DCTPP1, a reliable Q-biomarker for comprehensive evaluation of the quality of tripterygium glycoside tablets based on chemical references.

BACKGROUND: As first-line clinical drugs, tripterygium glycoside tablets (TGTs) often have inconsistent efficacy and toxic side effects, mainly due to inadequate quality control. Therefore, clinically relevant quality standards for TGTs are urgently required. PURPOSE: Based on chemical substances and considering pharmacological efficacy, we aimed to develop an effective quality evaluation method for TGTs. METHODS: Representative commercial samples of TGTs were collected from different manufacturers, and qualitative UHPLC/LTQ-Orbitrap-MS and quantitative UHPLC-MS/MS analysis methods were successfully applied to evaluate their quality similarities and differences based on their chemical properties. Then the anti-immunity, anti-inflammatory and antitumor activities of TGTs and related monomers were evaluated using Jurkat, RAW264.7, MIA PaCa-2, and PANC-1 as cellular models. Subsequently, we predicted and verified small molecule-DCTPP1 interactions via molecular docking using the established DCTPP1 enzymatic activity assay. Finally, we performed a gray relational analysis to evaluate the chemical characteristics and biological effects of TGTs produced by different manufacturers. RESULTS: We collected 24 batches of TGTs (D01-D24) from 5 manufacturers (Co. A, Co. B, Co. C, Co. D, Co. E) for quality evaluation. The chemical composition analysis revealed significant differences in the substance bases of the samples. The D02, D18-D20 samples from Co. B constituted a separate group that differed from other samples, mainly in their absence of diterpenoids and triterpenoids, including triptolide, triptophenolide, and triptonide. In vitro anti-immunity, antitumor and anti-inflammatory tests using the same TGT concentration revealed that, except for D02, D18-D20, the remaining 20 samples exhibited different degrees of anti-immunity, antitumor and anti-inflammatory activity. Our experiments verified that triptolide, triptophenolide, and triptonide were all DCTPP1 inhibitors, and that TGTs generally exhibited DCTPP1 enzyme inhibitory activity. Moreover, the inhibitory activity of D02, D18-D20 samples from Co. B was much lower than that of the other samples, with a nearly tenfold difference in IC50. Further comprehensive analysis revealed a high correlation between DCTPP1 enzyme inhibition activity and the anti-immunity and antitumor and anti-inflammatory activities of these samples. CONCLUSION: The established DCTPP1 enzymatic activity assay proved suitable for quantitative pharmacological and pharmaceutical analysis to complement the existing quality control system for TGTs and to evaluate their effectiveness.

Structural diversification of natural substrates modified by the O-methyltransferase AurJ from Fusarium Graminearum.

Aromatic polyketide and phenylpropanoid derivatives are a large class of natural products produced by bacteria, fungi, and plants. The O-methylation is a unique decoration that can increase structural diversity of aromatic compounds and improve their pharmacological properties, but the substrate specificity of O-methyltransferase hinders the discovery of more natural products with O-methylation through biosynthesis. Here, we reported that the O-methyltransferase AurJ from plant pathogenic fungus Fusarium graminearum could methylate a broad range of natural substrates of monocyclic, bicyclic, and tricyclic aromatic precursors, exhibiting excellent substrate tolerance. This finding will partly change our stereotype about the specificity of traditional methyltransferases, and urge us to mine more O-methyltransferases with good substrate tolerance and discover more methylated natural products for drug discovery and development through directed evolution and combinatorial biosynthesis.

Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.

Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.

Mining and Characterization of Indolethylamine N-Methyltransferases in Amphibian Toad Bufo gargarizans.

Strong, psychedelic indolethylamines (IAAs) are typically present in trace amounts in the majority of species, but they build up significantly in the skin of amphibian toads, especially N-methylated 5-hydroxytryptamine (5-HT) analogues. However, there is no pertinent research on the investigation of indoleamine N-methyltransferase (INMT) in amphibians, nor is there any adequate information on the key amino acids that influence the activity of known INMTs from other species. Herein, we focused on Bufo toad INMT (BINMT) for the first time and preliminarily identified BINMT 1 from the transcriptomes of Bufo gargarizans active on tryptamine, 5-HT, and N-methyl-5-HT. We established the enzyme kinetic characteristics of BINMT 1 and identified the essential amino acids influencing its activity via molecular docking and site-directed mutagenesis. Subsequently, we carried out sequence alignment and phylogenetic tree analysis on 43 homologous proteins found in the genome of B. gargarizans with BINMT 1 as the probe and selected seven of them for protein expression and activity assays. It was found that only three proteins possessing the highest similarity to BINMT 1 had INMT activity. Our research unveils the binding residues of BINMT for 5-HT analogues for the first time and initiates the study of INMTs in amphibian toads, serving as a tentative reference for further study of BINMT and providing insight into the comprehension of BINMT's catalytic mechanism and its role in the biosynthesis of 5-HT analogues in Bufo toads. It also contributes to the expansion of the INMT library to help explore and explain interspecies evolution in the future.

Interrater agreement between children's self-reported and their mothers' proxy-reported dental anxiety: a Chinese cross-sectional study.

BACKGROUND: Children's dental anxiety is common in dental clinics. This study aimed to determine the interrater agreement between children's self-reported and their mothers' proxy-reported dental anxiety and its affecting factors. METHODS: In this cross-sectional study performed in a dental clinic, primary school students and their mothers were assessed for enrollment eligibility. The Modified Dental Anxiety Scale plus Facial Image Scale (MDAS-FIS) was employed to test both the children's self-reported and their mothers' proxy-reported dental anxiety independently. The interrater agreement was analyzed using percentage agreement and the linear weighted kappa (k) coefficient. Factors affecting children's dental anxiety were analyzed using univariate and multivariate logistic regression models. RESULTS: One hundred children and their mothers were enrolled. The median ages of the children and mothers were 8.5 and 40.0 years old, respectively, and 38.0% (38/100) of the children were female. The scores of children's self-reported dental anxiety were significantly higher than their mothers' proxy-reported dental anxiety (MDAS-Questions 1-5, all p < 0.05); moreover, there was no agreement between the two groups in terms of all anxiety hierarchies (kappa coefficient = 0.028, p = 0.593). In the univariate model, a total of seven factors (age, gender, maternal anxiety, number of dental visits, mother's presence or absence, oral health status, and having siblings or not) were involved for analysis, and age [every 1-year increase, odds ratio (OR) = 0.661, 95% confidence interval (CI) = 0.514-0.850, p = 0.001], several dental visits (every 1 visit increase, OR = 0.409, 95% CI = 0.190-0.880, p = 0.022), and mother presence (OR = 0.286, 95% CI = 0.114-0.714, p = 0.007) were affecting factors. In the multivariate model, only age (every 1 year increase) and maternal presence were associated with 0.697-fold (95% CI = 0.535-0.908, p = 0.007) and 0.362-fold (95% CI = 0.135-0.967, p = 0.043) decreases in the risk of children's dental anxiety during dental visits and treatment, respectively. CONCLUSION: There was no significant agreement between elementary school students' self-reported dental anxiety and mothers' proxy ratings of children's dental anxiety, which suggests that self-reported dental anxiety by children should be encouraged and adopted, and the mother's presence during dental visits is strongly recommended.

Hydroxylation with Unusual Stereoinversion Catalyzed by an FeII /2-OG Dependent Oxidase and 3,6-Diene-2,5-diketopiperazine Formation in the Biosynthesis of Brevianamide†K.

Natural products with the 3,6-diene-2,5-diketopiperazine core are widely distributed in nature; however, the biosynthetic mechanism of 3,6-diene-2,5-diketopiperazine in fungi remains to be further elucidated. Through heterologous expression and biochemical investigation of an FeII /2-oxoglutarate-dependent oxidase (AspE) and a heme-dependent P450 enzyme (AspF), we report that AspE, AspF and subsequent dehydration account for the formation of the 3,6-diene-2,5-diketopiperazine substructure of brevianamide K from Aspergillus sp. SK-28, a symbiotic fungus of mangrove plant Kandelia candel. More interestingly, in-depth investigation of the enzymatic mechanism showed that AspE promotes hydroxylation of brevianamide Q with unprecedented stereoinversion through hydrogen atom abstraction and water nucleophilic attack from the opposite face of the resultant iminium cation intermediate.

Production of the antifungal biopesticide physcion through the combination of microbial fermentation and chemical post-treatment.

Physcion is an anthraquinone compound observed dominantly in medicinal herbs. This anthraquinone possesses a variety of pharmaceutically important activities and has been developed to be a widely used antifungal biopesticide. Herein, we report on the effective preparation of 3R-torosachrysone (4), a tetrahydroanthracene precursor of physcion, in Aspergillus oryzae NSAR1 by heterologous expression of related genes mined from the phlegmacins-producing ascomycete Talaromyces sp. F08Z-0631. Conditions for converting 4 into physcion were studied and optimized, leading to the development of a concise approach for extracting high-purity physcion from the alkali-treated fermentation broth of the 4-producing A. oryzae strain.

Mechanistic investigations of hirsutene biosynthesis catalyzed by a chimeric sesquiterpene synthase from Steccherinum ochraceum.

The high efficiency and elegance of terpene synthases is fascinating in constructing the molecular skeleton of complicated terpenoids with multiple chiral centers. Although the rapid development of sequencing technology has led to the discovery of an increasing number of terpene synthases, the cyclization mechanisms of some terpene synthases remains elusive. Here, we report that a chimeric sesquiterpene synthase from Steccherinum ochraceum is responsible for the biosynthesis of (+)-hirsutene, a linear triquinane sesquiterpene. Structural validation, and isotope labeling experiments demonstrate that the biosynthesis of (+)-hirsutene employs an unusual cyclization mode, involving three different cyclization processes (C1-C11, C2-C9, C3-C6), one intramolecular 1,2-hydride shift (C9-C10) and three successive 1,2-alkyl shifts to construct the 5-5-5 fused ring skeleton of (+)-hirsutene.

Unsymmetrically Regioselective Homodimerization Depends on the Subcellular Colocalization of Laccase/Fasciclin Protein in the Biosynthesis of Phlegmacins.

Phlegmacins are homodimeric dihydroanthracenone natural products featuring two torosachrysone monomers unsymmetrically conjugated by 7,10'-coupling. Herein, we report the identification and characterization of the biosynthetic gene cluster of phlegmacins in ascomycete Talaromyces sp. F08Z-0631. On the basis of the heterologous reconstitution of the phlegmacin pathway in Aspergillus oryzae, we demonstrated an unprecedented laccase-involved unsymmetrically regioselective oxidative coupling reaction. The association of laccase PhlC and the fasciclin partner protein PhlB was verified to be indispensable for the coupling activity. Intriguingly, both proteins can be transferred and located independently at the mitochondrial membrane. Notably, only their subcellular colocalization led to the occurrence of oxidative dimerization. These observations add new insights into the poorly understood catalytic mechanisms of various laccases involved in the biosynthesis of secondary metabolites, particularly those functioning with variable partners.

A Flavin-Dependent Monooxygenase Mediates Divergent Oxidation of Rifamycin.

Rifamycins have been clinically utilized against mycobacterial infections for more than 50 years; however, their biosynthesis has not been fully elucidated. Here, on the basis of in vivo gene deletions, in vitro enzyme assays, isotope labeling, and site-directed mutations, we found that a flavin-dependent monooxygenase encoded by a rifamycin biosynthetic gene cluster, Rif-Orf17, not only converted the naphthoquinone chromophore of rifamycin S into benzo-γ-pyrone but also linearized rifamycin SV through phenolic hydroxylation. Both oxidation routes lead to inactivation of rifamycins.

Triptolide: reflections on two decades of research and prospects for the future.

Covering: 2000 to 2020 Triptolide is a bioactive diterpene triepoxide isolated from Tripterygium wilfordii Hook F, a traditional Chinese medicinal plant whose extracts have been used as anti-inflammatory and immunosuppressive remedies for centuries. Although triptolide and its analogs exhibit potent bioactivities against various cancers, and inflammatory and autoimmune diseases, none of them has been approved to be used in the clinic. This review highlights advances in material sourcing, molecular mechanisms, clinical progress and new drug design strategies for triptolide over the past two decades, along with some prospects for the future course of development of triptolide.

A Glucose-Triptolide Conjugate Selectively Targets Cancer Cells under Hypoxia.

A major hurdle in the treatment of cancer is chemoresistance induced under hypoxia that is characteristic of tumor microenvironment. Triptolide, a potent inhibitor of eukaryotic transcription, possesses potent antitumor activity. However, its clinical potential has been limited by toxicity and water solubility. To address those limitations of triptolide, we designed and synthesized glucose-triptolide conjugates (glutriptolides) and demonstrated their antitumor activity in vitro and in vivo. Herein, we identified a lead, glutriptolide-2 with an altered linker structure. Glutriptolide-2 possessed improved stability in human serum, greater selectivity toward cancer over normal cells, and increased potency against cancer cells. Glutriptolide-2 exhibits sustained antitumor activity, prolonging survival in a prostate cancer metastasis animal model. Importantly, we found that glutriptolide-2 was more potent against cancer cells under hypoxia than normoxia. Together, this work provides an attractive glutriptolide drug lead and suggests a viable strategy to overcome chemoresistance through conjugation of cytotoxic agents to glucose.

LncRNA TTN-AS1 promotes the progression of oral squamous cell carcinoma via miR-411-3p/NFAT5 axis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common kind of squamous cell carcinoma of the head and neck, which is a threat to public health. Long noncoding RNAs (lncRNAs) are associated with the development of various diseases, including cancers. LncRNA titin antisense RNA 1 (TTN-AS1) is known as a crucial regulatory factor in several cancers. Nevertheless, the specific functions of TTN-AS1 in OSCC remains obscure. METHODS: The expression of TTN-AS1 in OSCC samples or cells was analyzed through qRT-PCR. Colony formation assay, EdU assay, flow cytometry assay, TUNEL assay and wound healing assay were conducted to estimate the functions of TTN-AS1 in OSCC cells. RIP and luciferase reporter assays were utilized to detect the interaction between TTN-AS1 and miR-411-3p as well as between miR-411-3p and NFAT5. RESULTS: TTN-AS1 expression was stronger in OSCC cells. Knockdown of TTN-AS1 effectively restrained cell proliferation and migration but had inductive role in apoptosis. Moreover, TTN-AS1 could function as the miR-411-3p sponge in OSCC and miR-411-3p exerted the inhibitory functions on OSCC cell growth. In addition, NFAT5 was proven as the target of miR-411-3p. Rescue assay indicated that overexpressing NFAT5 could reverse the inhibitory function of TTN-AS1 depletion on cell growth. CONCLUSION: lncRNA TTN-AS1 contributed to the progression of OSCC via miR-411-3p/NFAT5 axis.

Targeted Delivery and Sustained Antitumor Activity of Triptolide through Glucose Conjugation.

Triptolide, a key ingredient from the traditional Chinese medicinal plant thunder god vine, which has been used to treat inflammation and autoimmune diseases for centuries, has been shown to be an irreversible inhibitor of the XPB subunit of the transcription factor TFIIH and initiation of RNA polymerase II mediated transcription. The clinical development of triptolide over the past two decades has been limited by its toxicity and low water solubility. Herein, we report the development of a glucose conjugate of triptolide, named glutriptolide, which was intended to target tumor cells overexpressing glucose transporters selectively. Glutriptolide did not inhibit XPB activity in vitro but demonstrated significantly higher cytotoxicity against tumor cells over normal cells with greater water solubility than triptolide. Furthermore, it exhibited remarkable tumor control in vivo, which is likely due to sustained stepwise release of active triptolide within cancer cells. These findings indicate that glutriptolide may serve as a promising lead for developing a new mechanistic class of anticancer drugs.

Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide.

Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.

[Preparation of Tb3+, Yb3+ doped Y2O3 luminescent powders and near-infrared quantum cutting research].

Y2O3:Tb3+ and Y2O3:Tb3+, Yb3+ samples were prepared by co-precipitation method. The morphology, microstructure and fluorescence spectra at room temperature of samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and fluorescence spectrometer, The optimal process conditions of Y2O3:Tb3+ under different doping concentrations, annealing temperature, and pH value of the solution were obtained: Tb3+ concentration is 1.5%, annealing temperature is 1400 degrees C, an alkaline solution environment, and samples under 300 nm light excitation have the largest green light emission at 543 nm. The corresponding relation of Tb3+ ion level structure and transition properties and experimental spectra were analyzed in detail, and we explained the influence mechanism of process conditions and the fluorescence quenching process mainly effects luminous intensity of samples. The energy transfer from sensitizing ions Tb3+ to active ion Yb3+ was confirmed, it made the sample have considerable emitting light in the near-infrared region; the authors described the process of cooperation conversion luminescence between the two ions from the level transition angle, and also analyzed the system of fluorescence quenching process. Test results showed that the near infrared quantum cutting can effectively improve the luminous efficiency of doped ions, and will have broad application prospects in the silicon solar cells and other fields.

Self-resistance to an antitumor antibiotic: a DNA glycosylase triggers the base-excision repair system in yatakemycin biosynthesis.

Resistance is (not) futile: The yatakemycin biosynthetic gene cluster involves the ytkR2 gene, which encodes a protein with homology to a recently discovered bacterial DNA glycosylase. Genetic validation in vivo, biochemical assays, and in vitro mutagenesis studies revealed that YtkR2 confers resistance for the bacteria by specifically recognizing and cleaving the YTM-modified base (see scheme).

Characterization of yatakemycin gene cluster revealing a radical S-adenosylmethionine dependent methyltransferase and highlighting spirocyclopropane biosynthesis.

Yatakemycin (YTM), an antitumor natural product, represents the most potent member of a class of potent anticancer natural products including CC-1065 and duocarmycins. Herein we describe the biosynthetic gene cluster of YTM, which was identified by genome scanning of Streptomyces sp. TP-A0356. This cluster consists of 31 open reading frames (ORFs) and was localized to a 36 kb DNA segment. Moreover, its involvement in YTM biosynthesis was confirmed by cluster deletion, gene replacement, and complementation. Inactivation of ytkT, which encodes a radical S-adenosylmethionine (SAM) protein, created a mutant strain that failed to produce YTM but accumulated a new metabolite, which was structurally elucidated as a precursor that was related to the formation of the cyclopropane ring. More importantly, biochemical characterization of the radical SAM-dependent enzyme YtkT revealed that it is a novel C-methyltransferase and contributes to an advanced intermediate during formation of the cyclopropane ring through a radical mechanism in the YTM biosynthetic pathway. On the basis of in silico analysis, genetic experiments, structure elucidation of the novel intermediate, and biochemical characterization, a biosynthetic pathway for yatakemycin was proposed, which sets the stage to further investigate the novel enzymatic mechanisms and engineer the biosynthetic machinery for the production of novel analogues.

Diazo reagents with small steric footprints for simultaneous arming/SAR studies of alcohol-containing natural products via O-H insertion.

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely, an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O-H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated, and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of α-trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors.

XPB, a subunit of TFIIH, is a target of the natural product triptolide.

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.