Búsqueda de nuevas dianas terapéuticas en Acinetobacter baumannii mediante la identificación de genes metabólicos implicados en la virulencia e infección

Motivación: Las infecciones nosocomiales son un problema clínico de creciente importancia para la salud pública cuyo coste asociado es muy elevado [1]. Acinetobacter baumannii es un bacilo aerobio Gram negativo causante de un gran número de estas infecciones, provocando pneumonia y bacteremia con una alta mortalidad [2]. La emergencia global de cepas multirresistentes dificulta el tratamiento clínico de las infecciones y la industria farmacéutica ha reducido su interés en el desarrollo de nuevos antibióticos [3]. Por ello, es necesario identificar y caracterizar genes de A. baumannii para el desarrollo de nuevos antimicrobianos y vacunas.Métodos: Se usa una librería de 9000 mutantes de A. baumannii ATCC 17978 creada por mutagénesis aleatoria por la inserción del transposón EZ-Tn5<R6Kori/KAN>tnp en el genoma, lo que proporciona resistencia a kanamicina. Se realizó un screening en medio mínimo (M9), que simula las condiciones presentes en el huésped durante la infección, para determinar las cepas mutantes funcionalmente deficientes, y en distintos antibióticos, para caracterizar el mecanismo de resistencia de este patógeno.Resultados: 57 mutantes de 5100 no mostraron crecimiento en M9 tras la incubación a 37°C durante 5 días. Los productos génicos presentaban la siguiente localización: 50 proteínas citoplasmáticas (87,72%), 5 de la membrana citoplasmática (8,77%) y 2 proteínas con una localización subcelular desconocida (3,51%). De estas proteínas, 27 participan en el metabolismo de aminoácidos (47,37%), 8 en el metabolismo de carbohidratos (14,04%), 3 en la síntesis/reparación del ADN (5,26%), 2 en el procesamiento del ADN/ARN (3,51%), 1 es una proteína de un sistema de dos componentes (1,75%), 1 en la síntesis del peptidoglicano (1,75%), 1 en el metabolismo de ácidos grasos (1,75%), 9 en procesos de producción energética (15,79%), 1 en el transporte celular y 4 con función desconocida (7,02%). Además, en el screening de toda la librería en distintos antibióticos se han encontrado 2 genes de resistencia a fosfomicina y 5 a ciprofloxacina.Conclusiones: Se han identificado genes requeridos para el crecimiento de A.baumannii en condiciones de limitación de nutrientes. Las proteínas codificadas por estos genes son potenciales dianas para el desarrollo de nuevos agentes antimicrobianos. Además, se han identificado genes de resistencia a fosfomicina y ciprofloxacina, lo que permitirá caracterizar el mecanismo de resistencia de A.baumannii a estos antibióticos

Metal-catalyst-free gas-phase synthesis of long-chain hydrocarbons

Development of sustainable processes for hydrocarbons synthesis is a fundamental challenge in chemistry since these are of unquestionable importance for the production of many essential synthetic chemicals, materials and carbon-based fuels. Current industrial processes rely on non-abundant metal catalysts, temperatures of hundreds of Celsius and pressures of tens of bars. We propose an alternative gas phase process under mild reaction conditions using only atomic carbon, molecular hydrogen and an inert carrier gas. We demonstrate that the presence of CH2 and H radicals leads to efficient C-C chain growth, producing micron-length fibres of unbranched alkanes with an average length distribution between C23-C33. Ab-initio calculations uncover a thermodynamically favourable methylene coupling process on the surface of carbonaceous nanoparticles, which is kinematically facilitated by a trap-and-release mechanism of the reactants and nanoparticles that is confirmed by a steady incompressible flow simulation. This work could lead to future alternative sustainable synthetic routes to critical alkane-based chemicals or fuels

Interplay Among Different Fosfomycin Resistance Mechanisms in Klebsiella Pneumoniae

The objectives of this study were to characterize the role of the uhpT, glpT, and fosA genes in fosfomycin resistance in Klebsiella pneumoniae and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were used, and their genomes were sequenced. DuhpT, DglpT, and DfosA mutants were constructed from two isolates and K. pneumoniae ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512mg/liter, in vitro activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307mg/liter were evaluated with and without PPF (0.623mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024mg/liter. The addition of 0.623mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of fosA led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623mM). The lowest fosfomycin-resistant mutant frequencies were found in ΔfosA mutants, with decreases in frequency from 1.69×10-1 to 1.60×10-5 for 64mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of fosA and not with the addition of PPF. We conclude that fosA gene inactivation leads to a decrease in fosfomycin resistance in K. pneumoniae. The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations.Ministerio de Economía y Competitividad PI16/01824, REIPI RD12/0015/0010, EIPI RD16/0016/0001Junta de Andalucía PI-0044Innovative Medicines Initiative 115523, 115620, 11573

Predicting the onset and persistence of episodes of depression in primary health care. The predictD-Spain study: Methodology

Background: The effects of putative risk factors on the onset and/or persistence of depression remain unclear. We aim to develop comprehensive models to predict the onset and persistence of episodes of depression in primary care. Here we explain the general methodology of the predictD-Spain study and evaluate the reliability of the questionnaires used. Methods: This is a prospective cohort study. A systematic random sample of general practice attendees aged 18 to 75 has been recruited in seven Spanish provinces. Depression is being measured with the CIDI at baseline, and at 6, 12, 24 and 36 months. A set of individual, environmental, genetic, professional and organizational risk factors are to be assessed at each follow-up point. In a separate reliability study, a proportional random sample of 401 participants completed the test-retest (251 researcher-administered and 150 self-administered) between October 2005 and February 2006. We have also checked 118,398 items for data entry from a random sample of 480 patients stratified by province. Results: All items and questionnaires had good test-retest reliability for both methods of administration, except for the use of recreational drugs over the previous six months. Cronbach's alphas were good and their factorial analyses coherent for the three scales evaluated (social support from family and friends, dissatisfaction with paid work, and dissatisfaction with unpaid work). There were 191 (0.16%) data entry errors. Conclusion: The items and questionnaires were reliable and data quality control was excellent. When we eventually obtain our risk index for the onset and persistence of depression, we will be able to determine the individual risk of each patient evaluated in primary health car

Cytokine profile and anti-inflammatory activity of a standardized conditioned medium obtained by coculture of monocytes and mesenchymal stromal cells (PRS CK STORM)

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1B secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir/ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an anti-apoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence

Identifying comorbidities and lifestyle factors contributing to the cognitive profile of early Parkinson's disease

Background: Identifying modifiable risk factors for cognitive impairment in the early stages of Parkinson's disease (PD) and estimating their impact on cognitive status may help prevent dementia (PDD) and the design of cognitive trials. Methods: Using a standard approach for the assessment of global cognition in PD and controlling for the effects of age, education and disease duration, we explored the associations between cognitive status, comorbidities, metabolic variables and lifestyle variables in 533 PD participants from the COPPADIS study. Results: Among the overall sample, 21% of participants were classified as PD-MCI (n = 114) and 4% as PDD (n = 26). The prevalence of hypertension, diabetes and dyslipidemia was significantly higher in cognitively impaired patients while no between-group differences were found for smoking, alcohol intake or use of supplementary vitamins. Better cognitive scores were significantly associated with regular physical exercise (p < 0.05) and cognitive stimulation (< 0.01). Cognitive performance was negatively associated with interleukin 2 (Il2) (p < 0.05), Il6 (p < 0.05), iron (p < 0.05), and homocysteine (p < 0.005) levels, and positively associated with vitamin B12 levels (p < 0.005). Conclusions: We extend previous findings regarding the positive and negative influence of various comorbidities and lifestyle factors on cognitive status in early PD patients, and reinforce the need to identify and treat potentially modifiable variables with the intention of exploring the possible improvement of the global cognitive status of patients with PD

Hepatic galectin-3 is associated with lipid droplet area in non-alcoholic steatohepatitis in a new swine model

Non-alcoholic fatty liver disease (NAFLD) is currently a growing epidemic disease that can lead to cirrhosis and hepatic cancer when it evolves into non-alcoholic steatohepatitis (NASH), a gap not well understood. To characterize this disease, pigs, considered to be one of the most similar to human experimental animal models, were used. To date, all swine-based settings have been carried out using rare predisposed breeds or long-term experiments. Herein, we fully describe a new experimental swine model for initial and reversible NASH using cross-bred animals fed on a high saturated fat, fructose, cholesterol, cholate, choline and methionine-deficient diet. To gain insight into the hepatic transcriptome that undergoes steatosis and steatohepatitis, we used RNA sequencing. This process significantly up-regulated 976 and down-regulated 209 genes mainly involved in cellular processes. Gene expression changes of 22 selected transcripts were verified by RT-qPCR. Lipid droplet area was positively associated with CD68, GPNMB, LGALS3, SLC51B and SPP1, and negatively with SQLE expressions. When these genes were tested in a second experiment of NASH reversion, LGALS3, SLC51B and SPP1 significantly decreased their expression. However, only LGALS3 was associated with lipid droplet areas. Our results suggest a role for LGALS3 in the transition of NAFLD to NASH