Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.

Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives. ONE-SENTENCE SUMMARY: The enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

The Biosynthesis of Norsesquiterpene Aculenes Requires Three Cytochrome P450 Enzymes to Catalyze a Stepwise Demethylation Process.

Aculenes are a unique class of norsequiterpenes (C14 ) that are produced by Aspergillus aculeatus. The nordaucane skeleton in aculenes A-D may be derived from an ent-daucane precursor through demethylation, however, the enzymes involved remain unexplored. We identified the biosynthetic gene cluster and characterized the biosynthetic pathway based on gene inactivation, feeding experiments, and heterologous reconstitution in Saccharomyces cerevisiae and Aspergillus oryzae. We discovered that three cytochrome P450 monoxygenases are required to catalyze the stepwise demethylation process. AneF converts the 12-methyl group into a carboxylic acid and AneD installs the 10-hydroxy group for later tautomerization and stabilization. Finally, AneG installs an electron-withdrawing carbonyl group at the C-2 position, which triggers C-12 decarboxylation to yield the nordaucane skeleton. Additionally, a terpene cyclase (AneC) was found that forms a new product (dauca-4,7-diene).

Influence factors for successful corneal donation among Chinese adults: data from Nanjing between 2001 and 2012.

AIM: To investigate the factors that may influence the successful corneal donation among adults in China. METHODS: This retrospective study was conducted in 2012. The eligible participants were all the adults registered in Nanjing Red Cross Eye Bank to donate their corneas after death during the period of 2001 and 2012. Multivariate logistic regression models were applied to investigate the influence factors for successful donation, the outcome events. RESULTS: Totally, 210 of 328 (64.0%) registered potential donors successfully donated their corneas after death. The mean (SD) age at registration was 64.7 (12.5) for all participants, with 65.5 (10.1) and 63.2 (15.8) for successful and unsuccessful donors, respectively. With multivariate logistic regression analysis, five factors, the willingness of donation, age, education level, residence area, and cause of death were identified to be associated with successful corneal donation. CONCLUSION: The willingness of donation and some socio-demographic factors might substantially affect their successful donation after death for people who registered to donate corneas.

Hydrogen sulfide inhibits formaldehyde-induced endoplasmic reticulum stress in PC12 cells by upregulation of SIRT-1.

BACKGROUND: Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. OBJECTIVE: We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. PRINCIPAL FINDINGS: We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. CONCLUSION/SIGNIFICANCE: These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity.

Liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation for the repair of tarsal defects.

The objectives of this study were to compare the efficiency of three methods for repairing tarsal defects (glycerine-preserved, alcohol-preserved and cryopreserved tarsal plate and palpebral conjunctival transplantation) based on histopathological changes and apoptosis, and to evaluate the clinical effects of repairing tarsal defects by liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation. Forty-eight rabbit eyes with tarsal defects were equally divided into three groups and transplanted with tarsal plates and palpebral conjunctivae under the following conditions: Group 1, liquid nitrogen cryopreservation; Group 2, glycerine preservation; and Group 3, alcohol preservation. Histopathological changes in the transplants were observed by light microscopy 1 week, 1 month and 3 months postoperatively. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Clinically, transplantations of liquid nitrogen-cryopreserved tarsal plates and palpebral conjunctivae were performed in 30 eyes (29 cases) to repair tarsal defects caused by the excision of neoplasms. The postoperative inflammatory reaction and number of apoptotic cells in Group 1 were lower compared with those in Groups 2 and 3. Clinically, of the 30 eyes operated on, 14 were cured, 15 improved and 1 failed between 6 and 90 months of follow-up. Liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation is an easy, feasible and convenient procedure. Its effects are fairly favorable, with only a small rejection rate postoperatively. Therefore, it is an ideal method for repairing tarsal defects.

Mitomycin C "straight scleral tunnel incision"--trabeculectomy with a releasable suture.

OBJECTIVE: To evaluate the efficacy of "straight scleral tunnel incision" -trabeculectomy with a releasable suture supplemented with mitomycin C (MMC) on reducing intraocular pressure (IOP), complications, and corneal astigmatism in patients with primary angle-closure glaucoma (PACG). METHODS: Totally 217 acute or chronic PACG patients with occludable angle above 180 degrees and IOP above 21 mm Hg were divided into 3 groups. Patients in group A (98 cases, 128 eyes), B (71 cases, 95 eyes), and C (48 cases, 60 eyes) were treated with "straight scleral tunnel incision"-trabeculectomy with a releasable suture supplemented with MMC, releasable suture trabeculectomy with MMC, and trabeculectomy with MMC, respectively. IOP, complications, and surgically induced astigmatism (SIA) were evaluated preoperatively and up to 12 months postoperatively. RESULTS: IOP of 2 weeks after treatment was significantly lower than preoperative IOP in all the 3 groups (all P <0. 001). Success rates (IOP < or = 20 mm Hg) in group A, B, and C were 87.91%, 89.23%, and 83.72% respectively at 12 months after treatment (P = 0.256). The incidence of shallow anterior chamber and hypotony had no significant difference between group A and B, but both of them were lower than that in group C (P < 0.05). There were no significant differences in preoperative corneal astigmatism among the 3 groups. The corneal astigmatism after 2 weeks in group A (1.71 +/- 1.47D) was higher than that before operation (1.28 +/- 1.05D, P = 0.126). With 12 months gone, the astigmatism almost returned to preoperative levels. The corneal astigmatisms after 2 weeks in group B and C (1.99 +/- 1.20D and 2. 22 +/- 1.39D) were significantly higher than those before operation (1.20 +/- 0.85D and 1.18 +/- 0.93D, P = 0.002, P = 0.001), respectively. With 12 months gone, the mean astigmatisms in group B and C (1.87 +/- 0.91D and 1.90 +/- 1.16D) were still significantly higher than those before operation (P = 0.001, P = 0.003). The highest astigmatic polar values in group A, B, and C (1.00D, 1. 89D, and 1. 77D) occurred after 2 weeks, 1 month, and 1 month postoperation, respectively, which were significantly higher than those before operation (0.19 +/- 1.32D, 0.12 +/- 1.22D, and 0.17 +/- 1.25D, P < 0.01), respectively. With 12 months gone, they were 0. 03D, -0. 18D, and -0. 13D higher than those before operation, respectively. The rates of function bleb and thin-wall bleb were 71.43% and 26.37% in group A, 75.38% and 29.23% in group B, 72.09% and 25.58% in group C, respectively at 12 months after treatment There were no significant differences among the 3 groups. CONCLUSION: "Straight scleral tunnel incision" -trabeculectomy with a releasable suture supplemented with MMC can reduce complications and get satisfactory results in reducing IOP and SIA.