Electrostatics and the Assembly of an RNA Virus

Electrostatic interactions play a central role in the assembly of single-stranded RNA viruses. Under physiological conditions of salinity and acidity, virus capsid assembly requires the presence of genomic material that is oppositely charged to the core proteins. In this paper we apply basic polymer physics and statistical mechanics methods to the self-assembly of a synthetic virus encapsidating generic polyelectrolyte molecules. We find that (i) the mean concentration of the encapsidated polyelectrolyte material depends on the surface charge density, the radius of the capsid, and the linear charge density of the polymer but neither on the salt concentration or the Kuhn length, (ii) the total charge of the capsid interior is equal but opposite to that of the empty capsid, a form of charge reversal. Unlike natural viruses, synthetic viruses are predicted not to be under an osmotic swelling pressure. The design condition that self-assembly only produces filled capsids is shown to coincide with the condition that the capsid surface charge exceeds the desorption threshold of polymer surface adsorption. We compare our results with studies on the self-assembly of both synthetic and natural viruses.Comment: 41 pages, 4 figure

Enzymatic surface hydrolysis of PET : effect of structural diversity on kinetic properties of cutinases from thermobifida

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Financial support was also given from Sachsisches Staatsministerium fur Umwelt und Landwirtschaft, Germany. PET was kindly provided by Dr. Vincent Nierstrasz from Ghent University

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Natural deep eutectic solvents as multifuntional media for the valorization of agricultural wastes

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.Microbial Biotechnolog

Natural Deep Eutectic Solvents as Multifunctional Media for the Valorization of Agricultural Wastes

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H 2 O 2 to promote the epoxidation reaction. The proof-of-concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined. BT/Biocatalysi

Surface engineering of polyester-degrading enzymes to improve efficiency and tune specificity

Certain members of the carboxylesterase superfamily can act at the interface between water and water-insoluble substrates. However, nonnatural bulky polyesters usually are not efficiently hydrolyzed. In the recent years, the potential of enzyme engineering to improve hydrolysis of synthetic polyesters has been demonstrated. Regions on the enzyme surface have been modified by using site-directed mutagenesis in order to tune sorption processes through increased hydrophobicity of the enzyme surface. Such modifications can involve specific amino acid substitutions, addition of binding modules, or truncation of entire domains improving sorption properties and/or dynamics of the enzyme. In this review, we provide a comprehensive overview on different strategies developed in the recent years for enzyme surface engineering to improve the activity of polyester-hydrolyzing enzymes

Natural deep eutectic solvents as performance additives for peroxygenase catalysis

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase‐catalysed oxyfunctionalization reactions. Choline chloride‐based NADES are of particular interest as they can serve as solvent, enzyme‐stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof‐of‐concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.Microbial Biotechnolog

Natural Deep Eutectic Solvents as Performance Additives for Peroxygenase Catalysis

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase-catalysed oxyfunctionalization reactions. Choline chloride-based NADES are of particular interest as they can serve as solvent, enzyme-stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof-of-concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.BT/Biocatalysi