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1.
Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.
Lorent, C, Pelmenschikov, V, Frielingsdorf, S, Schoknecht, J, Caserta, G, Yoda, Y, Wang, H, Tamasaku, K, Lenz, O, Cramer, SP, et al
Angewandte Chemie (International ed. in English). 2021;(29):15854-15862
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Abstract
To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.
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2.
Detailed numerical study of the peak shapes of neutral analytes injected at high solvent strength in short reversed-phase liquid chromatography columns and comparison with experimental observations.
Pepermans, V, Chapel, S, Heinisch, S, Desmet, G
Journal of chromatography. A. 2021;:462078
Abstract
We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (2D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.
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3.
CompassR Yields Highly Organic-Solvent-Tolerant Enzymes through Recombination of Compatible Substitutions.
Cui, H, Jaeger, KE, Davari, MD, Schwaneberg, U
Chemistry (Weinheim an der Bergstrasse, Germany). 2021;(8):2789-2797
Abstract
The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β-hydrolase fold.
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4.
Revisiting the Self-Assembly of Highly Aromatic Phenylalanine Homopeptides.
Mayans, E, Alemán, C
Molecules (Basel, Switzerland). 2020;(24)
Abstract
Diphenylalanine peptide (FF), which self-assembles into rigid tubular nanostructures, is a very short core recognition motif in Alzheimer's disease β-amyloid (Aβ) polypeptide. Moreover, the ability of the phenylalanine (F or Phe)-homopeptides to self-assemble into ordered nanostructures has been proved. Within this context it was shown that the assembly preferences of this family of compounds is altered by capping both the N- and C-termini using highly aromatic fluorenyl groups (i.e., fluorenyl-9-methoxycarbonyl and 9-fluorenylmethyl ester, named Fmoc and OFm, respectively). In this article the work performed in the field of the effect of the structure and incubation conditions on the morphology and polymorphism of short (from two to four amino acid residues) Phe-homopeptides is reviewed and accompanied by introducing some new results for completing the comparison. Special attention has been paid to the influence of solvent: co-solvent mixture used to solubilize the peptide, the peptide concentration and, in some cases, the temperature. More specifically, uncapped (FF, FFF, and FFFF), N-capped with Fmoc (Fmoc-FF, Fmoc-FFF, and Fmoc-FFFF), C-capped with OFm (FF-OFm), and doubly capped (Fmoc-FF-OFm, Fmoc-FFF-OFm, and Fmoc-FFFF-OFm) Phe-homopeptides have been re-measured. Although many of the experienced assembly conditions have been only revisited as they were previously reported, other experimental conditions have been examined by the first time in this work. In any case, pooling the effect of highly aromatic blocking groups in a single study, using a wide variety of experimental conditions, allows a perspective of how the disappearance of head-to-tail electrostatic interactions and the gradual increase in the amount of π-π stacking interactions, affects the morphology of the assemblies. Future technological applications of Phe-homopeptides can be envisaged by choosing the most appropriate self-assemble structure, defining not only the length of the peptide but also the amount and the position of fluorenyl capping groups.
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5.
Adduct under Field-A Qualitative Approach to Account for Solvent Effect on Hydrogen Bonding.
Shenderovich, IG, Denisov, GS
Molecules (Basel, Switzerland). 2020;(3)
Abstract
The location of a mobile proton in acid-base complexes in aprotic solvents can be predicted using a simplified Adduct under Field (AuF) approach, where solute-solvent effects on the geometry of hydrogen bond are simulated using a fictitious external electric field. The parameters of the field have been estimated using experimental data on acid-base complexes in CDF3/CDClF2. With some limitations, they can be applied to the chemically similar CHCl3 and CH2Cl2. The obtained data indicate that the solute-solvent effects are critically important regardless of the type of complexes. The temperature dependences of the strength and fluctuation rate of the field explain the behavior of experimentally measured parameters.
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6.
Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.
Maenpuen, S, Pongsupasa, V, Pensook, W, Anuwan, P, Kraivisitkul, N, Pinthong, C, Phonbuppha, J, Luanloet, T, Wijma, HJ, Fraaije, MW, et al
Chembiochem : a European journal of chemical biology. 2020;(10):1481-1491
Abstract
We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.
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7.
Environmental and Genetic Factors Influencing Kidney Toxicity.
Lash, LH
Seminars in nephrology. 2019;(2):132-140
Abstract
The kidneys are a frequent target organ for toxicity from exposures to various environmental chemicals and agents. To understand the risk to human health from such exposures, it is important to consider both the underlying chemical and pathologic mechanisms and factors that may modify susceptibility to injury. Choices of exemplary environmental agents to review are based on those with selective effects on the kidneys and for which significant amounts of mechanistic and human data are available. These include the heavy metals cadmium and arsenic, fluoride, the organic solvents trichloroethylene and perchloroethylene, drinking water disinfection by-products haloacids, food and herbal drug contaminants aristolochic acid and melamine, and heat stress. Some common mechanistic features of all these diverse exposures are highlighted, and include oxidative stress and mitochondrial damage. Two major genetic factors that are discussed include genetic polymorphisms in plasma membrane transporters that catalyze uptake and accumulation or efflux and elimination of environmental chemicals, and genetic polymorphisms in bioactivation enzymes that generate toxic and reactive metabolites. Identification of methods to prevent environmental toxicant-associated kidney damage and understanding the genetic factors that influence kidney function and the kidney's response to exposures can be applied to refine risk assessments.
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8.
Chemical treatments for modification and immobilization to improve the solvent-stability of lipase.
Matsumoto, T, Yamada, R, Ogino, H
World journal of microbiology & biotechnology. 2019;(12):193
Abstract
Lipase is a lipolytic enzyme that catalyzes the hydrolysis of lipids and esterification reactions. Lipase has been utilized in industrial uses, food processing, and therapeutic applications as a biocatalyst. However, substrates of lipase are often insoluble in water, and this problem limits its utility. Lipases are also used in organic solvents where the solvent-stability of lipase is an important factor. There is a huge number of approaches that can be undertaken to improve the organic solvent-stability of lipases. For example, screening of solvent-tolerant lipase in nature and direct evolution of lipase using genetic engineering are some of the employed approaches. Here, we focus on approaches based on the chemical treatment of lipases for modification and immobilization. The solvent-stability of lipase was improved by the attachment of other molecules, such as surfactants, polymers, and carbohydrates. The immobilization of the enzyme is been known to be an effective approach for not only recycling the enzyme but also its stabilization. Several reports have demonstrated that the solvent-stability of lipase is also improved by immobilization. In this review, we provide an overview of the approaches used to improve the solvent-stability of lipase.
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9.
Solid dispersion technology as a strategy to improve the bioavailability of poorly soluble drugs.
Cid, AG, Simonazzi, A, Palma, SD, Bermúdez, JM
Therapeutic delivery. 2019;(6):363-382
Abstract
Over the last half-century, solid dispersions (SDs) have been intensively investigated as a strategy to improve drugs solubility and dissolution rate, enhancing oral bioavailability. In this review, an overview of the state of the art of SDs technology is presented, focusing on their classification, the main preparation methods, the limitations associated with their instability, and the marketed products. To fully take advantage of SDs potential, an improvement in their physical stability and the ability to prolong the supersaturation of the drug in gastrointestinal fluids is required, as well as a better scientific understanding of scale-up for defining a robust manufacturing process. Taking these limitations into account will contribute to increase the number of marketed pharmaceutical products based on SD technology.
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10.
Four types of human platelet lysate, including one virally inactivated by solvent-detergent, can be used to propagate Wharton jelly mesenchymal stromal cells.
Chen, MS, Wang, TJ, Lin, HC, Burnouf, T
New biotechnology. 2019;:151-160
Abstract
There is accumulating experimental evidence that human platelet lysate (HPL) made from platelet concentrates can replace fetal bovine serum (FBS) as a xeno-free clinical-grade supplement of growth media to expand mesenchymal stromal cells (MSCs). However, uncertainties exist in regard to impacts that various manufacturing methods of HPL can exert on the expansion and differentiation capacity of MSCs. In particular, there is a need to evaluate the possibility of implementing virus-inactivation treatment during HPL production to ensure optimal safety of industrial HPL pools. Expired human platelet concentrates from four different donors were pooled and subjected to freeze-thaw cycles (-80/+37 °C), followed or not by serum-conversion by calcium chloride, heat-treatment at 56 °C for 30 min, or solvent-detergent (S/D) virus inactivation. The concentrations of total proteins, growth factors and fibrinogen, and the chemical compositions of the HPLs were characterized. The impact of HPL supplementation on the cell morphology, doubling time, immunophenotype and trilineage differentiation capacity of Wharton jelly MSCs (WJMSCs) were compared over five passages, using FBS as a control and normalizing the protein content. Data showed that WJMSCs expanded equally well, exhibited a typical fibroblast morphology, had short doubling times, maintained their immunophenotypes, and differentiated into chondrocyte, osteocyte, and adipocyte lineages in all HPL-supplemented media, all of which were more effective than FBS. In conclusion, we found minimal detectable impact of the HPL manufacturing process, including S/D virus inactivation, on the suitability of expanding WJMSCs in vitro.