Block and gradient copoly(2-oxazoline) micelles : strikingly different on the inside

Herein, we provide a direct proof for differences in the micellar structure of amphiphilic diblock and gradient copolymers, thereby unambiguously demonstrating the influence of monomer distribution along the polymer chains on the micellization behavior. The internal structure of amphiphilic block and gradient co poly(2-oxazolines) based on the hydrophilic poly(2-methyl-2-oxazoline) (PMeOx) and the hydrophobic poly(2-phenyl-2-oxazoline) (PPhOx) was studied in water and water ethanol mixtures by small-angle X-ray scattering (SAXS), small angle neutron scattering (SANS), static and dynamic light scattering (SLS/DLS), and H-1 NMR spectroscopy. Contrast matching SANS experiments revealed that block copolymers form micelles with a uniform density profile of the core. In contrast to popular assumption, the outer part of the core of the gradient copolymer micelles has a distinctly higher density than the middle of the core. We attribute the latter finding to back-folding of chains resulting from hydrophilic hydrophobic interactions, leading to a new type of micelles that we refer to as micelles with a "bitterball-core" structure

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

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

Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability

Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-beta-1,4-xylanase from Bacillus subtilis under acidic conditions. Since the pH dependence of protein stability is governed by the ionisation states of the side chains of its titrable amino acid residues, we explored the strategy of changing pH-stability profiles by altering pK(a) values of key residues through in silico designed mutations. To this end, computational predictions and molecular modelling were carried out using the recently developed pKD software package. Four endoxylanase variants, in which the pK(a) values of either Asp4 and Asp11 or His149 were targeted to shift downwards through incorporation of three to five point mutations, were generated and recombinantly expressed in the cytoplasm of Escherichia coli. All four mutants showed considerably increased functional stability at acid pH levels. They retained similar to 30-70% and similar to 75-95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only similar to 23% and similar to 57%, respectively, for the wild-type enzyme under the experimental conditions. No acidophilic adaptation of the catalytic activity had occurred. In addition, their functional stability and catalytic activity profiles under different temperature and ionic strength conditions were significantly altered. These findings contribute to general understanding of the molecular mechanisms governing the pH-dependent stability of GH11 proteins, and hence they can be applied to enhance the stability and effectiveness of many GH11 endoxylanases used in industry today.status: publishe

Phage display based identification of novel stabilizing mutations in glycosyl hydrolase family 11 B. subtilis endoxylanase XynA

Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation temperature by 2-3 degrees C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges. (C) 2008 Elsevier Inc. All rights reserved.status: publishe

Fusarium graminearum xylanases show different functional stabilities, substrate specificities and inhibition sensitivities

When grown on arabinoxylan as the sole carbon Source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XyIA and XyIB are glycoside hydrolase family (GH) I I xylanases, while XyIC and XyID) belong to GH 10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 degrees C, respectively. interestingly, XyIC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XyIC and XyID hydrolysed xylotriose. The two GH 10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XyIC Substrate while XyIA and XyIB prefer sparsely substituted oat spelt xylan. XyIC and XyID were inhibited by xylanase inhibiting protein (XIP), while XyIA and XyIB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XyIC is a valuable candidate for use in biotechnological applications. (C) 2008 Elsevier Inc. All rights reserved.status: publishe

Truncated derivatives of a multidomain thermophilic GH10 xylanase from Thermotoga maritima reveal structure related activity profiles and substrate hydrolysis patterns

Efficient heteroxylan degradation in the context of economically feasible lignocellulosic biomass biorefining requires xylanolytic enzymes with optimal thermostability and specificity. Therefore, the structure activity relationship of a modular thermophilic glycoside hydrolase family 10 xylanase (xylanase A from Thermotoga maritima MSB8, rXTMA) was investigated through construction of six truncated derivatives, lacking at least one of the 2 N- and/or 2 C-terminal modules. The temperatures for optimal activity and stability of the xylanases were strongly influenced by the presence of the different modules and ranged from 60 to 80 °C and 50 to 80 °C, respectively. In contrast, the pH for optimal activity was only slightly affected (pH 6.0 to 7.0). The tested xylanases retained over 80% activity after 2 h pre-incubation at 50 °C between pH 5.0 and 11.0. Most unexpectedly, changes in the modular structure led to a 26-fold wide range of specific activities of the enzymes towards xylohexaose, while the activity towards insoluble polymeric heteroxylan was comparable for all but one xylanase. rXTMAΔC, lacking the C-terminal modules, had a 60% higher specific activity towards the latter substrate than the wild type enzyme. These results show that key properties of XTMA can be tuned to allow for optimal performance of the enzyme in biotechnological processes such as in the bioconversion of lignocellulosic biomass.status: publishe

Alteration of Bacillus subtilis XynA endoxylanase substrate selectivity by site-directed mutagenesis

Bacillus subtilis endoxylanase XynA was engineered by site-directed mutagenesis to alter its substrate selectivity, i.e. the ratio of its capacity to solubilise water-unextractable arabinoxylans (WU-AX) to its capacity to hydrolyse water-extractable AX (AE-AX). Four surface exposed aromatic residues were targeted for mutation to alanine, either individually (Y113 and W185) orjointly (F48, Y94, Y113 and W185), based on the general role of aromatic residues in the binding of endoxylanases to insoluble substrates and the hypothesis that they affect the activity of endoxylanases towards WU-AX. Determination of the substrate selectivity factors and WU-AX degradation profile of the wild type and mutant enzymes using an elaborate procedure requiring incubation of native wheat flour WU-AX and WE-AX with different enzyme levels showed that mutants W185A and 4mutA, the mutant containing all four mutations, displayed lower selectivity towards WU-AX than the wild type recombinant endoxylanase. Each of the endoxylanases had similar binding activity to wheat flour WU-AX and insoluble oat spelt xylan. (c) 2007 Elsevier Inc. All rights reserved.status: publishe