Nearly perfect kinetic resolution of racemic o-nitrostyrene oxide by AuEH2, a microsomal epoxide hydrolase from Aspergillus usamii, with high enantio- and regio-selectivity.

Only a few known epoxide hydrolases (EHs) displayed activity towards o-nitrostyrene oxide (4a), presumably owing to the large steric hindrance caused by o-nitro substituent. Therefore, excavating EHs with high activity and enantio- and/or regio-selectivity towards racemic (rac-) 4a is essential but challenging. Here, AuEH2 from Aspergillus usamii was expressed in E. coli BL21(DE3). E. coli/Aueh2, an E. coli transformant expressing AuEH2, possessed EH activities of 16.2-184 U/g wet cell towards rac-styrene oxide (1a) and its derivatives (2a-13a), and the largest enantiomeric ratio of 96 towards rac-4a. The regioselectivity coefficients, ßR and ßS, of AuEH2 were determined to be 99.2% and 98.9%, suggesting that it regiopreferentially attacks the Cß in the oxirane rings of (R)- and (S)-4a. Then, the nearly perfect kinetic resolution of 20 mM rac-4a in pure water was carried out using 20 mg/mL wet cells of E. coli/Aueh2 at 25 °C for 50 min, retaining (S)-4a with over 99% ees and 48.9% yields, while producing (R)-o-nitrophenyl-1,2-ethanediol (4b) with 95.3% eep and 49.8% yieldp. To elucidate the molecular mechanism of AuEH2 with high enantiopreference for (R)-4a, its crystal structure was solved by X-ray diffraction and the molecular docking of AuEH2 with (R)- or (S)-4a was simulated.

Highly regio- and enantio-selective hydrolysis of two racemic epoxides by GmEH3, a novel epoxide hydrolase from Glycine max.

A novel epoxide hydrolase from Glycine max, designated GmEH3, was excavated based on the computer-aided analysis. Then, gmeh3, a GmEH3-encoding gene, was cloned and successfully expressed in E. coli Rosetta(DE3). Among the ten investigated rac-epoxides, GmEH3 possessed the highest and best complementary regioselectivities (regioselectivity coefficients, αS = 93.7% and ßR = 97.2%) in the asymmetric hydrolysis of rac-m-chlorostyrene oxide (5a), and the highest enantioselectivity (enantiomeric ratio, E = 55.6) towards rac-phenyl glycidyl ether (7a). The catalytic efficiency (kcatS/KmS = 2.50 mM-1 s-1) of purified GmEH3 for (S)-5a was slightly higher than that (kcatR/KmR = 1.52 mM-1 s-1) for (R)-5a, whereas the kcat/Km (5.16 mM-1 s-1) for (S)-7a was much higher than that (0.09 mM-1 s-1) for (R)-7a. Using 200 mg/mL wet cells of E. coli/gmeh3 as the biocatalyst, the scale-up enantioconvergent hydrolysis of 150 mM rac-5a at 25 °C for 1.5 h afforded (R)-5b with 90.2% eep and 95.4% yieldp, while the kinetic resolution of 500 mM rac-7a for 2.5 h retained (R)-7a with over 99% ees and 43.2% yields. Furthermore, the sources of high regiocomplementarity of GmEH3 for (S)- and (R)-5a as well as high enantioselectivity towards rac-7a were analyzed via molecular docking (MD) simulation.

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.

Greatly enhancing the enantioselectivity of PvEH2, a Phaseolus vulgaris epoxide hydrolase, towards racemic 1,2-epoxyhexane via replacing its partial cap-loop.

To achieve the kinetic resolution and enantioconvergent hydrolysis of rac-1,2-epoxyhexane, the E value of PvEH2 was enhanced by substituting its partial cap-loop. Based on the experimental results reported previously and computer-aided analysis, the flexible and variable cap-loop, especially its middle segment, was speculated to be related to the catalytic properties of PvEH2. In view of this, four PvEH2's hybrids, Pv2St, Pv2Pv1, Pv2Vr1 and Pv2Vr2, were designed by substituting the middle segment (190EGMGSNLNTSMP201) of a cap-loop in PvEH2 with the corresponding ones in StEH, PvEH1, VrEH1 and VrEH2, respectively. Then, the hybrid-encoding genes, pv2st, pv2pv1, pv2vr1 and pv2vr2, were constructed by fusion PCR, and expressed in E. coli Rosetta(DE3). The expressed hybrid, Pv2St, displayed the highest specific activity of 35.3 U/mg protein towards rac-1,2-epoxyhexane. The corresponding transformant, E. coli/pv2st, exhibited the largest E value of 24.2, which was 11.5-fold that of E. coli/pveh2 expressing PvEH2. The scale-up kinetic resolution of 280 mM rac-1,2-epoxyhexane was carried out using 40 mg dry cells/mL of E. coli/pv2st at 25 °C for 4.5 h, retaining (S)-1,2-epoxyhexane with >99.5% ees and 36.9% yield. Additionally, the chemo-enzymatic enantioconvergent hydrolysis of rac-1,2-epoxyhexane using E. coli/pv2st followed by sulfuric acid produced (R)-hexane-1,2-diol with 73.0% eep and 86.5% yield.

A Case of Complete Remission from Advanced Gastric Adenocarcinoma with Synchronous Liver Metastasis in Response to EOX Chemotherapy.

Gastric cancer is a malignant tumor with a high degree of malignancy. Multiple liver metastases from gastric cancer (LMGCs) are common. However, the treatment of LMGCs is very difficult. It is rare to achieve complete remission (CR) and long-term survival after treatment. We present the case of a patient with gastric adenocarcinoma and multiple liver metastases who showed CR for more than 33 months after perioperative EOX (epirubicin, oxaliplatin, and capecitabine) combination chemotherapy with radical distal gastrectomy and resection of liver metastases. The patient is still in follow-up without tumor recurrence. These findings suggest that LMGC does not necessarily mean a poor prognosis; preoperative chemotherapy combined with surgery may be a good treatment option for LMGC in selected patients. Further studies are needed to support this treatment approach.

Relationship between HER2 and JAK/STAT-SOCS3 signaling pathway and clinicopathological features and prognosis of ovarian cancer.

OBJECTIVE: The study aims to explore the relationship between expressions of HER2 and JAK/STAT3-SOCS3 signaling pathway and clinicopathological features and prognosis of ovarian cancer (OC). METHODS: A total of 136 OC patients were collected. Immunohistochemistry was applied to measure the expressions of STAT3, p-STAT3, SOCS3, HER2 and p-HER2 in the tumor tissues and adjacent normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expressions of HER2, SOCS3 and STAT3 and western blotting was applied for protein expressions of HER2, p-HER2, SOCS3, STAT3 and p-STAT3 in the tumor tissues and adjacent normal tissues. Flow cytometry was used for the cell apoptosis in the blank, afatinib (A), ruxolitinib (R) and afatinib + ruxolitinib (A + R) groups. Follow-up was performed to explore relationship of HER2, SOCS3, and STAT3 expressions with survival time of OC patients. RESULTS: HER2, p-HER2, STAT3, and p-STAT3 expressions were higher while SOCS3 expression was lower in the tumor tissues. The positive expressions of STAT3, HER2, p-HER2 and p-STAT3 were lower while the positive expression of SOCS3 was higher in the adjacent normal tissues. The expressions of HER2, SOCS3, and p-STAT3 were associated with clinical stage and lymph node metastasis (LNM), and STAT3 expression has correlation with histological grade and LNM. The mRNA and protein expressions of HER2, STAT3 and p-STAT3 in the tumor tissues were higher than those in the adjacent normal tissues, but SOCS3 expression was significantly decreased. The positive expressions of HER2, p-HER2 and STAT3, the negative expression of SOCS3 and pathological stages were important risk factors for the prognosis of patients with OC. CONCLUSION: Our study showed that the expressions of HER2, STAT3, and SOCS3 are associated with the progression of OC, and higher expressions of HER2 and STAT3 and lower expression of SOCS3 predict poor prognosis of OC.