Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Structural Characterization of the Complex between Hen Egg-White Lysozyme and Zr(IV) -Substituted Keggin Polyoxometalate as Artificial Protease

Successful co-crystallization of a noncovalent complex between hen egg-white lysozyme (HEWL) and the monomeric Zr(IV) -substituted Keggin polyoxometalate (POM) (Zr1 K1), (Et2 NH2 )3 [Zr(PW11 O39 )] (1), has been achieved, and its single-crystal X-ray structure has been determined. The dimeric Zr(IV) -substituted Keggin-type polyoxometalate (Zr1 K2), (Et2 NH2 )10 [Zr(PW11 O39 )2 ] (2), has been previously shown to exhibit remarkable selectivity towards HEWL hydrolysis. The reported X-ray structure shows that the hydrolytically active Zr(IV) -substituted Keggin POM exists as a monomeric species. Prior to hydrolysis, this monomer interacts with HEWL in the vicinity of the previously identified cleavage sites found at Trp28-Val29 and Asn44-Arg45, through water-mediated H-bonding and electrostatic interactions. Three binding sites are observed at the interface of the negatively charged Keggin POM and the positively charged regions of HEWL at: 1) Gly16, Tyr20, and Arg21; 2) Asn44, Arg45, and Asn46; and 3) Arg128.status: publishe

Structural characterization of the complex between hen egg-white lysozyme and ZrIV^{IV} -Substituted Keggin polyoxometalate as artificial protease

International audienceSuccessful co-crystallization of a noncovalent complex between hen egg-white lysozyme (HEWL) and the monomeric Zr$^{IV}$-substituted Keggin polyoxometalate(POM) (Zr1K1), (Et$_2$NH$_2$)$_3$ [Zr(PW$_{11}$O$_{39}$)] (1), has been achieved, and its single-crystal X-ray structure has been determined. The dimeric Zr$^{IV}$-substituted Keggin-type polyoxometalate (Zr1K2), (Et$_2$NH$_2$)$_{10}$[Zr(PW$_{11}$O$_{39}$)$_2$] (2), has been previously shown to exhibit remarkable selectivity towards HEWL hydrolysis. The reported X-ray structure shows that the hydrolytically active Zr$^{IV}$-substituted Keggin POM exists as a monomeric species. Prior to hydrolysis, this monomer interacts with HEWL in the vicinity of the previously identified cleavage sites found at Trp28-Val29 and Asn44-Arg45, through water-mediated H-bonding and electrostatic interactions. Three binding sites are observed at the interface of the negatively charged Keggin POM and the positively charged regions of HEWL at: 1) Gly16, Tyr20, and Arg21; 2) Asn44, Arg45, and Asn46; and 3) Arg128

CRYSTAL STRUCTURES OF RSEGFP AND RSGREEN0.7 REVEAL PHOTOSWITCHING IN EGFP-DERIVED FLUORESCENT PROTEINS

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Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies

International audienceAbstract Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography

How does Bacillus thuringiensisBacillus\ thuringiensis crystallize such a large diversity of toxins?

International audienceBacillus thuringiensis (Bt) is a natural crystal-making bacterium. Bt diversified into many subspecies that have evolved to produce crystals of hundreds of pesticidal proteins with radically different structures. Their crystalline form ensures stability and controlled release of these major virulence factors. They are responsible for the toxicity and host specificity of Bt, explaining its worldwide use as a biological insecticide. Most research has been devoted to understanding the mechanisms of toxicity of these toxins while the features driving their crystallization have long remained elusive, essentially due to technical limitations. The evolution of methods in structural biology, pushing back the limits in size of amenable protein crystals now allows access to be gained to structural information hidden within natural crystals of such toxins. In this review, we present the main parameters that have been identified as key drivers of toxin crystallization in Bt, notably in the light of recent discoveries driven by structural biology studies. Then, we develop how the future evolution of structural biology will hopefully unveil new mechanisms of Bt toxin crystallization, opening the door to their hijacking with the aim of developing a versatile in vivo crystallization platform of high academic and industrial interest

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

International audienceThe development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different scientific fields, including toxinology and structural biology, as well as from industry, notably for biotechnological and medical applications. As a natural crystal-making bacterium, Bacillus thuringiensis (Bt) has evolved through millions of years to produce hundreds of highly structurally diverse pesticidal proteins as micrometer-sized crystals. The long-term stability of Bt protein crystals in aqueous environments and their specific and controlled dissolution are characteristics that are particularly sought after. In this article, we explore whether the crystallization machinery of Bt can be hijacked as a means to produce (micro)crystalline formulations of proteins for three different applications: (i) to develop new bioinsecticidal formulations based on rationally improved crystalline toxins, (ii) to functionalize crystals with specific characteristics for biotechnological and medical applications, and (iii) to produce microcrystals of custom proteins for structural biology. By developing the needs of these different fields to figure out if and how Bt could meet each specific requirement, we discuss the already published and/or patented attempts and provide guidelines for future investigations in some underexplored yet promising domains

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies

β-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins.status: publishe

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies

International audienceβ-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins

Structure–function dataset reveals environment effects within a fluorescent protein model system

A previous version of this manuscript has been deposited on a preprint server (https://doi.org/10.1101/2020.09.25.313528)International audienceAnisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined thisusing fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering method