Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.

Combined toxicity of therapeutic pharmaceuticals to duckweed, Lemna minor.

Pharmaceuticals, which are designed to be biologically active at low concentrations, are found in surface waters, meaning aquatic organisms can be exposed to complex mixtures of pharmaceuticals. In this study, the adverse effects of four pharmaceuticals, 17α-ethynylestradiol (synthetic estrogen), methotrexate (anticancer drug), diclofenac (nonsteroidal anti-inflammatory drug) and fluoxetine (antidepressant), and their binary mixtures at mg/L concentrations were assessed using the 7-day Lemna minor test, with both apical and biochemical markers evaluated. The studied biochemical markers included chlorophyll a, chlorophyll b, carotenoids and oxidative stress enzymes catalase, glutathione-S-transferase and glutathione reductase, with effects compared to solvent controls. The adverse effects on Lemna minor were dose-dependent for frond number, surface area, relative chlorophyll content and activity of glutathione S-transferase for both individual pharmaceuticals and binary mixtures. According to the individual toxicity values, all tested pharmaceuticals can be considered as toxic or harmful to aquatic organisms, with methotrexate considered highly toxic. The most sensitive endpoints for the binary mixtures were photosynthetic pigments and frond surface area, with effects observed in the low mg/L concentration range. The concentration addition model and toxic unit approach gave similar mixture toxicity predictions, with binary mixtures of methotrexate and fluoxetine or methotrexate and 17α-ethynylestradiol exhibiting synergistic effects. In contrast, mixtures of diclofenac with fluoxetine, 17α-ethynylestradiol or methotrexate mostly showed additive effects. While low concentrations of methotrexate are expected in surface water, chronic ecotoxicological data for invertebrates and fish are lacking, but this is required to better assess the environmental risk of methotrexate.

Addressing challenges in providing a reliable ecotoxicology data for graphene-oxide (GO) using an algae (Raphidocelis subcapitata), and the trophic transfer consequence of GO-algae aggregates.

The graphene oxide (GO) due to its exceptional structure, physicochemical and mechanical properties is a very attractive material for industry application. Even though, the unique properties of GO (e.g. structure, size, shape, etc.) make the risk assessment of this nanomaterial very challenging in comparison with conventional ecotoxicology studies required by regulators. Thus, there is a need for standardized characterization techniques and methodology to secure a high quality/reliable data on the ecotoxicology of GO, and to establish environmentally acceptable levels. Herein, authors address the crucial quality criteria when evaluating the ecotoxicology of GO using an algae (Raphidocelis subcapitata) and a shrimp (Paratya australiensis). This study provides a detail characterization and modification of the used GO, robust quantification and a suspension stability in different media for ecotoxicology studies. It was observed that under the same exposure conditions the behavior of GO and the estimated outcomes (IC50 values) in modified algae media differed in comparison to the referent media. Further to that, the adverse effects of GO on the algae cell structure and the potential uptake of GO by the algae cells were examined using the TEM with different staining techniques to avoid artefacts. Shrimps which were exposed to GO-algae aggregates via the food intake did not indicate stress or accumulation of GO. Our work presents an important insight to necessity of establishing a benchmark ecotoxicology assays for GO (e.g. characterization techniques, choice of media, etc.) and providing a reliable data to be used by regulators in risk assessment of two-dimensional (2D) nanomaterials.

Conformational Analyses of Physiological Binary and Ternary Copper(II) Complexes with l-Asparagine and l-Histidine; Study of Tridentate Binding of Copper(II) in Aqueous Solution.

This study explores the structural properties and energy landscapes of the physiologically important bis(l-asparaginato)copper(II) [Cu(l-Asn)2] and (l-histidinato)(l-asparaginato)copper(II) [Cu(l-His)(l-Asn)]. The conformational analyses in the gas phase and implicitly modeled water medium, and magnetic parameters of electron paramagnetic resonance spectra were attained using density functional theory calculations. The apical CuII coordination and hydrogen bonding were analyzed. Predicted lower-energy structures enabled the confirmation and, for apical bonding, also the refinement of structural proposals from literature. Available experimental results were indecisive regarding the amido-group binding in the CuII equatorial plane in solutions, but the examination of the relative stability of Cu(l-Asn)2 conformers in 30 binding modes confirms the glycine-like mode as the most stable one. Previously reported experimental results for Cu(l-His)(l-Asn) were interpreted for l-His to have a tridentate histamine-like mode. However, the aqueous conformers with l-His in the glycinato mode are also predicted to have low energies, which does not contradict the tridentate l-His binding. The predicted magnetic parameters of conformers with an apical oxygen atom (intramolecular or from a water molecule) can reproduce the experimental data. An extent of conformational flexibility and abundance of l-His-containing ternary copper(II) amino acid complexes under physiological conditions may be related.

Microbial levan and pullulan as potential protective agents for reducing adverse effects of copper on Daphnia magna and Vibrio fischeri.

Microbial polysaccharides, due to their unique physiochemical properties, have found application in the food industry, cosmetics, pharmacy and medicine. In the environment, microbes can use polysaccharides to alleviate the adverse effects of heavy metals in their close proximity. This adaptive property shows interesting potential for bioremediation. Herein, the effects of the exopolysaccharides (EPS) levan, produced by the bacterium Bacillus licheniformis NS032 and pullulan, produced by the fungus Aureobasidium pullulans CH-1 in the presence of copper (Cu2+) have been investigated for the first time on antioxidant enzyme activity, respiration and Cu2+ bioaccumulation of Daphnia magna as well as the bioluminescence of Vibrio fischeri. Both EPS decreased toxicity of Cu2+ in the acute test with D. magna. The activity of catalase (CAT) was significantly diminished after acute exposure to Cu2+ in comparison to treatments with Cu2+ and EPS, while in the prolonged acute exposure the CAT activity did not show statistically significant (P ≤ 0.05) differences between treatments with and without the EPS. According to ICP-MS results, during prolonged acute exposure of neonates, the bioaccumulation of Cu2+ in treatments without the EPS was 52.03 µg/g of biomass (wet), while in treatments with EPS, the bioaccumulation was lower by one order of magnitude. The respiration of neonates during acute exposure to Cu2+ with or without the EPS was monitored using the MicroOxymax respirometer, and the results show the EPS can positively effect the respiration. In the case of bacterial bioluminescence, the toxicity of Cu2+ decreased in treatments with EPS (30 min EC10) from 3.54 mg/L to 140.61 mg/L (levan) and 45.00 mg/L (pullulan). This study demonstrates protective effect of EPS against Cu2+ toxicity on D. magna and V. fischeri, and opens the door for further investigation of potential application of levan and pullulan in bioremediation of heavy metals and mitigation of their adverse effects in the aquatic environment.

Engineering the Slow Photon Effect in Photoactive Nanoporous Anodic Alumina Gradient-Index Filters for Photocatalysis.

In this study, we explore for the first time the capabilities of nanoporous anodic alumina gradient-index filters (NAA-GIFs) functionalized with titanium dioxide (TiO2) photoactive layers to enhance photon-to-electron conversion rates and improve the efficiency of photocatalytic reactions by "slow photon" effect. A set of NAA-GIFs was fabricated by sinusoidal pulse anodization, in which a systematic modification of various anodization parameters (i.e., pore widening time, anodization period, and anodization time) enables the fine-tuning of the photonic stopband (PSB) of these nanoporous photonic crystals (PCs) across the spectral regions. The surface of NAA-GIFs was chemically modified with photoactive layers of TiO2 to create a composite photoactive material with precisely engineered optical properties. The photocatalytic performance of TiO2-functionalized NAA-GIFs was assessed by studying the photodegradation of three model organic dyes (i.e., methyl orange, Rhodamine B, and methylene blue) with well-defined absorption bands across different spectral regions under simulated irradiation conditions. Our study demonstrates that when the edges of characteristic PSB of TiO2-modified NAA-GIFs are completely or partially aligned with the absorption band of the organic dyes, the photodegradation rate is enhanced due to "slow photon" effect. A rational design of the photocatalyst material with respect to the organic dye is demonstrated to be optimal to speed up photocatalytic reactions by an efficient management of photons from high-irradiance spectral regions. This provides new opportunities to develop high-performing photocatalytic materials for efficient photocatalysis with broad applicability.

Ecotoxicology of manufactured graphene oxide nanomaterials and derivation of preliminary guideline values for freshwater environments.

The unique physical and chemical properties of graphene-based nanomaterials (GNMs) have inspired a diverse range of scientific and industrial applications. The market value of GNMs is predicted to reach $US 1.3 billion by 2023. Common to many nanomaterials, an important and unresolved question is the environmental consequences of the increases in GNMs use. The current deficiencies in studies reporting ecotoxicology data for GNMs include differences in analytical methodologies for quantification, no standardized test guidelines, differences in morphology of GNMs, the lack of Chemical Abstract Service numbers, and the quality of the reported data. The assessment of potential adverse effects on aquatic organisms typically relies on guideline values based on species sensitivity distributions (SSDs) of toxicity data. We present preliminary water quality guideline values for graphene oxide NMs in freshwaters. Data include 10 species from 7 phyla (bacteria and fungi were not included). The most sensitive organism was found to be the freshwater shrimp Palaemon pandaliformis. The derived guideline values for 99, 95, 90, and 80% species protection were 350, 600, 830, and 1300 µg/L, respectively. These results will contribute to the regulatory derivations of future water quality guideline values for graphene-based NMs. Environ Toxicol Chem 2018;37:1340-1348. © 2018 SETAC.

Calculating the geometry and Raman spectrum of physiological bis(L-histidinato)copper(II): an assessment of DFT functionals for aqueous and isolated systems.

Reliable density functional theory (DFT) calculations can be performed in conjuction with spectroscopic measurements to elucidate the structural properties of physiologically important bis(amino acidato)copper(II) compounds in solutions. They can provide insight into the influence of intermolecular interactions on the molecular geometry in the crystal lattice or solution when compared with a DFT gas-phase minimum. Our previous paper [Markovic et al. (2014) Eur J Inorg Chem 198] reported the DFT-determined geometries and Raman spectra for different conformers of physiological bis(L-histidinato)copper(II) with 20 explicit water molecules, as calculated using the B3LYP functional. The present study examined the reliability of those B3LYP results by applying the M06 functional instead, as it should better account for noncovalent interactions. The water molecules were positioned more compactly around the complex by M06 than by B3LYP. The accuracies of the two functionals when compared to relevant experimental data showed that M06 was better at reproducing in-plane Cu-N bond lengths but B3LYP gave more accurate axial Cu-O distances. Both functionals reproduced the experimental Raman spectrum at pH 8 to similar levels of accuracy and provided precise information on the Cu(II) coordination mode and conformation in aqueous solution. Additionally, we assessed several DFT and DFT-D functionals (BP86, B3LYP, B3LYP-D, M06, M06 L, wB97XD, mPW2PLYPD) by using them to model the geometries of experimental bis(L-histidinato)copper(II) crystalline conformations as isolated systems, and then benchmarking the results against those from high-level second-order pertubation Møller-Plesset (MP2) calculations. Although this assessment resulted in an equivocal conclusion because the MP2 results for the isolated complex were inconsistent with the corresponding DFT outcomes, it does provide new information on future benchmark options.

Insights into the Hydrogen-Atom Transfer of the Blue Aroxyl.

An experimental and theoretical study on hydrogen-atom transfer dynamics in the hydrogen-bonded substituted phenol/phenoxyl complex of the blue aroxyl (2,4,6-tri-tert-butylphenoxyl) is presented. The experimental exchange dynamics is determined in different organic solvents from the temperature-dependent alternating line-width effect in the continuous-wave ESR spectrum. From bent Arrhenius plots, effective tunnelling contributions with parallel heavy-atom motion are concluded. To clarify the transfer mechanism, reaction paths for different conformers of the substituted phenol/phenoxyl complex are modelled theoretically. Various DFT and post-Hartree-Fock methods including multireference methods are applied. From the comparison of experimental and theoretical data it is concluded that the system favours concerted hydrogen-atom transfer along a parabolic reaction path caused by heavy-atom motion.

The Important Role of the Hydroxyl Group on the Conformational Adaptability in Bis(l-threoninato)copper(II) Compared to Bis(l-allo-threoninato)copper(II): Quantum Chemical Study.

Detailed structural properties of physiological bis(amino acidato)copper(II) complexes are generally unknown in solutions. This paper examines how stereochemical differences between the essential amino acid l-threonine and its diastereomer l-allo-threonine, which is rarely present in nature, may affect relative stabilities of bis(l-threoninato)copper(II) and bis(l-allo-threoninato)copper(II) in the gas phase and aqueous solution. These amino acids can bind to Cu(II) via the nitrogen and carboxylato oxygen atoms, the nitrogen and hydroxyl oxygen atoms, and the carboxylato and hydroxyl oxygen atoms. We term these coordination modes G, No, and Oo, respectively. The density functional theory (DFT) calculations with the B3LYP functional of the conformational landscapes for all possible coordination modes of both complexes revealed their very similar stability in the gas phase and in aqueous solution. The conformational analyses resulted in 196 and 267 conformers of isolated copper(II) chelates with l-threonine and l-allo-threonine, respectively. The G-G coordination mode is the most stable, both in the gas phase and aqueous solution. Very similar energy values of the lowest-energy solvated cis and trans G-G conformers in implicitly accounted water medium are in accord with the experimental results that these isomers are present in aqueous solution at physiological pH values. The transition-state structures, activation Gibbs free energies, and reaction rates calculated using DFT/B3LYP and MP2 for the transformations from the most stable cis G-G and trans Oo-G conformers to trans G-G ones for the first time reveal several alternate coordination-mode transformation mechanisms in the copper(II) complexes with amino acids other than glycine. The trans Oo-G conformers are kinetically more stable than cis G-G ones in the gas phase. The only significant difference found between the two complexes is a more suitable position of the hydroxyl group in physiological bis(l-threoninato)copper(II) to form intramolecular hydrogen bonds, which may restrain its conformational space.

Microwave-hydrothermal method for the synthesis of composite materials for removal of arsenic from water.

Composite material Zr-doped TiO2, suitable for the removal of arsenic from water, was synthetized with fast and simple microwave-hydrothermal method. Obtained material, Zr-TiO2, had uniform size and composition with zirconium ions incorporated into crystal structure of titanium dioxide. Synthetized composite material had large specific surface area and well-developed micropore and mesopore structure that was responsible for fast adsorption of As(III) and As(V) from water. The influence of pH on the adsorption capacity of arsenic was studied. The kinetics and isotherm experiments were also performed. The treatment of natural water sample containing high concentration of arsenic with composite material Zr-TiO2 was efficient. The concentration of arsenic was reduced to the value recommended by WHO.

Graphene Aerogels Decorated with α-FeOOH Nanoparticles for Efficient Adsorption of Arsenic from Contaminated Waters.

Arsenic (As) is the world's most hazardous chemical found in drinking water of many countries; therefore, there is an urgent need for the development of low-cost adsorbents for its removal. Here, we report a highly versatile and synthetic route for the preparation of a three-dimensional (3D) graphene-iron oxide nanoparticle aerogel composite for the efficient removal of As from contaminated water. This unique three-dimensional (3D) interconnected network was prepared from natural graphite rocks with a simple reaction, without the use of harsh chemicals, which combines with the exfoliation of graphene oxide (GO) sheets via the reduction of ferrous ion to form a graphene aerogel composite decorated with iron oxide nanoparticles. The prepared adsorbent showed outstanding absorption performance for the removal of As from contaminated water, because of its high surface-to-volume ratio and characteristic pore network in the 3D architecture. The performed case study using real drinking water contaminated with As under batch conditions showed successful removal of arsenic to the concentration recommended by the World Health Organisation (WHO).

Phytoextraction of metals by Erigeron canadensis L. from fly ash landfill of power plant "Kolubara".

The objectives of this study were to determine the concentrations of Pb, Cd, As, Cr, Cu, Co, Ni, Zn, Ba, Fe, Al and Ag in Erigeron canadensis L. growing on fly ash landfill of power plant "Kolubara", Serbia. The content of each element was determined in every part of plant separately (root, stalk and inflorescence) and correlated with the content of elements in each phase of sequential extraction of fly ash. In order to ambiguously select the factors that are able to decidedly characterize the particular part of plant, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed. The bioconcentration factors and translocation factors for each metal were calculated in order to determine the feasibility of the use of plant E. canadensis L. for phytoremediation purpose. There were strong positive correlations between metals in every part of plant samples, and metals from pseudo total form of sequential extraction indicate that the bioavailability of elements in fly ash is similarly correlated with total form. Retained Al, Fe, Cr and Co in the root indicate its suitability for phytostabilization. This plant takes up Cd and Zn from the soil (bioconcentration factors (BCFs) greater than 1), transporting them through the stalk into the inflorescence (translocation factors (TFs) higher than 1). Regarding its dominance in vegetation cover and abundance, E. canadensis L. can be considered adequate for phytoextraction of Cd and Zn from coal ash landfills at Kolubara.

Application of non-thermal plasma reactor and Fenton reaction for degradation of ibuprofen.

Pharmaceutical compounds have been detected frequently in surface and ground water. Advanced Oxidation Processes (AOPs) were reported as very efficient for removal of various organic compounds. Nevertheless, due to incomplete degradation, toxic intermediates can induce more severe effects than the parent compound. Therefore, toxicity studies are necessary for the evaluation of possible uses of AOPs. In this study the effectiveness and capacity for environmental application of three different AOPs were estimated. They were applied and evaluated for removal of ibuprofen from water solutions. Therefore, two treatments were performed in a non-thermal plasma reactor with dielectric barrier discharge with and without a homogenous catalyst (Fe(2+)). The third treatment was the Fenton reaction. The degradation rate of ibuprofen was measured by HPLC-DAD and the main degradation products were identified using LC-MS TOF. Twelve degradation products were identified, and there were differences according to the various treatments applied. Toxicity effects were determined with two bioassays: Vibrio fischeri and Artemia salina. The efficiency of AOPs was demonstrated for all treatments, where after 15 min degradation percentage was over 80% accompanied by opening of the aromatic ring. In the treatment with homogenous catalyst degradation reached 99%. V. fischeri toxicity test has shown greater sensitivity to ibuprofen solution after the Fenton treatment in comparison to A. salina.

Combined experimental and computational study of cis-trans isomerism in bis(L-valinato)copper(II).

Heating of polycrystalline cis aquabis(L-valinato)copper(II) at 90 °C resulted in a dehydrated powder. Recrystallization from aqueous solution of the obtained product yielded anhydrous trans bis(L-valinato)copper(II). The X-ray crystal and molecular structures of trans bis(L-valinato)copper(II) and cis aquabis(L-valinato)copper(II) are presented. Molecular modeling calculations were attempted to resolve factors that influenced the isomerization and crystallization of either the aqua cis- or the anhydrous trans-isomer. Conformational analyses of trans- and cis-isomers were completed in vacuo and in crystal by molecular mechanics, and in aqueous solution by molecular dynamics (MD) simulations using the same force field. Although the conformers with trans-configuration are the most stable in vacuo, those with cis-configuration form more favorable intermolecular interactions. Consequently, both cis- and trans-isomers are predicted to be present in aqueous solution. According to the crystal structure simulations and predictions, cis-isomer requires water molecules to form energetically more stable crystal packings than trans-isomer. The MD modeling of the self-assembly of 16 bis(L-valinato)copper(II) complexes in aqueous solution for the first time predicted the crystallization nucleus formation to proceed from monomers to oligomers by Cu-to-O(carboxylato) and/or N-H···O(carboxylato) weak bonds; these oligomers then bind together via water molecules until they acquire the right positions for noncovalent bonding like in the experimental crystal structures. Fifty-nanosecond MD simulations accomplished for a system consisting of equal numbers of complexes and water molecules at 298 and 370 K suggested complete cis-to-trans transformation at the higher temperature. Prevalence of either cis- or trans-conformers in water upon dissolvation may explain the crystallization results.