Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions

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 < 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

CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain

International audienceThe multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presentingits chemokine domain (CD) to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 incirculating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior

Evaluation of aminopyrrolidine amide to improve chloride transport in CFTR-defective cells

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Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A

International audienceFerroportin 1 (FPN1) is a major facilitator superfamily transporter that is essential for proper maintenance of human iron homeostasis at the systemic and cellular level. FPN1 dysfunction leads to the progressive accumulation of iron in reticuloendothelial cells, causing hemochromatosis type 4A (or ferroportin disease), an autosomal dominant disorder that displays large phenotypic heterogeneity. Although crystal structures have unveiled the outward- and inward-facing conformations of the bacterial homolog Bdellovibrio bacteriovorus Fpn (or Bd2019) and calcium has recently been identified as an essential cofactor, our molecular understanding of the iron transport mechanism remains incomplete. Here, we used a combination of molecular modeling, molecular dynamics simulations, and Ala site-directed mutagenesis, followed by complementary in vitro functional analyses, to explore the structural architecture of the human FPN1 intracellular gate. We reveal an interdomain network that involves 5 key amino acids and is likely very important for stability of the iron exporter facing the extracellular milieu. We also identify inter- and intradomain interactions that rely on the 2 Asp84 and Asn174 critical residues and do not exist in the bacterial homolog. These interactions are thought to play an important role in the modulation of conformational changes during the transport cycle. We interpret these results in the context of hemochromatosis type 4A, reinforcing the idea that different categories of loss-of-function mutations exist. Our findings provide an unprecedented view of the human FPN1 outward-facing structure and the particular function of the so-called "gating residues" in the mechanism of iron export.-Guellec, J., Elbahnsi, A., Le Tertre, M., Uguen, K., Gourlaouen, I., Férec, C., Ka, C., Callebaut, I., Le Gac, G. Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A

The Spectra of Disease-Causing Mutations in the Ferroportin 1 (SLC40A1) Encoding Gene and Related Iron Overload Phenotypes (Hemochromatosis Type 4 and Ferroportin Disease)

International audienceSLC40A1 is the sole iron export protein reported in mammals and is a key player in both cellular and systemic iron homeostasis. This unique iron exporter, which belongs to the major facilitator superfamily, is predominantly regulated by the hyposideremic hormone hepcidin. SLC40A1 dysfunction causes ferroportin disease, and autosomal dominant iron overload disorder characterized by cellular iron retention, principally in reticuloendothelial cells, correlating with high serum ferritin and low to normal transferrin saturation. Resistant to hepcidin, SLC40A1 mutations are rather associated with elevated plasma iron and parenchymal iron deposition, a condition that resembles HFE-related hemochromatosis and is associated with more clinical complications. With very few exceptions, only missense variations are reported at the SLC40A1 locus; this situation increasingly limits the establishment of pathogenicity. In this mutation update, we provide a comprehensive review of all the pathogenic or likely pathogenic variants, variants of unknown significance, and benign or likely benign SLC40A1 variants. The classification is essentially determined using functional, structural, segregation, and recurrence data. We furnish new information on genotype-phenotype correlations for loss-of-function, gain-of-function, and other SLC40A1 variants, confirming the existence of wide clinical heterogeneity and the potential for misdiagnosis. All information is recorded in a locus-specific online database

Extracorporeal membrane oxygenation for immunocompromised children with acute respiratory distress syndrome: a French referral center cohort

International audienceBackground: Immunocompromised children are likely to develop a refractory acute respiratory distress syndrome (ARDS). The usefulness of providing extracorporeal life support (ECLS) to these patients is a subject of debate. The aim of our study was to report the outcomes and to compare factors associated with mortality between immunocompromised and non-immunocompromised children supported with veno-venous ECMO. Methods: We performed a retrospective monocentric study in the French pediatric ECMO center of Armand Trousseau Hospital, including all pediatric patients aged from 1 month to 18 years requiring ECLS for ARDS. Results: Between 2007 and 2018, one hundred and eleven (111) patients underwent ECMO for respiratory failure; among them twenty-five (25) were immunocompromised. Survival rate at 6 months after intensive care discharge was significantly lower for immunocompromised patients compared to non-immunocompromised ones (41.7% vs. 62.8%; p = 0.04). ARDS severity was similar between the 2 groups. Fungal pneumonias were reported only in immunocompromised patients (12.5% versus 0% in the control group; p = 0.001). Bleeding complications were significantly more frequent in the immunocompromised group and blood product transfusions were also more frequently required in this group. Conclusion: Six months after intensive care discharge, survival rate of immunocompromised children supported with ECMO for pediatric ARDS is lower than for nonimmunocompromised patients. But, the expectation for a favorable outcome is real and it is worth it if their condition is likely to be compatible with a good long-term quality of life

Feasibility of open abdomen surgery treatment for near fatal necrotizing enterocolitis in preterm infants

International audienceIntroduction: Necrotizing Enterocolitis (NEC) remained a dramatic complication leading to death or neonatal morbidities in preterms. For some, Intra-Abdominal Hypertension (IAH) and Abdominal Compartment Syndrome worsened the multi-organ failure. An open abdomen surgery could be an alternative to conventional surgical treatment to move beyond this stage.Objectives: To retrospectively describe the clinical course, pre- and post-operative features of preterms suffering from severe NEC with IAH treated by open abdomen surgery and referred to our center from October 2007 to September 2019. Our secondary objective is to identify various risk factors for mortality in this population.Methods: Data on neonatal, clinical, biological, pre and post-operative features and outcome were collected. Univariate analyses were performed to compare their pre and post-operative features stratifying on outcome.Results: Among 29 included patients, 14 (48%) survived to discharge without short bowel syndrome. Death was associated with an earlier postnatal age at NEC (16.3 ± 9.1 versus 31.3 ± 25.9 days; p = 0.004) and followed a withdrawal of treatment in 60% of cases. Surgery was associated with a significant improvement of respiratory and hemodynamic features (decrease of mean ventilator pressure from 13.1 ± 5.4 to 11.3 ± 4.0 cmH2O, p < 0.001), oxygen requirement (mean FiO2 decreased from 65.0% ± 31.2 to 49.0% ± 24.6, p < 0.001) and inotropic score (from 38.6 ± 70.1 to 29.9 ± 64.3, p < 0.001). In the survival group, pre and post-operative findings exhibited a significant increase of serum lactate concentrations from 2.7 ± 1.6 to 11.0 ± 20.3 mmol/L (p = 0.02) but a similar pH.Conclusion: Open abdomen surgery could be considered to rescue preterms with near fatal NEC. IAH and Abdominal Compartment Syndrome in these preterms should be investigated through further studies

The Inhibition of the Membrane-Bound Transcription Factor Site-1 Protease (MBTP1) Alleviates the p.Phe508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Defects in Cystic Fibrosis Cells

International audienceCystic Fibrosis (CF) is present due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the most frequent variant being p.phe508del. The CFTR protein is a chloride (Cl-) channel which is defective and almost absent of cell membranes when the p.Phe508del mutation is present. The p.Phe508del-CFTR protein is retained in the endoplasmic reticulum (ER) and together with inflammation and infection triggers the Unfolded Protein Response (UPR). During the UPR, the Activating Transcription Factor 6 (ATF6) is activated with cleavage and then decreases the expression of p.Phe508del-CFTR. We have previously shown that the inhibition of the activation of ATF6 alleviates the p.Phe508del-CFTR defects in cells overexpressing the mutated protein. In the present paper, our aim was to inhibit the cleavage of ATF6, and thus its activation in a human bronchial cell line with endogenous p.Phe508del-CFTR expression and in bronchial cells from patients, to be more relevant to CF. This was achieved by inhibiting the protease MBTP1 which is responsible for the cleavage of ATF6. We show here that this inhibition leads to increased mRNA and p.Phe508del-CFTR expression and, consequently, to increased Cl-efflux. We also explain the mechanisms linked to these increases with the modulation of genes when MBTP1 is inhibited. Indeed, RT-qPCR assays show that genes such as HSPA1B, CEBPB, VIMP, PFND2, MAPK8, XBP1, INSIG1, and CALR are modulated. In conclusion, we show that the inhibition of MBTP1 has a beneficial effect in relevant models to CF and that this is due to the modulation of genes involved in the disease

Glomerular Hyper- and Hypofiltration During Acute Circulatory Failure

International audienceObjective: To assess glomerular filtration rate in the early phase of acute circulatory failure by measuring iohexol plasma clearance.Design: Interventional prospective multicentric study.Setting: Three French ICUs in tertiary teaching hospitals.Patients: Patients with acute circulatory failure within 12 hours after ICU admission.Interventions: IV administration of a nontoxic 5-mL dose of iohexol. Collection of nine arterial blood samples over 24 hours for iohexol plasma concentration measurements. Iohexol clearance calculation with a population pharmacokinetic model. Iohexol clearance was an estimation of the mean glomerular filtration rate over 24 hours.Measurements and Main Results: Among 99 included patients, we could calculate iohexol clearance for 85. The median iohexol clearance was 31 mL/min (interquartile range, 16–44). According to iohexol clearance, 41 patients (48%) had severe hypofiltration (clearance, 130 mL/min). Urinary creatinine clearance underestimated renal impairment in one patient out of two; the bias of iohexol clearance toward 24-hour urinary creatinine clearance over the same period was –18.1 mL/min (limits of agreement, –73.5 to 37.4).Conclusions: We demonstrated the feasibility of iohexol clearance measurement in unstable critically ill patients. Normal kidney function is exceptional during the early phase of acute circulatory failure. Glomerular filtration rate estimation by urinary creatinine clearance frequently fails to detect renal impairment. Hyperfiltration is very infrequent