Combination of 2DE and LC for plant proteomics analysis

The use of analytical biochemical techniques with different separation properties allows us to better understand the proteome. To demonstrate this we have used two different methodologies to analyze embryos from a Tunisian cultivar of durum wheat (Triticum durum Desf.), variety Oum Rabiaa. We compared conventional 2D electrophoresis with liquid-phase chromatography. Our results show that a similar number of proteins were detected with both techniques. However, analysis of protein resolution at different pH ranges showed significant differences. By using a large pH gradient we observed that liquid chromatography presents higher resolution at extreme pH, either acidic or basic. Conversely, 2DE is more resolutive at intermediate pH (pH 5–6.5). Taking these results in consideration, we propose that 2DE and liquid chromatography are complementary methods to analyze complex protein extracts and can be used in parallel to acquire a wider perspective and a better understanding of the embryo proteome.This work was supported by Bio 2009-13044-Co2-01 from MICIN.Peer reviewe

Functional characterization of DHN-5, a dehydrin showing a differential phosphorylation pattern in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance

Water-deficit stress caused by drought and soil salinization adversely affects plant growth and crop productivity. Dehydrins are involved in the adaptation to water and osmotic stress. We have identified a wheat dehydrin named DHN-5 that is closely related to the maize RAB17. The full-length cDNA of Dhn-5 gene encodes a putative protein of 227 amino acids and contains 2 conserved lysine-rich-K-segment (EKKGIMDKIKEKLPG) repeats preceded by a stretch of eight serine residues, characteristic of group 2 LEA family. The Northern blot analyses showed a strong accumulation of Dhn-5 transcript in mature wheat embryos and to a lesser extent in ABA and salt-treated seedlings. Interestingly, DHN-5 protein accumulated differentially in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance. By using specific dehydrin antibodies and 2D immunoblot analysis on proteins extracted from mature embryos in these two varieties, a differential phosphorylation pattern of DHN-5 was observed. In the resistant variety (R), beside a basic protein spot, a series of acidic spots were detected whereas in the sensitive variety (S) the acidic spots were weakly detectable. These acidic forms correspond to highly phosphorylated forms of DHN-5, which can be removed by alkaline phosphatase treatment. Accumulation of phosphorylated DHN-5 mainly in the R variety suggests a role of P-DHN-5 in preservation of cell integrity during late embryogenesis and desiccation. Subcellular localization of the DHN-5:GFP fusion protein indicated that DHN-5 would be primarily nuclear, suggesting a nuclear role in wheat osmotic stress response. The observed differential phosphorylation pattern of DHN-5 in the resistant and sensitive wheat varieties could be used as a basis for a molecular screen of tolerance/sensitivity to drought and salt stresses in wheat germplasm.This work was supported jointly by grants from the Ministry of Scientific Research, Technology and Development of Competencies, Tunisia and the Agence Espagnole de cooperation Internationale (AECI) Officina Técnica de Cooperación, Spain.Peer reviewe

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na+ and K+ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.This work was supported jointly by grants from the Ministry of Higher Education, Scientific Research and Technology, Tunisia and the Agence Espagnole de cooperation Internationale (AECI) Officina Técnica de Cooperación, Spain.Peer reviewe

Memory based cuckoo search algorithm for feature selection of gene expression dataset

Cancer prediction has been shown to be important in the cancer research area. This importance has prompted many researchers to review machine learning-approaches to predict cancer outcome using gene expression dataset. This dataset consists of many genes (features) which can mislead the prediction ability of the machine learning methods, as some features may lead to confusion or inaccurate classification. Since finding the most informative genes for cancer prediction is challenging, feature selection techniques are recommended to pick important and relevant features out of large and complex datasets. In this research, we propose the Cuckoo search method as a feature selection algorithm, guided by the memory-based mechanism to save the most informative features that are identified by the best solutions. The purpose of the memory is to keep track of the selected features at every iteration and find the features that enhance classification accuracy. The suggested algorithm has been contrasted with the original algorithm using microarray datasets and the proposed algorithm has been shown to produce good results as compared to original and contemporary algorithms

Healthcare-associated infections in patients with severe COVID-19 supported with extracorporeal membrane oxygenation: a nationwide cohort study

International audienceBackground Both critically ill patients with coronavirus disease 2019 (COVID-19) and patients receiving extracorporeal membrane oxygenation (ECMO) support exhibit a high incidence of healthcare-associated infections (HAI). However, data on incidence, microbiology, resistance patterns, and the impact of HAI on outcomes in patients receiving ECMO for severe COVID-19 remain limited. We aimed to report HAI incidence and microbiology in patients receiving ECMO for severe COVID-19 and to evaluate the impact of ECMO-associated infections (ECMO-AI) on in-hospital mortality. Methods For this study, we analyzed data from 701 patients included in the ECMOSARS registry which included COVID-19 patients supported by ECMO in France. Results Among 602 analyzed patients for whom HAI and hospital mortality data were available, 214 (36%) had ECMO-AI, resulting in an incidence rate of 27 ECMO-AI per 1000 ECMO days at risk. Of these, 154 patients had bloodstream infection (BSI) and 117 patients had ventilator-associated pneumonia (VAP). The responsible microorganisms were Enterobacteriaceae (34% for BSI and 48% for VAP), Enterococcus species (25% and 6%, respectively) and non-fermenting Gram-negative bacilli (13% and 20%, respectively). Fungal infections were also observed (10% for BSI and 3% for VAP), as were multidrug-resistant organisms (21% and 15%, respectively). Using a Cox multistate model, ECMO-AI were not found associated with hospital death (HR = 1.00 95% CI [0.79–1.26], p = 0.986). Conclusions In a nationwide cohort of COVID-19 patients receiving ECMO support, we observed a high incidence of ECMO-AI. ECMO-AI were not found associated with hospital death. Trial registration number NCT04397588 (May 21, 2020)

Veno-Arterial Extracorporeal Membrane Oxygenation for Circulatory Failure in COVID-19 Patients: Insights from the ECMOSARS Registry

International audienceObjectives: The clinical profile and outcomes of patients with Covid-19 who require veno-arterial or veno-venous-arterial extracorporeal membrane oxygenation (VA-ECMO - VAV-ECMO) are poorly understood. We aimed to describe the characteristics and outcomes of these patients and to identify predictors of both favorable and unfavorable outcomes.Methods: ECMOSARS is a multicenter, prospective, nationwide French registry enrolling patients who require VV/VA-ECMO in the context of Covid-19 infection (652 patients at 41 centers). We focused on 47 patients supported with VA- or VAV-ECMO for refractory cardiogenic shock.Results: Median age was 49. 14% of patients had a prior diagnosis of heart failure. The most common etiologies of cardiogenic shock were acute pulmonary embolism (30%), myocarditis (28%), and acute coronary syndrome (4%). E-CPR (Extracorporeal Cardiopulmonary Resuscitation) occurred in 38%. In-hospital survival was 28% in the whole cohort, and 43% when E-CPR patients were excluded. ECMO cannulation was associated with significant improvements in pH and FiO2 on day one, but non-survivors showed significantly more severe acidosis and higher FiO2 than survivors at this point (p = 0.030 and p = 0.006). Other factors associated with death were greater age (p = 0.02), higher BMI (p = 0.03), E-CPR (p = 0.001), non-myocarditis etiology (p = 0.02), higher serum lactates (p = 0.004), epinephrine (but not noradrenaline) use before initiation of ECMO (p = 0.003), hemorrhagic complications (p = 0.001), greater transfusion requirements (p = 0.001), and more severe SAVE and SAFE scores (p = 0.01 and p = 0.03).Conclusions: We report the largest focused analysis of VA- and VAV-ECMO recipients in Covid-19. Although relatively rare, the need for temporary mechanical circulatory support in these patients is associated with poor prognosis. However, VA-ECMO remains a viable solution to rescue carefully selected patients. We identified factors associated with poor prognosis and suggest that E-CPR is not a reasonable indication for VA-ECMO in this population

Bleeding and thrombotic events in patients with severe COVID-19 supported with extracorporeal membrane oxygenation: a nationwide cohort study

International audiencePurpose: To describe bleeding and thrombotic events and their risk factors in patients receiving extracorporeal membrane oxygenation (ECMO) for severe coronavirus disease 2019 (COVID-19) and to evaluate their impact on in-hospital mortality.Methods: The ECMOSARS registry included COVID-19 patients supported by ECMO in France. We analyzed all patients included up to March 31, 2022 without missing data regarding bleeding and thrombotic events. The association of bleeding and thrombotic events with in-hospital mortality and pre-ECMO variables was assessed using multivariable logistic regression models.Results: Among 620 patients supported by ECMO, 29% had only bleeding events, 16% only thrombotic events and 20% both bleeding and thrombosis. Cannulation site (18% of patients), ear nose and throat (12%), pulmonary bleeding (9%) and intracranial hemorrhage (8%) were the most frequent bleeding types. Device-related thrombosis and pulmonary embolism/thrombosis accounted for most of thrombotic events. In-hospital mortality was 55.7%. Bleeding events were associated with in-hospital mortality (adjusted odds ratio (adjOR) = 2.91[1.94-4.4]) but not thrombotic events (adjOR = 1.02[0.68-1.53]). Intracranial hemorrhage was strongly associated with in-hospital mortality (adjOR = 13.5[4.4-41.5]). Ventilation duration before ECMO ≥ 7 days and length of ECMO support were associated with bleeding. Thrombosis-associated factors were fibrinogen ≥ 6 g/L and length of ECMO support.Conclusions: In a nationwide cohort of COVID-19 patients supported by ECMO, bleeding incidence was high and associated with mortality. Intracranial hemorrhage incidence was higher than reported for non-COVID patients and carried the highest risk of death. Thrombotic events were less frequent and not associated with mortality. Length of ECMO support was associated with a higher risk of both bleeding and thrombosis, supporting the development of strategies to minimize ECMO duration