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/)

Réseau de régulation de la transcription des gènes du système protéolytique de lactococcus lactis

International audienceNetwork of regulation of gene transcription of the proteolytic system of Lactococcus lactis . The proteolytic system of lactococci that allows degradation of caseins and proteins of milk is complex. Milk proteins contain all amino acids necessary for growth of lactic acid bacteria. The proteolytic system consists of an extracellularly located proteinase, transport systems for di-tripeptides and oligopeptides and a multitude of intracellular peptidases. Expression of 13 genes was followed by transcriptional fusions in presence of different peptide sources. Transcription of 6 genes is repressed in media containing peptides and that of 4 genes (pepN, pepC, prtP and opp-pepO1 operon) by dipeptides containing one of the 3 branched amino acids (isoleucine, leucine and valine). Repression of gene transcription required that regulatory peptides are translocated into the cell and degraded in amino acids. Cell factors involved in this regulation were identified in derepressed mutants obtained by random mutagenesis by transposition. DtpT, a di-tripeptides transporter and CodY, homologous of the Bacillus subtilis pleiotropic regulator of transcription were the most frequently inactivated proteins. pepC, pepN and opp-pepO1 transcription is not repressed in codY and dtpT mutant. These genes of the proteolytic system belong to a same regulon since their expression is repressed by CodY regulator depending on intracellular concentration of branched amino acids or derivative products of them.Lactococcus lactis possède un système protéolytique complexe pour dégrader les caséines, protéines majoritaires du lait qui contiennent tous les acides aminés nécessaires à sa croissance. Ce système comprend une protéase de paroi extracellulaire, trois systèmes de transport spécifiques des di-tripeptides et des oligopeptides et de nombreuses peptidases localisées à l'intérieur de la cellule. L'influence de la source de peptides sur l'expression de 13 gènes de ce système a été caractérisée grâce à des fusions transcriptionnelles. La transcription de 6 de ces gènes est réprimée en présence d'une source riche en peptides et pour 4 d'entre eux (pepN, pepC, prtP et l'opéron opp-pepO1) par des dipeptides qui contiennent au moins un des trois acides aminés branchés (leucine, isoleucine et valine). Pour permettre la répression de la transcription, les dipeptides doivent être transportés et hydrolysés en acides aminés dans la cellule. Des facteurs cellulaires impliqués dans la régulation de l'expression de ces différents gènes ont été identifiés dans des mutants déréprimés, obtenus par mutagenèse aléatoire par transposition. Il s'agit du transporteur des di- et tripeptides (DtpT) et d'une protéine homologue à la protéine CodY, qui est un régulateur pléiotrope de la transcription de Bacillus subtilis. Nous avons montré que les gènes pepC, pepN et l'opéron opp-pepO1 constituent un régulon dont l'expression est réprimée par CodY en fonction de la concentration intracellulaire en acides aminés branchés ou d'un produit de leur catabolisme

Human Immunodeficiency Virus Type 1 Nef Expression Prevents AP-2-Mediated Internalization of the Major Histocompatibility Complex Class II-Associated Invariant Chain▿ †

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2

Dynamics of HIV-Containing Compartments in Macrophages Reveal Sequestration of Virions and Transient Surface Connections

International audienceDuring HIV pathogenesis, infected macrophages behave as ‘‘viral reservoirs’’ that accumulate and retain virions withindedicated internal Virus-Containing Compartments (VCCs). The nature of VCCs remains ill characterized and controversial.Using wild-type HIV-1 and a replication-competent HIV-1 carrying GFP internal to the Gag precursor, we analyzed thebiogenesis and evolution of VCCs in primary human macrophages. VCCs appear roughly 14 hours after viral proteinsynthesis is detected, initially contain few motile viral particles, and then mature to fill up with virions that become packedand immobile. The amount of intracellular Gag, the proportion of dense VCCs, and the density of viral particles in theirlumen increased with time post-infection. In contrast, the secretion of virions, their infectivity and their transmission to Tcells decreased overtime, suggesting that HIV-infected macrophages tend to pack and retain newly formed virions intodense compartments. A minor proportion of VCCs remains connected to the plasma membrane overtime. Surprisingly, livecell imaging combined with correlative light and electron microscopy revealed that such connections can be transient,highlighting their dynamic nature. Together, our results shed light on the late phases of the HIV-1 cycle and reveal some ofits macrophage specific feature

A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response

International audienceThe biocontrol agent Rhodococcus erythropolis disrupts virulence of plant and human Gram-negative pathogens by catabolizing their N-acyl-homoserine lactones. This quorum-quenching activity requires the expression of the qsd (quorum-sensing signal degradation) operon, which encodes the lactonase QsdA and the fatty acyl-CoA ligase QsdC, involved in the catabolism of lactone ring and acyl chain moieties of signaling molecules, respectively. Here, we demonstrate the regulation of qsd operon expression by a TetR-like family repressor, QsdR. This repression was lifted by adding the pathogen quorum signal or by deleting the qsdR gene, resulting in enhanced lactone degrading activity. Using interactomic approaches and transcriptional fusion strategy, the qsd operon derepression was elucidated: it is operated by the binding of the common part of signaling molecules, the homoserine lactone ring, to the effector-receiving domain of QsdR, preventing a physical binding of QsdR to the qsd promoter region. To our knowledge, this is the first evidence revealing quorum signals as inducers of the suitable quorum-quenching pathway, confirming this TetR-like protein as a lactone sensor. This regulatory mechanism designates the qsd operon as encoding a global disrupting pathway for degrading a wide range of signal substrates, allowing a broad spectrum anti-virulence activity mediated by the rhodococcal biocontrol agent. Understanding the regulation mechanisms of qsd operon expression led also to the development of biosensors useful to monitor in situ the presence of exogenous signals and quorum-quenching activity

Virus-Containing Compartments fill up with time.

<p>(<i>A</i>) Quantification of sparse versus dense VCCs at 3 or 7 dpi. Live macrophages infected with HIV Gag-iGFP ΔEnv were visualized by spinning disk confocal microscopy. Taking into account the compartments with a diameter superior to 1 µm, we considered a VCC as sparse when internal movement was detected; otherwise it was counted as a dense VCC. Values presented are means +/− SD of the percentage of sparse versus dense compartments calculated from 4 donors. Statistical significance was calculated for the differences between day 3 and 7 (<i>p</i> = 0.0003). (<i>B</i>) Quantification of viral density within VCCs at 3 and 7 dpi. Macrophages were infected with HIV NLAD8 and fixed at 3 or 7 dpi. Then, samples were embedded in epon for electron microscopy. For each VCC identified, the size of the VCCs and the number of particles per VCC were quantified. The graph represents the distribution of the viral particle density of the VCCs at both time points. <i>(C)</i> Time-lapse imaging of VCCs. Macrophages infected with HIV Gag-iGFP ΔEnv virus were imaged at 4 dpi during 2 h. Three-dimensional images were acquired every 5 min with a spinning disc microscope. Here are presented 2 snapshots (left panels) from the Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069450#pone.0069450.s010" target="_blank">Video S5</a> and their corresponding 3D reconstructions (right panels). A Gag-iGFP+ compartment is slowly filled. Note that two compartments fuse between 1∶30 and 2∶00 h. Bar 2 µm.</p

Characterization of the Virus-Containing Compartments.

<p>(<i>A</i>) Dynamic imaging of the HIV cycle in macrophages. Macrophages infected with HIV Gag-iGFP virus were imaged from 1 to 8 dpi. To minimize photo-cytotoxicity, images were acquired every 15 min with an epifluorescent Biostation microscope (see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069450#pone.0069450.s006" target="_blank">Video S1</a>). Here are presented 4 snapshots from the movie at the indicated times post infection. Epifluorescent and corresponding transmission images are presented. Bar 20 µm. Bottom diagram. Schematic representation of Gag expression in infected macrophages at four morphologically distinct stages corresponding to the images presented above. (I) Gag is not yet expressed (II) Gag is detected in the cytosol in a diffuse pattern (III) Gag concentrates in internal compartments that accumulate and are in motion, and (IV) macrophages become more motile. (<i>B</i>) Heterogeneity of the VCCs in primary macrophages. Spinning disk confocal micrograph of the central region of a macrophage infected for 5 days with HIV Gag-iGFP ΔEnv. Orthogonal plans (xz and yz) from the red cross are also presented. The arrow points to a compartment that, by time-lapse analysis (see corresponding Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069450#pone.0069450.s007" target="_blank">Video S2</a>), contained few Gag+ structures moving fast in the lumen, while the arrowhead indicates a compartment that appeared full and still. Bar 5 µm. (<i>C</i>) VCCs exhibit heterogeneous density of virions. Ultrathin cryosections of macrophages infected with HIV-1 NLAD8 for 7 days were prepared and labeled for p17 with protein A coupled to gold 10 nm. Two examples of VCCs are shown, one is packed with virions (right), the other (left) only contains a few virions. Bar 500 nm.</p

Virus-Containing Compartments connection to the plasma membrane.

<p>(<i>A</i>) Only a subset of the VCCs is rapidly accessible to Dex-TR. Confocal micrographs of living macrophages infected with HIV Gag-iGFPΔEnv virus for 3 days were imaged immediately after addition of 3 kDa Dex-TR (40 µg/ml). (<i>B</i>) Quantification of the VCCs accessible to Dex-TR at 3 or 7 dpi. Percentages of connected/unconnected VCCs according to dextran labeling were quantified at 3 and 7 dpi. About one-third of the VCCs were connected to the plasma membrane, independently of the time post infection. Histograms are means of experiments performed on 3 donors (more than 200 VCCs were analyzed for each time point).</p

Transient connection of the Virus-Containing Compartment with the plasma membrane.

<p>(A–C) Macrophages were infected for 4 days and imaged as in Fig. 3C. (A) Imaging was first performed after addition of a 10 kD Dex-A546 (t = 0 min, upper panel). A 10 kD Dex-A647 was added 40 min later, and imaging of the same cell was carried out (t = 40 min, lower panel). Several Gag-iGFP compartments are visible and appear accessible to both dextrans (green arrows) but others remain inaccessible to second dextran (red arrows). (<i>B</i>) Confocal imaging from 4 to 5 dpi, was performed immediately after addition of Dex-A546 (t = 0 min, upper panel). A Gag-iGFP+ sparse compartment containing Dex-A546 can be seen. After 11 hours (t = 11 h, lower panel), the same cell was exposed to Dex-A647 and imaged immediately after. The Gag-iGFP+ compartment appeared denser but remained negative for Dex-A647. (<i>C</i>) Correlative light and EM of the very same cell show that the GFP signal observed in light microscopy corresponds to a <i>bona fide</i> dense VCC containing viral particles.</p

Heterogeneity of the Virus-Containing Compartments.

<p>(A–B). Analysis of the VCC by photobleaching experiments. FRAP of Gag-iGFP in the VCC or cytosol of infected macrophages. Live macrophages infected for 4 days with HIV Gag-iGFP ΔEnv were imaged by spinning disk confocal microscopy every sec for 5 sec, then bleached at maximum laser power for 20 msec. Right after photobleaching, images were acquired every 500 ms for 30 sec (panel B, Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069450#pone.0069450.s009" target="_blank">Video S4</a>) or 500 ms for 30 sec and then every 10 sec for 5 min (panel A, Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069450#pone.0069450.s008" target="_blank">Video S3</a>). On the left panels, images from the Movies represent Gag-iGFP, before, right after, and 5 min (A) or 500 ms (B) after photobleaching. On the right panels, average intensity of the regions of FRAP (red circles and lines) and controls (blue and green circles and lines), are represented as a function of time. Gag-iGFP in the cytosol quickly recovers after photobleaching, which is not the case in VCCs. Bar 5 µm. Data are representative of two independent experiments performed with two donors.</p