Evaluación del metabolismo del glutatión en la tolerancia a metales pesados

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 14-02-2014Se estudiaron las respuestas de estrés oxidativo inducido por cadmio (Cd) y mercurio (Hg), característico síntoma de toxicidad por metales, en plantas de alfalfa crecidas en un medio semi-hidropónico con diferentes dosis de metal (0, 3, 10 y 30 μM) durante una semana. Ambos metales produjeron alteraciones en enzimas antioxidantes, siendo el Hg más tóxico que el Cd, destacando la inhibición de la actividad glutatión reductasa (GR), mientras que con Cd hubo mayor síntesis de fitoquelatinas (PCs). Estás diferencias en los mecanismos de toxicidad nos llevó a plantear la posibilidad de identificar las firmas de estrés específicas para elementos tóxicos (Cd, Hg y arsénico (As); 0, 6 y 30 μM) en la planta metalofita Silene vulgaris. Tras una semana de exposición, se confirmó que la inhibición de GR era un indicador específico de estrés por Hg, siendo de nuevo importante la producción de PCs en plantas tratadas con Cd. Asimismo, el Cd produjo fotoinhibición y alteración en proteínas relacionadas con el aparato fotosintético, lo que pudo estar relacionado con la mayor translocación de este metal a hoja. La evidente implicación del GSH y otros biotioles en la respuesta a metal(loide)s tóxicos nos llevó a plantear el experimentos funcionales para caracterizar en detalle su contribución en las respuestas Cd y Hg, para lo que utilizaron mutantes de Arabidopsis thaliana con niveles alterados de GSH y biotioles frente al ecotipo silvestre (Col- 0). Se trabajó con tres alelos mutantes de la enzima γ-glutamilcisteina sintetasa (γECS), pad2-1, cad2-1, y rax1-1 (acumulan 20, 30 y 45% del GSH detectado en Col-0, respectivamente); y un mutante defectivo en fitoquelatina sintasa (PCS) cad1-3 (que no acumula PCs). Se diseñaron dos aproximaciones distintas, por un lado se infiltraron hojas de plantas Col-0, cad2-1, rax1-1 y cad1-3 o se cultivaron en un sistema hidropónico puro con Cd y Hg (0, 3, 10 o 30 μM) durante 24-72 h. Se observó que pequeñas diferencias en el contenido de GSH hace que la planta se enfrente al estrés de diferente manera, teniendo comportamientos similares a Col-0 en rax1-1, mientras que pad2-1 mostró la mayor sensibilidad. También se estudió el perfil transcripcional de genes de la ruta de asimilación de azufre y metabolismo del GSH en plantas de Arabidopsis Col-0, mutante de γECS y cad1-3, tratadas con un nivel moderado de Hg (3 μM) durante 72 h. Se confirmó que los mutantes cad2-1 y pad2-1 fueron particularmente sensibles a Hg, al tiempo que es importante el nivel de GSH en el patrón de expresión génica. Nuestros datos apoyan la noción de un umbral crítico de concentración de GSH, observada en los mutantes rax1-1 con niveles de GSH más parecidos a Col-0, que permite tolerancia a Cd y Hg. Se ha puesto de manifiesto la importancia del GSH en la tolerancia, tanto a nivel del ajuste metabólico de la respuesta, como en la capacidad de absorción y transporte de metales y metaloides tóxicos, observándose respuestas específicas para cada contaminante. Esta información puede ser útil para la optimización de estrategias de descontaminación de suelos contaminados por elementos tóxicos mediante fitorremediación.We studied the responses of oxidative stress induced by cadmium (Cd) and mercury (Hg), a characteristic symptom of metal toxicity, in alfalfa plants grown on a semi-hydroponic medium with different doses of metal (0, 3, 10 and 30 μM) for a week. Both metals produced alterations in antioxidant enzymes, being Hg more toxic than Cd, in particular the strong inhibition of glutathione reductase (GR) activity, while there was greater synthesis of phytochelatins (PCs) with Cd. The observed differences in the mechanisms of toxicity led us to identifying the specific stress signatures for different toxic elements (Cd, Hg and arsenic (As); 0, 6 and 30 μM) in the metallophyte Silene vulgaris. After a week of exposure, we confirmed that GR inhibition was a specific indicator of stress by Hg, being Cd a potent inductor of PCs production. Cadmium also caused photoinhibition and alteration in proteins related to the photosynthetic apparatus, which could be related to increased translocation of this metal to the shoot. The involvement of GSH and other biothiols in response to toxic metal(loid)s prompted us to perform functional experiments to characterize in detail its contribution in the responses to Cd and Hg, using mutants of Arabidopsis thaliana with altered levels of GSH and biothiols compared with the wild type (Col-0). We studied three mutant alleles of the enzyme γ-glutamylcysteine synthetase (γECS), pad2-1, cad2-1and rax1-1 (that contain 20, 30 and 45% of Col-0 GSH levels, respectively); and a mutant defective in phytochelatin synthase (PCS) cad1-3 (does not accumulate PCs). Two different approaches were designed, on the one hand infiltrated leaves of plants Col-0, cad2-1, rax1-1 and cad1-3, or were cultivated in a pure hydroponic system with Cd and Hg (0, 3, 10 or 30 μM) for 24-72 h. It was observed that small differences in the content of GSH makes the plant to face stress differently, showing similar behaviors Col-0 and rax1 -1, while pad2-1 was more sensitivity. It was also studied the transcriptional profile of genes of the assimilation of sulfur pathway and GSH metabolism in Arabidopsis Col-0, the γECS mutant plants of and cad1-3, treated with a moderate level of Hg (3 μM) for 72 h. It was confirmed that the cad2-1 and pad2-1 mutants were particularly sensitive to Hg, at the time that the level of GSH is important for the gene expression profile. Our data support the notion of a critical threshold of GSH concentration, observed in the rax1-1 mutant with GSH levels more similar to Col-0, which allows tolerance to Cd and Hg. It has been highlighted the importance of GSH in tolerance, both at the level of metabolic adjustment of the response, and at the level of toxic metal and metalloids absorption and transport, with specific responses for each pollutant. This information can be useful for the optimization of the decontamination strategies of soils polluted with toxic elements using phytoremediation technologies

Breeding and scientific advances in the fight against Dutch elm disease - will they allow the use of elms in forest restoration?

Revisión de los avances científicos y de producción de planta con fin de evaluar el potencial en la recuperación del olmo común

The Early Oxidative Stress Induced by Mercury and Cadmium Is Modulated by Ethylene in Medicago sativa Seedlings

Cadmium (Cd) and mercury (Hg) are ubiquitous soil pollutants that promote the accumulation of reactive oxygen species, causing oxidative stress. Tolerance depends on signalling processes that activate different defence barriers, such as accumulation of small heat sock proteins (sHSPs), activation of antioxidant enzymes, and the synthesis of phytochelatins (PCs) from the fundamental antioxidant peptide glutathione (GSH), which is probably modulated by ethylene. We studied the early responses of alfalfa seedlings after short exposure (3, 6, and 24 h) to moderate to severe concentration of Cd and Hg (ranging from 3 to 30 μM), to characterize in detail several oxidative stress parameters and biothiol (i.e., GSH and PCs) accumulation, in combination with the ethylene signalling blocker 1-methylcyclopropene (1-MCP). Most changes occurred in roots of alfalfa, with strong induction of cellular oxidative stress, H2O2 generation, and a quick accumulation of sHSPs 17.6 and 17.7. Mercury caused the specific inhibition of glutathione reductase activity, while both metals led to the accumulation of PCs. These responses were attenuated in seedlings incubated with 1-MCP. Interestingly, 1-MCP also decreased the amount of PCs and homophytochelatins generated under metal stress, implying that the overall early response to metals was controlled at least partially by ethylen

Learning Criteria and Evaluation Metrics for Textual Transfer between Non-Parallel Corpora

We consider the problem of automatically generating textual paraphrases with modified attributes or stylistic properties, focusing on the setting without parallel data (Hu et al., 2017; Shen et al., 2017). This setting poses challenges for learning and evaluation. We show that the metric of post-transfer classification accuracy is insufficient on its own, and propose additional metrics based on semantic content preservation and fluency. For reliable evaluation, all three metric categories must be taken into account. We contribute new loss functions and training strategies to address the new metrics. Semantic preservation is addressed by adding a cyclic consistency loss and a loss based on paraphrase pairs, while fluency is improved by integrating losses based on style-specific language models. Automatic and manual evaluation show large improvements over the baseline method of Shen et al. (2017). Our hope is that these losses and metrics can be general and useful tools for a range of textual transfer settings without parallel corpora

Attenuation of mercury phytotoxicity with a high nutritional level of nitrate in alfalfa plants grown hydroponically

Mercury (Hg) is one of the most dangerous pollutant heavy metals to the environment, which causes several toxic effects in plants upon accumulation, such as induction of oxidative stress. Nitrate (NO3 – ) is the prevalent form to incorporate nitrogen (N) in higher plants, through its reduction to nitrite (NO2 – ) by the enzyme nitrate reductase (NR). We studied the physiological alterations caused by Hg (0, 6 and 30 µM) in alfalfa plants grown at two different levels of NO3 – : low, (2 mM; LN), and high (12 mM; HN) for one week using a semi-hydroponic culture system. Several parameters of oxidative stress such as lipid peroxidation, chlorophyll content, biothiol concen tration, and ascorbate peroxidase (APX) and glutathione reductase (GR) activities showed that HN plants were less affected by Hg. Nitrate reductase activity and NO3 – concentration were also altered under Hg stress, with lower impact in plants nourished with high NO3 – . Our results highlight the importance of the NO3 – nutritional status to improve tolerance to toxic metals like H

Dissecting the molecular responses potentially involved in the tolerance of two Ulmus minor

Experimento molecular que sugiere que la tolerancia de Ulmus minor a la grafiosis está relacionada a la expresión diferencial de algunos genes como aquellos relacionados con GO:000960

Contribution of phytochelatins to cadmium tolerance in peanut plants

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H2O2) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.Fil: Bianucci, Eliana Carolina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Sobrino Plata, Juan. No especifíca;Fil: Carpena Ruiz, Ramón O.. Universidad Autónoma de Madrid; EspañaFil: Del Carmen Tordable, María. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Fabra, Adriana Isidora. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Hernández, Luis E.. No especifíca;Fil: Castro, Stella Maris. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Risk factors for infections caused by carbapenem-resistant Enterobacterales: an international matched case-control-control study (EURECA)

Cases were patients with complicated urinary tract infection (cUTI), complicated intraabdominal (cIAI), pneumonia or bacteraemia from other sources (BSI-OS) due to CRE; control groups were patients with infection caused by carbapenem-susceptible Enterobacterales (CSE), and by non-infected patients, respectively. Matching criteria included type of infection for CSE group, ward and duration of hospital admission. Conditional logistic regression was used to identify risk factors. Findings Overall, 235 CRE case patients, 235 CSE controls and 705 non-infected controls were included. The CRE infections were cUTI (133, 56.7%), pneumonia (44, 18.7%), cIAI and BSI-OS (29, 12.3% each). Carbapenemase genes were found in 228 isolates: OXA-48/like, 112 (47.6%), KPC, 84 (35.7%), and metallo-beta-lactamases, 44 (18.7%); 13 produced two. The risk factors for CRE infection in both type of controls were (adjusted OR for CSE controls; 95% CI; p value) previous colonisation/infection by CRE (6.94; 2.74-15.53; <0.001), urinary catheter (1.78; 1.03-3.07; 0.038) and exposure to broad spectrum antibiotics, as categorical (2.20; 1.25-3.88; 0.006) and time-dependent (1.04 per day; 1.00-1.07; 0.014); chronic renal failure (2.81; 1.40-5.64; 0.004) and admission from home (0.44; 0.23-0.85; 0.014) were significant only for CSE controls. Subgroup analyses provided similar results. Interpretation The main risk factors for CRE infections in hospitals with high incidence included previous coloni-zation, urinary catheter and exposure to broad spectrum antibiotics

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Wood development regulators involved in apical growth in Pinus canariensis

The shoot apical meristem is responsible of seasonal length increase in plants. In woody plants transition from primary to secondary growth is also produced during seasonal apical growth. These processes are controlled by different families of transcription factors. Levels of transcriptomic activity during apical growth were measured by means of a cDNA microarray designed from sequences related to meristematic activity in Pinus canariensis. The identification of differentially expressed genes was performed using a time-course analysis. A total of 7170 genes were differentially expressed and grouped in six clusters according to their expression profiles. We identified master regulators, such as WUSCHEL-like HOMEOBOX (WOX), to be involved in the first stages of apical development, i.e. growth of primary tissues, while other transcription factors, such as Class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) and KNOTTED-like (KNOX) and BEL1-like (BELL) HOMEODOMAIN proteins, were found to be induced during last stages of apical seasonal development, already with secondary growth. Our results reveal the main expression patterns of these genes during apical development and the transition from primary to secondary stem growth. In particular, the regulatory factors identified play key roles in controlling stem architecture and constitute candidate genes for the study of other development processes in conifers.Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu