A multifunctional hydrogel loaded with two nanoagents improves the pathological microenvironment associated with radiation combined with skin wounds.

Persistent oxidative stress and recurring waves of inflammation with excessive reactive oxygen species (ROS) and free radical accumulation could be generated by radiation. Exposure to radiation in combination with physical injuries such as wound trauma would produce a more harmful set of medical complications, which was known as radiation combined with skin wounds (RCSWs). However, little attention has been given to RCSW research despite the unsatisfactory therapeutic outcomes. In this study, a dual-nanoagent-loaded multifunctional hydrogel was fabricated to ameliorate the pathological microenvironment associated with RCSWs. The injectable, adhesive, and self-healing hydrogel was prepared by crosslinking carbohydrazide-modified gelatin (Gel-CDH) and oxidized hyaluronic acid (OHA) through the Schiff-base reaction under mild condition. Polydopamine nanoparticles (PDA-NPs) and mesenchymal stem cell-secreted small extracellular vesicles (MSC-sEV) were loaded to relieve radiation-produced tissue inflammation and oxidation impairment and enhance cell vitality and angiogenesis individually or jointly. The proposed PDA-NPs@MSC-sEV hydrogel enhanced cell vitality, as shown by cell proliferation, migration, colony formation, and cell cycle and apoptosis assays in vitro, and promoted reepithelization by attenuating microenvironment pathology in vivo. Notably, a gene set enrichment analysis of proteomic data revealed significant enrichment with adipogenic and hypoxic pathways, which play prominent roles in wound repair. Specifically, target genes were predicted based on differential transcription factor expression. The results suggested that MSC-sEV- and PDA-NP-loaded multifunctional hydrogels may be promising nanotherapies for RCSWs. STATEMENT OF SIGNIFICANCE: The small extracellular vesicle (sEV) has distinct advantages compared with MSCs, and polydopamine nanoparticles (PDA-NPs), known as the biological materials with good cell affinity and histocompatibility which have been reported to scavenge ROS free radicals. In this study, an adhesive, injectable, self-healing, antibacterial, ROS scavenging and amelioration of the radiation related microenvironment hydrogel encapsulating nanoscale particles of MSC-sEV and PDA-NPs (PDA-NPs@MSC-sEV hydrogel) was synthesized for promoting radiation combined with skin wounds (RCSWs). GSEA analysis profiled by proteomics data revealed significant enrichments in the regulations of adipogenic and hypoxic pathways with this multi-functional hydrogel. This is the first report of combining this two promising nanoscale agents for the special skin wounds associated with radiation.

Metabolic engineering of Escherichia coli for the production of (R)-α-lipoic acid.

OBJECTIVE: To increase the production of (R)-α-lipoic acid directly from octanoic acid using engineered Escherichia coli with the regeneration of S-adenosylmethionine. RESULTS: The biosynthesis of (R)-α-lipoic acid (LA) in E. coli BL21(DE3) is improved by co-expression of lipoate-protein ligase A (LplA) from E. coli MG1655 and lipoate synthase (LipA) from Vibrio vulnificus. The engineered strain produces 20.99 µg l-1 of LA in shake flask cultures. The titers of LA are increased to 169.28 µg l-1 after the optimization of the medium components and fermentation conditions. We find that the [4Fe-4S] cluster is important for the activity of LipA and co-expression of iscSUA promotes the regeneration of the [4Fe-4S] cluster and leads to the highest LA titer of 589.30 µg l-1. CONCLUSION: The method described here can be widely applied for the biosynthesis of (R)-α-lipoic acid and other metabolites.

Visible spectrophotometric assay for characterization of ω-transaminases.

Omega-transaminases (ω-TAs) have attracted considerable interest for their use in asymmetric synthesis of chiral amines with a high degree of optical purity and yield. The rapid evaluation of the characteristics of newly identified or engineered ω-TAs is important for industrial applications. In this study, a visible spectrophotometric assay was developed for rapid quantitative determination of ω-TA activities based on the transamination of 2-(4-nitrophenyl)ethan-1-amine to generate a red product (E)-N-(4-nitrophenethyl)-2-(4-nitrophenyl)ethen-1-amine. After various co-solvents were evaluated, dimethyl sulfoxide (DMSO) was considered optimal because the red product exhibits good solubility and retains its original color. The red product dissolved in DMSO has its highest absorbance at 465 nm, and its concentration has a good linear relationship with the absorbance. A spectrophotometric assay was established and validated using conventional HPLC analysis (<10% divergence). This method was then used to characterize an ω-TA from thermophilic Geobacillus thermoleovorans and an ω-TA obtained from the metagenome of a soda lake. The results demonstrated that ω-transaminase enzymatic properties could be characterized simply, rapidly, and at low cost, using this newly established visible spectrophotometric assay method.

[Cloning, expression, directed evolution in vitro and structural simulation of ß-glycosidase from Bacillus subtilis].

OBJECTIVE: To further study physiological functions and structure of ß-glycosidase, we cloned the bglC gene of Bacillus subtilis and expressed it in E. coli BL21 (DE3), followed by the characterization and structural simulation of the enzyme. METHODS: We amplified the bglC gene and transferred it into E. coli BL21 (DE3), then we obtained a mutant with higher hydrolytic activity by directed evolution. After purifying the enzymes through a nickel-nitrilotriacetic acid agarose column, we characterized the wild-type and mutant enzymes. By means of CD spectrum, Native-PAGE and protein 3-D structure modeling, we analyzed the higher structure of the ß-glycosidase. RESULTS: We got one mutant enzyme BS-GLY_M1 (A242T/T385A/S425L) with improved hydrolytic activity by directed evolution and screening. The specific activity of wild-type enzyme was 9.7 U/mg, with optimum temperature at 60 degrees C and optimum pH at 7.0. The specific activity of BS-GLY_M1 was 17. 1U/mg, with optimum temperature at 55 degrees C and optimum pH at 7.0. Moreover, the half-life time of the mutant enzyme at 55 degrees C was 3.5 h, 2 h longer than that of wild-type enzyme. Furthermore, the catalytic efficiency (K(m)/K(cat)) of BS-GLY_M1 on the substrates 4-nitrophenyl-ß-galactoside, lactose, and arbutin improved obviously. The polymer forms of the enzyme under the native conditions were of dimer and tetramer, but the dimer was the most probable functional unit. Result of structural simulation also showed slight changes occurred in the tertiary structure of the mutant enzyme, which may be the main reason for the enhanced thermal stability and catalytic efficiency of BS-GLY _M1. [ CONCLUSION: ß-glycosidase from Bacillus subtilis could be expressed in E. coli BL21 (DE3), meanwhile its hydrolysis efficiency could be further improved by directed evolution.

Transferring chromosome DNA fragments from multiple donor cells into a host strain for yeast strain improvement.

Based on a common biological phenomenon - homologous recombination - a novel method was developed by transferring chromosome DNA fragments extracted from multiple donor cells into a host strain. Through this method of transferring DNA fragments, foreign DNA fragments are introduced into one host cell and multiple positive traits from multiple strains may be integrated into the host strain. We first confirmed its feasibility in both prokaryotic and eukaryotic cells by selecting reverse mutants to prototrophy from auxotrophic strains through receiving chromosomal DNA fragments of wild-type parental strains. We then applied this method to Saccharomyces cerevisiae to improve its ethanol and temperature tolerance. We introduced donor chromosome DNA fragments from different S. cerevisiae strains with improvements in ethanol or temperature tolerance into a common strain S. cerevisiae and obtained a strain with much superior ethanol and temperature tolerance. The results showed that the Transferring DNA Fragments method provides a new way for strain breeding.

A continuous and adsorptive bioprocess for efficient production of the natural aroma chemical 2-phenylethanol with yeast.

Natural 2-phenylethanol is a high value aroma chemical and can be produced from l-phenylalanine via Ehrlich pathway by yeasts. Due to serious product inhibition, the space-time yield is usually low. A continuous approach using macroporous resin as in situ adsorbent made it possible that the quantity and viability of the cells were improved simultaneously. With Saccharomyces cerevisiae sp. strain R-UV3, the highest space-time yield of 0.90 gL(-1)h(-1) reported so far was obtained.

[Preparation and identification of OmpW monoclonal antibodies].

AIM: To prepare and characterize the mouse monoclonal antibodies against Vibrio parahaemolyticus OmpW. METHODS: The OmpW amino acid sequence from three diseased Vibrio was analyzed by Bioinformatics. Mice were immunized with r-OmpW which was highly expressed and purified in E.coli. Five Vibrio(Va, Vp, Vh, Vv, Van) were chosen as antigen for mAb selection.The characters of the anti-OmpW monoclonal antibodies were studied by Western blot, Flow Cytometry, indirect immunofluorescence. RESULTS: OmpW was testified a highly conservative membrane protein.Three clones of anti-OmpW mAb was obtained. The Ig subclass of the mAb secreted from fused cell S5C10 was IgG3, which of the titer was 4.6×10(4);. The mAb could specifically recognize Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, which could not react with Pseudomonas flurosecens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli. CONCLUSION: The mAb could specially recognize five diseased Vibrio, which is a useful tool for the further study of the diagnosis of Vibrio.

Synergistic inhibition effect of 2-phenylethanol and ethanol on bioproduction of natural 2-phenylethanol by Saccharomyces cerevisiae and process enhancement.

Natural 2-phenylethanol (PEA) could be produced on a large scale by way of bioconversion with yeast from l-phenylalanine. In this work the synergistic inhibition effect of the target product PEA and the byproduct ethanol on the bioconversion rate by Saccharomyces cerevisiae R-UV3 was systematically studied and a new kinetic model with an item representing the synergistic effect was proposed. Optimization strategies to repress the inhibition effect of PEA and ethanol were carried out in the mode of fed-batch culture with ISPR. The glucose concentration was regulated at the level of 0.2±0.1g/L by controlling a suitable respiratory quotient on line, which could limit the accumulation of the ethanol lower than 10g/L. In the presence of resin FD0816 with a weight of 10% of the medium, PEA was removed from the broth and the overall PEA concentration and the space-time yield reached 13.7g/L and 0.39g L(-1) h(-1) respectively. The semi-continuous process with ISPR was performed, in which the replacement of the resin was operated repeatedly when the aqueous PEA was over 2.7g/L and bioconversion continued until the bioactivity of the yeast cells declined, consequently achieving a final overall PEA concentration of 32.5g/L and a space-time yield of 0.45g L(-1) h(-1).

Effects of a chemically derived homo zwitterionic polysaccharide on immune activation in mice.

In this study, a chemically modified homo zwitterionic polysaccharide (ZPS), sulfated chitosan, was used to examine its effects on murine immune response. The results showed that homoZPS could markedly promote the proliferation of both splenic T/B cells and adhesive cells. In particular, flow cytometry assay demonstrated that the sulfated chitosan could non-specifically activate CD3(+) and CD8(+) T cells proliferation in vitro. The effectiveness of sulfated chitosan as adjuvant was tested using bovine serum albumin (BSA) and diphtheria toxin (DT) as antigens and compared with that of aluminum hydroxide. The levels of specific antibodies to BSA and DT significantly increased after homoZPS vaccination. Both homoZPS and aluminum hydroxide adjuvants could protect mice against the attack of DT from edemas of spleen and tail. The present findings demonstrated the chemically derived homoZPS could be a potential candidate in the development of T-lymphocyte dependent vaccine adjuvants.

[Cloning and expression of Vitreoscilla hemoglobin in Streptomyces aureofaciens].

Vitreoscilla hemoglobin Gene was cloned in Streptomyces aureofaciens through E. coli-Streptomyces shuttle plasmids constructed by both pIJ699-pUC19(vhb) and pIJ702-pBR322(vhb). Under low dissolved oxygen conditions, expression of hemoglobin enhanced CTC yield of engineering strain more about 40%-60% that that of control by improving oxygen transmission in cells. Hemoglobin expression by Promoter of tetracycline resistance gene was more effective for oxygen transmission than that by its native promoter regulated by dissolved oxygen concentrations in environments of locally low concentrations of dissolved oxygen.

[Study on effect of Vitreoscilla hemoglobin gene expression on growth and metabolism of Streptomyces aureofaciens].

Vitreoscilla hemoglobin gene was expressed in S. aureofaciens through promoter of tetracycline resistance gene. Characteristics of S. aureofaciens growth and metabolism were studied in 1 m3 fermenter. In high dissolved oxygen concentrations, expression of hemoglobin gene had little effect on growth and metabolism of S. aureofaciens, and there were no obvious differences between engineering strain and control. The chlortetracycline of engineering strain was 22905 u/mL and of control was 22896 u/mL respectively. Under conditions of low dissolved oxygen, expression of hemoglobin enhanced growth, maintenance of energetic mycelium configuration and chlortetracycline yield of S. aureofaciens: the mycelium concentrations of engineering strain were more about 5%-10% and yield was more 11.4% than control.