Mechanistic characterisation of a fungal fusicoccane-type diterpene synthase involved in the biosynthesis of talaro-7,13-diene.

The cyclisation mechanism of the fungal fusicoccane (FC)-type diterpene synthase (DTS) TadA was investigated by extensive isotopic labelling experiments, and the pH-dependency of the product selectivity of this enzyme was explored. These studies provide new insights into the cyclisation mechanisms of FC-type DTSs.

Identification of three novel P450 enzymes involved in the oxidative modification of a newly discovered fusicoccane diterpene.

Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5-8-5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2-5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5-8-5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis.

ROS-responsive MSC-derived Exosome Mimetics Carrying MHY1485 Alleviate Renal Ischemia Reperfusion Injury through Multiple Mechanisms.

Renal ischemia reperfusion (IR) injury is a prevalent inflammatory nephropathy in surgeries such as renal transplantation or partial nephrectomy, damaging renal function through inducing inflammation and cell death in renal tubules. Mesenchymal stromal/stem cell (MSC)-based therapies, common treatments to attenuate inflammation in IR diseases, fail to exhibit satisfying effects on cell death in renal IR. In this study, we prepared MSC-derived exosome mimetics (EMs) carrying the mammalian target of the rapamycin (mTOR) agonist to protect kidneys in proinflammatory environments under IR conditions. The thioketal-modified EMs carried the mTOR agonist and bioactive molecules in MSCs and responsively released them in kidney IR areas. MSC-derived EMs and mTOR agonists protected kidneys synergistically from IR through alleviating inflammation, apoptosis, and ferroptosis. The current study indicates that MSC-TK-MHY1485 EMs (MTM-EM) are promising therapeutic biomaterials for renal IR injury.

Upregulation of NPC1 and its association with poor prognosis in gastric cancer.

BACKGROUND: The Niemann-Pick disease type C1 (NPC1) protein plays a pivotal role in lipid transport, particularly free cholesterol, within lysosomal/late endosomal membranes. Previous studies have highlighted NPC1 as a promising target for cholesterol trafficking and cancer therapy. Nevertheless, the expression of NPC1 in gastric cancer (GC) and its clinical implications remain unexplored. This study aims to investigate NPC1 expression in GC and its correlation with patient prognosis. METHODS: NPC1 expression levels in GC and normal tissues were assessed using the GEPIA database, and survival analysis was conducted via Kaplan‒Meier Plotter. Evaluation of potential biological effects of NPC1 in GC by protein-protein interaction network and GO, KEGG bioenrichment analysis. Immunohistochemistry was performed on surgical samples collected from 306 GC patients. Correlations between NPC1 expression, clinical characteristics, and patient prognosis were analyzed. RESULTS: NPC1 mRNA expression was elevated in GC tissues compared to normal tissues (P < 0.05) and significantly associated with poorer prognosis. In our cohort of 306 patients, NPC1 exhibited significant upregulation in GC versus adjacent normal tissues (P = 0.031). High NPC1 expression correlated with adverse clinical characteristics, including lymph node metastasis, distant metastasis, and advanced TNM stage (all P < 0.05). Patients with high NPC1 expression experienced notably shorter overall survival (P < 0.001), particularly in stages III and IV (P = 0.003). Multivariate Cox regression analysis identified high NPC1 expression as an independent prognostic factor for GC patients (HR 1.57, 95% CI 1.14-2.18, P = 0.006). Lastly, an optimized nomogram incorporating NPC1, tumor size, and TNM stage was constructed. CONCLUSIONS: NPC1 expression is upregulated in GC and serves as a pivotal prognostic factor for adverse outcomes in GC patients.

Exosome-mimetic vesicles derived from fibroblasts carrying matrine for wound healing.

Background: Chronic skin wounds are a leading cause of hospital admissions and reduced life expectancy among older people and individuals with diabetes. Delayed wound healing is often attributed to a series of cellular abnormalities. Matrine, a well-studied component found in Sophora flavescens, is recognized for its anti-inflammatory effects. However, its impact on wound healing still remains uncertain. This study aims to explore the potential of matrine in promoting wound healing. Methods: In this study, we utilized gradient extrusion to produce fibroblast-derived exosome-mimetic vesicles as carriers for matrine (MHEM). MHEM were characterized using transmission electron microscopy and dynamic light scattering analysis. The therapeutic effect of MHEM in wound healing was explored in vitro and in vivo. Results: Both matrine and MHEM enhanced the cellular activity as well as the migration of fibroblasts and keratinocytes. The potent anti-inflammatory effect of matrine diluted the inflammatory response in the vicinity of wounds. Furthermore, MHEM worked together to promote angiogenesis and the expression of transforming growth factor ß and collagen I. MHEM contained growth factors of fibroblasts that regulated the functions of fibroblasts, keratinocytes and monocytes, which synergistically promoted wound healing with the anti-inflammatory effect of matrine. Conclusions: MHEM showed enhanced therapeutic efficacy in the inflammatory microenvironment, for new tissue formation and angiogenesis of wound healing.

Molecular crosstalk between circadian clock and NLRP3 inflammasome signaling in Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Research has recently found that both animal models and patients with PD have circadian dysfunction, accompanied by abnormal expression of circadian genes and proteins, which implies that the circadian clock plays a crucial role in PD etiopathogenesis. In addition, a strong relationship between NLRP3 inflammasome signaling and PD has been observed. Meanwhile, the activation of the NLRP3 inflammasome is highly relevant to dysfunctions of the molecular clock. Therefore, alleviating the neuroinflammation caused by NLRP3 inflammasome signaling by adjusting the abnormal molecular clock may be a potential strategy for preventing and treating PD. In this article, we have reviewed the potential or direct relationship between abnormalities of the circadian clock and NLRP3 inflammasome signaling in PD.

Biosynthesis of fungal terpenoids.

Covering: up to August 2023Terpenoids, which are widely distributed in animals, plants, and microorganisms, are a large group of natural products with diverse structures and various biological activities. They have made great contributions to human health as therapeutic agents, such as the anti-cancer drug paclitaxel and anti-malarial agent artemisinin. Accordingly, the biosynthesis of this important class of natural products has been extensively studied, which generally involves two major steps: hydrocarbon skeleton construction by terpenoid cyclases and skeleton modification by tailoring enzymes. Additionally, fungi (Ascomycota and Basidiomycota) serve as an important source for the discovery of terpenoids. With the rapid development of sequencing technology and bioinformatics approaches, genome mining has emerged as one of the most effective strategies to discover novel terpenoids from fungi. To date, numerous terpenoid cyclases, including typical class I and class II terpenoid cyclases as well as emerging UbiA-type terpenoid cyclases, have been identified, together with a variety of tailoring enzymes, including cytochrome P450 enzymes, flavin-dependent monooxygenases, and acyltransferases. In this review, our aim is to comprehensively present all fungal terpenoid cyclases identified up to August 2023, with a focus on newly discovered terpenoid cyclases, especially the emerging UbiA-type terpenoid cyclases, and their related tailoring enzymes from 2015 to August 2023.

Echinacoside exerts neuroprotection via suppressing microglial α-synuclein/TLR2/NF-κB/NLRP3 axis in parkinsonian models.

BACKGROUND: Echinacoside (ECH), a natural active compound, was found to exert neuroprotection in Parkinson's disease (PD). However, the underlying molecular mechanisms remain controversial. PURPOSE: This study aimed to explore the roles of ECH in PD and its engaged mechanisms. CONCLUSION: In vivo, MPTP was adapted to construct subacute PD mouse model to explore the regulation of ECH on NLRP3 inflammasome. In vitro, α-synuclein (α-syn)/MPP+ was used to mediate the activation of NLRP3 inflammasome in BV2 cells, and the mechanism of ECH regulation of it was explored with molecular docking, immunofluorescence, Western blotting, and small molecule inhibitors. CONCLUSION: The activation of microglial NLRP3 inflammasome could be evoked by MPTP in vitro, but its toxic metabolite MPP+ alone cannot trigger the activation of NLRP3 inflammasome in vitro, which requires α-synuclein (α-syn) priming. Exogenous α-syn could evoke microglial TLR2/NF-κB/NLRP3 axis, playing the priming role in MPP+ -mediated NLRP3 inflammasome activation. ECH can suppress the upregulation of α-syn in MPTP-treated mice and BV2 microglia. It can also suppress the activation of the TLR2/NF-κB/NLRP3 axis induced by α-syn. CONCLUSION: ECH exerts neuroprotective effects by downregulating the TLR2/NF-κB/NLRP3 axis via reducing the expression of α-syn in the PD models.

Baicalin exerts neuroprotective actions by regulating the Nrf2-NLRP3 axis in toxin-induced models of Parkinson's disease.

Baicalin, a potent anti-oxidative and anti-inflammatory flavonoid compound derived from Scutellaria baicalensis, has emerged as a neuroprotective agent. However, the mechanisms by which baicalin is neuroprotective in Parkinson's disease (PD) remain unclear. In this research, α-syn/MPP+ and MPTP were used to establish PD models in BV2 cells and C57BL/6 mice, respectively. The effect and mechanism of action of baicalin in PD were investigated by Western blotting, RT-qPCR, ELISA, Immunohistochemistry (IHC) staining, Immunofluorescence (IF) staining, HPLC and methods. Results demonstrate that baicalin mitigates oxidative stress, microglia activation and inflammatory response caused by α-syn/MPP+ and MPTP. It protects against dopaminergic neuron loss and relieves motor deficits. Meanwhile, baicalin not only significantly up-regulates the expression of Nrf2 and its downstream antioxidant enzyme, but also suppresses the activation of NLRP3 inflammasome simultaneously. Notably, the beneficial effects of baicalin in PD treatment are blocked by Nrf2 knockdown. This research reveals that baicalin may exert neuroprotective effects in PD treatment by suppressing the activation of NLRP3 inflammasome and it is dependent on the Nrf2-mediated antioxidative response.

Low temperature, mechanical wound, and exogenous salicylic acid (SA) can stimulate the SA signaling molecule as well as its downstream pathway and the formation of fruiting bodies in Flammulina filiformis.

Low temperature (LT) and mechanical wound (MW), as two common physics methods, have been empirically used in production to stimulate the primordia formation of Flammulina filiformis, which is typically produced using the industrial production mode. However, the detailed effect on the fruiting body formation and important endogenous hormones and signaling pathways in this process is poorly understood. In this study, LT, MW, their combination, i.e., MW + LT, and low concentration of SA (0.1 mM SA) treatments were applied to the physiologically mature mycelia of F. filiformis. The results showed that the primordia under the four treatments began to appear on the 5th-6th days compared with the 12th day in the control (no treatment). The MW + LT treatment produced the largest number of primordia (1,859 per bottle), followed by MW (757), SA (141), and LT (22), compared with 47 per bottle in the control. The HPLC results showed that the average contents of endogenous SA were significantly increased by 1.3 to 2.6 times under four treatments. A total of 11 SA signaling genes were identified in the F. filiformis genome, including 4 NPR genes (FfNpr1-4), 5 TGA genes (FfTga1-5), and 2 PR genes (FfPr1-2). FfNpr3 with complete conserved domains (ANK and BTB/POZ) showed significantly upregulated expression under all four above treatments, while FfNpr1/2/4 with one domain showed significantly upregulated response expression under the partial treatment of all four treatments. FfTga1-5 and FfPr1-2 showed 1.6-fold to 8.5-fold significant upregulation with varying degrees in response to four treatments. The results suggested that there was a correlation between "low temperature/mechanical wound-SA signal-fruiting body formation", and it will help researchers to understand the role of SA hormone and SA signaling pathway genes in the formation of fruiting bodies in fungi.

Codelivery of ERCC2 small interfering RNA and cisplatin with macrophage-derived mimetic nanovesicles for enhanced bladder cancer treatment.

Cisplatin-based chemotherapy plays a vital role in the management of muscle-invasive bladder cancer (MIBC); however, off-tumor toxicity and resistance often lead to cancer recurrence and eventual treatment failure. The loss of function of the nucleotide excision repair gene excision repair cross-complementing rodent repair deficiency gene 2 ( ERCC2 ) in cancer cells correlates with sensitivity to cisplatin, while its overexpression causes cisplatin resistance. Small interfering RNA (siRNA) knockdown of ERCC2 combined with cisplatin treatment may improve therapeutic outcomes in patients with bladder cancer. Here, we aimed to develop macrophage-derived mimetic nanovesicles (MNVs) as a nanoplatform for the simultaneous delivery of cisplatin and ERCC2 siRNA for enhancing the efficacy of bladder cancer chemotherapy. The cellular uptake, gene down-regulation, tumor inhibition effects, and biosafety of the synthesized nanodrugs (MNV-Co) as a synergistic therapeutic strategy for MIBC were evaluated in vitro and in vivo . The results indicated high efficacy of MNV-Co against MIBC and low off-tumor toxicity. Furthermore, by down-regulating ERCC2 mRNA and protein levels, MNV-Co improved chemosensitivity, promoted cancer cell apoptosis, and effectively suppressed tumor growth. This study presents a potential approach for delivering cisplatin and ERCC2 siRNA concurrently to treat bladder cancer using a biomimetic nanosystem.

Refinement of the Fusion Tag PagP for Effective Formation of Inclusion Bodies in Escherichia coli.

Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high ß-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation "hot spots" (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the ß-strands. Substitution of these prolines by residues with high ß-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable ß-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.

Isolation of new compounds related to xyloketals biosynthesis implies an alternative pathway for furan-fused-chromene formation.

In fungi, there is a rare group of natural products harboring the 2,3,3a,9a-tetrahydro-4H-furo[2,3-b]chromene skeleton, represented by xyloketal B, which display a wide range of biological activities and have drawn significant attention. In this work, four new analogues simpliketals A-D (1-4), as well as two other new compounds simplilactones A and B (5 and 6), were isolated from Simplicillium sp. AHK071-01. Their structures were elucidated by extensive NMR spectroscopic methods, 13C NMR calculation, single-crystal X-ray diffraction, and ECD calculation. In addition, five known compounds (7-11) including alboatrin (7) were also obtained. Based on the structural similarity of the above compounds, we inferred that compounds 5, 6, and 8-11 might be biosynthetically related with 1-4 and 7, which allowed us to propose an alternative biosynthetic route to generate the furan-fused chromene skeleton of this class of compounds, instead of a previously presumed polyketide-terpenoid hybrid pathway. Finally, cytotoxicity assays showed that 1-4 exhibited weak inhibitory activity on PANC-1 cells and that 2 and 3 possessed moderate activity against SH-SY5Y cells.

Acyl carrier protein tag can enhance tobacco etch virus protease stability and promote its covalent immobilisation.

Fusion expression is widely employed to enhance the solubility of recombinant proteins. However, removal of the fusion tag is often required due to its potential impact on the structure and activity of passenger proteins. Tobacco etch virus (TEV) protease is widely used for this purpose due to its stringent sequence recognition. In the present work, fusion to the acyl carrier protein from E. coli fatty acid synthase (ACP) significantly increased the yield of recombinant soluble TEV, and the ACP tag also greatly improved TEV stability. The cleavage activity of TEV was not affected by the ACP fusion tag, and ACP-TEV retained high activity, even at unfavourable pH values. Moreover, ACP-TEV could be efficiently modified by co-expressed E. coli holo-ACP synthase (AcpS), leading to covalent attachment of 4'-phosphopantetheine (4'-PP) group to ACP. The sulfhydryl group of the long, flexible 4'-PP chain displayed high specific reactivity with iodoacetyl groups on the solid support. Thus, TEV could be immobilised effectively and conveniently via the active holo-ACP, and immobilised TEV retained high cleavage activity after a long storage period and several cycles of reuse. As a low-cost and recyclable biocatalyst, TEV immobilised by this method holds promise for biotechnological research and development. KEY POINTS: • The ACP tag greatly increased the soluble expression and stability of TEV protease. • The ACP tag did not affect the cleavage activity of TEV. • The holo-ACP Tag effectively mediated the covalent immobilisation of TEV.

Amelioration effect of water extract from Ganoderma resinaceum FQ23 solid-state fermentation fungal substance with high-yield ergothioneine on anxiety-like insomnia mice.

Herein, a solid-state fermentation (SSF) system of Ganoderma resinaceum FQ23 with high-yield ergothioneine (EGT) was established, and the amelioration effect of the water extract from its fungal substance on anxiety-like insomnia mice was studied. The content of EGT in the G. resinaceum FQ23 SSF fungal substance increased to 1.146 ± 0.066 mg g-1 DW in the optimization tests. Besides EGT, the common functional components of the water extract from the G. resinaceum FQ23 SSF fungal substance (GSW) were determined, including triterpenoids, polysaccharides, phenols, proteins and amino acids. The animal experiments showed that GSW could alleviate the anxiety-like behavior, improve the antioxidant capacity and protect the organ structure of the anxiety-like insomnia mice. With an increase in the dose of GSW given to the anxiety-like insomnia mice, their serum 5-HT and GABA levels increased, HPA axis hormone levels significantly decreased, BDNF level notably increased, and the response level of the BDNF/CREB signaling pathway was significantly enhanced, indicating that GSW may improve neuroendocrine regulation and neuroprotection in anxiety-like insomnia mice. A 30-times dose of GSW had no acute toxicity in the normal mice. Therefore, the SSF fungal substance of G. resinaceum FQ23 is a potential dietary source for improving sleep. It can be used as a solid drink to help people who are poor sleepers and as a substitute for tea or coffee to help people who are like to drink tea or coffee and cannot sleep.

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme.

Fusicoccane-type terpenoids are a subgroup of diterpenoids featured with a unique 5-8-5 ring system. They are widely distributed in nature and possess a variety of biological activities. Up to date, only five fusicoccane-type diterpene synthases have been identified. Here, we identify a two-gene biosynthetic gene cluster containing a new fusicoccane-type diterpene synthase gene tadA and an associated cytochrome P450 gene tadB from Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D2O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known fusicoccane-type diterpene synthases, in which a neutral intermediate is firstly formed and then protonated by an environmental proton. In addition, we demonstrate that the associated cytochrome P450 enzyme TadB is able to catalyze multiple oxidation of talaro-7,13-diene to yield talaro-6,13-dien-5,8-dione.

High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma.

Aims: Colon adenocarcinoma (COAD) is responsible for 90% of all colorectal cancer cases and is one of the most common causes of cancer-related deaths worldwide. ATP6V1s (cytosolic V1 domain of vacuolar adenosine triphosphatase) participate in the biological process of transporting hydrogen ions and are implicated in tumor growth and metastasis. ATP6V1C2 as a family member has been documented to associate with esophageal carcinoma and renal clear cell carcinoma, while its roles in COAD remain elusive. Methods: The expression status, potential molecular mechanism, and prognostic value of ATP6V1C2 in COAD were investigated using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. In addition, its biological roles in COAD were explored through in vitro studies. Results: ATP6V1C2 showed a significantly higher expression level in COAD compared with matched non-cancerous tissues. High expression of ATP6V1C2 predicted a shorter overall survival both in TCGA and GEO COAD datasets, and ATP6V1C2 was identified as an independent factor associated with overall survival in COAD. Bioinformatic analyses showed that high expression of ATP6V1C2 was associated with high epithelial-mesenchymal transition (EMT) score and Wnt signaling pathway was significantly enriched from differentially expressed genes between ATP6V1C2-high and -low group. We also found that high expression of ATP6V1C2 could decrease pathway activity of CD8 T effector implicated in tumor microenvironment (TME). In vitro study revealed that ATP6V1C2 knockdown resulted in aberrant expression of Wnt- and EMT-related genes and inhibited COAD cell proliferation and growth. Conclusion: This is the first study to reveal the molecular functions of ATP6V1C2 in COAD. Our study suggests that overexpressed ATP6V1C2 might promote EMT by activating Wnt signaling pathway, resulting in cancer metastasis and poor prognosis. This study paves the way for understanding potential molecular mechanisms and therapeutic perspectives in COAD.

Construction and Validation of a Ferroptosis-Related Prognostic Model for Gastric Cancer.

BACKGROUND: Gastric cancer (GC), an extremely aggressive tumor with a very different prognosis, is the third leading cause of cancer-related mortality. We aimed to construct a ferroptosis-related prognostic model that can be distinguished prognostically. METHODS: The gene expression and the clinical data of GC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO). The ferroptosis-related genes were obtained from the FerrDb. Using the "limma" R package and univariate Cox analysis, ferroptosis-related genes with differential expression and prognostic value were identified in the TCGA cohort. Last absolute shrinkage and selection operator (LASSO) Cox regression was applied to shrink ferroptosis-related predictors and construct a prognostic model. Functional enrichment, ESTIMATE algorithm, and single-sample gene set enrichment analysis (ssGSEA) were applied for exploring the potential mechanism. GC patients from the GEO cohort were used for validation. RESULTS: An 8-gene prognostic model was constructed and stratified GC patients from TCGA and meta-GEO cohort into high-risk groups or low-risk groups. GC patients in high-risk groups have significantly poorer OS compared with those in low-risk groups. The risk score was identified as an independent predictor for OS. Functional analysis revealed that the risk score was mainly associated with the biological function of extracellular matrix (ECM) organization and tumor immunity. CONCLUSION: In conclusion, the ferroptosis-related model can be utilized for the clinical prognostic prediction in GC.

The Oxidation Cascade of a Rare Multifunctional P450 Enzyme Involved in Asperterpenoid A Biosynthesis.

The cytochrome P450 enzymes (P450s or CYPs) are heme-containing enzymes which catalyze a wide range of oxidation reactions in nature. In our previous study, a rare multifunctional P450 AstB was found, which can dually oxidize two methyl groups (C-19 and C-21) of preasperterpenoid A to asperterpenoid A with 3-carboxyl and 11-hydroxymethyl groups. However, the oxidation order of C-19 and C-21 catalyzed by AstB is unclear. In order to reveal this oxidation order, probable pathways catalyzed by AstB were proposed, and the oxidation order of C-19 and C-21 was obtained by quantum chemistry calculations. The potential intermediates (three new asperterpenoids D-F, 1-3) were obtained through the chemical investigation on the extract of the transformant strain and chemical conversions, which were used as the standards to detect their existences in the extract of the transformant strain with HPLC-MS. Combined with the quantum chemistry calculation and the HPLC-MS analysis, the catalyzed order of AstB in asperterpenoid A biosynthesis was revealed. Furthermore, the mPTPB inhibition of obtained asperterpenoids was evaluated, and the results showed that 3-carboxyl and the oxidation station of C-21 would be the key factors for mPTPB inhibition of asperterpenoids.

Integrated meta-analysis, network pharmacology, and molecular docking to investigate the efficacy and potential pharmacological mechanism of Kai-Xin-San on Alzheimer's disease.

CONTEXT: Kai-Xin-San (KXS) has been used to treat Alzheimer's disease (AD) for thousands of years. However, no quantitative data regarding AD treatment using KXS are available. Moreover, its active compounds and mechanism of action for the treatment of AD remain largely unclear. OBJECTIVES: To evaluate the efficacy and the potential pharmacological mechanisms of KXS in AD treatment. MATERIALS AND METHODS: A systematic collection of KXS experiments was conducted from PubMed, Web of Science, Embase, CNKI, VIP, and Wanfang Data up to February, 2020. Review Manager 5 software was used for meta-analysis. In network pharmacology, components of KXS were screened, AD-related genes were then identified and the 'component-target-pathway' network constructed. Molecular docking was finally employed for in silico simulation matching between representative KXS compounds and their target genes. RESULTS: Meta-analysis revealed that KXS improves the cognitive benefits in AD models by reducing the time of escape latency (SMD = -16.84) as well as increasing the number of cross-platform (SMD = 2.56) and proportion of time in the target quadrant (SMD = 7.52). Network pharmacology identified 25 KXS active compounds and 44 genes targets. DRD2, MAOA, ACHE, ADRA2A and CHRM2 were core target proteins. Besides, 22 potential pathways of KXS were identified, like cholinergic synapses, the cGMP/PKG pathway and calcium signalling. Molecular docking showed that stigmasterol, aposcopolamine and inermin can closely bind three targets (ACHE, ADRA2A and CHRM2). DISCUSSION AND CONCLUSION: These findings suggest that KXS exerts effect on AD through multi-target, multi-component and multi-pathway mechanism. Future studies may explore the active components of KXS.