Removal Of Monomers Research Articles

Insights into Error Control Mechanisms in Biological Processes: Copolymerization and Enzyme-Kinetics Revisited.

The high fidelity observed in biological information processing ranging from replication to translation has stimulated significant research efforts to clarify the underlying microscopic picture. Theoretically, several approaches to analyze the error rates have been proposed. The copolymerization theory describes the addition and removal of monomers at the growing tip of a copolymer, leading to a closed set of nonlinear equations. On the other hand, enzyme-kinetics approaches formulate linear equations of biochemical networks, describing transitions between discrete chemical states. However, it is still unclear whether the error values computed by the two approaches agree. Moreover, there are conflicting interpretations on whether the error is under thermodynamic or kinetic discrimination control. In this work, we examine the error rate in persistent copying biochemical processes by specifically analyzing both theoretical approaches. The initial disagreement of the results between the two theories motivated us to rederive the formula for the error rate in the kinetic model. The error computed with the new method resulted in excellent agreement between both theoretical approaches and with Monte Carlo simulations. Furthermore, our theoretical analysis shows that the kinetic discrimination controls the error, even when the energy difference between adding the right and wrong products is very small. Our theoretical investigation gives important insights into the physical-chemical properties of complex biological processes by providing the quantitative framework to evaluate them.

Thermodynamic limits of the depolymerization of poly(olefin)s using mechanochemistry.

Mechanochemistry is a promising approach for chemical recycling of commodity plastics, and in some cases depolymerization to the monomer(s) has been reported. However, while poly(olefin)s comprise the largest share of global commodity plastics, mechanochemical depolymerization of these polymers in standard laboratory-scale ball mill reactors suffers from slow rates. In this work, the observed reactivities of poly(styrene), poly(ethylene) and poly(propylene) are rationalized on the basis of thermodynamic limitations of their depolymerization by depropagation of free radical intermediates. In addition, subsequent phase partitioning equilibria for the removal of monomers from the reactor via a purge gas stream are discussed for these polymers. For poly(styrene), a typical vibratory ball mill supplies just enough energy for its depolymerization to be driven by either thermal hotspots or adiabatic compression of the impact site, but the same energy supply is far from sufficient for poly(propylene) and poly(ethylene). Meanwhile, removal of styrene from the reactor is thermodynamically hindered by its lower volatility, but this is not an issue for either propylene or ethylene. The implications of these thermodynamic limitations for mechanochemical reactor design and potential for mechanocatalytic processes are highlighted.

Studies of caprolactam and cyclic oligomer content, molecular weight, and thermal properties during melt post-polycondensation of polycaprolactam in the film state

In industrial application of polycaprolactam, the content of caprolactam monomer and oligomers, as well as their average molecular weight are the key factors. In this work, a liquid-chromatography/time-of-flight mass spectrometry (LC-TOF-MS) method and an advanced polymer chromatography/multi-angle laser light scattering/refractive index detector (APC-MALLS-RID) hyphenated technique have been developed as a quantitative analysis method for monomer and cyclic oligomers, and a means of determining the molecular weight of polycaprolactam. The melt post-polycondensation reaction characteristics of polycaprolactam films and the variation in monomers and cyclic oligomers (C2–C7) content have been systematically investigated. The results showed excellent repeatability and stability of quantitative test results on monomers and cyclic oligomers in polycaprolactam. The content of monomers and cyclic oligomers in the polycaprolactam decreased from 10.21% to 1.52% at 260 °C and 50 Pa for 60 min, and the average molecular weight increased from 1.53×104 g/mol to 2.36×104 g/mol. Additionally, the results of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) experiments showed that the thermal stability and crystallization properties of polycaprolactam were improved after the film melt post-polycondensation reaction. Thus, this melt post-polycondensation method can realize the efficient removal of monomers and cyclic oligomers (compared with the traditional hot water extraction method) and afford high-quality polycaprolactam with high average molecular weight.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.

Enhancing the Genetic Diversity of FXI Deficiency: Structural Variants and New Cross Reactive Positive Variants

Introduction Factor XI (FXI) is a procoagulant element that circulates in plasma as a homodimer. It participates in both intrinsic and extrinsic coagulation pathways, as it is activated by both FXIIa and thrombin. Elevated levels of FXI are significantly associated with a high risk of thrombosis. FXI deficiency, considered a rare disorder in Caucasians, increases the risk of bleeding and protects against thrombosis. The identification of new naturally occurring genetic defects that cause FXI deficiency may help to identify new key domains and residues in the folding, secretion, dimerization and function of this molecule, as well as define its clinical impact. The aim of this study was to identify and characterize new genetic defects causing FXI deficiency. Methods We studied 91 unrelated Caucasian families with FXI deficiency (FXI:C<70%) identified mainly by prolonged aPTT. The entire F11 gene was sequenced by high-throughput sequencing (NGS). Structural variants (SVs), identified by MLPA, were characterized by nanopore long read sequencing using real-time enrichment methods on a MinION device, and validated by PCR and Sanger sequencing. Plasma FXI was characterized by functional methods using chromogenic and coagulation assays, and by immunological methods under both basal and silica-activated conditions. In addition, recombinant expression of wild-type (WT) and selected FXI variants was performed in HEK293. Results Thirty-one different F11 variants were identified. Three of them were novel SVs, being characterized at the nucleotide level by nanopore sequencing. They included two partial duplications, one involving exons 8-9 and the other exon 14; and one complete gene deletion spanning 7 MB including 142 genes. This was a de novo mutation originating in the paternal allele potentially during spermatogenesis. Interestingly, in all cases, repetitive elements were detected at the breakpoints. We also identified two small INDELs and 26 SNVs with different consequences (splicing defects, N=2; nonsense, N=3; and missense, N=21). We highlight 6 missense variants that, without affecting secretion (cross-reactive material, CRM+), produced FXI:C deficiency. Three of them were located in the catalytic domain; p.Pro538Leu, p.Glu565Lys and p.Cys599Tyr. Two variants disrupted the disulfide bridges p.Phe295Cys and p.Cys339Phe. The p.Phe295Cys was identified in a hemizygous state in a patient with severe FXI deficiency (FXI:C=4%). It produced two aberrant circulating FXI dimers through a potential shuffling of intramolecular disulfide bonds that were also observed in the recombinant protein. p.Cys339Phe affecting the residue involved in homodimerization was identified in 2 unrelated subjects with mild FXI deficiency. These individuals had FXI monomers in plasma that were activated by silica. Recombinant expression of this variant secreted only monomers, whereas WT expression in HEK293 secreted FXI dimers and monomers in the same proportion. The p.Arg268His variant located in the Apple 3 domain was identified in two subjects, one of them in homozygous state. Of note, we also detected 6 carriers of a variant affecting the same residue but causing CRM- deficiency (p.Arg268Cys). Most cases had not or only mild bleeding events which were not related to the mutation, type of deficiency, or FXI levels, although cases with severe deficiency presented more events. We point out a severe haemorrhage with fatal consequences during COVID-19 infection in a heterozyous carrier of the CRM+ p.Arg268His. Conclusions We confirm the genetic heterogeneity causing FXI deficiency in Caucasians. SVs affecting F11, probably caused by recombination of repetitive elements, are heterogeneous in type and extent and may not be detected by conventional short-read sequencing methods. Although most F11 variants cause type I defects, we have identified 6 type II or CRM+ deficiencies, and describe the first CRM+ variant affecting the Apple 3 domain. The data show that mutations in the catalytic domain or in aberrant disulfide bonds can allow secretion of inactive dimer variants. In addition, for the first time we describe the detection of monomeric variants in plasma. This, together with the similar secretion of WT FXI dimers and monomers in HEK293, suggests the relevance of Cys339 for the removal of WT monomers from the circulation.

Osmotic-Enhanced-Photoelectrochemical Hydrogen Production Based on Nanofluidics

Osmotic-Enhanced-Photoelectrochemical Hydrogen Production Based on Nanofluidics

Solvent-Free Production by Extrusion of Bio-Based Poly(glycerol-co-diacids) Sheets for the Development of Biocompatible and Electroconductive Elastomer Composites.

Faced with growing global demand for new potent, bio-based, biocompatible elastomers, the present study reports the solvent-free production of 13 pure and derived poly(glycerol-co-diacid) composite sheets exclusively using itaconic acid, sebacic acid, and 2,5-furandicarboxylic acid (FDCA) with glycerol. Herein, modified melt polycondensation and Co(II)-catalyzed polytransesterification were employed to produce all exploitable prepolymers, enabling the easy and rapid manufacturing of elastomer sheets by extrusion. Most of our samples were loaded with 4 wt% of various additives such as natural polysaccharides, synthetic polymers, and/or 25 wt% sodium chloride as porogen agents. The removal of unreacted monomers and acidic short oligomers was carried out by means of washing with NaHCO3 aqueous solution, and pH monitoring was conducted until efficient sheet surface neutralization. For each sheet, their surface morphologies were observed by Field-emission microscopy, and DSC was used to confirm their amorphous nature and the impact of the introduction of every additive. The chemical constitution of the materials was monitored by FTIR. Then, cytotoxicity tests were performed for six of our most promising candidates. Finally, we achieved the production of two different types of extrusion-made PGS elastomers loaded with 10 wt% PANI particulates and 4 wt% microcrystalline cellulose for adding potential electroconductivity and stability to the material, respectively. In a preliminary experiment, we showed the effectiveness of these materials as performant, time-dependent electric pH sensors when immersed in a persistent HCl atmosphere.

Study on Recrystallization Process of Poly(DL-lactide)/Poly(ethylene glycol) Diblock Copolymer

Poly (DL-lactide)/Poly (ethylene glycol) diblock copolymer is successfully synthesized with mPEG and DL-LA as main raw materials, and the residual monomers of the polymer were removed by recrystallization. FT-IR is used to characterize the structure of the polymer. Gas chromatography is used to determine the residual monomers and residual solvent of the polymer. The solubility of polymer in different solvents was determined, and the effect of different solvent systems on the removal of residual monomers in copolymer were discussed. The results showed that the Poly(DL-lactide)/Poly(ethylene glycol) diblock copolymer was recrystallized with acetone/ isopropyl ether solvent system has the best ability to remove residual monomers, and the residual monomers of the obtained polymer are less than 0.01 wt%, the total residual solvent is only 0.24 wt%, and the productivity is as high as 95.5 wt%.

Bioinspired Interface Design of Sewable, Weavable, and Washable Fiber Zinc Batteries for Wearable Power Textiles

AbstractWearable electronics have great demands for flexibility, high‐performance, and customized batteries that require the significant advancement of renewable and weavable power fiber design. Here, weavable, sewable, and washable aqueous zinc batteries (AZBs)‐based power fibers are developed by the synergistic interfacial design of the quinone‐rich polydopamine as organic redox‐active cathodes and nano‐binders on the carbon substrate simultaneously. By the removal of soluble monomers or oligomers and the boosted ratios of the quinone groups, the polydopamine polymers as organic redox‐active cathodes in AZBs deliver large specific capacity (372.3 mA h g−1 at 50 mA g−1), excellent toughness, and long‐term cyclic durability (80% retention over 1700 cycles at 1000 mA g−1). Moreover, the power textiles with custom‐tailored patterns can be prepared by the direct use of the fiber electrodes as the threads for a sewing and loom machine. The sewn or woven power textiles display stable electrochemical performances that can power various electronic devices under different bending conditions, even after washing for 3 h. This work provides great potential for large‐scale production of AZBs with high‐performance for next‐generation wearable energy‐storage devices.

Removal of Aβ Oligomers from the Blood: A Potential Therapeutic System for Alzheimer's Disease.

PurposeAmyloid-β protein (Aβ) is one of the causative proteins of Alzheimer’s disease. We have been developing extracorporeal blood Aβ-removal systems as a method for enhancing Aβ clearance from the brain. We reported previously that medical adsorbents and hemodialyzers removed Aβ monomers from peripheral blood, which was associated with influx of Aβ monomers from the brain into the bloodstream. Our intent here was to develop a method to promote clearance of Aβ oligomers and to provide an estimate of the molecular size of intact Aβ oligomers in plasma.MethodsTwo hollow-fiber devices with different pore sizes (Membranes A and B) were evaluated as removers of Aβ oligomers with human plasma in vitro. The concomitant removal of Aβ oligomers and monomers was investigated by using Membrane B and hexadecyl alkylated cellulose beads or polysulfone hemodialyzers. Double-filtration plasmapheresis with Membrane A was investigated as an approach for the removal of plasma Aβ oligomers in humans.ResultsAβ oligomers were effectively removed by both Membranes A and B. The increase of Aβ oligomers in plasma was observed just after the removal of plasma Aβ oligomers in humans. The intact molecular size of major Aβ oligomers in the plasma was estimated to be larger than albumin at approximately 60 kDa or more. Additionally, the concomitant removal of Aβ monomers and oligomers evoked dissociation of larger Aβ oligomers into smaller ones and monomers.ConclusionAβ oligomers were cleared from plasma both in vitro and in human subjects by using hollow-fiber membranes with large pores, indicating that their intact sizes were mostly larger than 60 kDa. Aβ oligomers in peripheral circulation were increased after some clearances in human. Further investigation will determine whether the Aβ oligomers detected in circulation after clearance were via influx from the brain.

Effects of triethylene glycol dimethacrylate and hydroxyethyl methacrylate on macrophage polarization.

To evaluate the effects of hydrophilic dental resin monomers, triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA), on the polarization of a human monocyte cell line (THP-1). THP-1 cells were treated with resin monomers at noncytotoxic concentrations for 48h and were analysed for CD86 and CD206 expressions using flow cytometry. The cells were stimulated for polarization in the presence of resin monomers (co-treatment) or after treatment with monomers (pre-treatment). CD86 and CD206 mRNA in co-treated cells was evaluated using quantitative real-time polymerase chain reaction. The release of TNF-α and TGF-β by pre-treated and co-treated cells was assessed using enzyme-linked immunosorbent assay. Morphological changes of macrophages during polarization were observed using bright-field microscopy. One-way analysis of variance was used for statistical analysis. TEGDMA (1mmolL-1 ) and HEMA (2mmolL-1 ) did not induce CD86 and CD206 expressions in THP-1 cells but rather inhibited their expressions in the co-treated cells. The inhibitory effects also appeared at the transcription level. However, the expression of surface markers was not affected by pre-treatment with resin monomers. The release of TNF-α and TGF-β by M1- and M2-stimulated cells, respectively, was suppressed by co-treatment (P<0.05). Microscopic studies revealed that co-treatment with resin monomers suppressed polarization-associated morphological changes such as cell volume increase. TEGDMA and HEMA inhibited macrophage polarization to both M1 and M2 at the transcription level, and the inhibitory effects disappeared upon the removal of resin monomers from the cell culture.

Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.22-μm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 μm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (<100 nm), suppressed the aggregate growth. The presented findings demonstrate that a removal treatment with a specific adsorbent of non-native IgG conformers enables long-term stable storage of therapeutic IgG molecules and will facilitate mitigation of the immunogenicity of IgG preparations.

Studies on the properties of RO membranes for salt and boron removal: Influence of thermal treatment methods and rinsing treatments

The physicochemical properties and performance of thin film composite (TFC) membranes depend on its synthesis conditions. In this study, TFC membranes were fabricated using interfacial polymerization (IP) method and the effects of heat treatment methods and post-IP rinsing (prior to heat curing step) on the membrane performances were investigated. Keeping the substrate in minimal heat exposure could prevent substrate pore annealing that potentially reduces the membrane water permeability. Evidently, the membranes with only polyamide (PA) layer being heat-treated exhibited >250% enhancement in pure water flux (PWF) compared to the membranes where both PA and substrate layer were heat-treated. Also, the membranes rinsed with pure n-hexane tended to display higher PWF without significantly decreasing solute rejection, possibly due to the reduced membrane cross-linking following the removal of unreacted monomers and excess solvent from the membrane surface. The membrane performance became practically the same after post-IP rinsing, regardless of the solvent used in the IP reaction, suggesting that the disparity in the membrane performance is mainly caused by the difference in solvent evaporation rate during heat treatment. The variations in solvent surface tension and viscosity during the IP reaction meanwhile did not play a key role affecting PA layer chemistry and performance.

Sustainable commercial nanocrystalline cellulose manufacturing process with acid recycling

Nanocrystalline cellulose (NCC) is a biomaterial having potential applications in a wide range of industries. It is industrially produced by concentrated acid hydrolysis of cellulosic materials. In this process, the sulfuric acid rich liquor can be concentrated and reused. However, removal of sugar monomers and oligomers is necessary for such recycling. Membrane and ion exchange technology can be employed to remove sugars; however, such technologies are not efficient in meeting the quality required to recycle the acid solution. As a part of the present study, activated carbon (AC) has been evaluated as an adsorbent for sugar removal from the acidic solution generated during commercial nanocrystalline cellulose manufacturing process. Almost complete removal of sugar can be achieved by this approach. The maximum sugar removal observed during this study was 3.4g/g of AC. Based on this finding, a sustainable method has been proposed for commercial nanocrystalline cellulose manufacturing.

Poly(methyl methacrylate-co-butyl acrylate-co-acrylic acid): Physico-chemical characterization and targeted dye sensitized solar cell application

A random poly(methyl methacrylate-co-butyl acrylate-co-acrylic acid) [P(MMA-co-BA-co-AA)] was synthesized via emulsion polymerization utilizing thermally-activated persulfate radicals. The successful synthesis of high molecular weight copolymer at high monomer conversion rate was confirmed by NMR study. Various techniques were employed to characterize the structure, electrical and thermal properties of copolymer. The removal of unreacted monomers from the end product by casting technique was rationalized from the disappearance of CC FTIR characteristic peak in the cast film. The drying of the emulsion above the glass transition temperature (Tg=24°C) yields an amorphous film with smooth surface morphology. The suitability of the formed continuous film as a base polymer for iodide-based electrolytes in dye sensitized solar cell (DSSC) fabrication was investigated. The polymeric solid state electrolytes (PSSE) were formulated by varying the doping concentrations of sodium iodide (NaI) in fixed ratio of iodine (I2) and mixtures of ethylene carbonate: propylene carbonate (EC:PC, 1:1). The DSSC fabricated using PSSE with 15wt.% NaI showed the highest solar-to-electrical energy conversion efficiency (η) of 3.22% under 100mWcm−2 sunlight intensity. This implies that the physico-chemical properties of the synthesized copolymer are suitable for the development of photoelectrochemical device.

Kinetics of formation of bile salt micelles from coarse-grained Langevin dynamics simulations.

We examine the mechanism of formation of micelles of dihydroxy bile salts using a coarse-grained, implicit solvent model and Langevin dynamics simulations. We find that bile salt micelles primarily form via addition and removal of monomers, similarly to surfactants with typical head-tail molecular structures, and not via a two-stage mechanism - involving formation of oligomers and their subsequent aggregation to form larger micelles - originally proposed for bile salts. The free energy barrier to removal of single bile monomers from micelles is ≈2kBT, much less than what has been observed for head-tail surfactants. Such a low barrier may be biologically relevant: it allows for rapid release of bile monomers into the intestine, possibly enabling the coverage of fat droplets by bile salt monomers and subsequent release of micelles containing fats and bile salts - a mechanism that is not possible for ionic head-tail surfactants of similar critical micellar concentrations.

Isolation and analysis of amyloid-β 1-42 monomer and oligomers in liquid droplets using an immunoaffinity membrane.

Monomeric molecules such as amyloid-β can aggregate and transform into oligomeric and fibrous forms, which are implicated in the development and progression of Alzheimer's disease. Novel analytical techniques for the formation of oligomers are required to examine the neurotoxic amyloid-β oligomers involving fibrils. After isolating amyloid-β monomer 1-42 using a biotinylated antibody bound to membrane-immobilized avidin (immunoaffinity membrane), their masses were determined by MALDI-TOF MS. Fluorometric determination of more than 0.5μM of aggregated amyloid-β in pipette droplets was performed after aggregation and dilution of 1mM amyloid-β. Thus, large (>105nm) amyloid-β oligomers in microliter volumes of fluids were isolated using the immunoaffinity membrane and quantitatively analyzed after removal of amyloid-β monomers and small oligomers by non-denaturing electrophoresis. In addition, amyloid-β oligomers were specifically isolated from a mixture of human plasma and aggregated amyloid-β and then fluorometrically analyzed. Our results show that the combination of immunoaffinity membrane-binding and fluorescence determinations together with one drop analysis could be used to isolate and detect huge neurotoxic amyloid-β oligomers such as fibrils in plasma samples.

Thermosensitive hybrid hydrogels with silica nanoparticle-cross-linked polymer networks

A simple methodology for the preparation of thermosensitive organic–inorganic hybrid hydrogels using silica nanoparticle-mediated polymer networks is described. A thermosensitive copolymer poly[N-isopropylacrylamide-co-(3-methacryloxypropyltrimethoxysilane)], (pNS), with reactive side chains (SiOCH3) was first synthesized by free radical polymerization using N-isopropylacrylamide (NIPAAm) and 3-methacryloxypropyltrimethoxysilane (MAPTS). Then, simple mixing of the aqueous solution of this copolymer with silica nanoparticle (SiP) suspensions at room temperature led to the formation of thermosensitive hybrid hydrogels cross-linked with silica nanoparticles (SiP) which did not require any other processing like washing for the removal of unreacted monomers and initiators. The effects of SiP content on gelation abilities, temperature-responsive behaviors, swelling and deswelling kinetics, and mechanical properties of the hydrogels were investigated. The results showed that transparent hybrid hydrogels with adjustable network structures were obtained within a few minutes to a couple of hours depending on the concentration of the copolymers and the silica nanoparticles. The hybrid hydrogels exhibited a lower critical solution temperature (LCST) of around 33°C with no significant deviation from conventional poly(N-isopropylacrylamide) hydrogels; the LCST was not significantly affected by the concentration of silica nanoparticles (which in these systems serve as the cross-linkers). In addition, the hydrogels showed significantly large equilibrium swelling ratios, improved mechanical strength, and suitable deswelling behavior, which can easily be tuned by varying the composition of the hybrid hydrogels.

Recombinant Cannabinoid Type 2 Receptor in Liposome Model Activates G Protein in Response to Anionic Lipid Constituents

Human cannabinoid type 2 (CB(2)) receptor expressed in Escherichia coli was purified and successfully reconstituted in the functional form into lipid bilayers composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesteryl hemisuccinate (CHS). Reconstitution was performed by detergent removal from the protein/lipid/detergent mixed micelles either on an adsorbent column, or by rapid dilution to below the critical micelle concentration of detergent followed by removal of detergent monomers on a concentrator. Proteoliposomes prepared at a protein/phospholipid/CHS molar ratio of 1/620-650/210-220 are free of detergent as shown by (1)H NMR, have a homogeneous protein/lipid ratio shown by isopycnic gradient ultracentrifugation, and are small in size with a mean diameter of 150-200 nm as measured by dynamic light scattering. Functional integrity of the reconstituted receptor was confirmed by quantitative binding of (2)H-labeled agonist CP-55,940-d(6) measured by (2)H magic angle spinning NMR, as well as by activation of G protein. The efficiency of G protein activation by agonist-bound CB(2) receptor was affected by negative electric surface potentials of proteoliposomes controlled by the content of anionic CHS or POPS. The activation was highest at an anionic lipid content of about 50 mol %. There was no correlation between the efficiency of G protein activation and an increase of hydrocarbon chain order induced by CHS or cholesterol. The results suggest the importance of anionic lipids in regulating signal transduction by CB(2) receptor and other class A GPCR. The successful reconstitution of milligram quantities of pure, functional CB(2) receptor enables a wide variety of structural studies.

Removal of monomer delignification products by laccase from Trametes versicolor

The influence of a laccase from Trametes versicolor on the removal of phenolic monomers in liquid hot water pretreated wheat straw supernatants (LHW-S) was examined. Beside the total phenol content derived by Folin–Ciocalteu (FC-) assay, phenolic monomers were measured via headspace-solid phase micro-extraction (HS-SPME)/GC–MS. A notable decrease of the phenols was achieved using 0.2 and 0.5U/mL laccase whilst higher dosage showed no improvement. Nearly all kind of monomer phenolic compounds identified in the LHW-S were found to be removed after 24h. However, acetophenone and 4-hydroxybenzaldehyde (HBA) were obviously not affected by laccase. Summarizing, three laccase reaction groups (LRG) of phenolic monomers could be classified: immediate removal (LRG-A), degradation after 1day (LRG-B), no effect of laccase (LRG-C). Additionally, HS-SPME/GC was found to be a powerful tool to study the reaction of laccase and phenolic monomers in complex lignocellulose derived solutions.