Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization.

A novel immobilization matrix for the entrapment of viable whole-cell Baeyer–Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer–Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements

Propagation rate coefficient for sulfobetaine monomers by PLP-SEC

Zwitterionic polymers have been extensively used in the biomedical field, however, the knowledge about the kinetics and mechanism of radical polymerization of their monomers is limited. This work introduces the first propagation rate coefficients, kp's, determined by pulsed-laser polymerization in conjunction with size-exclusion chromatography for the methacrylate-type sulfobetaine monomers N-(methacryloylaminopropyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium betaine (SBAm) and N-(methacryloyloxyethyl)-N,N-dimethyl-N-(3-sulfopropyl) betaine (SBE). The kp values are determined in aqueous solutions for monomer concentrations between 5 and 50 wt % and temperatures between 15 and 80 °C. The propagation rate for SBE is by about a factor of two higher than that for SBAm. The solvent effect manifested by the kp dependence on monomer concentration is stronger for SBAm than for SBE. The kp decreases upon increasing the monomer concentration up to 20 wt % followed by independence of kp on further increased monomer concentration. The anti-polyelectrolyte effect and the significance of hydrogen bonding interactions on kp values are illustrated by polymerizations in the presence of NaCl and organic co-solvents, respectively.This work was supported by VEGA Grant Agency under the contract No. 2/0198/14, and by the Slovak Research and Development Agency under the contract No. APVV-14-0858. Mrs. Eva Hipka from the Polymer Institute of the Slovak Academy of Sciences is thanked for the SEC experiments. Dr. Anatoly Nikitin from the ILIT RAS, Russia, is acknowledged for the discussion on the possible influence of low termination rate limit on propagation rate coefficients. Prof. Dr. Michael Buback from the Georg-August University in Göttingen, Germany, is thanked for providing valuable comments to the manuscript.Scopu

Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

International audienceLipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P) H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE

Fluorescence properties of Chlorella sp. algae

Water quality and its fast and reliable monitoring is the challenge of the future. Design of appropriate biosensors that would be capable of non-invasive identification of water pollution is an important prerequisite for such challenge. Chlorophylls are pigments, naturally presented in all plants that absorb light. The main forms of chlorophyll in algae are chlorophyll a and chlorophyll b, other pigments include xantophylls and beta-carotenes. Our aim was to characterize endogenous fluorescence of the Chlorella sp. algae, present naturally in drinking water. We recorded spatial, spectral and lifetime fluorescence distribution in the native algae. We noted that the fluorescence was evenly distributed in the algae cytosol, but lacked in the nucleus and reached maximum at 680-690 nm. Fluorescence decay of chlorella sp. was double-exponential, and clearly shorter than that of its isolated pigments. For the first time, fluorescence lifetime image of the algae is presented. Study of the fluorescence properties of algae is aimed at the improvement of water supply contamination detection and cleaning

Viscoelastic and photo-actuation studies of composites based on polystyrene-grafted carbon nanotubes and styrene-b-isoprene-b-styrene block copolymer

Photoactuating composites based on the linear triblock copolymer polystyrene-b-polyisoprene-b-polystyrene (SIS) were prepared by incorporation of polystyrene-modified multiwalled carbon nanotubes (MWCNT-PS). Modification of MWCNT was performed by surface-initiated atom transfer radical polymerization (SI ATRP) of styrene. The presence of the polystyrene chains on the MWCNT surface facilitated their dispersion in the SIS matrix. Improved interactions of the modified MWCNT-PS compared to neat MWCNT were confirmed by dynamic mechanical analysis. The activation energy of glass transition of the polystyrene phase in the MWCNT-PS/SIS composite increased significantly compared to the neat SIS matrix, while the incorporation of neat MWCNT to the SIS matrix disturbed the physical cross-linking of the SIS and degraded its elastic properties. The photo-actuation ability of the MWCNT-PS/SIS composite was proved using atomic force microscopy. © 2013 Elsevier Ltd. All rights reserved.Slovak Research and Development Agency APVV [APVV-0109-10]; Centre of Excellence SAS for Functionalized Multiphase Materials (FUN-MAT); European Commission [228916]; DAAD [50755038]; European Regional Development Fund; National Budget of Czech Republic, within the framework of project Centre of Polymer Systems [CZ.1.05/2.1.00/03.0111]; [TACR TE01020118

Effect of Dexamethasone on Thermoresponsive Behavior of Poly(2-Oxazoline) Diblock Copolymers

Thermoresponsive polymers play an important role in designing drug delivery systems for biomedical applications. In this contribution, the effect of encapsulated hydrophobic drug dexamethasone on thermoresponsive behavior of diblock copolymers was studied. A small series of diblock copoly(2-oxazoline)s was prepared by combining thermoresponsive 2-n-propyl-2-oxazoline (nPrOx) and hydrophilic 2-methyl-2-oxazoline (MeOx) in two ratios and two polymer chain lengths. The addition of dexamethasone affected the thermoresponsive behavior of one of the copolymers, nPrOx20-MeOx180, in the aqueous medium by shifting the cloud point temperature to lower values. In addition, the formation of microparticles containing dexamethasone was observed during the heating of the samples. The morphology and number of microparticles were affected by the structure and concentration of copolymer, the drug concentration, and the temperature. The crystalline nature of formed microparticles was confirmed by polarized light microscopy, confocal Raman microscopy, and wide-angle X-ray scattering. The results demonstrate the importance of studying drug/polymer interactions for the future development of thermoresponsive drug carriers

Visualization of carbon nanotubes dispersion in composite by using confocal laser scanning microscopy

Nanocomposites of polystyrene-block-polyisoprene-block-polystyrene triblock copolymer (SIS) and various types of neat and polystyrene-modified carbon nanotubes (CNT-PS) were prepared and distribution of the CNT-PS throughout the polymer matrix was evaluated using confocal laser scanning microscopy (CLSM). The Nanoamor MWCNT-PS with highest thickness of 25-60 nm were readily visualized using both reflection mode without necessity of fluorescent labelling and fluorescent mode after addition of free dye to the nanocomposite. Visualization of Nanocyl MWCNT-PS with thickness of 8-18 nm and SWCNT-PS was achieved after covalent labelling of the CNT-PS with benzothioxanthene fluorescent dye. The CLSM can serve as a non-invasive method for evaluation of quality of dispersion of nanofillers on quite large area and at various depth of polymer film. © 2016 Elsevier Ltd. All rights reserved.50755038, DAAD, German Academic Exchange ServiceVEGA grant agency [2/0112/13]; Slovak Research and Development Agency APVV [APVV-14-0891]; SAS-MOST JRP [2014-9]; DAAD project [50755038]; Ministry of Education, Youth and Sports of the Czech Republic - Program NPU I [LO1504

Progress in emerging techniques for characterization of immobilized viable whole-cell biocatalysts

Proper understanding of viable cell physiology during biocatalytic processes, progress in development of biocompatible immobilization techniques, as well as introduction of new non-invasive characterization techniques are the main prerequisites for the successful development of novel whole-cell biocatalysts. Selection of the optimal immobilized viable cell biocatalyst is possible when standardised characterization techniques are employed. The development of these techniques requires advanced analytics and a multidisciplinary effort. This review provides a summary on progress in development of characterization methods and emerging tools for determination of morphology, physiology, and biocatalytic efficiency of immobilized viable whole cells.Slovak Grant Agency for Science VEGA [2/0090/16]; Slovak Research and Development Agency [APVV-15-0227]; Research and Development Operational Programme - ERDF [26240220079