Genetic transformation is a fundamental tool in molecular biology research of medicinal plants. Tailoring transgenic technologies to each distinct medicinal plant would necessitate a substantial investment of time and effort. Here, we present a simple hairy root transformation method that does not require sterile conditions, utilizing Agrobacterium rhizogenes strain K599 and the visible RUBY reporter system. Transgenic hairy roots were obtained for six tested medicinal plant species, roots or rhizomes of which have recognized medicinal value, spanning four botanical families and six genera (Platycodon grandiflorus, Atractylodes macrocephala, Scutellaria baicalensis, Codonopsis pilosula, Astragalus membranaceus, and Glycyrrhiza uralensis). Furthermore, two previously identified Glycyrrhiza uralensis UGTs that convert liquiritigenin into liquiritin in heterologous systems were studied in planta using the method. Our results indicate that overexpression of GuUGT1 but not GuUGT10 and Cas9-mediated knockout of GuUGT1 profoundly influenced the accumulation of liquiritin and isoliquiritin in licorice roots. Therefore, the method described here represents a simple, rapid and widely applicable hairy root transformation method that enables fast gene functional study in medicinal plants.

OBJECTIVE: In Pakistan, traditional medicines are an important component of the medical system, with numerous varieties and great demands. However, due to the scattered resources and the lack of systematic collection and collation, adulteration of traditional Pakistani medicine (TPM) is common, which severely affects the safety of their medicinal use and the import and export trades. Therefore, it is urgent to systematically organize and unify the management of TPM and establish a set of standards and operable methods for the identification of TPM. METHODS: We collected and organized the information on 128 TPMs with regard to their medicinal parts, efficacy, usage, and genetic material, based on Pakistan Hamdard Pharmacopoeia of Eastern Medicine: Pharmaceutical Codex. The genetic information of TPM is summarized from national center for biotechnology information (NCBI) and global pharmacopoeia genome database (GPGD). Furthermore, we utilized bioinformatics technology to supplement the chloroplast genome (cp-genome) data of 12 TPMs. To build the web server, we used the Linux + Apache + MySQL + PHP (LAMP) system and constructed the webpage on a PHP: Hypertext Preprocessor (PHP) model view controller (MVC) framework. RESULTS: We constructed a new genomic database, the traditional Pakistani medicine genomic database (TPMGD). This database comprises five entries, namely homepage, medicinal species, species identification, basic local alignment search tool (BLAST), and download. Currently, TPMGD contains basic profiles of 128 TPMs and genetic information of 102 TPMs, including 140 cytochrome c oxidase subunit I (COI) sequences and 119 mitochondrial genome sequences from Bombyx mori, 1 396 internal transcribed spacer 2 (ITS2) sequences and 1 074 intergenic region (psbA-trnH) sequences specific to 92 and 83 plant species, respectively. Additionally, TPMGD includes 199 cp-genome sequences of 82 TPMs. CONCLUSION TPMGD is a multifunctional database that integrates species description, functional information inquiry, genetic information storage, molecular identification of TPM, etc. The database not only provides convenience for TPM information queries but also establishes the scientific basis for the medication safety, species identification, and resource protection of TPM.

Objective To reveal the pharmacodynamic material basis of Kaixinsan by using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)and the integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function".Methods Through a review of literature,databases,and previous studies,the chemical components of ginseng,polygala,poria,and acorus were systematically cataloged.A qualitative analysis method for the chemical constituents in the aqueous extract of Kaixinsan was developed,allowing for the identification of its chemical components.A qualitative analysis for rat plasma based on HPLC-MS/MS was established,which was applied to analyze the plasma exposure component spectrum following oral administration of Kaixinsan aqueous extract in rats.Aerobic respiration was evaluated using a seahorse cell energy metabolism analyzer,and the effect of key components of Kaixinsan on mitochondrial aerobic respiration was assessed.Results Four main types of components were identified in the Kaixinsan aqueous extract,including saponins,oligosaccharide esters,xanthones,and triterpenes,comprising a total of 231 identified compounds.Analysis of rat plasma 30 minutes after gavage with Kaixinsan identified 55 compounds.The analysis revealed that ginsenoside Rg1,3,6'-disinapoylsucrose,polygalaxanthone Ⅲ and poricoic acid B could significantly enhance mitochondrial respiratory capacity using in vitro cellular assays to detect aerobic respiration of four main components entered blood.Conclusions Saponins,oligosaccharide esters,xanthones,and triterpenes may be the material basis for the pharmacological effect of Kaixinsan by improving mitochondrial function.The integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function"provides a new approach for in-depth exploration of the material basis underlying the efficacy of traditional Chinese medicine.

We conducted a prospective study to assess the non-inferiority of adjuvant chemotherapy alone versus adjuvant concurrent chemoradiotherapy (CCRT) as an alternative strategy for patients with early-stage (FIGO 2009 stage IB-IIA) cervical cancer having risk factors after surgery. The condition was assessed in terms of prognosis, adverse effects, and quality of life. This randomized trial involved nine centers across China. Eligible patients were randomized to receive adjuvant chemotherapy or CCRT after surgery. The primary end-point was progression-free survival (PFS). From December 2012 to December 2014, 337 patients were subjected to randomization. Final analysis included 329 patients, including 165 in the adjuvant chemotherapy group and 164 in the adjuvant CCRT group. The median follow-up was 72.1 months. The three-year PFS rates were both 91.9%, and the five-year OS was 90.6% versus 90.0% in adjuvant chemotherapy and CCRT groups, respectively. No significant differences were observed in the PFS or OS between groups. The adjusted HR for PFS was 0.854 (95% confidence interval 0.415-1.757; P = 0.667) favoring adjuvant chemotherapy, excluding the predefined non-inferiority boundary of 1.9. The chemotherapy group showed a tendency toward good quality of life. In comparison with post-operative adjuvant CCRT, adjuvant chemotherapy treatment showed non-inferior efficacy in patients with early-stage cervical cancer having pathological risk factors. Adjuvant chemotherapy alone is a favorable alternative post-operative treatment.
Female ; Humans ; Uterine Cervical Neoplasms/drug therapy* ; Prospective Studies ; Quality of Life ; Neoplasm Staging ; Chemoradiotherapy ; Chemotherapy, Adjuvant/adverse effects* ; Adjuvants, Immunologic ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Retrospective Studies

Objective To investigate the clinical effect of transcatheter arterial chemoembolization (TACE) combined with microwave ablation (MWA) (TACE-MWA) versus repeat resection (RR) in the treatment of recurrent hepatocellular carcinoma (RHCC). Methods A total of 178 patients with RHCC who were admitted to The Second People's Hospital of Neijiang from June 2015 to September 2020 were enrolled, and according to the treatment modality, they were divided into RR group with 64 patients and TACE-MWA group with 114 patients. Baseline demographic data, liver function, and tumor conditions before treatment were recorded, and the patients were followed up to October 2021 to compare postoperative overall survival (OS) time and recurrence-free survival (RFS) time between the two groups. Subgroup analysis based on recurrence pattern (recurrence time and tumor size) was performed, and the influencing factors for prognosis were analyzed. The independent samples t -test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data; the Kaplan-Meier method was used for postoperative survival rate, the Log-rank test was used for survival difference analysis, and a multivariate Cox regression analysis was used to investigate independent risk factors for survival. Results The multivariate analysis showed that tumor diameter, alpha-fetoprotein (AFP) level, alanine aminotransferase, albumin, and time to recurrence were independent prognostic factors for OS (all P < 0.05), and AFP level and time to recurrence were independent prognostic factors for RFS (both P < 0.05). For RHCC with late recurrence (> 2 years), there were significant differences between the two groups in median OS (54.0 months vs 36.0 months, χ 2 =6.171, P =0.013) and median RFS (28.0 months vs 21.0 months, χ 2 = 5.211, P =0.022). For RHCC with a tumor diameter of ≤5 cm, there was a significant difference in median OS between the two groups (33.0 months vs 27.0 months, χ 2 =6.447, P =0.011). Conclusion RR has a similar clinical effect to TACE-MWA in RHCC with early recurrence or a tumor diameter of > 5 cm, but RR should be the first choice for RHCC with late recurrence or a tumor diameter of ≤5 cm.

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.

Objective To construct a recombinant adeno-associated virus(rAAV)vector containing a human anti-epidermal growth factor receptor(anti-EGFR)single-chain variable fragment antibody gene,and observe its inhibitory effects on pancreatic cancer cell lines.Methods Human anti-EGFR single-chain variable fragment antibody gene was inserted into the Kpn I and Bgl Ⅱ sites to construct a rAAV-anti EGFR vector,and then rAAV1-EGFP group and rAAV1-anti EGFR group were established.The expression of anti-EGFR antibody was observed.Antibody expression was detected by Western blot,and the inhibition and apoptosis rates of human pancreatic cancer cell lines(PCT-3,SW1990,Capan-1,ASPC-1,MiaPaCa-2 and PANC-1 cells)were detected by CCK-8 assay and flow cytometry,respectively.All data were analyzed using the t test.Results The results of Western blot assay demonstrated that anti-EGFR antibody was expressed in 6 pancreatic cancer cell lines.The inhibition rates of rAAV1-EGFP and rAAVl-anti EGFR on pancreatic ASPC-1 cells were 1.1%± 2.4% and 15.1%±3.5%,respectively,with a significant difference between the 2 groups(t =6.598,P ＜0.05).The apoptosis rates of PANC-1 cells were 7.0% ± 3.0% in the rAAV1-EGFP group and 1 1.4% ± 2.5% in the rAAV1-anti EGFR group,with no significant difference between the 2 grouvs(t = 1.952,P ＞0.05).The apoptosis rates of SW1990,ASPC-1,Capan-1,PCT-3,MiaPaCa-2 cells were 1.1% ± 0.8%,1.5% ± 0.7%,1.7% ± 1.2%,1.1%±0.7% and 2.2% ± 1.1% in the rAAV1-EGFP group,and 17.6% ± 2.2%,46.9% ± 3.9%,20.0% ±2.8%,12.1% ± 1.6% and 31.1% ±2.5% in the rAAV1-anti EGFR group,respectively,with significant differences between the 2 groups(t = 12.208,19.846,10.405,10.909,18.327,P ＜0.05).Conclusions A rAAV-anti EGFR vector with human anti-EGFR single-chain variable fragment antibody gene was constructed.Anti-EGFR antibody has obvious inhibition effects on pancreatic cancer cell lines.