Membrane insertion of soluble CLIC1 into active chloride channels is triggered by specific divalent cations

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+ and Zn2+ trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+ leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+ mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+ and Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENT CLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+ and Zn2+ binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition

The formation of vortex loops (strings) in continuous phase transitions

The formation of vortex loops (global cosmic strings) in an O(2) linear sigma model in three spatial dimensions is analyzed numerically. For over-damped Langevin dynamics we find that defect production is suppressed by an interaction between correlated domains that reduces the effective spatial variation of the phase of the order field. The degree of suppression is sensitive to the quench rate. A detailed description of the numerical methods used to analyze the model is also reported.Comment: LaTeX, 17 pages, 6 eps figures 2 references and a footnote adde

A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels

The chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl− efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl− efflux in a pH-dependent manner. Together, our results identify a two- step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl− efflux

Essential Emergency and Critical Care: a consensus among global clinical experts.

BACKGROUND: Globally, critical illness results in millions of deaths every year. Although many of these deaths are potentially preventable, the basic, life-saving care of critically ill patients are often overlooked in health systems. Essential Emergency and Critical Care (EECC) has been devised as the care that should be provided to all critically ill patients in all hospitals in the world. EECC includes the effective care of low cost and low complexity for the identification and treatment of critically ill patients across all medical specialties. This study aimed to specify the content of EECC and additionally, given the surge of critical illness in the ongoing pandemic, the essential diagnosis-specific care for critically ill patients with COVID-19. METHODS: In a Delphi process, consensus (>90% agreement) was sought from a diverse panel of global clinical experts. The panel iteratively rated proposed treatments and actions based on previous guidelines and the WHO/ICRC's Basic Emergency Care. The output from the Delphi was adapted iteratively with specialist reviewers into a coherent and feasible package of clinical processes plus a list of hospital readiness requirements. RESULTS: The 269 experts in the Delphi panel had clinical experience in different acute medical specialties from 59 countries and from all resource settings. The agreed EECC package contains 40 clinical processes and 67 requirements, plus additions specific for COVID-19. CONCLUSION: The study has specified the content of care that should be provided to all critically ill patients. Implementing EECC could be an effective strategy for policy makers to reduce preventable deaths worldwide

Mutually Beneficial Combination of Molecular Dynamics Computer Simulations and Scattering Experiments

We showcase the combination of experimental neutron scattering data and molecular dynamics (MD) simulations for exemplary phospholipid membrane systems. Neutron and X-ray reflectometry and small-angle scattering measurements are determined by the scattering length density profile in real space, but it is not usually possible to retrieve this profile unambiguously from the data alone. MD simulations predict these density profiles, but they require experimental control. Both issues can be addressed simultaneously by cross-validating scattering data and MD results. The strengths and weaknesses of each technique are discussed in detail with the aim of optimizing the opportunities provided by this combination

Thyroid Hormone Therapy for Older Adults with Subclinical Hypothyroidism.

BACKGROUND: The use of levothyroxine to treat subclinical hypothyroidism is controversial. We aimed to determine whether levothyroxine provided clinical benefits in older persons with this condition. METHODS: We conducted a double-blind, randomized, placebo-controlled, parallel-group trial involving 737 adults who were at least 65 years of age and who had persisting subclinical hypothyroidism (thyrotropin level, 4.60 to 19.99 mIU per liter; free thyroxine level within the reference range). A total of 368 patients were assigned to receive levothyroxine (at a starting dose of 50 μg daily, or 25 μg if the body weight was <50 kg or the patient had coronary heart disease), with dose adjustment according to the thyrotropin level; 369 patients were assigned to receive placebo with mock dose adjustment. The two primary outcomes were the change in the Hypothyroid Symptoms score and Tiredness score on a thyroid-related quality-of-life questionnaire at 1 year (range of each scale is 0 to 100, with higher scores indicating more symptoms or tiredness, respectively; minimum clinically important difference, 9 points). RESULTS: The mean age of the patients was 74.4 years, and 396 patients (53.7%) were women. The mean (±SD) thyrotropin level was 6.40±2.01 mIU per liter at baseline; at 1 year, this level had decreased to 5.48 mIU per liter in the placebo group, as compared with 3.63 mIU per liter in the levothyroxine group (P<0.001), at a median dose of 50 μg. We found no differences in the mean change at 1 year in the Hypothyroid Symptoms score (0.2±15.3 in the placebo group and 0.2±14.4 in the levothyroxine group; between-group difference, 0.0; 95% confidence interval [CI], -2.0 to 2.1) or the Tiredness score (3.2±17.7 and 3.8±18.4, respectively; between-group difference, 0.4; 95% CI, -2.1 to 2.9). No beneficial effects of levothyroxine were seen on secondary-outcome measures. There was no significant excess of serious adverse events prespecified as being of special interest. CONCLUSIONS: Levothyroxine provided no apparent benefits in older persons with subclinical hypothyroidism. (Funded by European Union FP7 and others; TRUST ClinicalTrials.gov number, NCT01660126 .)

A quantitative assay to study the lipid selectivity of membrane-associated systems using solution NMR

The activity of membrane proteins and compounds that interact with the membrane is modulated by the surrounding lipid composition. However, there are no simple methods that determine the composition of these annular phospholipids in eukaryotic systems. Herein, we describe a simple methodology that enables the identification and quantification of the lipid composition around membrane-associated compounds using SMA-nanodiscs and routine $^1H-^{31}P NMR$

Alternative pathway dysregulation in tissues drives sustained complement activation and predicts outcome across the disease course in COVID-19

Complement, a critical defence against pathogens, has been implicated as a driver of pathology in COVID-19. Complement activation products are detected in plasma and tissues and complement blockade considered for therapy. To delineate roles of complement in immunopathogenesis, we undertook the largest comprehensive study of complement in an COVID-19 to date, a comprehensive profiling of 16 complement biomarkers, including key components, regulators and activation products, in 966 plasma samples from 682 hospitalised COVID-19 patients collected across the hospitalisation period as part of the UK ISARIC4C study. Unsupervised clustering of complement biomarkers mapped to disease severity and supervised machine learning identified marker sets in early samples that predicted peak severity. Compared to heathy controls, complement proteins and activation products (Ba, iC3b, terminal complement complex) were significantly altered in COVID-19 admission samples in all severity groups. Elevated alternative pathway activation markers (Ba and iC3b) and decreased alternative pathway regulator (properdin) in admission samples associated with more severe disease and risk of death. Levels of most complement biomarkers were reduced in severe disease, consistent with consumption and tissue deposition. Latent class mixed modelling and cumulative incidence analysis identified the trajectory of increase of Ba to be a strong predictor of peak COVID-19 disease severity and death. The data demonstrate that early-onset, uncontrolled activation of complement, driven by sustained and progressive amplification through the alternative pathway amplification loop is a ubiquitous feature of COVID-19, further exacerbated in severe disease. These findings provide novel insights into COVID-19 immunopathogenesis and inform strategies for therapeutic intervention

Risk of adverse outcomes in patients with underlying respiratory conditions admitted to hospital with COVID-19: a national, multicentre prospective cohort study using the ISARIC WHO Clinical Characterisation Protocol UK

Background: Studies of patients admitted to hospital with COVID-19 have found varying mortality outcomes associated with underlying respiratory conditions and inhaled corticosteroid use. Using data from a national, multicentre, prospective cohort, we aimed to characterise people with COVID-19 admitted to hospital with underlying respiratory disease, assess the level of care received, measure in-hospital mortality, and examine the effect of inhaled corticosteroid use. Methods: We analysed data from the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study. All patients admitted to hospital with COVID-19 across England, Scotland, and Wales between Jan 17 and Aug 3, 2020, were eligible for inclusion in this analysis. Patients with asthma, chronic pulmonary disease, or both, were identified and stratified by age (<16 years, 16–49 years, and ≥50 years). In-hospital mortality was measured by use of multilevel Cox proportional hazards, adjusting for demographics, comorbidities, and medications (inhaled corticosteroids, short-acting β-agonists [SABAs], and long-acting β-agonists [LABAs]). Patients with asthma who were taking an inhaled corticosteroid plus LABA plus another maintenance asthma medication were considered to have severe asthma. Findings: 75 463 patients from 258 participating health-care facilities were included in this analysis: 860 patients younger than 16 years (74 [8·6%] with asthma), 8950 patients aged 16–49 years (1867 [20·9%] with asthma), and 65 653 patients aged 50 years and older (5918 [9·0%] with asthma, 10 266 [15·6%] with chronic pulmonary disease, and 2071 [3·2%] with both asthma and chronic pulmonary disease). Patients with asthma were significantly more likely than those without asthma to receive critical care (patients aged 16–49 years: adjusted odds ratio [OR] 1·20 [95% CI 1·05–1·37]; p=0·0080; patients aged ≥50 years: adjusted OR 1·17 [1·08–1·27]; p<0·0001), and patients aged 50 years and older with chronic pulmonary disease (with or without asthma) were significantly less likely than those without a respiratory condition to receive critical care (adjusted OR 0·66 [0·60–0·72] for those without asthma and 0·74 [0·62–0·87] for those with asthma; p<0·0001 for both). In patients aged 16–49 years, only those with severe asthma had a significant increase in mortality compared to those with no asthma (adjusted hazard ratio [HR] 1·17 [95% CI 0·73–1·86] for those on no asthma therapy, 0·99 [0·61–1·58] for those on SABAs only, 0·94 [0·62–1·43] for those on inhaled corticosteroids only, 1·02 [0·67–1·54] for those on inhaled corticosteroids plus LABAs, and 1·96 [1·25–3·08] for those with severe asthma). Among patients aged 50 years and older, those with chronic pulmonary disease had a significantly increased mortality risk, regardless of inhaled corticosteroid use, compared to patients without an underlying respiratory condition (adjusted HR 1·16 [95% CI 1·12–1·22] for those not on inhaled corticosteroids, and 1·10 [1·04–1·16] for those on inhaled corticosteroids; p<0·0001). Patients aged 50 years and older with severe asthma also had an increased mortality risk compared to those not on asthma therapy (adjusted HR 1·24 [95% CI 1·04–1·49]). In patients aged 50 years and older, inhaled corticosteroid use within 2 weeks of hospital admission was associated with decreased mortality in those with asthma, compared to those without an underlying respiratory condition (adjusted HR 0·86 [95% CI 0·80−0·92]). Interpretation: Underlying respiratory conditions are common in patients admitted to hospital with COVID-19. Regardless of the severity of symptoms at admission and comorbidities, patients with asthma were more likely, and those with chronic pulmonary disease less likely, to receive critical care than patients without an underlying respiratory condition. In patients aged 16 years and older, severe asthma was associated with increased mortality compared to non-severe asthma. In patients aged 50 years and older, inhaled corticosteroid use in those with asthma was associated with lower mortality than in patients without an underlying respiratory condition; patients with chronic pulmonary disease had significantly increased mortality compared to those with no underlying respiratory condition, regardless of inhaled corticosteroid use. Our results suggest that the use of inhaled corticosteroids, within 2 weeks of admission, improves survival for patients aged 50 years and older with asthma, but not for those with chronic pulmonary disease. Funding: National Institute for Health Research, Medical Research Council, NIHR Health Protection Research Units in Emerging and Zoonotic Infections at the University of Liverpool and in Respiratory Infections at Imperial College London in partnership with Public Health England