Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus.

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin.

BACKGROUND: Isoquercitrin (quercetin-3-O-ß-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards. RESULTS: Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three ß-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor. CONCLUSION: The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.

Full-length transcriptome, proteomics and metabolite analysis reveal candidate genes involved triterpenoid saponin biosynthesis in Dipsacus asperoides.

Dipsacus asperoides is a traditional medicinal herb widely used in inflammation and fracture in Asia. Triterpenoid saponins from D. asperoides are the main composition with pharmacological activity. However, the biosynthesis pathway of triterpenoid saponins has not been completely resolved in D. asperoides. Here, the types and contents of triterpenoid saponins were discovered with different distributions in five tissues (root, leaf, flower, stem, and fibrous root tissue) from D. asperoides by UPLC-Q-TOF-MS analysis. The discrepancy between five tissues in D. asperoides at the transcriptional level was studied by combining single-molecule real-time sequencing and next- generation sequencing. Meanwhile, key genes involved in the biosynthesis of saponin were further verified by proteomics. In MEP and MVA pathways, 48 differentially expressed genes were identified through co-expression analysis of transcriptome and saponin contents, including two isopentenyl pyrophosphate isomerase and two 2,3-oxidosqualene ß-amyrin cyclase, etc. In the analysis of WGCNA, 6 cytochrome P450s and 24 UDP- glycosyltransferases related to the biosynthesis of triterpenoid saponins were discovered with high transcriptome expression. This study will provide profound insights to demonstrate essential genes in the biosynthesis pathway of saponins in D. asperoides and support for the biosynthetic of natural active ingredients in the future.

Analysis of Prognostic Risk Factors of Endoscopic Submucosal Dissection (ESD) and Curative Resection of Gastrointestinal Neuroendocrine Neoplasms.

Objective: To explore the prognostic risk factors of ESD curative resection of gastrointestinal-neuroendocrine neoplasms (GI-NENs). Methods: A total of 97 patients treated with ESD successfully in our hospital were selected, their surgical site, size, number of resection lesions, operation time, intraoperative complications (such as bleeding and perforation), and treatment status were recorded, and the number of hemostatic clamps used after the postoperative follow-up results and the independent risk factors for ESD complications were obtained through the comparison between the noncomplication group and the ESD complication group using regression analysis. Results: A total of 97 patients with gastrointestinal neuroendocrine tumors were treated with ESD. 61 were males, 36 were females, the ratio of male to female was 1.7 : 1, onset age was 20-78 years old, and median onset age was 50 years old. In 81 cases, tumors were located in the stomach, 10 in the duodenum, and 6 in the rectum. A total of 103 lesions were detected by endoscopy, including 1 case with 2 sites in the stomach, 5 cases with 2 sites in the rectum, and the rest were single. The tumor diameter was 0.3 ∼ 2.5 cm, and the median diameter was 0.6 cm; there were 25 sites with a diameter less than 5 cm. There were 57 places with 10 mm, 16 places with 10-15 mm, and 5 places with >15 mm. All ESD operations were performed in one piece, with a total resection rate of 100%; 89.6% (60/67) of postoperative pathology showed negative basal, and 90.3% (56/62) showed negative resection margin, with a complete resection rate of 88.9% (48/54). ESD's operation time is 6 ∼ 66 min, and the median time is 18 min. During the operation, 5 cases had small amount of bleeding, 3 cases were perforated, 2 cases of delayed postoperative bleeding, 1 case of bleeding was caused by the patient's failure to follow the advice of the doctor to eat a large amount of solid food too early, and 1 case of delayed perforation (all recovered and discharged). ESD operation that bled, age, gender, and perforation location, pathological grade, pathological classification, tumor diameter, tumor surface, operation time, number of titanium clips, origin, echo uniformity, and echo level were statistically insignificant (P > 0.05). Postoperative bleeding was related to the operation time (P=0.017), but it was not an independent risk factor for postoperative bleeding (P=0.118; OR, 0.226; 95% CI, 0.035-1.461). 59 cases were followed up by endoscopy after the operation, and recurrence or no new tumors were found. Conclusion: ESD is an effective and safe treatment method for gastrointestinal neuroendocrine tumors with a diameter of 1-2 cm without invading the muscularis propria. The intraoperative complications seem to have little relationship with the patient; postoperative delayed bleeding is closely related to the ESD operation time but it is not an independent risk factor.

pH-responsive magnolol nanocapsule-embedded magnolol-grafted-chitosan hydrochloride hydrogels for promoting wound healing.

Bacterial infection and oxidative stress seriously delay wound healing. Here, magnolol-grafted-chitosan hydrochloride (CSCl-MAG) was synthesized and crosslinked with genipin to form hydrogel (CSMH). By embedding magnolol-loaded chitosan nanocapsules (MCNGs) into CSMH, we designed a novel nanocapsule- embedded hydrogel dressing (MCNG-H) with sustained antibacterial and antioxidant properties. The swelling, rheology, cytotoxicity, antibacterial and antioxidant properties of MCNG-H were studied. MCNG-H exhibited good swelling properties and biocompatibility. The antibacterial and antioxidant assays in vitro demonstrated the corporative effect of CSM-H and MCNGs in inhibiting bacteria and eliminating reactive oxygen species (ROS). Moreover, MCNG-H revealed the pH-responsive release profiles of loaded MAG, and ensured sustained release of MAG with the dual protective effects of MCNG and CSMH, which helped to maintain long-term antibacterial and antioxidant activities. Finally, MCNG-H significantly accelerated wound healing in a splinted excisional dermal wound model, promising a competitive dressing for the treatment of infected wounds.

Integrated transcriptome and proteome analyses reveal candidate genes for ginsenoside biosynthesis in Panax japonicus C. A. Meyer.

Panax japonicus C. A. Meyer is a plant of the Araliaceae family, and its rhizomes can be used as dietary supplements. It is extremely rich in bioactive components ginsenosides with benefits to human health. However, the underlying mechanisms of ginsenosides biosynthesis in Panax japonicus remains poorly understood. Therefore, a comprehensive analysis of the metabolites, transcriptome, and proteome was conducted to investigate ginsenoside metabolism of Panax japonicus. Here, three types of ginsenosides were found to exhibited tissue-specific distribution using the liquid chromatography-mass spectrometry method. Next, differentially expressed gene analysis revealed that transcript levels of ginsenosides biosynthetic genes have significant differences between differential samples. In addition, correlation analysis showed that the ginsenosides content was closely related to the expression level of 29 cytochrome P450s and 92 Uridine diphosphate-glycosyltransferases. Finally, phylogenetic analysis was performed for the target proteins to conduct preliminary studies on their functions and classification. This study provides insight into the dynamic changes and biosynthetic pathway of ginsenosides and offers valuable information on the metabolic regulation of Panax japonicus.

Copper metal-organic framework embedded carboxymethyl chitosan-g-glutathione/polyacrylamide hydrogels for killing bacteria and promoting wound healing.

Bacterial infection and its induced oxidative stress as major clinical challenge during wound healing call for an urgent response for the development of medical dressings with multi-functions, such as antioxidant and antibacterial. To meet this demand, copper metal organic framework nanoparticles (HKUST NPs) and carboxymethyl chitosan-g-glutathione (CMCs-GSH) were synthesized and characterized. By embedding HKUST NPs into PAM/CMCs-GSH hydrogel (AOH), we developed a novel hydrogel dressing (HKUST-Hs) with dual effects of antibacterial and antioxidant. The morphology, swelling behavior, oxidation resistance and antibacterial properties of HKUST-Hs were investigated as well as the slow-release behavior of copper ions. Full-thickness cutaneous wound model of rats was created to assess the promoting effect of HKUST-Hs on wound healing. We found that HKUST NPs could be well dispersed in HKUST-Hs by shielding the positive charge of copper ions, and thus copper ions released were uniformly distributed and chelated with CMCs-GSH to promote the swelling stability of HKUST-Hs. Also, HKUST-Hs exhibited good free radical scavenging ability in vitro antioxidant assay. Meanwhile, a gradient sustained-release system of copper ions was formed in HKUST-Hs owing to the inhibition of HKUST NPs to copper release and the chelation of CMCs-GSH, which effectively inhibited the explosive release of copper ions and prolonged the release period, thereby reducing cytotoxicity. In vitro antibacterial test demonstrated there was synergistic antibacterial effect between the slow-released copper ions and CMCs-GSH, which improved the antibacterial activity and antibacterial persistence of HKUST-Hs. Finally, HKUST-Hs accelerated wound healing in vivo by continuously killing bacteria and inhibiting oxidative stress.

LncRNA NLIPMT Inhibits Tumorigenesis in Esophageal Squamous-Cell Carcinomas by Regulating miR-320/Survivin Axis.

BACKGROUND: LncRNA has been widely investigated for decades and plays critical roles in the progression of cancer. However, lncRNA NLIPMT, as a novel non-coding RNA, only was studied in breast cancer. This study aimed to explore the role of NLIPMT in esophageal squamous-cell carcinomas (ESCC). MATERIALS AND METHODS: NLIPMT, miR320 and survivin mRNA in ESCC tissues (or non-tumor tissue) were detected by qRT-PCR. Dual-luciferase reporter assay was performed to assess the relationship between miR-320 and survivin. In ESCC cell lines KYSE510 and ECA109, miR-320 mimic and expression vectors carrying NLIPMT and survivin were used. Cell cycle, apoptosis, proliferation and migration were detected by flow cytometry, CCK-8, transwell assay, respectively. NIPMT, miR-320 and survivin expression were measured by qRT-PCR and Western blotting. RESULTS: NLIPMT was downregulated in ESCC and predicted poor survival of ESCC patients. NLIPMT was positively correlated with miR-320 and negatively correlated with survivin in ESCC tumor tissues. Dual-luciferase reporter assay showed that miR-320 directly regulated survivin. qRT-PCR and Western blotting showed that NLIPMT promoted miR-320 expression and inhibited survivin expression via up-regulating miR-320. Moreover, both NLIPMT and miR-320 overexpression inhibited cell proliferation and migration and promoted cell cycle arrest and apoptosis in ESCC cells, while their effects were abolished by survivin overexpression. CONCLUSION: We demonstrate that NLIPMT inhibits cell proliferation and migration and promotes cell cycle arrest and apoptosis in ESCC cells by regulating the miR-320/survivin axis. NLIPMT may be a novel prognosis biomarker in ESCC patients.