Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Screening and expression analysis of transcription factors involved in genuineness of Codonopsis pilosula in Shanxi / 中国中药杂志

This study aims to mine the transcription factors that affect the genuineness of Codonopsis pilosula in Shanxi based on the transcriptome data of C. pilosula samples collected from Shanxi and Gansu, and then analyze the gene expression patterns, which will provide a theoretical basis for the molecular assisted breeding of C. pilosula. Gene ontology(GO) functional annotation, conserved motif prediction, and gene expression pattern analysis were performed for the differential transcription factors predicted based on the transcriptome data of C. pilosula from different habitats. A total of 61 differentially expressed genes(DEGs) were screened out from the transcriptome data. Most of the DEGs belonged to AP2/ERF-ERF family, with the conserved motif of [2X]-[LG]-[3X]-T-[3X]-[AARAYDRAA]-[3X]-[RG]-[2X]-A-[2X]-[NFP]. Forty-three of the DEGs showed significantly higher gene expression in C. pilosula samples from Shanxi than in the samples from Gansu, including 11 genes in the AP2/ERF-ERF family, 5 genes in the NAC fa-mily, 1 gene in the bHLH family, and 2 genes in the RWP-RK family, while 18 transcription factors showed higher expression levels in the samples from Gansu. GO annotation predicted that most of the DEGs were enriched in GO terms related to transcriptional binding activity(103), metabolic process(26), and stress response(23). The expression of transcription factor genes, CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 was higher in the samples from Shanxi and in the roots of C. pilosula. CpNAC92, CpbHLH128, and CpRAP2-7 responded to the low temperature, temperature difference, and iron stresses, while CpNAC100 only responded to low temperature and iron stresses. The screening and expression analysis of the specific transcription factors CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 in C. pilosula in Shanxi laid a theoretical foundation for further research on the mechanism of genuineness formation of C. pilosula.

Identification and analysis of glycosyltransferase in Codonopsis pilosula from different regions based on transcriptome / 药学学报

Lu Dangshen is the geoherb in Shanxi Province. The content of Codonopsis pilosula polysaccharides (CPP) in Lu Dangshen is more than that in other Codonopsis Radix from other regions. Glycosyltransferase is the key enzyme for the synthesis of bioactive components, such as CPP and tangshenoside I. Based on the transcriptome data of C. pilosula [Codonopsis pilosula (Franch.) Nannf.] from different producing areas, this study carried out functional annotation of GO and KEGG, conservative domain analysis, phylogenetic tree analysis and expression pattern analysis of glycosyltransferase genes in C. pilosula to provides a theoretical basis for exploring the mechanism of genuineness formation in Lu Dangshen. In this study, 98 glycosyltransferase genes were screened and identified, which belonged to GT family 1, GT family 2, GT family 90 and other families. By GO functional annotation, it was found that most of the glycosyltransferase genes had catalytic activity. Analysis of KEGG functional annotation showed that C. pilosula glycosyltransferase was mainly involved in glycan organism and terpenoid and polyketone metabolism. Among them, conserved domain of 42 glycosyltransferase genes in GT family 1 was [X]-W-[2X]-Q-[3X]-[LH]-[5X]-[FLTHCGWNS]-[2X]-E-[4X]-[GVP]-[4X]-P-[4X]-Q-[2X]-[NAK]. Phylogenetic tree analysis based on the glycosyltransferase sequence in Arabidopsis thaliana showed that C. pilosula glycosyltransferases were mainly located in Arabidopsis thaliana UGT73, 72 and 85 branches. Gene expression pattern analysis showed that expression of CpUGT73AH2 was higher in Lu Dangshen than that in Baitiaodang and could respond to drought and low temperature stress. In conclusion, a glycosyltransferase gene CpUGT73AH2, which is involved in the metabolism of terpenoids and polyketides and can respond to environmental stress, was screened from the C. pilosula glycosyltransferase family 1, which was used to further study the role of C. pilosula glycosyltransferase in Lu Dangshen. It laid a theoretical foundation for further study on the role of C. pilosula glycosyltransferase in the formation of Lu Dangshen.

Clone and expression of CpGAPDH gene in Codonopsis pilosula / 中国中药杂志

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.