Near-perfect kinetic resolution of o-methylphenyl glycidyl ether by RpEH, a novel epoxide hydrolase from Rhodotorula paludigena JNU001 with high stereoselectivity.

In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA gene Rpeh-encoding epoxide hydrolase (RpEH) of Rhodotorula paludigena JNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) of Rpeh was 1236 bp encoding RpEH of 411 amino acids and was heterologously expressed in Escherichia coli BL21(DE3). The substrate spectrum of expressed RpEH showed that the transformant E. coli/Rpeh had excellent enantioselectivity to 2a, 3a, and 5a-10a, among which E. coli/Rpeh had the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E = 101) for 8a, and its regioselectivity coefficients, αR and ßS, toward (R)- and (S)-8a were 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL of E. coli/Rpeh, the near-perfect kinetic resolution of rac-8a at a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8a with more than 99% enantiomeric excess (ees) and 46.7% yield and producing (S)-8b with 93.2% eep and 51.4% yield with high space-time yield (STY) for (R)-8a and (S)-8b were 30.6 and 37.3 g/L/h.

Near-perfect kinetic resolution of racemic p-chlorostyrene oxide by SlEH1, a novel epoxide hydrolase from Solanum lycopersicum with extremely high enantioselectivity.

An open reading frame of sleh1, a gene encoding for a novel epoxide hydrolase from Solanum lycopersicum (SlEH1), was amplified by RT-PCR and expressed in E. coli BL21(DE3). The substrate spectrum assay showed that E. coli/sleh1 had EH activities towards all tested substrates except for racemic (rac-) 5a, and the highest enantiomeric ratio (E > 200) towards rac-2a, retaining (R)-2a with 99.1% ees and 49.2% yields and affording (R)-2b with 89.8% eep and 46.7% yieldp. Besides, E. coli/sleh1 also hydrolyzed of rac-7a-9a with moderate regioselectivities, producing (S)- or (R)-7b-9b with 40.5-51.3% eep and 69.4-75.2% yieldp. The pH optimum and stability of the purified SlEH1 were 7.5 and at a range of 6.5-8.5, and it was thermostable at or below 40 °C. Its catalytic efficiency (kcatS/KmS = 7.49 mM-1 s-1) for (S)-2a was much higher than that for (R)-2a. The gram-scale kinetic resolution of 150 mM rac-2a was carried out by E. coli/sleh1 at 20 °C for 8 h, producing (R)-2a with 98.2% ees and 45.3% overall yields after purification by silica gel column chromatography. Furthermore, the source of extremely high enantioselectivity of SlEH1 towards rac-2a was analyzed by molecular docking simulations.

Construction of chitosan/ZnO nanocomposite film by in situ precipitation.

In the present work, flexible chitosan/ZnO nanocomposite films were prepared by a green and facile method through in situ precipitation of nano-ZnO (nZnO) in the chitosan film. Zn(Ac)2 was added in chitosan solution to provide Zn2+, thus Zn2+ was fixed in the chitosan matrix and converted into nZnO through interaction with NaOH with heating. The structure and properties of the hybrid films were characterized by Field emission scanning electron microscope (FESEM), atomic force microscope (AFM), Fourier transform infra-red (FT-IR), X-ray diffraction (XRD) and tensile testing. The results indicated that there was strong coordination interaction existed between Zn2+ and chitosan matrix for the good dispersion of nZnO in the chitosan film. Furthermore, nZnO distributed evenly in the chitosan and aggregated to form micro-nano-binary hierarchical structure, mimicking lotus leaf structure. Therefore, this work provides an effective way to prepare biocompatible and antibacterial chitosan/ZnO nanocomposite films, showing potential applications in the fields of antibacterial packaging and dressings.

Hyaluronic acid-chitosan nanoparticles encoding CrmA attenuate interleukin-1ß induced inflammation in synoviocytes in vitro.

Osteoarthritis (OA) is a common degenerative joint disease characterized by inflammation of synoviocytes and degradation of cartilage. In the present study, hyaluronic acid/chitosan (HA/CS) nanoparticles were used as a vehicle for gene therapy of OA, and the cytokine response modifier A (CrmA) pDNA was proposed as the target gene. The HA/CS/pCrmA nanoparticles were prepared and the characteristics of the nanoparticles were examined. The nanoparticles were spherical, and the smallest size was obtained with the HA:CS weight ratio of 1:4. The release analysis exhibited a constant release over 29 days. The pDNA was completely combined with HA/CS nanoparticles and the HA/CS nanoparticles protected pDNA from degradation. Subsequently, rat synoviocytes were transfected with HA/CS/pDNA nanoparticles, and the results demonstrated that the HA/CS nanoparticles were able to improve the transfection capacity of pDNA. The cytotoxicity of the HA/CS/pDNA nanoparticles was additionally detected using a MTS assay to ensure that the HA/CS nanoparticle was a safe carrier. To additionally investigate the effects of HA/CS/pCrmA nanoparticles on synoviocytes in OA, the MMP­3 and MMP­13 gene expression levels were detected at the gene and protein expression levels. These results indicated that the HA/CS/pCrmA nanoparticles attenuated interleukin­1ß­mediated inflammation in synoviocytes. It was concluded that the HA/CS/pCrmA nanoparticles may provide a novel approach to the treatment of OA.

Chondroprotective Effects of Hyaluronic Acid-Chitosan Nanoparticles Containing Plasmid DNA Encoding Cytokine Response Modifier A in a Rat Knee Osteoarthritis Model.

BACKGROUND/AIMS: Interleukin (IL)-1ß plays an essential role in the pathophysiology of osteoarthritis (OA). Cytokine response modifier A (CrmA) can prevent the generation of active IL-1ß. This study aimed to explore the chondroprotective effects of hyaluronic acid-chitosan nanoparticles containing plasmid DNA encoding CrmA (HA/CS-CrmA) in a rat OA model. METHODS: HA/CS-CrmA nanoparticles were synthesized through the complex coacervation of cationic polymers. The characteristics, toxicity, and transfection of the nanoparticles were investigated. Furthermore, the potential effects of HA/CS-CrmA nanoparticles were evaluated via a rat anterior cruciate ligament transection (ACLT) model of OA. Cartilage damage and synovial inflammation were assessed by safranin O/fast green and hematoxylin and eosin staining. Type II collagen in cartilage was measured by immunohistochemistry, and the expression levels of IL-1ß, matrix metalloproteinase (MMP)-3, and MMP-13 in synovial tissue were detected by western blot. RESULTS: The HA/CS-CrmA nanoparticles, which effectively entrapped plasmid DNA, showed an adequate size (100-300 nm) and a regular spherical shape. The nanoparticles safely transfected synoviocytes and released plasmid DNA in a sustained manner over 3 weeks. Additionally, HA/CS-CrmA nanoparticles significantly inhibited cartilage damage, synovial inflammation, and the loss of type II collagen induced by ACLT. The expression levels of IL-1ß, MMP-3, and MMP-13 in synovial tissue were dramatically down-regulated by HA/CS-CrmA nanoparticles. CONCLUSIONS: These results suggested that HA/CS-CrmA nanoparticles could attenuate cartilage destruction and protect against early OA by inhibiting synovial inflammation via inhibition of IL-1ß generation.

Tristetraprolin: A novel target of diallyl disulfide that inhibits the progression of breast cancer.

Diallyl disulfide (DADS), a volatile component of garlic oil, has various biological properties, including antioxidant, antiangiogenic and anticancer effects. The present study aimed to explore novel targets of DADS that may slow or stop the progression of breast cancer. First, xenograft tumor models were created by subcutaneously injecting MCF-7 and MDA-MB-231 breast cancer cells into nude mice. Subsequently, western blot analysis was performed to investigate the expression of tristetraprolin (TTP), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in the xenograft tumors, and cell cultures. Tablet cloning, Transwell and wound healing assays revealed that DADS treatment significantly inhibited the proliferation, invasion and migration of breast cancer cells. In addition, DADS treatment led to significant downregulation of uPA and MMP-9 protein expression, but significantly upregulated TTP expression in vivo and in vitro. Knocking down TTP expression using small interfering RNA reversed the aforementioned effects of DADS, which suggests TTP is a key target of DADS in inhibiting the progression of breast cancer.