Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

[Effect of a novel pH-responsive tertiary amine monomer dodecylmethylaminoethyl methacrylate modified resin adhesive on biofilm formation of Streptococcus mutans and Lactobacillus casei in vitro].

Objective: To explore the application prospect of a new pH-responsive tertiary amine monomer dodecylmethylaminoethyl methacrylate (DMAEM) modified resin adhesive (DMAEM@RA) in the prevention and treatment of secondary caries. Methods: Five percents DMAEM was added to the resin adhesive to synthesize DMAEM@RA for modifying. Streptococcus mutans (Sm) and Lactobacillus casei (Lc) biofilms were cultured on resin adhesive and DMAEM@RA, respectively. The culture systems were set up at pH=7.4, 6.0, 5.5, and 5.0. The antimicrobial activity of DMAEM@RA was evaluated by quantitative PCR. The effects of DMAEM@RA on biofilm thickness, bacterial amount, and extracellular polysaccharides were studied by scanning electron microscope (SEM) and extracellular polysaccharide staining. Real-time fluorescence quantitative PCR was used to study the effect of DMAEM@RA on the expression levels of cariogenic genes in Sm. Results: DMAEM@RA could significantly reduce the amount of Sm and Lc under acidic conditions, especially Lc. At pH=5.0, the logarithm value of co-cultured Sm bacteria [lg (CFU/ml)] in DMAEM@RA group (7.58±0.01) was significantly lower than that in control group (7.87±0.03) (t=14.32, P<0.001), and the logarithm value of Lc bacteria [lg (CFU/ml)] (7.29±0.04) was also significantly lower than that in control group (7.93±0.15) (t=6.93, P=0.002). SEM observed that the bacteria decreased and the cell fragments appeared in DMAEM@RA group. In addition, DMAEM@RA significantly reduced the biomass of extracellular polysaccharides in the dual-species biofilm under acidic conditions. At pH=5.0, the biomass of extracellular polysaccharides in DMAEM@RA group [(25.13±3.14) mm3/mm2] was significantly lower than that in the control group [(42.66±7.46) mm3/mm2] (t=3.75, P=0.020). DMAEM@RA could significantly up-regulate the expressions of gtfB and gtfC genes in Sm under acidic conditions. At pH=5.0, gtfB and gtfC genes were significantly up-regulated by (14.64± 0.44) times and (2.99±0.20) times, respectively (t=-42.74, P<0.001; t=-13.55, P<0.001). Conclusions: The DMAEM@RA has a good antibacterial effect under acidic conditions, demonstrating that it has a good potential to prevent the occurrence and development of secondary caries.

TMT-based quantitative proteomic analysis reveals the underlying mechanisms of glycidyl methacrylate-induced 16HBE cell malignant transformation.

Glycidyl methacrylate (GMA) has been widely used as tackifying/crosslinking copolymer monomer in the industrial section. Occupational and environmental exposure to GMA is inevitable. GMA is classified as a Group 2 A carcinogen. However, it still lacks a sufficient understanding of its carcinogenicity at the protein level. The major pathways and players during the malignant transformation process remain unknown. In this study, we first established and characterized a malignant transformation model using human bronchial epithelial (16HBE) cells exposed to 8 µg/mL GMA. Then the proteomics approach, western-blot analysis as well as quantitative PCR (qPCR) analysis were employed to investigate its underlying mechanisms of carcinogenicity. Our results showed that the 16HBE cells exposed to GMA and passaged to the 40th generation had undergone a malignant transformation. Proteomic analysis revealed that 123 proteins were significantly up-regulated while 160 proteins were down-regulated during the process of malignant transformation. Importantly, further pathway analysis identified the extracellular matrix-receptor (ECM-receptor) interaction pathway to be one of the major players mediating the process and most of the differentially expressed proteins (DEPs) were up-regulated, including two vital proteins, CD44 and MMP14, as well as members from integrin family. These results provide direct proteomic evidence that DEPs related to the ECM-receptor interaction pathway play an active role in reinforcing the carcinogenicity of GMA. The findings of this study might deepen our understanding of the underlying mechanisms of GMA carcinogenicity and thus facilitate the risk assessment of GMA.

Implementation of Photosensitive, Injectable, Interpenetrating, and Kartogenin-Modified GELMA/PEDGA Biomimetic Scaffolds to Restore Cartilage Integrity in a Full-Thickness Osteochondral Defect Model.

Cartilage defects caused by mechanical tear and wear are challenging clinical problems. Articular cartilage has unique load-bearing properties and limited self-repair ability. The current treatment methods, such as microfractures and autogenous cartilage transplantation to repair full-thickness cartilage defects, have apparent limitations. Tissue engineering technology has the potential to repair cartilage defects and directs current research development. To enhance the regenerative capacities of cartilage in weight-bearing areas, we attempted to develop a biomimetic scaffold loaded with a chondroprotective factor that can recreate structure, restore mechanical properties, and facilitate anabolic metabolism in larger joint defects. For enhanced spatial control over both bone and cartilage layers, it is envisioned that biomaterials that meet the needs of both tissue components are required for successful osteochondral repair. We used gelatin methacrylate (GELMA) and polyethylene glycol diacrylate (PEGDA) light-cured dual-network cross-linking modes that can significantly increase the mechanical properties of scaffolds and are capable of restoring function and prolonging the degradation time. Once the hydrogel complex was injected into the osteochondral defect, in situ UV light curing was applied to seamlessly connect the defect repair tissue with the surrounding normal cartilage tissue. The small molecule active substance kartogenin (KGN) can promote cartilage repair. We encapsulated KGN in biomimetic scaffolds so that, as the scaffold degrades, scaffold-loaded KGN was slowly released to induce endogenous mesenchymal stem cells to home and differentiate into chondrocytes to repair defective cartilage tissue. Our experiments have proven that, compared with the control group, GELMA/PEGDA + KGN repaired cartilage defects and restored cartilage to hyaline cartilage. Our study suggests that implementing photosensitive, injectable, interpenetrating, and kartogenin-modified GELMA/PEDGA biomimetic scaffolds may be a novel approach to restore cartilage integrity in full-thickness osteochondral defects.

Mechanosensor YAP cooperates with TGF-ß1 signaling to promote myofibroblast activation and matrix stiffening in a 3D model of human cardiac fibrosis.

Cardiac fibrosis is characterized by a maladaptive remodeling of the myocardium, which is controlled by various inflammatory pathways and cytokines. This remodeling is accompanied by a significant stiffening of the matrix, which may contribute to further activate collagen synthesis and scar formation. Evidence suggests that TGF-ß1 signaling, the main pro-fibrotic pathway in cardiac fibrosis, might cooperates with the Hippo transcriptional pathway by activating YAP. To directly test the cooperation of mechanical cues and paracrine signaling in cardiac fibrosis, we developed a 3D model of cardiac extracellular matrix remodeling by generating tissue blocks with Gelatin Methacrylate, a bioink with tunable stiffness, and human cardiosphere-derived stromal cells. Using this strategy, we assessed the cooperation of TGF-ß1 and YAP transcriptional factor to matrix compaction. Using mechanical compression tests, Masson's trichrome staining, immunofluorescence, and RT-qPCR, we demonstrate that pharmacological inhibition of YAP complex reverts almost completely the pro-compaction phenotype and the matrix-remodeling activity of cells treated with TGF-ß1. Our data show a direct connection between the classical pro-fibrotic signaling driven by TGF-ß1 and the mechanically activated pathways under the control of YAP in cardiac remodeling. Treatment with the elective drug targeting YAP is sufficient to override this cooperation with potential benefits for anti-fibrotic therapeutic applications. STATEMENT OF SIGNIFICANCE: Heart failure is a pathology in continuous growth worldwide, characterized by a progressive fibrosis, which decreases the pumping efficiency of the heart. Experimental evidences suggest that fibroblasts, normally responsible for the turnover of the cardiac matrix, are involved in myocardial fibrosis by differentiating into 'myofibroblasts'. These cells remodel extensively the cardiac extracellular matrix and deposit abundant collagen with a consequent increase in stiffness. In the present contribution, we propose a new 3D model of cell-mediated cardiac extracellular matrix stiffening to investigate the mechano-chemical mechanisms underlying the onset of the pathology. We also consolidate a pharmacological treatment able to prevent the pathological activation of fibroblasts with potential benefits for anti-fibrotic treatment of the failing heart.

Antifouling poly(PEGMA) grafting modified titanium surface reduces osseointegration through resisting adhesion of bone marrow mesenchymal stem cells.

As an alternative strategy to achieve the desired bone augmentation, tenting screw technology (TST) has considerably broadened the indications for implant treatment. Titanium tenting screws are typically used in TST to maintain the space for bone regeneration. However, a high degree of osteogenic integration complicate titanium tenting screw removal and impact the bone healing micro-environment. Previous efforts have been focused on modifying titanium surfaces to enhance osseointegration while ignoring the opposite process. Due to the vital role of bone marrow mesenchymal stem cells (BMSCs) in bone regeneration, it might be feasible to reduce osseointegration around titanium tenting screws by resisting the adhesion of BMSCs. Herein, poly(ethylene glycol)methyl ether methacrylate (poly(PEGMA)) with an optimal length of PEG chain was incorporated with a Ti surface in terms of surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP). The cell apoptosis analysis showed that the new surface would not induce the apoptosis of BMSCs. Then, the adhesive and proliferative behaviors of BMSCs on the surface were analyzed which indicated that the poly(PEGMA) surface could inhibit the proliferation of BMSCs through resisting the adhesion process. Furthermore, in vivo experiments revealed the presence of the poly(PEGMA) on the surface resulted in a lower bone formation and osseointegration compared with the Ti group. Collectively, this dense poly(PEGMA) surface of Ti may serve as a promising material for clinical applications in the future. STATEMENT OF SIGNIFICANCE: The poly(ethylene glycol)methyl ether methacrylate (poly(PEGMA)) with an optimal length of PEG chain was grafted onto a Ti surface by surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP). The PEGMA surface could reduce the osteogenic integration by preventing the adhesion of cells, resulting in a lower pullout force of the modified implant and thereby desirable and feasible applications in dental surgery.

Graded-Three-Dimensional Cell-Encapsulating Hydrogel as a Potential Biologic Scaffold for Disc Tissue Engineering.

BACKGROUND: Intervertebral disk (IVD) degeneration, which can cause lower back pain, is a major predisposing factor for disability and can be managed through multiple approaches. However, there is no satisfactory strategy currently available to reconstruct and recover the natural properties of IVDs after degeneration. As tissue engineering develops, scaffolds with embedded cell cultures have proved critical for the successful regeneration of IVDs. METHODS: In this study, an integrated scaffold for IVD replacement was developed. Through scanning electron microscopy and other mechanical measurements, we characterized the physical properties of different hydrogels. In addition, we simulated the physiological structure of natural IVDs. Nucleus pulposus (NP) cells and annulus fibrosus-derived stem cells (AFSCs) were seeded in gelatin methacrylate (GelMA) hydrogel at different concentrations to evaluate cell viability and matrix expression. RESULTS: It was found that different concentrations of GelMA hydrogel can provide a suitable environment for cell survival. However, hydrogels with different mechanical properties influence cell adhesion and extracellular matrix component type I collagen, type II collagen, and aggrecan expression. CONCLUSION: This tissue-engineered IVD implant had a similar structure and function as the native IVD, with the inner area mimicking the NP tissue and the outer area mimicking the stratified annulus fibrosus tissue. The new integrated scaffold demonstrated a good simulation of disc structure. The preparation of efficient and regeneration-promoting tissue-engineered scaffolds is an important issue that needs to be explored in the future. It is hoped that this work will provide new ideas and methods for the further construction of functional tissue replacement discs.

2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells.

Extracellular ATP (adenosine triphosphate) and transient receptor potential ankyrin 1 (TRPA1) channels are involved in calcium signaling in odontoblasts and dental pain. The resin monomer 2-hydroxyethyl methacrylate (HEMA), used in dental restorative procedures, is related to apoptotic cell death via oxidative stress. Although the TRPA1 channel is highly sensitive to reactive oxygen species (ROS), the effect of HEMA-induced ROS on ATP release to the extracellular space and the TRPA1 channel has not been clarified in human dental pulp. In this study, we investigated the extracellular ATP signaling and TRPA1 activation by HEMA-derived ROS in immortalized human dental pulp cells (hDPSC-K4DT). Among the ROS-sensitive TRP channels, TRPA1 expression was highest in undifferentiated hDPSC-K4DT cells, and its expression levels were further enhanced by osteogenic differentiation. In differentiated hDPSC-K4DT cells, 30 mM HEMA increased intracellular ROS production and ATP release, although 3 mM HEMA had no effect. Pretreatment with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone) or TRPA1 antagonist HC-030031 suppressed HEMA-induced responses. These results suggest that ROS production induced by a higher dose of HEMA activates the TRPA1 channel in human dental pulp cells, leading to ATP release. These findings may contribute to the understanding of the molecular and cellular pathogenesis of tertiary dentin formation and pain in response to dental biomaterials.

Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer.

Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.

Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors.

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

Biomimetic Joint Paint for Efficient Cartilage Repair by Simultaneously Regulating Cartilage Degeneration and Regeneration in Pigs.

Irregular partial-thickness cartilage defect is a common pathogenesis of osteoarthritis (OA) with no available treatment in clinical practice. Currently, cartilage tissue engineering is only suitable for a limited area of full-thickness cartilage defect. Here, we design a biomimetic joint paint for the intractable partial-thickness cartilage defect repair. The joint paint, composed of a bridging layer of chondroitin sulfate and a surface layer of gelatin methacrylate with hyaluronic acid, can quickly and tightly adhere to the cartilage defect by light activation. Being treated by the joint paint, the group of rabbit and pig models with partial-thickness cartilage defects showed a restoration of a smooth cartilage surface and the preservation of normal glycosaminoglycan content, whereas the untreated control group exhibited serious progressive OA development. This paint treatment functions by prohibiting chondrocyte apoptosis, maintaining chondrocyte phenotype, and preserving the content of glycosaminoglycan in the partial-thickness cartilage defects. These findings illustrated that the biomimetic joint paint is an effective and revolutionary therapeutics for the patients with noncurable partial-thickness cartilage defects.

Bacterial Redox Potential Powers Controlled Radical Polymerization.

Microbes employ a remarkably intricate electron transport system to extract energy from the environment. The respiratory cascade of bacteria culminates in the terminal transfer of electrons onto higher redox potential acceptors in the extracellular space. This general and inducible mechanism of electron efflux during normal bacterial proliferation leads to a characteristic fall in bulk redox potential (Eh), the degree of which is dependent on growth phase, the microbial taxa, and their physiology. Here, we show that the general reducing power of bacteria can be subverted to induce the abiotic production of a carbon-centered radical species for targeted bioorthogonal molecular synthesis. Using two species, Escherichia coli and Salmonella enterica serovar Typhimurium as model microbes, a common redox active aryldiazonium salt is employed to intervene in the terminal respiratory electron flow, affording radical production that is mediated by native redox-active molecular shuttles and active bacterial metabolism. The aryl radicals are harnessed to initiate and sustain a bioorthogonal controlled radical polymerization via reversible addition-fragmentation chain transfer (BacRAFT), yielding a synthetic extracellular matrix of "living" vinyl polymers with predetermined molecular weight and low dispersity. The ability to interface the ubiquitous reducing power of bacteria into synthetic materials design offers a new means for creating engineered living materials with promising adaptive and self-regenerative capabilities.

Molecular docking of metal ion immobilized ligands to proteins in affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) has become a widespread analytical and preparative separation method for therapeutic proteins, peptides nucleic acids, hormones, and enzymes. N-Methacryloyl-l-histidine Methyl Ester (MAH) monomer is recently used as a synthesized affinity ligand in IMAC. It is capable of chelating with many transition metal ions such as Zn2+ , Ni2+ , and Cu2+ ions through its histidine residue. In this way, proteins can bind selectively to these immobilized metal ions through MAH as a ligand in affinity chromatography. In this study, we applied the computational docking method on the interactions that occur between the MAH monomer and its complexes with Zn2+ ions as ligands and protein molecules as targets. MAH monomer was drawn and created using the Avogadro software as an optimization tool. Human insulin (Ins) molecule and horse heart cytochrome C (Cyt C) were selected as target proteins to interact with MAH monomer as affinity ligand. Automated docking software AutoDock v4.2 was used for docking of MAH monomer to Ins and Cyt C, respectively. The affinity ligand complexes with Zn2+ ions bound to one, two, and three moles of MAH were studied and compared separately. The lowest binding energies of Ins and Cyt C proteins in 1:1 mol ratio of MAH-Zn2+ were found as (-4.14) and (-4.92) kcal/mol, respectively.

A novel amino cellulose derivative using ATRP method: Preparation, characterization, and investigation of its antibacterial activity.

In this study, we prepared a novel amino cellulose derivative (benzyl cellulose-g-poly [2-(N,N-Dimethylamino)ethyl methacrylate]) via a homogeneous ATRP method. The successful synthesis of the novel amino cellulose was confirmed by FT-IR and 1H NMR. This study addressed the different characteristics of the prepared polymer including the thermal stability, solubility, and X-ray diffraction pattern. The antibacterial activity of the synthesized cellulose derivative was investigated using the diffusion disk method against both gram-negative (Escherichia coli, Salmonella enterica) and gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria. Based on the inhibition zone, it was confirmed that the prepared benzyl cellulose-g-PDMAEMA possesses acceptable antibacterial activity against Escherichia coli, Salmonella enterica, and Staphylococcus aureus while Bacillus subtilis is resistant to the prepared polymer. Also according to the inhibition zone, it was shown that benzyl cellulose-g-PDMAEMA has more impact on E. coli and Salmonella enterica than Staphylococcus aureus. Molecular dynamics simulation was also used to study the interaction of the synthesized cellulose derivative with a model membrane which presented atomistic details of the polymer-lipid interactions. According to the results obtained from the molecular dynamics simulation, the polymer was able to destabilize the structure of the membrane and clearly express its signs of degradation.

In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer.

PURPOSE: The development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR. METHODS: Four positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively. RESULTS: Cellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen). CONCLUSION: Positively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

Exploiting arginine distributions for the selective and efficient depletion of arginine-rich plasma proteins.

The number and arrangement of arginine (Arg) residues in protein chains contribute greatly to the selective capturing of proteins on a designed adsorbent consisting of organic phosphate functionalized fibrous SiO2 microspheres, and the efficient depletion of high abundance Arg-rich protein species from human plasma is achieved.

Engineered citrate synthase improves citramalic acid generation in Escherichia coli.

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.

Fabrication of a porous polymer membrane enzyme reactor and its enzymatic kinetics study in an artificial kidney model.

A porous polymer membrane-based d-amino acid oxidase (DAAO) reactor was developed that mimicked enzymatic activity in a renal ischemia model. Using glycidyl methacrylate as a biocompatible reactive monomer, poly(styrene-glycidyl methacrylate) was synthesized via a reversible addition fragment chain transfer polymerization technique. The prepared porous polymer membrane was used as a support to effectively immobilize DAAO. Compared to DAAO modified on nonporous polymer membrane and free DAAO in solution, the constructed porous polymer membrane-based DAAO enzyme reactor displayed 3-fold and 19-fold increase in enzymolysis efficiency, respectively. In addition, a chiral ligand exchange capillary electrophoresis system for DAAO was used to study DAAO enzymatic kinetics with d,l-methionine as the substrate. The proposed porous polymer membrane-based enzyme reactor showed excellent performance both on reproducibility and stability. Moreover, the enzyme reactor was successfully applied to mimic DAAO activity in a renal ischemia model. These results demonstrated that the enzyme could be efficiently immobilized onto a porous polymer membrane as an enzyme reactor and has great potential in mimicking the enzymatic activity in kidney.

Gellan gum and its methacrylated derivatives as in situ gelling mucoadhesive formulations of pilocarpine: In vitro and in vivo studies.

Gellan gum was chemically modified by the reaction with methacrylic anhydride to produce derivatives with 6, 14 and 49% methacrylation. The structure and substitution degrees of these derivatives were confirmed by 1H NMR- and FTIR-spectroscopy. These derivatives are more hydrophobic compared to pristine gellan and form turbid solutions in water. In vitro study performed with formulations of sodium fluorescein containing gellan gum and its methacrylated derivatives indicated that methacrylation enhances their retention on bovine conjunctival mucosa. In vivo experiments with the formulations of pilocarpine hydrochloride containing gellan gum and methacrylated derivatives have demonstrated that all polymers enhance the drug effect significantly, but best performance is observed for the polysaccharide with 6% methacrylation.

Enzymatic Synthesis of 2-(ß-Galactosyl)-ethyl Methacrylate by ß-Galactosidase from Pyrococcus woesei and Application for Glycopolymer Synthesis and Lectin Studies.

Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(ß-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed KD values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.