Synthesis of biocompatible BSA-GMA and two-photon polymerization of 3D hydrogels with free radical type I photoinitiator.

Although the development of three-dimensional (3D) printing technology is growing rapidly in the biomedical field, it remains a challenge to achieve arbitrary 3D structures with high resolution and high efficiency. Protein hydrogels fabricated by two- photon polymerization (TPP) have excellent mechanical properties, high precision, and 3D architecture. However, a large number of the amino acid group in bovine serum albumin (BSA) would be consumed when the protein-based hydrogels use dyes of free radical type II photoinitiators. In this study, we use glycidyl methacrylate (GMA) to modify BSA molecules to obtain a series of BSA-GMA materials, allowing the protein material to be two-photon polymerized with a water-soluble free radical type I photoinitiator. The precisely controllable 3D structure of the BSA-GMA hydrogel was fabricated by adjusting the concentration of the precursor solution, the degree of methacrylation, and the processing parameters of the TPP technique. Importantly, BSA-GMA materials are free of acidic hazardous substances. Meanwhile, the water-soluble initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphite (LAP) allows TPP on the vinyl group of the GMA chain and thus without consuming its amino acid group. The as-prepared BSA-GMA hydrogel structure exhibits excellent autofluorescence imaging, pH responsiveness, and biocompatibility, which would provide new avenues for potential applications in tissue engineering and biomedical fields to meet specific biological requirements.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.

Wetting of Cell Aggregates on Microdisk Topography Structures Achieved by Maskless Optical Projection Lithography.

Cell aggregates as a 3D culture model can effectively mimic the physiological processes such as embryonic development, immune response, and tissue renewal in vivo. Researches show that the topography of biomaterials plays an important role in regulating cell proliferation, adhesion, and differentiation. It is of great significance to understand how cell aggregates respond to surface topography. Herein, microdisk array structures with the optimized size are used to investigate the wetting of cell aggregates. Cell aggregates exhibit complete wetting with distinct wetting velocities on the microdisk array structures of different diameters. The wetting velocity of cell aggregates reaches a maximum of 293 µm h-1 on microdisk structures with a diameter of 2 µm and is a minimum of 247 µm h-1 on microdisk structures of 20 µm diameter, which suggests that the cell-substrates adhesion energy on the latter is smaller. Actin stress fibers, focal adhesions (FAs), and cell morphology are analyzed to reveal the mechanisms of variation of wetting velocity. Furthermore, it is demonstrated that cell aggregates adopt climb and detour wetting modes on small and large-sized microdisk structures, respectively. This work reveals the response of cell aggregates to micro-scale topography, providing guidance for better understanding of tissue infiltration.

Micropattern of Silver/Polyaniline Core-Shell Nanocomposite Achieved by Maskless Optical Projection Lithography.

With the development of device miniaturization, a flexible and fast preparation method is in demand for achieving microstructures with desired patterns. We develop a novel photoreduction-polymerization method for preparing conductive metal-polymer patterns. Ag/polyaniline (PANI) nanocomposites have been successfully synthesized by maskless optical projection lithography (MOPL) technology, which is based on multiphoton absorption and the localized surface plasmon resonance (LSPR) effect. The individualized design and synthesis of the nanocomposite patterns at the micro-nano scale are flexibly realized on a variety of substrates. The surface-enhanced Raman scattering (SERS) effect of Rhodamine 6G (R6G) is demonstrated on the microstructure of a square maze-shaped Ag/PANI nanocomposite. The electrical conductivity of the as-prepared nanocomposite is obtained. The preparation protocol proposed in this study opens up new avenues for the fabrication of micro-nano devices such as sensors and detectors.

Modulation of Cell Behavior by 3D Biocompatible Hydrogel Microscaffolds with Precise Configuration.

Three-dimensional (3D) micronano structures have attracted much attention in tissue engineering since they can better simulate the microenvironment in vivo. Two-photon polymerization (TPP) technique provides a powerful tool for printing arbitrary 3D structures with high precision. Here, the desired 3D biocompatible hydrogel microscaffolds (3D microscaffold) with structure design referring to fibroblasts L929 have been fabricated by TPP technology, particularly considering the relative size of cell seed (cell suspension), spread cell, strut and strut spacing of scaffold. Modulation of the cell behavior has been studied by adjusting the porosity from 69.7% to 89.3%. The cell culture experiment results reveal that the obvious modulation of F-actin can be achieved by using the 3D microscaffold. Moreover, cells on 3D microscaffolds exhibit more lamellipodia than those on 2D substrates, and thus resulting in a more complicated 3D shape of single cell and increased cell surface. 3D distribution can be also achieved by employing the designed 3D microscaffold, which would effectively improve the efficiency of information exchange and material transfer. The proposed protocol enables us to better understand the cell behavior in vivo, which would provide high prospects for the further application in tissue engineering.

Ionic Carbazole-Based Water-Soluble Two-Photon Photoinitiator and the Fabrication of Biocompatible 3D Hydrogel Scaffold.

Two-photon polymerization of a three-dimensional (3D) hydrogel structure has been widely applied in biological tissue engineering. For improving the biocompatibility of hydrogel structures, a new kind of ionic carbazole water-soluble photoinitiator was prepared to realize the fabrication of a 3D hydrogel structure in aqueous phase. 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC) and cucurbit[7]uril (CB7) have been employed to generate a complex with better water solubility by host-guest interactions. The binding ratio of the complex was demonstrated to be 1:1 through the characterization of isothermal titration calorimetry (ITC). The two-photon absorption (TPA) cross section of the complex increases to 2500 GM compared with the 750 GM of the BMVMC molecule. Then, an aqueous-phase photoresist was obtained using the CB7/BMVMC complex as the photoinitiator and poly(ethylene glycol) diacrylate (PEGda) as the hydrogel monomer. Two-photon fabrication capability in aqueous phase has been studied using the as-prepared photoresist. A low laser threshold of 3.7 mW as well as a high resolution of 180 nm are achieved. Benefiting from the fluorescence properties of the photoinitiator, we can achieve the confocal fluorescence images without any assistance of fluorescent probes. Subsequently, a 3D engineered hydrogel scaffold microstructure was fabricated by the two-photon polymerization technology, whose biocompatibility was demonstrated by culturing the structure with living cells of L929. The BMVMC-CB7 complex and the as-prepared photoresist are demonstrated to have good biocompatibility, which is prospective for further application in tissue engineering.

Complete genome sequence of the bacterium Methylovorus sp. strain MP688, a high-level producer of pyrroloquinolone quinone.

Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.

Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25.

Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.

Production and radioprotective effects of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ) was produced by fermentation of the Methylovorus sp. MP688 strain and purified by ion-exchange chromatography, crystallization and recrystallization. The yield of PQQ reached approximately 125 mg/L and highly pure PQQ was obtained. To determine the optimum dose of PQQ for radioprotection, three doses (2 mg/kg, 4 mg/kg, 8 mg/kg) of PQQ were orally administrated to the experimental animals subjected to a lethal dose of 8.0 Gy in survival test. Survival of mice in the irradiation + PQQ (4 mg/kg) group was found to be significantly higher in comparison with the irradiation and irradiation + nilestriol (10 mg/kg) groups. The numbers of hematocytes and bone marrow cells were measured for 21 days after sublethal 4 Gy gamma-ray irradiation with per os of 4 mg/kg of PQQ. The recovery of white blood cells, reticulocytes and bone marrow cells in the irradiation + PQQ group was faster than that in the irradiation group. Furthermore, the recovery of bone marrow cell in the irradiation + PQQ group was superior to that in irradiation + nilestriol group. Our results clearly indicate favourable effects on survival under higher lethal radiation doses and the ability of pyrroloquinoline quinine to enhance haemopoietic recovery after sublethal radiation exposure.

[The role of calcium-sensing receptor on ischemia/reperfusion-induced rat cardiomyocyte apoptosis].

OBJECTIVE: To investigate the role of calcium-sensing receptor (CaR) on ischemia/reperfusion-induced rat cardiomyocyte apoptosis. METHODS: The isolated rat hearts were subjected to 40 min ischemia followed by 2h of reperfusion with or without CaR agonist GdCl3 at the beginning of reperfusion. Control hearts (without ischemia) and ischemic hearts (40 min ischemia without reperfusion) served as controls. The protein expressions of CaR, Bcl-2 and cyt C were detected by Western blot. Cardiomyocyte apoptosis was detected by TUNEL. Mitochondrial potential (Deltaphim) was detected by laser confocal microscopy. RESULTS: Compared to controls groups, the expressions of CaR and apoptotic cells were significantly increased, Deltaphim and expressions of mitochondria cyt C and Bcl-2 were significantly reduced in ischemia/reperfusion hearts with or without GdCl3. CONCLUSION: CaR was involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion via mitochondrial pathway.

[Establishment of the screening method and isolation of PQQ producing strains].

Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli, being a candidate for enzymic detection of PQQ, is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21 (DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method, over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC, absorption spectra assay, and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences, overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95% . The highest value is with two strains of Methylovorus, which reached at 99%.

[Integration and expression of sdh gene in Escherichia coli].

The chloramphenicol-resistant cassette with short shared sequences of ptsG gene on both ends was PCR-generated from plasmid pKF3 and ligated to pMD18-T to construct pMD18-PC. The sdh gene for sorbose dehydrogenase was generated from plasmid pQE60-SDH and inserted into pMD18-PC, then pMD18-PC-SDH was constructed. It was digested with Pvu II and the target fragment ptsG1-cat-sdh-ptsG2 was recovered and electroporated into Escherichia coli JM109/pKD46. Homologous-recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombination. The detection result showed that the integron JM109s was of sorbose dehydrogenase activity. The PCR products assay using the upstream and downstream sequences of ptsG gene as primers and JM109s genomic DNA as template, indicated that sdh gene had been integrated at the ptsG gene site in Escherichia coli.

[Knockout of the ptsG gene in Escherichia coli and cultural characterization of the mutants].

Metabolic engineering provide powerful tools for the systematic manipulation of cellular metabolic activities. The ptsG gene for glucose-specific transporter Enzyme II CBGlc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette with short shared sequences on both ends generated by PCR was electroporated into Escherichia coli DH5alpha and JM109. Recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombinase functions. Therefore, the ptsG gene was disrupted to construct the mutants called DH5alphaP and JM109P. There was no difference between the mutants and parent strains in LB media.However, in LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass, together with exploiting more glucose. The maximal cell density obtained with DH5alphaP was approximately 3 times more than that of DH5alpha, then the result of JM109P increased fourfold. The products of recombinant protein TNF respectively accounted for 24.3% of total cellular protein in DH5alphaP with A600 8.28 and 20.8% of total cellular protein in JM109P with A600 7.62. The specific volume expression amount of TNF was greater in the ptsG mutant than in its parent strain. These results demonstrate that the ptsG-mutant strains will be available for high cell-density culture.

[Expression of hydantoin hydrolase gene in Escherichia coli].

Hydantoin hydrolase with responsibility for the ring opening of hydantoin is one of the components of hydantoin utility enzymes of Arthrobacter BT801 which can convert 5-benzylhydantoin into L-phenylalanine. The expression of hydantoin hydrolase gene (hyuH) is very important in elucidation of mechanisms of bio-catalysis and its application in asymmetry synthesis of amino acids. To improve the production and activity of the enzyme, the hydantoin hydrolase gene was amplified by PCR and cloned into E. coli by using vector pT221. The hydantoin hydrolase gene was highly expressed in E. coli BL21 (DE3) under the control of T7 promoter. A protein band about 50kD was detected by SDS-PAGE in the recombinant cell lysate. The objective protein in BL21 (DE3)/pT221-hyuH accounted for 40% of total cellular protein, mostly in soluble form. The products in the recombinant strain showed biological activity.

[Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli].

Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.

[Construction of gene library of Arthrobacter BT801 and isolation & expression of hydantoinase gene].

Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.

[The construction of shuttle vectors of Brevibacillus brevis-Escherichia coli].

The 5' region of the cell wall protein(CWP) gene containing multiple tandem promoters and the signal peptide-coding sequence was isolated by PCR from Br. brevis 50, and used to construct the shuttle vector pBKE50, which included the replication origin of pUB110 and the erythromycin-resistance gene of pGK12. The alpha-amylase gene of Bacillus subtilis 168 was ligated to pBKE50, producing plasmid pBKE50/alpha-amy. After the resulting plasmid was introduced into Br. brevis 50, soluble and biologically active alpha-amylase was secreted directly into the culture medium. The expression level of alpha-amylase in the recombinant Br. brevis 50 was twice higher than that of the donor strain.