Network Pharmacology, Molecular Docking, and Experimental Validation on Guiluoshi Anzang Decoction Against Premature Ovarian Insufficiency.

BACKGROUND AND OBJECTIVES: Premature Ovarian Insufficiency (POI) is a disease suffered by women under the age of 40 when ovarian function has declined, seriously affecting both the physical and mental health of women. Guiluoshi Anzang decoction (GLSAZD) has been used for a long time and has a unique therapeutic effect on improving ovarian function. This study aims to investigate the mechanism of GLSAZD in treating POI through network pharmacology, molecular docking, and experimental verification. METHODS: In this study, the active ingredients of Guiluoshi Anzang Decoction and the targets of POI were obtained from TCMSP, BATMANN-TCM, Uniprot, GeneCards, and other databases, and network pharmacology analysis was performed. Molecular docking was conducted to validate the affinity of the main active ingredient of GLSAZD to key POI targets. A POI SD rat model was established, and HE staining, ELISA, Real-time PCR, and Western blot experiments were performed to verify the predicted core targets and the therapeutic effects. RESULTS: 10 core targets and the top 5 ingredients were screened out. Molecular docking showed core targets AKT1, CASP3, TNF, TP53, and IL6 had stable binding with the core 5 ingredients quercetin, kaempferol, beta-sitosterol, luteolin, and Stigmasterol. GO and KEGG enrichment analysis demonstrated the mechanism involved in the positive regulation of gene expression, PI3K-AKT signaling pathway, and apoptosis signaling pathways. Animal experiments indicated GLSAZD could up-regulate the protein expression of p-PI3K and p-AKT1 and the mRNA expression of STAT3 and VEGF, down-regulate TP53 and Cleaved Caspase-3 protein expression in rat`s ovarian tissues and serum TNF-α and IL-6 protein levels, activate PI3K-AKT signaling pathway and inhibit the apoptosis signaling pathway. CONCLUSION: GLSAZD treats POI through multi-component, multi-target, and multi-pathway approaches. This study provided evidence for its clinical application in treating POI and shed light on the study of traditional medicine of the Guangxi Zhuang Autonomous Region in China.

Surface Oxygen Deficiency Enabled Spontaneous Antiprotein Fouling in WO3 Nanosheets for Biosensing in Biological Fluids.

Biofouling deteriorates the performance of sensors operated in biofluids. Protein adsorption is believed to be the first step of biofouling, which also reduces biocompatibility by further inducing cell adhesion, platelet activation, and even inflammation. Current studies of antifouling coatings are focused on polymers and hydrogels, which have succeeded in remaining resistant to protein adsorption, but their application on sensor electrodes is limited due to low conductivity and biocompatibility. Here, we report a spontaneous antibiofouling strategy for sensor electrodes by controlling oxygen vacancies in WO3 nanosheets. Irreversible adsorption of proteins was reduced by 76% in unprocessed human plasma when electrodes were coated with WO3 rich in surface oxygen vacancy. These electrodes maintained 91% of the initial current density after 1 month of incubation in human plasma.

Nanotoxicity of tungsten trioxide nanosheets containing oxygen vacancy to human umbilical vein endothelial cells.

Because of the excellent performance in photochemistry, WO3 is increasingly applied in the field of biology and medicine. However, little is known about the mechanism of WO3 cytotoxicity. In this work, WO3 nanosheets with oxygen vacancy are synthesized by solvothermal method, then characterized and added to culture medium of human umbilical vein endothelial cells (HUVECs) with different concentrations. We characterized and analyzed the morphology of nano-WO3 by transmission electron microscopy and calculated the specific data of oxygen vacancy by XPS. It is the first time the effect of WO3-x on cells that WO3-x can cause oxidative stress in HUVEC cells, resulting in DNA damage and thus promoting apoptosis. Transcriptome sequencing is performed on cells treated with low and high concentrations of WO3-x, and a series of key signals affecting cell proliferation and apoptosis are detected in differentially expressed genes, which indicates the research direction of nanotoxicity. The expression levels of key genes are also verified by quantitative PCR after cell treatment with different concentrations of WO3-x. This work fills the gap between the biocompatibility of nano WO3-x materials and molecular cytology and paves the way for investigating the mechanism and risks of oxygen vacancy in cancer therapy.

Chromosome-level genome assembly and functional characterization of terpene synthases provide insights into the volatile terpenoid biosynthesis of Wurfbainia villosa.

Wurfbainia villosa is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of China. Its dried fruits (called Fructus Amomi) are broadly used in traditional Chinese medicine for curing gastrointestinal diseases and are rich in volatile terpenoids. Here, we report a high-quality chromosome-level genome assembly of W. villosa with a total size of approximately 2.80 Gb, 42 588 protein-coding genes, and a very high percentage of repetitive sequences (87.23%). Genome analysis showed that W. villosa likely experienced a recent whole-genome duplication event prior to the W. villosa-Zingiber officinale divergence (approximately 11 million years ago), and a recent burst of long terminal repeat insertions afterward. The W. villosa genome enabled the identification of 17 genes involved in the terpenoid skeleton biosynthesis pathway and 66 terpene synthase (TPS) genes. We found that tandem duplication events have an important contribution to the expansion of WvTPSs, which likely drove the production of volatile terpenoids. In addition, functional characterization of 18 WvTPSs, focusing on the TPS-a and TPS-b subfamilies, showed that most of these WvTPSs are multi-product TPS and are predominantly expressed in seeds. The present study provides insights into the genome evolution and the molecular basis of the volatile terpenoids diversity in W. villosa. The genome sequence also represents valuable resources for the functional gene research and molecular breeding of W. villosa.

Research on germplasm diversity of Amomum villosum. Lour in genuine producing area.

BACKGROUND: Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum. METHODS: The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations. RESULTS: The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565. CONCLUSION: There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.

Tanshinone IIA enhances the ovarian reserve and attenuates ovarian oxidative stress in aged mice.

BACKGROUND: Tanshinone IIA (TSA), a major lipophilic component extracted from the roots of Salvia miltiorrhiza Bunge, has been widely used in China for its various biological activities. However, its effect on ovarian reserve in aged mice was not studied elsewhere. OBJECTIVES: This study aimed to explore the effect of TSA on the ovarian reserve of aged mice as well as young mice. Forty weeks old mice (N = 40) were considered as aged group compared to 4 weeks old mice (N = 40), and these groups were subdivided into four subgroups (N = 10) to receive different doses of TSA (0, 10, 20, and 40 µg/g/day). METHODS: The effect of TSA was evaluated by counting follicular number by histological examination. Basal serum levels of FSH, LH, E2, and anti-Mullerian hormone (AMH) were measured by ELISA. Moreover, the expression levels of antioxidant genes (CAT, Nrf2, GPX1), gap junction (Cx37), ERK1/2, and Smad5 family gene were examined at both mRNA (qPCR) and protein levels (western blot). RESULTS: Follicular number, level of AMH and E2, and the expression of CAT, Nrf2, and GPX1 genes increased significantly (p < 0.05) in aged mice administrated with medium (20 µg/g/day) and high (40 µg/g/day) doses of TSA, whereas FSH and LH levels were significantly low compared to low dose (10 µg/g/day) and control (0 µg/g/day) aged subgroups. However, we did not observe any effect of all doses of TSA on young mice. CONCLUSIONS: Administration of TSA with medium and high doses up-regulates the expression of antioxidative genes, reduces the oxidative injury, increases levels of AMH, and E2 levels that are relatively comparable to those in young mice, and consequently results in a healthy oocyte development.

Point-of-care COPD diagnostics: biomarkers, sampling, paper-based analytical devices, and perspectives.

Chronic obstructive pulmonary disease (COPD) has become the third leading cause of global death. Insufficiency in early diagnosis and treatment of COPD, especially COPD exacerbations, leads to a tremendous economic burden and medical costs. A cost-effective and timely prevention requires decentralized point-of-care diagnostics at patients' residences at affordable prices. Advances in point-of-care (POC) diagnostics may offer new solutions to reduce medical expenditures by measuring salivary and blood biomarkers. Among them, paper-based analytical devices have been the most promising candidates due to their advantages of being affordable, biocompatible, disposable, scalable, and easy to modify. In this review, we present salivary and blood biomarkers related to COPD endotypes and exacerbations, summarize current technologies to collect human whole saliva and whole blood samples, evaluate state-of-the-art paper-based analytical devices that detect COPD biomarkers in saliva and blood, and discuss existing challenges with outlooks on future paper-based POC systems for COPD diagnosis and management.

Mitigating hook effect in one-step quantitative sandwich lateral flow assay by timed conjugate release.

Sandwich lateral flow assay (LFA) is one of the most successfully commercialized paper-based biosensors, which offers a rapid, low-cost, one-step assay. Despite its advantages, conventional sandwich LFA is fundamentally limited by the high-dose "hook" effect-a phenomenon that occurs at very high analyte concentrations and results in false-negative results. In this paper, we present a novel strategy of automatic timed detection antibody release to mitigate the hook effect in sandwich LFA without additional manual steps. We introduced an intermediate pad treated with saturated sucrose solution to regulate the flow between the nitrocellulose membrane and the conjugate pad in order to delay the reaction between detection antibodies and analytes. Using C-reactive protein (CRP) as a representative analyte, we demonstrated that our strategy exhibited a range of detection 10 times wider than that of our conventional LFA, without sacrificing the limit of detection. Comparing to other published strategies, our work could offer a one-step, cost-effective approach that is closely unified with the benefits of the LFA.

Research on the reproductive biological characteristics of Amomum villosum Lour. and Amomum longiligulare T. L. Wu.

OBJECTIVE: To explore the influence of biological characteristics on the yield of Amomum villosum Lour. and Amomum longiligulare T. L. Wu, to find an effective pollen viability evaluation method and storage method to solve the problem of the low yield of Amomum plants. METHODS: Five germplasm of Amomum plants were used to investigate the effects of the phenological phase, pollen viability, and stigma receptivity on natural and artificial fruit set. RESULTS: Amomum longiligulare T. L. Wu showed late flowering, and its natural pollination rate is higher than that of Amomum villosum Lour. In all germplasm, the artificial pollination rate and fruit setting rate are more than 3 times higher than that under natural conditions. Fruits begin to drop seven days after successful pollination, and the fruit drop is basically stable after one month. The hybridization verification showed that TTC method was simpler and more accurate than in vitro germination method. Optimal storage conditions for pollen are 4°C and high humidity. After 36 h of storage, pollen can still be used for artificial cross-pollination or as hybrid parents. CONCLUSION: The special biological characteristics are the fundamental reason for the low natural pollination rate of Amomum plants. The accurate measurement method of Amomum plants pollen is the TTC method, and storage at 4°C and high humidity can increase the yield, which was six times that of the natural yield.

Exploring antimicrobial mechanism of essential oil of Amomum villosum Lour through metabolomics based on gas chromatography-mass spectrometry in methicillin-resistant Staphylococcus aureus.

Amomum villosum Lour (A. villosum Lour) has medicinal properties and has been widely used in China for many years. Herein we aimed to investigate the antibacterial mechanism and the metabolome variation caused by A. villosum Lour essential oil (EO) in methicillin-resistant Staphylococcus aureus (MRSA). The metabolite profile of MRSA was acquired, and metabolic pathways were assessed for significant alterations caused upon treating bacterial cells with EO, the antibacterial mechanism of EO was further investigated in combination with multiple experiments. Metabolomics analysis revealed that 72 metabolites and 10 pathways were significantly affected. EO specifically disrupted amino acid metabolism and the tricarboxylic acid (TCA) cycle, and also inhibited adenosine triphosphate (ATP) and reactive oxygen species (ROS) synthesis. Furthermore, the activities of pivotal enzymes involved in the TCA cycle were suppressed. Increased ROS levels could decrease the sensitivity of MRSA to EO, improving the survival of EO-treated MRSA cells. Our data indicate that A. villosum Lour EO causes metabolic dysfunction in MRSA, leading to reduced ROS levels, disruption of the TCA cycle, inhibition of ATP synthesis, and suppression of the activities of key enzymes.

[Study on ecological stereoscopic cultivation mode of Amomum villosum-Dimocarpus longan].

In order to set up a technical standard for planting Amomum villosum in wood forest in the future, we analyzed the relationship between the ecological factors and the yield of A. villosum planted in five Dimocarpus imocarpus longan wood forests and five miscellaneous wood forests in Yangchun city, to find out the dominant factors that affect the yield of A. villosum. The results showed that agricultural measures of fertilization, artificial irrigation and removing the old plants were positively correlated with the yield of A. villosum, the pesticide spraying and soil pH value were negatively correlated with the yield of A. villosum. But the effects of ecological factors on the yield were not significantly. High yield regions are generally located in the ravine, two sides of mountain stream and other places where water is more adequate. The slope of cultivated field with high yield is generally less than 30°, lighting and ventilation are more appropriate; soil type is generally sandy or loam, shade density is generally about 50%, and pollinators are many in quantity and variety. And we found that there was a large difference in mineral nutrient contents of soils among ten plantations. Results indicate that the yield of A. villosum is determined by the combination of each ecological factor. Suitable light intensity, moisture, ventilation and reasonable fertilization are conductive to increase the yield of A. villosum, but the use of pesticides and soil alkalization hinder the increase of A. villosum production. Too high shade density and the abuse of pesticides may be the main reason for limiting the yield of A. villosum planted in D. longan wood forests. This study has obtained key techniques of the ecological stereoscopic cultivation mode of A. villosum-D. longan, which lays a theoretical foundation for the guidance of farmers in planting A. villosum in the D. Longan forest in the future.

HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells.

Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5'-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at -149 to -3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1.

Synthesis of α,α-difluoromethylene alkynes by palladium-catalyzed gem-difluoropropargylation of aryl and alkenyl boron reagents.

gem-Difluoropropargyl bromides are versatile intermediates in organic synthesis, but have rarely been employed in transition-metal-catalyzed cross-coupling reactions. The first palladium-catalyzed gem-difluoropropargylation of organoboron reagents with gem-difluoropropargyl bromides is now reported. The reaction proceeds under mild reaction conditions with high regioselectivity; it features a broad substrate scope and excellent functional-group compatibility and thus provides an attractive approach for the synthesis of complex fluorinated molecules, in particular for drug discovery and development.

Nickel-catalyzed cross-coupling of functionalized difluoromethyl bromides and chlorides with aryl boronic acids: a general method for difluoroalkylated arenes.

Transition-metal-catalyzed difluoroalkylation of aromatics remains challenging despite the importance of difluoroalkylated arenes in medicinal chemistry. Herein, the first successful example of nickel-catalyzed difluoroalkylation of aryl boronic acids is described. The reaction allows access to a variety of functionalized difluoromethyl bromides and chlorides, and paves the way to highly cost-efficient synthesis of a wide range of difluoroalkylated arenes. The notable features of this protocol are its high generality, excellent functional-group compatibility, low-cost nickel-catalyst, and practicality for gram-scale production, thus providing a facile method for applications in drug discovery and development.

Characterization and functional analysis of the genes encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase and 1-deoxy-D-xylulose-5-phosphate synthase, the two enzymes in the MEP pathway, from Amomum villosum Lour.

A DXR gene, AvDXR (GenBank accession no. FJ459894), and a DXS gene, AvDXS (GenBank accession no. FJ455512), were isolated from the leaves of Amomum villosum, one of the most well-known and authentic herbs in South China. The 1,749-bp full-length cDNA of AvDXR encoded a peptide of 472 amino acids, and the 2,347-bp full-length cDNA of AvDXS encoded a peptide of 715 amino acids. The deduced amino acid sequences of the AvDXR and AvDXS proteins share high homology with DXRs and DXSs from other plant species, and AvDXS belongs to class 1 plant DXS. The characterization based on bioinformatic analysis indicated that the AvDXR and AvDXS encoded functional proteins as DXR and DXS, respectively. The functional color assay in Escherichia coli with pAC-BETA implied that AvDXR and AvDXS encoded functional proteins that manipulated the biosynthesis of isoprenoid precursors. Both AvDXR and AvDXS were expressed extensively in the leaves, stems, roots, pericarps and seeds of A. villosum. AvDXS expression was similar in all tissues investigated, whereas higher levels of AvDXR were observed in the fruits, the main part for the accumulation of volatile oil in this plant. AvDXR was transformed into tobacco to confirm its function further. Overexpression of AvDXR in transgenic T1 generation tobacco increased DXR activity, photosynthetic pigment content and volatile isoprenoid components, and the increase of photosynthetic pigment content was consistent with the AvDXR transcription level. This study demonstrated that AvDXR plays important role in isoprenoid biosynthesis and it is useful for metabolic engineering.

A novel receptor kinase involved in jasmonate-mediated wound and phytochrome signaling in maize coleoptiles.

We identified a gene of maize (Zea mays L.) that is transcriptionally activated in decapitated coleoptiles. The amino acid sequence deduced from its full-length cDNA indicated that the identified gene encodes a novel leucine-rich-repeat receptor-like kinase. The gene is named WOUND-RESPONSIVE AND PHYTOCHROME-REGULATED KINASE1 (WPK1) based on the findings of this study. Database searches revealed two and three homologs of WPK1 for Arabidopsis thaliana and rice, respectively. These homologs occurred along with WPK1 on a phylogenetic branch separated from all reported receptor kinases. We uncovered that the level of WPK1 transcripts is up-regulated rapidly and transiently in response to wounding and red light. The response to red light was reversible by far-red light, indicating that it is mediated by phytochrome. Applied jasmonic acid activated the expression of WPK1, while ethylene, salicylic acid and abscisic acid had no such effect. These results strongly suggested that WPK1 is a component of the jasmonate-mediated signaling that participates in both wound-induced defensive and phytochrome-mediated photomorphogenetic responses. Furthermore, it was found that both wounding and red light up-regulate the transcript level of ZmAOS, a gene for the jasmonate biosynthesis enzyme allene oxide synthase, and that auxin inhibits the expression of WPK1 but not of ZmAOS. We present a model of jasmonate-mediated signaling to explain the results obtained.