Effect of electrolytes and surfactants on the thermoseparation of an ethylene oxide–propylene oxide random copolymer in aqueous solution

The thermoseparation of aqueous solutions of Breox 50 A 1000, an ethylene oxide–propylene oxide 50:50 (w/w) random copolymer, was studied. The cloud-point diagram for Breox in water solution and the effects of electrolytes and surfactants on the cloud-point temperature (CPT) were determined. The Breox concentration in both phases after the thermoseparation was followed with a reversed-phase HPLC method. The effects of separation temperature and additives on phase composition were evaluated.     

Partitioning of peptides and recombinant protein–peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structure

Genetic engineering has been used for fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in an aqueous two-phase system. The system was composed of dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer, EO30PO70. Peptides containing tryptophan, proline, arginine or aspartate residues were fused at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale extraction. The target protein and peptide tags were partitioned separately and then together in the fusion proteins in order to gain increased understanding of the influence of certain amino acid residues on the partitioning. The salt K₂SO₄ was used to reduce the charge dependent salt effects on partitioning and to evaluate the contribution to the partition coefficient from the hydrophobic–hydrophilic properties of the amino acid residues. The effect of Trp on peptide partitioning was independent of the difference in primary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only determined by the number of Trp. The effect of the charged residues, Arg and Asp, was dependent on the surrounding residues, i.e. if they were situated next to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fusion proteins. The effect of the salts sodium perchlorate and triethylammonium phosphate on the partitioning was also studied. The salt effects observed for the peptides were qualitatively similar to the effects observed for the fusion proteins.     

Ethylene oxide and propylene oxide random copolymer/sodium chloride aqueous two-phase systems: wetting and adsorption on dodecyl-agarose and polystyrene

Liquid/liquid partition chromatography is a mild yet powerful separation method for a variety of biological materials. This work demonstrates that it should be feasible to immobilize an ethylene oxide-propylene oxide (EO/PO) random copolymer solution and to use a solution of NaCl equilibrated against the polymer solution as the mobile-phase (poly (EO-PO) [P(EO-PO)] and NaCl form two aqueous phases known as aqueous two-phase systems). Three random copolymers with different molecular weights and EO/PO ratios were used. Dodecyl-agarose and polystyrene were tested as possible supports. The wetting energies of the aqueous two-phase systems on these two kinds of surfaces were calculated as well as contact angles for each phase on the same surfaces. Finally, the thickness of P(EO-PO) adsorption layers on polystyrene lattices were measured by dynamic light scattering. Contact angle measurements indicate that indeed some EO/PO copolymers preferentially wet hydrophobic substrates, forming thin films.     

Genotoxic effects of ethylene oxide and propylene oxide: a comparative study

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5–10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Effect of salts and surfactants on the partitioning of Fusarium solani pisi cutinase in aqueous two-phase systems of thermoseparating ethylene oxide/propylene oxide random copolymer and hydroxypropyl starch

An aqueous two-phase system composed by a thermoseparating random copolymer of ethylene oxide/propylene oxide 50/50 (%w/w), Breox, and hydroxypropyl starch – Reppal PES 100 was evaluated for the partitioning of Fusarium solani pisi recombinant cutinase. The effect of several additives on the partitioning of pure cutinase was evaluated. Micelles of sodium dodecanoate provided a ten-fold increase of the partitioning coefficient (K=9) and recovery yields of 60-75%. The phase diagrams of the systems composed of Breox, Reppal and sodium dodecanoate were determined and it was found that in systems with high surfactant concentrations, the binodal was moved to lower polymer concentrations, enabling a two-phase system with 6% (w/w) of each polymer.     

Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide--propylene oxide copolymers

Endoglucanases (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide-propylene oxide random copolymer (EO(50)PO(50)) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)(4) tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)(2) tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)(4) improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)(2).     

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.     

A 1,2‐propylene oxide sensor utilizing cataluminescence on CeO2 nanoparticles

A simple and sensitive gas sensor was proposed for the determination of 1,2‐propylene oxide (PO) based on its cataluminescence (CTL) by oxidation in the air on the surface of CeO₂ nanoparticles. The luminescence characteristics and optimal conditions were investigated in detail. Under optimized conditions, the linear range of the CTL intensity versus the concentration of PO was 10–150 ppm, with a correlation coefficient (r) of 0.9974 and a limit of detection (S/N = 3) of 0.9 ppm. The relative standard deviation for 40 ppm PO was 1.2% (n = 7). There was no or only weak response to common foreign substances including acetone, formaldehyde, ethyl acetate, acetic acid, chloroform, propanol, carbon tetrachloride, ether and methanol. There was no significant change in the catalytic activity of the sensor for 100 h. The proposed method was simple and sensitive, with a potential of detecting PO in the environment and industry. Copyright © 2014 John Wiley & Sons, Ltd.     

Why iron–dithiocarbamates ensure detection of nitric oxide in cells and tissues

The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping.     

Xylanase mass transfer studies in aqueous two-phase systems using spray and sieve plate columns

Aqueous two-phase systems (ATPSs) have long been used for biomolecule partitioning; these systems offer the possibility of using continuous or semicontinuous extraction processes. They require relatively simple equipment like spray or sieve plate columns that can be adapted for use in ATPSs. The aim of this work was to study the semicontinuous extraction of a model enzyme, xylanase, in spray and sieve plate columns, since, unlike centrifugal contactors, the cost of construction and maintenance of this equipment is low and it is easy to operate. For the spray column, the dispersed phase hold-up and overall mass transfer coefficients K(D) a were evaluated for different column heights and for different superficial velocities of the dispersed phase (light phase). Results indicated that an increase in superficial velocity in the range of 0-0.18 mm/s of the dispersed phase had a positive effect on K(D) a and on hold-up in all column heights studied, 75, 161 and 246 mm. For the same superficial velocity of the dispersed phase, the larger the hold-up was, the shorter the column. For the sieve plate column, the effects of the superficial velocity of the dispersed phase and the number of plates were also studied. Results showed that the K(D) a and hold-up increased with an increase in both parameters. The selectivity of separation of xylanase and BSA (model contaminant) was very high, since 60% of the enzyme was extracted in the light phase, whereas no significant amount of BSA was extracted. The possibility of using the sieve plate column in continuous operation for enzyme extraction was studied because previous work had only addressed the semicontinuous extraction of enzyme. The residence time distribution of the PEG phase using different superficial velocities of the salt phase was studied in continuous operation. The time required to reach the steady state was 40 min, and 70% of the xylanase was recovered. It was found that the Modified Power Spline software was well adjusted to the experimental results.     

A Composite Gel–Polymer/Glass–Fiber Electrolyte for Sodium‐Ion Batteries

An integrated preparation of a low‐cost composite gel–polymer/glass–fiber electrolyte with poly(vinylidene fluoride‐co‐hexafluoropropylene) (PVDF‐HFP) reinforced by a glass–fiber paper and modified by a polydopamine coating to tune the mechanical and surface properties of PVDF‐HFP is shown to be applicable to a sodium‐ion battery. The composite polymer matrix exhibits excellent mechanical strength and thermal stability up to 200 °C. After saturating with a liquid electrolyte, a wide electrochemical window and high ionic conductivity is obtained for the composite gel–polymer/glass–fiber electrolyte. When tested in a sodium‐ion battery with Na₂MnFe(CN)₆ as cathode, the rate capability, cycling performance, and coulombic efficiency are significantly improved. The results suggest that the composite polymer electrolyte is a very attractive separator for a large‐scale battery system where safety and cost are the main concerns.     

Dispersal capacity of Haematopota spp. and Stomoxys calcitrans using a mark–release–recapture approach in Belgium

The dispersion potential of mechanical vectors is an important factor in the dissemination of pathogens. A mark–release–recapture experiment was implemented using two groups (unfed and partially fed) of the Tabanidae (Diptera) (Haematopota spp.) and biting Muscidae (Diptera) (Stomoxys calcitrans) most frequently collected in Belgium in order to evaluate their dispersion potential. In total, 2104 specimens of Haematopota spp. were collected directly from horses and 5396 S. calcitrans were collected in a cattle farm using hand‐nets. Some of these insects were partially fed in vitro and all were subsequently coloured. Overall, 67 specimens of S. calcitrans (1.2%) and 17 of Haematopota spp. (0.8%) were recaptured directly on horses. Stomoxys calcitrans flew maximum distances of 150 m and 300 m when partially fed and unfed, respectively. Haematopota spp. travelled maximum distances of 100 m and 200 m when partially fed and unfed, respectively. Segregation measures seem essential in order to reduce the risk for pathogen transmission. A distance of 150 m appears to be the minimum required for segregation to avoid the risk for mechanical transmission, but in areas of higher vector density, this should probably be increased.     

Boosting Organic–Metal Oxide Heterojunction via Conjugated Small Molecules for Efficient and Stable Nonfullerene Polymer Solar Cells

Charge events across organic–metal oxide heterointerfaces routinely occur in organic electronics, yet strongly influence their overall performance and stability. They become even more complicated and challenging for the heterojunction conditions in polymer solar cells (PSCs), especially when nonfullerene acceptors with varied energetics are employed. In this work, an effective interfacial strategy that utilizes novel small molecule self‐assembled monolayers (SAMs) is developed to improve the electronic and electric, as well as chemical properties of organic–zinc oxide (ZnO) interfaces for nonfullerene PSCs. It is revealed that the tailored SAMs with well‐controlled energy levels and molecular dipoles can effectively optimize the energetic barrier and work function (WF) of heterointerface for optimal electron extraction. In addition, the introduction of SAMs atop of ZnO facilitates not only acceptor segregation near the n‐contact interface, but also passivation of the photocatalytic activities for ZnO, to improve overall performance and photo stability of the derived nonfullerene PSCs. Overall, the methodology and structure–property relationship revealed herein would be beneficial for a wide range of hybrid electronics.     

Study of Aedes albopictus dispersal in Rome,Italy, using sticky traps in mark–release–recapture experiments

We report the results of three mark–release–recapture experiments carried out in an urban area in Rome, Italy, to study the active dispersal of Aedes albopictus (Diptera: Culicidae). The 4.3% recapture rate obtained supports the use of sticky traps in MRR experiments to study the dispersal of Ae. albopictus females. Most fluorescent dust‐marked females were recaptured at the gravid stage at 50–200 m from the release sites during the first 9 days after release. The average of daily‐MDTs (Mean Distance Traveled) was 119 m and the maximum observed distance travelled ranged from 199 m to 290 m in the three replicates. These data provide the first information about the dispersal of Ae. albopictus in a temperate European area and appear to be consistent with the few data available on this subject from other urban areas, where dispersal was constrained by physical barriers. Although caution should be taken in generalizing these results, they should be considered when planning control activities in urban areas in Italy, as well as in other European countries. This is particularly relevant if control is intended to interrupt pathogen transmission in cases of possible arbovirus epidemics, such as the Chikungunya outbreak that occurred in Ravenna, Italy in 2007.     

A test of a field‐based 15N–nitrous oxide pool dilution technique to measure gross N2O production in soil

Soils are both a major source and sink of nitrous oxide (N₂O), but the proportion of soil N₂O production released to the atmosphere (termed the N₂O yield) is poorly constrained due to the difficulty in measuring gross N₂O production. The quantification of gross N₂O fluxes would greatly improve our ability to predict N₂O dynamics across the soil‐atmosphere interface. We report a new approach, the ¹⁵N₂O pool dilution technique, to measure rates of gross N₂O production and consumption under laboratory and field conditions. In the laboratory, gross N₂O production and consumption compared well between the ¹⁵N₂O pool dilution and acetylene inhibition methods whereas the ¹⁵NO₃^? tracer method measured significantly higher rates. In the field, N₂O emissions were not significantly affected by increasing chamber headspace concentrations up to 100 ppb ¹⁵N₂O. The pool dilution model estimates of ¹⁴N₂O and ¹⁵N₂O concentrations as well as net N₂O fluxes fit observed data very well, suggesting that the technique yielded robust estimates of gross N₂O production. Estimated gross N₂O consumption rates were underestimated relative to rates calculated as the difference between gross and net N₂O production rates, possibly due to heterogeneous and/or inadequate tracer diffusion to deeper layers in the soil profile. Gross N₂O production rates were high, averaging 8.4 ± 3.2 mg N m^?2 day^?1, and were most strongly correlated to mineral nitrogen concentrations and denitrifying enzyme activity (R² = 0.73). Gross N₂O production rates varied spatially, with the highest rates in soils with the best drainage and the highest mineral N availability. Estimated and calculated N₂O consumption rates constrained the average N₂O yield from 0.70 to 0.84. Our results demonstrate that the ¹⁵N₂O pool dilution technique can provide well‐constrained estimates of N₂O yields and field rates of gross N₂O production correlated to soil characteristics, improving our understanding of terrestrial N₂O dynamics.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.