6712-6717. Tamayo, R., Pratt, J.T., and Camilli, A. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu Rev Microbiol 61:131-148

Verbeia: Revista de estudios filológicos Nº0

Verbeia nace con la finalidad de contagiarnos con la pasión de la Filología. Durante este año hemos crecido, nuestro Comité Científico aumenta y con él las esperanzas de estabilidad. Todos sabemos lo que cuesta llegar hasta aquí, y hoy llegamos con artículos escritos por profesores e investigadores de distintas universidades del planeta

Overview of recent TJ-II stellarator results

The main results obtained in the TJ-II stellarator in the last two years are reported. The most important topics investigated have been modelling and validation of impurity transport, validation of gyrokinetic simulations, turbulence characterisation, effect of magnetic configuration on transport, fuelling with pellet injection, fast particles and liquid metal plasma facing components. As regards impurity transport research, a number of working lines exploring several recently discovered effects have been developed: the effect of tangential drifts on stellarator neoclassical transport, the impurity flux driven by electric fields tangent to magnetic surfaces and attempts of experimental validation with Doppler reflectometry of the variation of the radial electric field on the flux surface. Concerning gyrokinetic simulations, two validation activities have been performed, the comparison with measurements of zonal flow relaxation in pellet-induced fast transients and the comparison with experimental poloidal variation of fluctuations amplitude. The impact of radial electric fields on turbulence spreading in the edge and scrape-off layer has been also experimentally characterized using a 2D Langmuir probe array. Another remarkable piece of work has been the investigation of the radial propagation of small temperature perturbations using transfer entropy. Research on the physics and modelling of plasma core fuelling with pellet and tracer-encapsulated solid-pellet injection has produced also relevant results. Neutral beam injection driven Alfvénic activity and its possible control by electron cyclotron current drive has been examined as well in TJ-II. Finally, recent results on alternative plasma facing components based on liquid metals are also presented.ISSN:0029-5515ISSN:1741-432

Evolution over Time of Ventilatory Management and Outcome of Patients with Neurologic Disease∗

OBJECTIVES: To describe the changes in ventilator management over time in patients with neurologic disease at ICU admission and to estimate factors associated with 28-day hospital mortality. DESIGN: Secondary analysis of three prospective, observational, multicenter studies. SETTING: Cohort studies conducted in 2004, 2010, and 2016. PATIENTS: Adult patients who received mechanical ventilation for more than 12 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among the 20,929 patients enrolled, we included 4,152 (20%) mechanically ventilated patients due to different neurologic diseases. Hemorrhagic stroke and brain trauma were the most common pathologies associated with the need for mechanical ventilation. Although volume-cycled ventilation remained the preferred ventilation mode, there was a significant (p < 0.001) increment in the use of pressure support ventilation. The proportion of patients receiving a protective lung ventilation strategy was increased over time: 47% in 2004, 63% in 2010, and 65% in 2016 (p < 0.001), as well as the duration of protective ventilation strategies: 406 days per 1,000 mechanical ventilation days in 2004, 523 days per 1,000 mechanical ventilation days in 2010, and 585 days per 1,000 mechanical ventilation days in 2016 (p < 0.001). There were no differences in the length of stay in the ICU, mortality in the ICU, and mortality in hospital from 2004 to 2016. Independent risk factors for 28-day mortality were age greater than 75 years, Simplified Acute Physiology Score II greater than 50, the occurrence of organ dysfunction within first 48 hours after brain injury, and specific neurologic diseases such as hemorrhagic stroke, ischemic stroke, and brain trauma. CONCLUSIONS: More lung-protective ventilatory strategies have been implemented over years in neurologic patients with no effect on pulmonary complications or on survival. We found several prognostic factors on mortality such as advanced age, the severity of the disease, organ dysfunctions, and the etiology of neurologic disease