Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Le complexe AKIN, homologue de SNF1, chez A. thaliana (caractérisation de sous-unités non catalytiques et étude préliminaire de sa régulation et de ses fonctions)

Le complexe hétérotrimérique Ser/Thr kinase SNF1 de levure a été particulièrement conservé au cours de l'évolution, ces protéines étant présentes chez les mammifères (AMPK) et les plantes (SnRK1). De manière générale, ces kinases semblent impliquées dans les réponses cellulaires aux contraintes environnementales, nutritionnelles ou aux changements métaboliques. Dans un premier temps, nous avons caractérisé deux nouvelles sous-unités non catalytiques de type b et g chez A.thaliana, AKINb3 et AKINbg interagissant avec les sous-unités du complexe AKIN. Ces sous-unités atypiques, jamais décrites chez les mammifères et la levure, semblent spécifiques des végétaux. Dans un deuxième temps, une étude exhaustive des interactions entre tous les membres du complexe AKIN a montré qu'en théorie plusieurs complexes composés de l'assemblage de ces différentes sous-unités pourraient coexister dans la plante. De plus, l'analyse de l'expression de ces gènes au cours du développement d'A.thaliana et en réponse à différentes contraintes environnementales indique qu'un niveau de régulation de ce complexe kinase pouvait être réalisé par la transcription différentielle des sous-unités non catalytiques. Ce modèle est cohérent avec la multitude de cibles et de fonctions dans lesquelles le complexe SnRK1 semble impliqué chez les végétaux. Enfin, la recherche de partenaires de la sous-unité AKINbg a permis d'isoler des protéines potentiellement impliquées dans les réactions plantes/microorganismes. Par ailleurs, des résultats préliminaires semblent impliquer le(s) complexe(s) AKIN dans une vole de signalisation "sucre" chez les végétaux.The yeast SNFI1 heterotrimeric Ser/Thr kinase complex has been remarkably well conserved during the course of evolution, these proteins being present in mammals (AMPK) and plants (SnRK1). These kinases are usually considered as implicated in cell responses to environmental, nutritional and metabalic changes. We have characterized two novel non-catalytic b- and g-subunit in A.thaliana, AKINb3 and AKINbg interacting with the known AKIN subunits. These atypical protein present features, never described in mammals and yeasts, and could be specific to plants. A two-hybrid global analysis has been performed between the different a-, b- and g-type subunits. The data suggest that many complexes composed by a combination of generally three subunits could coexist in the plant. Moreover, the analysis of gene expression during the course of development of A.thaliana or in response to various environmental conditions indicate that one level of regulation of the kinase could be through the differential transcription of the non-catalytic subunits. This model is coherent with the large number of targets and functions implicating SnRK1 complexes. Finally, the search for partners of AKINbg subunit allowed us to isolate proteins potentially implicated in plant/pathogens interactions. Furthermore, preliminary data suggest the implication of AKIN complexe(s) in one sugar signalling pathway in plants.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Roles of Very-Long-chain fatty acid in the development of Arabidopsis thaliana

International audienceVery long chain fatty acids (VLCFAs) are necessary for the synthesis of triacylglycerols, epicuticular waxes and sphingolipides which are well known to be essential for seed storage and plant structure. Interestingly, studies showed that VLCFAs are also essential for plant development being involved in several cellular processes such as membrane trafficking, cell division and cell differentiation (1-3). The VLCFAs are elongated in the endoplasmic reticulum by the elongase complex composed of four enzymes. The acyl-CoA dehydratase involved in the third step was recently identified as PASTICCINO2 (PAS2) (4). The pas2 mutants show strong defects such as lost of cellular adherence, defects in division plate formation and vesicular dynamic (1, 4). However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressors screen with an A. thaliana cDNA library was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of PHS1, the yeast PAS2 ortholog, prevents growth and induce cytokinesis defects. We selected Arabidopsis genes able to restore growth in selective conditions by acting either directly on the VLCFAs synthesis or by bypassing the VLCFA requirement for cell division and growth. Results from the screen will be presented and in particular the identification of potentially a new dehydratase involved in VLCFAs elongation

β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit1[C][OA]

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each β-type subunit in plants

AKINbeta3, a plant specific SnRK1 protein, is lacking domains present in yeast and mammals non-catalytic ?-subunits

International audienceThe SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic β-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINβ3, a novel protein related to AKINβ subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (β-GBD) identified in the mammalian AMPKβ1. In spite of its unusual features, AKINβ3 complements the yeast sip1Δsip2Δgal83Δ mutant. Moreover, interactions between AKINβ3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINβ3 is a β-type subunit. A search for β-type subunits revealed the existence of β3-type proteins in other plant species. Furthermore, we suggest that the AKINβ3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINβ3-type subunits, involved in several functions among which some could be plant specific

Elevated Levels of MYB30 in the Phloem Accelerate Flowering in Arabidopsis through the Regulation of FLOWERING LOCUS T

In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of flowering locus T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT. CO-independent activation by high MYB30 expression results in FT levels that remain below those observed in the wild-type plants, which show an additive CO-dependent activation. In contrast, twin sister of FT (TSF) is repressed in plants expressing high levels of MYB30 in the phloem. In transient assays, MYB30 and CO additively increase the activity of a reporter construct driven by a 1 kb FT promoter. Acceleration of flowering by MYB30 does not require the presence of salicylic acid and is independent of FLC. Taken together, increased levels of MYB30, which was reported to be induced in response to the perception of pathogens, can accelerate flowering and MYB30 may thus be a candidate to mediate cross-talk between gene networks involved in biotic stress perception and flowering time

Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in \u3ci\u3eArabidopsis\u3c/i\u3e

Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C \u3e 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes

AKINβγ Contributes to SnRK1 Heterotrimeric Complexes and Interacts with Two Proteins Implicated in Plant Pathogen Resistance through Its KIS/GBD Sequence

The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the β- and γ-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical βγ-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the β-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the βγ-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINβγ clearly interacts with AKINβ-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINβγ gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINβγ expression. A two-hybrid screen performed with AKINβγ as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINβγ and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINβγ-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions

CONSTANS and the CCAAT Box Binding Complex Share a Functionally Important Domain and Interact to Regulate Flowering of Arabidopsis

The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression