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 explore the effect of personalized training based on early start Denver model (ESDM) combined with applied behavior analysis (ABA) in children with autism spectrum disorder (ASD) .Methods:From January 2021 to January 2023, convenience sampling was used to select 90 children with ASD who received treatment at the Shaoxing Seventh People's Hospital and their caregivers as research subjects. The children were divided into two groups using a random number table method in a 1∶1 ratio. The control group received routine rehabilitation, while the observation group was treated with personalized training based on ESDM combined with ABA. Both groups were intervened for six months. Children's Autism Rating Scale (CARS), Autism Behavior Checklist (ABC), Gesell Developmental Schedule (GDS), Sign-Significate Relation (S-S) Language Development Delay Assessment, Social Life Ability Scale for Infant-Junior Middle School Students (S-M), and Modified Barthel Index (MBI) were used to evaluated the effects of intervention on two groups of ASD children before and after intervention.Results:After six months of intervention, the CARS and ABC scores of ASD children in the observation group were lower than those in the control group, and the scores of GDS dimensions and total developmental quotient, S-S Language Development Delay Assessment score, S-M score, and MBI score were higher than those in the control group, and the differences were statistically significant (all P<0.05) . Conclusions:Personalized training based on ESDM combined with ABA has a good effect, which can improve loneliness symptoms of children with ASD, alleviate cognitive, language, and behavioral dysfunction, enhance social adaptation ability and activities of daily living.

Objective:The Nomogram prediction model of secondary insomnia in adolescent depression patients was constructed to provide reference and basis for clinical prevention of secondary insomnia in adolescent depression patients.Methods:A total of 158 adolescent depression patients who were treated in the Department of Pediatric Psychiatry of Shaoxing Seventh People′s Hospital from May 2021 to March 2022 were selected as the research objects by convenient sampling method. According to the Pittsburgh Sleep Quality Index (PSQI) score, the group was divided into a normal sleep group (PSQI ≤ 7 points) and a secondary insomnia group (PSQI>7 points). A cross-sectional survey was conducted using the Hamilton Depression Scale, Hamilton Anxiety Scaleand Social Support Rating Scale. Multivariate binary Logistic regression was used to analyze the influencing factors of secondary insomnia and construct a Nomogram prediction model for secondary insomnia in adolescents with depression. The accuracy of the model was judged by receiver operating characteristic curve. In addition, 70 adolescent depression patients who were treated in Shaoxing Seventh People′s Hospital from May 2022 to March 2023 were selected for success rate prediction. The results were compared with the actual observed results, and the fitting degree of the model was evaluated by Hosmer-Lemeshow test.Results:There were 72 cases in the normal sleep group, including 40 males and 32 females, aged (15.34 ± 2.62) years old, and 86 cases in the secondary insomnia group, including 51 males and 35 females, aged (14.89 ± 2.13) years old. The results of multivariate analysis showed that depression ( OR=6.381, 95 % CI 1.548-26.295, P<0.05), anxiety ( OR=6.248, 95 % CI 1.445-27.005, P<0.05) and social support ( OR=0.586, 95 % CI 0.346-0.994, P<0.05) were independent influencing factors for the occurrence of secondary insomnia in the two groups. On this basis, a risk prediction model was established. The AUC was 0.999, the sensitivity was 98.80 %, the specificity was 98.60 %, and the Youden index was 0.974. The total prediction accuracy of the external validation model was 99.80 %. Conclusions:The Nomogram prediction model of secondary insomnia in adolescent depression patients has excellent predictive efficacy. The degree of depression, anxiety and social support are the main influencing factors of secondary insomnia in patients. Medical staff should provide more effective psychological support for patients.

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 estab lish fingerprint of Duzhong butiansu pill s，analyze its chemical pattern recognition ，and determine the contents of 7 components in Duzhong butiansu pills ，so as to provide reference for the quality control of the preparation. METHODS ：HPLC method was adopted. The determination was performed on Pntulips BP-C 18 Plus column with 0.2％ phosphoric acid water-acetonitrile as mobile phase （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was set at 330 nm，and column temperature was 35 ℃. The sample size was 20 μL. With paeonol as the reference，the HPLC fingerprints of 12 batches of Duzhong butiansu pills （S1-S12） were established with Similarity Evaluation System for TCM Chromatographic Fingerprint （2012 edition）； common peaks were determined and the similarity was evaluated. The chromatographic peaks were identified by comparing with the reference substance. SPSS 21.0 and SIMCA 13.0 software were used for cluster analysis and principal component analysis ，and 22 common peaks were evaluated. The contents of the identified components in 12 batches of samples was determined by the above HPLC method. RESULTS ：A total of 22 common peaks were identified in the HPLC fingerprint of 12 batches of Duzhong butiansu pills ，and the similarity was no loss than 0.960. There were 7 chemical components identified ，which were gallic acid （peak 1），chlorogenic acid （peak 3），liquiritoside（peak 6），hyperoside （peak 7），verbascoside（peak 8），icariin（peak 14）and paeonol （peak 15）. Among the 12 batches of samples ，S1，S3-S5，S7， S9 and S 11 were classified as one category ，S2，S10 and S 124Y091 were clustered into one category ，S6 was one category and S was one category. The 22 common peaks were divided into three principal components. The characteristic value （15.130） and contribution rate （68.775％） of principal component 1 were the largest ，and the score coefficients of peak 3（0.305）and peak 4（0.298）were the highest. Among 12 batches of samples，the cont ents of above 7 components were 18.196 231.951 3，0.000 6-0.049 4，0.234 8-0.415 9，0.039 5-0.079 1，0.053 5-0.249 3，0.000 5-0.000 8，0.646 4-1.146 9 mg/g，respectively. CONCLUSIONS：HPLC fingerprint of Duzhong butiansu pills is established successfully. Twelve batches of samples are clustered into 4 category. Peak 3（chlorogenic acid ）and peak 4（unknown）may be the important factors causing the difference of samples. The content of gallic acid is the highest among the 7 components.

Objective to investigate the emotional state and childhood trauma among users of amphetaminetype-stimulants under compulsory seclusion rehabilitation in Shaoxing.Methods A cross-sectional design was conducted on 140 users of ATS in Shaoxing compulsory isolation drug rehabilitation center,collecting general information using homemade questionnaires.Using the Patient Health Questionnaire (PHQ-9),Generalized Anxiety Disorder Scale (GAD-7) and Mood Disorder Questionnaire(MDQ) to investigate the emotional state of users of ATS.The Childhood Trauma Questionnaire (CTQ) was applied to explore the childhood trauma experiences of users of ATS.Results Among the 140 users of ATS,84 (60％) users had emotional problems,24 users (17.14％) of anxiety disorder,22 users (15.72％) of depression,27 users (19.28％) of depression and anxiety disorder.66 users (47.1％)experienced at least one childhood trauma.The most common childhood trauma is the physical neglect,60 users (42.9％),followed by emotional neglect in 23 users(16.4％),again sexual abuse in 18 users (12.9％),emotional abuse and physical abuse is relatively fewer.Conclusion Among users of ATS under compulsory seclusion rehabilitation,emotional problems is prevalent.Proper emotional intervention and treatment in users of ATS would promote their mental health,thereby reducing the re-use of ATS,better return to society life.

A total of 105 patients with first-episode schizophrenia (male =51,female =54) and 99 normal controls (male =51,female =48) were included into this retrospective case-control study.Childhood trauma questionnaire-28 item short form (CTQ-SF) was used to assess the experience of childhood abuse.The result of binary logistic regression showed that emotional abuse (β =0.630,P ＜ 0.05) and emotional neglect(β =0.270,P ＜ 0.05) were included into the final model of predicting schizophrenia.It indicates that patients with first-episode schizophrenia experienced more early life stress than controls.Particularly emotional abuse and emotional neglect may play important roles in the onset of schizophrenia.

Objective To explore the genetic mechanism of Yang-deficiency constitution by detecting genomic copy number variations (CNVs). Methods Thirty cases of Yang-deficiency constitution and 30 cases of balanced constitution were included according to the standards of Classification and Determination of Constitution in Traditional Chinese Medicine. DNA was extracted from white blood cells in peripheral blood. A genome-wide association study was conducted by using Affymetrix SNP 6.0 platform. CNVs of each sample were analyzed using PennyCNV software. The Yang-deficiency constitution-specific copy number variation regions (CNVRs) of each autosome were identified. CNVR-related genes and their annotations were searched at online Human Genome Browser. Results The mean number of CNVs in balanced constitution group was 12.63±3.39, ranging from 8 to 20. After stepwise elimination of two Yang-deficiency constitution subjects, the mean number of CNVs in Yang-deficiency constitution group was 15.04±8.95, ranging from 2 to 38. A total of 26 CNVRs were identified from 28 Yang-deficiency constitution subjects, including 19 duplicated CNVRs, 6 deleted CNVRs, and 1 mixed type CNVR. Most CNVRs were shared by a few Yang-deficiency constitution subjects, and only 7 CNVRs were shared by more than 5 Yang-deficiency constitution subjects. The functions of representative genes in Yang-deficiency constitution-specific CNVRs were related with extracellular and intracellular signal transduction, metabolic regulation, and immune response, etc. Conclusion Yang-deficiency constitution subjects have some specific genomic CNVs, which might result in Yang-deficiency constitution phenotypes by influencing the expression of genes associated with extracellular and intracellular signal transduction, material metabolism (energy metabolism), and immune response, etc.

To explore the analysis methods for composite traditional Chinese medicine (TCM) constitutions.