A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

Lactones in the Synthesis of Prostaglandins and Prostaglandin Analogs

In the total stereo-controlled synthesis of natural prostaglandins (PGs) and their structural analogs, a vast class of compounds and drugs, known as the lactones, are encountered in a few key steps to build the final molecule, as: δ-lactones, γ-lactones, and 1,9-, 1,11-, and 1,15-macrolactones. After the synthesis of 1,9-PGF_2α and 1,15-PGF_2α lactones, many 1,15-lactones of E₂, E₃, F₂, F₃, A₂, and A₃ were found in the marine mollusc Tethys fimbria and the quest for understanding their biological role stimulated the research on their synthesis. Then 1,9-, 1,11-, and 1,15-PG lactones of the drugs were synthesized as an alternative to the corresponding esters, and the first part of the paper describes the methods used for their synthesis. The efficient Corey procedure for the synthesis of prostaglandins uses the key δ-lactone and γ-lactone intermediates with three or four stereocenters on the cyclopentane fragment to link the PG side chains. The paper describes the most used procedures for the synthesis of the milestone δ-Corey-lactones and γ-Corey-lactones, their improvements, and some new promising methods, such as interesting, new stereo-controlled and catalyzed enantioselective reactions, and methods based on the chemical/enzymatic resolution of the compounds in different steps of the sequences. The many uses of δ-lactones not only for the synthesis of γ-lactones, but also for obtaining 9β-halogen-PGs and halogen-substituted cyclopentane intermediates, as synthons for new 9β-PG analogs and future applications, are also discussed.     

Impact strength elastomer composites based on polystyrene components separated from waste electrical and electronic equipment

The reuse of plastic components of waste electrical and electronic equipment (WEEE) is an important concern both for environmental issues and to preserve the material resources, with minimum energy consumption. Considering that polystyrene fraction was reported as approximate 80% of the total amount of WEEE plastic, this article aims to evaluate the recycling of this fraction, without separation by components, by melt compounding with styrene-butadiene block-copolymer (SBS) and hydrogenated and maleinized SBS, the blend of the two elastomers acting both as an impact modifier and compatibilizer. The composites are characterized by mechanical analysis, impact tests, dynamic mechanical analysis, differential scanning calorimetry, thermogravimetric analysis, scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction. The recycling conditions of the polystyrene fraction as composites without eliminating the WEEE additives for improved UV and flame resistance, with physical mechanical properties comparable to those of high-impact polystyrene resulted from the study. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48329.     

Compressive properties of pristine and SiC-Te-added MgB2 powders,green compacts and spark-plasma-sintered bulks

Pristine and (SiC+Te)-added MgB₂ powders, green and spark plasma sintered (SPS) compacts were investigated from the viewpoint of quasi-static and dynamic (Split-Hopkinson Pressure Bar, SHPB) compressive mechanical properties The amount of the additive (SiC+Te) was selected to be the optimum one for maximization of the superconducting functional parameters. Pristine and added MgB₂ show very similar compressive parameters (tan δ, fracture strength, Vickers hardness, others) and fragment size in the SHPB test. However, for the bulk SPSed samples the ratio of intergranular to transgranular fracturing changes, the first one being stronger in the added sample. This is reflected in the quasi-static K_IC that is higher for the added sample. Despite this result, sintered samples are brittle and have roughly similar fragmentation behavior as for brittle engineering ceramics. In the fragmentation process, the composite nature of our samples should be considered with a special focus on MgB₂ blocks (colonies) that show the major contribution to fracturing. The Glenn-Chudnovsky model of fracturing under dynamic load provides the closest values to our experimental fragment size data.     

Enhanced UV- and visible-light driven photocatalytic performances and recycling properties of graphene oxide/ZnO hybrid layers

Light induced catalytic processes have attracted significant attention during the last years for wastewater treatment due to their efficiency in decomposition of organic contaminants. In this study we report the synthesis of graphene oxide (GO)/ZnO hybrid layers with high photocatalytic efficiency using laser radiation. The results show that the hybrid layers exhibit much improved photodecomposition efficiency as compared to pure GO or ZnO both under UV and visible-light irradiation. The enhanced photocatalytic efficiency of the hybrid as compared to the reference pure ZnO and GO layers was attributed to the contribution of GO to the separation and transport of the photogenerated charge carriers. Additionally, under visible light irradiation the organic molecules can act as first sensitizers in the degradation process. The recyclability of the layers was also investigated through repetitive photodegradation cycles under UV- or visible-light irradiation. After consecutive degradation runs, the hybrid photocatalyst layers were still stable and retained high degradation efficiency, ensuring reusability. The photocatalytic activity of the layers was correlated with the gradual change of their chemical structure during consecutive degradation cycles. Owing to the high photodegradation efficiency, reusability, and ease of recovery the synthesised hybrid layers consisting of easily available materials are suitable for environmental purification applications.     

Double Variational Binding—(SMILES) Conformational Analysis by Docking Mechanisms for Anti-HIV Pyrimidine Ligands

Variational quantitative binding–conformational analysis for a series of anti-HIV pyrimidine-based ligands is advanced at the individual molecular level. This was achieved by employing ligand-receptor docking algorithms for each molecule in the 1,3-disubstituted uracil derivative series that was studied. Such computational algorithms were employed for analyzing both genuine molecular cases and their simplified molecular input line entry system (SMILES) transformations, which were created via the controlled breaking of chemical bonds, so as to generate the longest SMILES molecular chain (LoSMoC) and Branching SMILES (BraS) conformations. The study identified the most active anti-HIV molecules, and analyzed their special and relevant bonding fragments (chemical alerts), and the recorded energetic and geometric docking results (i.e., binding and affinity energies, and the surface area and volume of bonding, respectively). Clear computational evidence was also produced concerning the ligand-receptor pocket binding efficacies of the LoSMoc and BraS conformation types, thus confirming their earlier presence (as suggested by variational quantitative structure-activity relationship, variational-QSAR) as active intermediates for the molecule-to-cell transduction process.     

Synthesis and characterization of a titanium phosphate-tellurite glass for Faraday rotators

This work is focused on investigation of thermal, structural, optical, magnetic, and magneto-optical properties of novel titanium phosphate-tellurite glass applied as Faraday rotators. The glass belonging to the system 35Li₂O–10Al₂O₃–5TiO₂–45P₂O₅–5TeO₂ was prepared by a nonconventional wet route of raw materials processing, followed by melting-quenching-annealing steps. Some important physical properties of the investigated glass have been measured and calculated, providing knowledge related to glass compactness, electronic structure, glass forming capability, etc. XRD analysis evidenced an amorphous network structure of the investigated glass. The optical absorption in the Vis domain is mainly due to Ti³⁺ ions and Te₂ clusters formed during the glass melting process. A relatively low optical absorption is noticed over 600 nm that activates a significant Faraday magneto-optical effect. Photoluminescence bands in the blue, red, and infrared domains are observed, caused by Te₂ clusters formed during the glass melting process. The magnetization in dependency on applied magnetic field reveals a complex behavior of the glass, depending on temperature. Thus, it is found a ferromagnetic behavior up to 2000 Oe, a paramagnetic component up to 40 000 Oe, followed by a diamagnetic one over 40 000 Oe. Faraday rotation angle and Verdet constant values in the visible domain are correlated with the reduced TeO₂ content of the glass.     

Intravital FRET: Probing Cellular and Tissue Function in Vivo

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.