Yu Lan Piao

Control of the intracellular levels of prostaglandin E2 through inhibition of the 15-hydroxyprostaglandin dehydrogenase for wound healing

Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much experimental data suggests that prostaglandin E₂ (PGE₂) plays a role in the prevention of excessive scarring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibitors significantly increased PGE₂ levels in A549 cells. In our continuing attempts to develop highly potent 15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28, 29, and 30 demonstrated IC₅₀ values of 0.048, 0.020, 0.038 and 0.048 μM, respectively. They also increased levels of PGE₂ in A549 cells. Especially, compound 28 significantly increased level of PGE₂ at 260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved wound healing with respect to control.

Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation.

Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

Characterization, stability, and antioxidant activity of Salicornia herbaciea seed oil

We investigated the physicochemical properties, chemical composition, stability and antioxidant activity from seed oil of Salicornia herbaciea grown in Korea. The density, refractive index, acid value, peroxide value, iodine value, saponification value, and unsaponifiable matter of oil were 0.91mg/mL, 1.48 at 20 °C, 1.89mg KOH/g oil, 10.20 mEq/kg oil, 1.08 g I/g oil, 216.21 mg KOH/g oil, and 2.60%, respectively. The major fatty acids were linoleic acid (43.73%), oleic acid (19.81%), arachidic acid (13.52%), and palmitic acid (11.84%), respectively. The oil contained high levels of α-tocopherol (249.2 mg/kg oil), followed by δ-tocopherol (89.3 mg/kg), and γ-tocopherol (75.6 mg/kg oil). The oil was found to have high levels of β-sitosterol (94.5mg/kg oil) and stigmasterol (65.7mg/kg oil), respectively. The total phenol, chlorophyll and β-carotene content of oil was 15.2, 94.5, and 8.2 mg/kg oil, respectively. The oil had good oxidative stability during 60 days of storage in a dark area at 50 °C. The maximum degradation rates of the oil were observed at 242.3 °C (9.5%/min), 382.6 °C (5.2%/min), and 440.7 °C (1.3%/min), respectively, where the rate of the weight decrease increased to a maximum up to this point. The ABTS radical scavenging activity of the oil was increased from 50.2 to 71.8% when the oil concentration extracted by methanol was increased from 100 to 300 μg/mL. This study suggests that S. herbaciea seed oil has potential use in functional foods, cosmetics or pharmaceuticals.

Comparative study on the antioxidant and nitrite scavenging activity of fruiting body and mycelium extract from Pleurotus ferulae

We investigated the effects of the antioxidant and the nitrite scavenging activities of the extracts from Pleurotus ferulae fruiting body grown on the solid state using corn cob and activated bleaching earth (CCABE media) and its mycelium grown in the liquid state. The total phenol and polysaccharide concentrations in hot water extract of fruiting body were approximately 3.6- and 4.3-fold higher than those of the mycelium. Using the hot water extract of fruiting body, the maximum DPPH radical scavenging activity at 9 mg/mL, hydroxyl radical scavenging activity at 12mg/mL, reducing power at 12 mg/mL, and chelating ability at 12 mg/mL were obtained, 80.5%, 72.4%, 0.99 OD (700 nm), and 77.0%, respectively. However, in the case of hydrogen peroxide scavenging activity, the ethanol extract was the highest, 78.7% at 12 mg/mL. The maximum nitrite scavenging activity was obtained, 89.7% at 6 mg/mL of hot water extract from fruiting body. Hot water extracts were more effective than ethanol extracts in scavenging activity on DPPH radicals and hydroxyl radical scavenging, reducing power, and chelating activity of ferrous, whereas ethanol extracts were more effective in hydrogen peroxide scavenging activity as evidenced by their lower EC50 values. These results indicate that the hot water extract of P. ferulae fruiting body using CCABE media has good potential to be used as a source of materials or additives for oxidation suppressant in food, cosmetics and drug compositions.

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: a computational and experimental study.

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.

Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation.

Administration of an efficient alginate lyase (AlgL) or AlgL mutant may be a promising therapeutic strategy for treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Nevertheless, the catalytic activity of wild‐type AlgL is not sufficiently high. It is highly desired to design and discover an AlgL mutant with significantly improved catalytic efficiency against alginate substrates. For the purpose of identifying an AlgL mutant with significantly improved catalytic activity, in this study, we first constructed and validated a structural model of AlgL interacting with substrate, providing a better understanding of the interactions between AlgL and its substrate. Based on the modeling insights, further enzyme redesign and experimental testing led to discovery of AlgL mutants, including the K197D/K321A mutant, with significantly improved catalytic activities against alginate and acetylated alginate in ciprofloxacin‐resistant P. aeruginosa (CRPA) biofilms. Further anti‐biofilm activity assays have confirmed that the K197D/K321A mutant with piperacillin/tazobactam is indeed effective in degrading the CRPA biofilms. Co‐administration of the potent mutant AlgL and an antibiotic (such as a nebulizer) could be effective for therapeutic treatment of CRPA‐infected patients with cystic fibrosis. Proteins 2016; 84:1875–1887.

Cell-based biological evaluations of 5-(3-bromo-4-phenethoxybenzylidene)thiazolidine-2,4-dione as promising wound healing agent

Recent studies have focused on prostaglandin E₂ (PGE₂) because PGE₂ regulates vertebrate hematopoietic stem cell induction and engraftment. PGE₂ acts through EP2 and EP4 receptors to mediate regeneration and hematopoietic stem cell (HSC) development via the Wnt signaling pathway. Previously we reported that inhibitors of 15-PGDH can control the intracellular levels of PGE₂. Therefore, we developed new potent 15-PGDH inhibitor, 5-(3-bromo-4-phenethoxybenzylidene)thiazolidine-2,4-dione (TD191), with an IC₅₀ of 4 nM and tested cell-based wound healing effects. This compound significantly increased the level of PGE₂ (451 pg/mL) in A549 cells, which was about 7-fold higher than that of control. HaCaT cells exposed to TD191 showed significantly improved wound healing after 48 h in scratch wound healing test, whereas treatment of TD191 to the fibroblast Hs27 cells slightly decreased cell growth compared with control. SCL is a basic helix-loop-helix transcription factor that is an essential for HSC development. By qPCR, SCL expression in HaCaT cells was 2-fold enhanced after addition of TD191, while treatment of TD191 into fibroblast Hs27 cells was not significantly changed the expression levels of the gene. This data provides in vitro evidence that TD191 may have utility for the therapeutic management of wound healing without scar formation.