Polygonati Rhizoma demonstrates significant potential for addressing both chronic and hidden hunger. The supply of high-quality seedlings is a primary factor influencing the development of the Polygonati Rhizoma industry. Warm water soaking is often used in agriculture to promote the rapid germination of seeds, while its application and molecular mechanism in Polygonati Rhizoma have not been reported. To rapidly obtain high-quality seedlings, this study treated Polygonatum kingianum var. grandifolium seeds with sand storage at low temperatures, warm water soaking, and cultivation temperature gradients. The results showed that the culture at 25 ℃ or sand storage at 4 ℃ for 2 months rapidly broke the seed dormancy of P. kingianum var. grandifolium, while the culture at 20 ℃ or sand storage at 4 ℃ for 1 month failed to break the seed dormancy. Soaking seeds in 60 ℃ warm water further increased the germination rate, germination potential, and germination index. Specifically, the seeds soaked at 60 ℃ and cultured at 25 ℃ without sand storage treatment(Aa25) achieved a germination rate of 78. 67%±1. 53% on day 42 and 83. 40%±4. 63% on day 77. The seeds pretreated with sand storage at 4 ℃ for 2 months, soaked in 60 ℃ water, and then cultured at 25 ℃ achieved a germination rate comparable to that of Aa25 on day 77. Transcriptomic analysis indicated that warm water soaking might promote germination by triggering reactive oxygen species( ROS), inducing the expression of heat shock factors( HSFs) and heat shock proteins( HSPs), which accelerated DNA replication, transcript maturation, translation, and processing, thereby facilitating the accumulation and turnover of genetic materials. According to the results of indoor controlled experiments and field practices, maintaining a germination and seedling cultivation environment at approximately 25 ℃ was crucial for the one-year seedling cultivation of P. kingianum var. grandifolium.
Germination ; Seedlings/genetics* ; Water/metabolism* ; Seeds/metabolism* ; Polygonatum/genetics* ; Temperature ; Plant Proteins/genetics* ; Plant Dormancy

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

OBJECTIVE: To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. METHODS: The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. RESULTS: Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CONCLUSIONS CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.
Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-met/metabolism* ; Liver Neoplasms/drug therapy* ; Signal Transduction/drug effects* ; Animals ; Humans ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Amphibian Venoms/therapeutic use* ; Cell Line, Tumor ; Neoplasm Metastasis ; Cell Survival/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Neoplasm Invasiveness ; Mice, Inbred BALB C ; Mice, Nude ; Mice ; Male ; Bufanolides/therapeutic use* ; Protein Precursors ; Prothrombin ; Biomarkers

Objective:To analyze the genetic characteristics of the VWF gene c.7332G＞A nonsense mutation and explore its molecular pathogenesis.Methods:Phenotypic diagnosis of the proband was performed using VWF:Ag,VWF:RCo,FⅧ:C and multimeric analysis.The probands were genotyped by NGS whole-exome sequencing,and the sequencing results were validated by sanger sequencing.The family members were genotyped by Sanger sequencing.The VWF gene c.7332G＞A nonsense mutant plasmid was constructed.After transfection,the function of VWF gene c.7332G＞A mutant plasmid was verified at cell level in vitro.The mRNA level was detected by qRT-PCR,and the expression level of protein was detected by Western blot,the function of multimerization was verified by the multimeric analysis.Results:VWF:Ag and VWF:RCo were all less than 3％in the proband,and the multimeric analysis showed multimer deficiency.The proband was diagnosed as type 3 VWD.The homozygous nonsense mutation of VWF gene c.7332G＞A was detected by gene sequencing.The VWF mRNA level of the mutant plasmid was decreased,and the VWF protein expression in the cell supernatant was decreased,the mutant protein was truncated and the function of VWF multimerization was impaired.Conclusion:A homozygous mutation in exon 43 of VWF gene,c.7332G＞A,was responsible for the probands type 3 VWD in the proband.The mutation caused a decrease in the relative level of VWF mRNA and protein,and impaired the function of VWF multimerization.

With the rapid development of generative artificial intelligence(GAI)technologies,their widespread application in academic research and writing is continuously expanding the boundaries of sci-entific inquiry.However,this trend has also raised a series of ethical and regulatory challenges,inclu-ding issues related to authorship,content authenticity,citation accuracy,and accountability.In light of the growing involvement of AI in generating academic content,establishing an open,controllable,and trustworthy ethical governance framework has become a key task for safeguarding research integrity and maintaining trust within the academic community.This expert consensus outlines ethical requirements across key stages of AI-assisted academic writing-including topic selection,data management,citation practices,and authorship attribution.It aims to clarify the boundaries and ethical obligations surrounding AI use in academic writing,ensuring that technological tools enhance efficiency without compromising in-tegrity.The goal is to provide guidance and institutional support for building a responsible and sustainable research ecosystem.

AIM To establish a UPLC-ESI-MS/MS method for analyzing the toxic material basis of 95％ethanol cold soaked ultrasonic extract(EC),95％ethanol heated reflux extract(EH)and water decoction extract(WD)from Dryopteris crassirhizoma Nakai.METHODS The analysis was performed on a 25 ℃ thermostatic agilent ZORBAX RRHD StableBond C18 column(2.1 mm×150 mm,1.8 μm),with the mobile phase comprising of methanol-0.2％formic acid flowing at 0.30 mL/min,and heated electrospray ion source was adopted in positive and negative ion scanning.Compounds were identified by Compound Discover 3.3 software combined with the database and related literature,and the main differential components were screened by Heatmap cluster analysis and partial least squares discriminant analysis.RESULTS 72 compounds were identified(22 phloroglucinols,19 flavonoids,8 phenylpropanoids,6 terpenoids and 17 other components).The main toxic differential components were phloroglucinols such as flavaspidic acid AB,didemethylpseudoaspidin AA and filixic acid PBP,flavonoids such as(-)-epicatechin,(-)-epigallocatechin,cianidanol,and other compounds such as indole-3-carboxaldehyde.CONCLUSION This method can rapidly,effectively and comprehensively characterize the main chemical composition of D.crassirhizoma,and provide a reference for the study of its pharmacological mechanism.

Objective:To analyze the genetic characteristics of the VWF gene c.7332G＞A nonsense mutation and explore its molecular pathogenesis.Methods:Phenotypic diagnosis of the proband was performed using VWF:Ag,VWF:RCo,FⅧ:C and multimeric analysis.The probands were genotyped by NGS whole-exome sequencing,and the sequencing results were validated by sanger sequencing.The family members were genotyped by Sanger sequencing.The VWF gene c.7332G＞A nonsense mutant plasmid was constructed.After transfection,the function of VWF gene c.7332G＞A mutant plasmid was verified at cell level in vitro.The mRNA level was detected by qRT-PCR,and the expression level of protein was detected by Western blot,the function of multimerization was verified by the multimeric analysis.Results:VWF:Ag and VWF:RCo were all less than 3％in the proband,and the multimeric analysis showed multimer deficiency.The proband was diagnosed as type 3 VWD.The homozygous nonsense mutation of VWF gene c.7332G＞A was detected by gene sequencing.The VWF mRNA level of the mutant plasmid was decreased,and the VWF protein expression in the cell supernatant was decreased,the mutant protein was truncated and the function of VWF multimerization was impaired.Conclusion:A homozygous mutation in exon 43 of VWF gene,c.7332G＞A,was responsible for the probands type 3 VWD in the proband.The mutation caused a decrease in the relative level of VWF mRNA and protein,and impaired the function of VWF multimerization.

AIM To screen the material basis for Rhei Radix et Rhizoma on improving human umbilical vein endothelial cell(HUVECs)injury before and after carbonization.METHODS The HPLC fingerprints were established,after which difference analysis was performed by orthogonal partial least squares discriminant analysis.The LPS-induced HUVECs injury model was established,then NO,MDA levels and SOD activity were detected.Gray correlation analysis and partial least squares regression were adopted in the investigation of spectrum-effect relationship,and active constituents were screened.RESULTS There were 22 and 20 common peaks in the fingerprints for 17 batches of raw products and carbonized products,respectively,along with the similarities of more than 0.8.Twelve main difference components were observable,among which gallic acid,5-hydroxymethylfurfural,catechin,aloe rhodopsin-8-O-β-D-glucoside,rhubarbic acid-8-O-β-D-glucoside and sennosides A were identified.The carbonized products demonstrated stronger effect on improving HUVECs injury than the raw product.The correlations of common peaks were more than 0.5 in the fingerprints for raw products and carbonized products,and peaks 3,5,11,12,15,19,23 exhibited significant effects on their efficacy(VIP values＞1).CONCLUSION This accurate,reliable and reproducible method can provide a basis for clarifying the material basis for hemostatic efficacy of Rhei Radix et Rhizoma and its carbonized product.