[Screening and expression analysis of transcription factors involved in genuineness of Codonopsis pilosula in Shanxi].

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.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.

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.

Micro-segmental analysis of the entry pathway and distribution of zolpidem in hair from different scalp regions after a single dose.

Introduction: Hair testing is well established for the assessment of past drug exposure; however, more research is needed to understand drug incorporation mechanisms and drug entry pathways into hair. Method: In this study, a micro-segmental LC-MS/MS method was used to analyze a 0.4 mm segment of hair after a single oral administration of zolpidem. Five single hairs were plucked at 1 day, 3 days, 7 days, and 28 days after administration from the vertex posterior of three subjects, and 5 single hairs were also plucked from the parietal, left temporal, and right temporal regions of the head at 28 days. Results and discussion: Proximal S1 (0-0.4 mm) in hair plucked at 1 day had the highest level of zolpidem at 1.5-2.4 pg/mm; much lower concentrations (< 1 pg/mm) were detected at proximal S2-S8 (0.4-3.2 mm). The drug concentration decreased gradually in S1 for 7 days after drug intake and disappeared by 28 days, suggesting that the drug from the bloodstream initially combined with the hair follicle and then gradually moved to the hair tip as the hair grew. The zolpidem concentration-hair segment profiles exhibited a large peak (root side) and a small peak (tip side) for the four sampling times in all three subjects, indicating that drug incorporation in the hair bulb occurred mainly from the blood but probably also entered the hair through sweat and sebum. Zolpidem was also detected in all hairs from the vertex posterior in all three subjects but was not detected in 1 hair from the parietal region and 2 hairs from the left temporal region. The consistency in drug detection, drug concentration level, and peak position was better in hair from the vertex posterior than from the other three regions, indicating that the vertex posterior is a suitable sampling region for estimating drug intake.

LC-MS-MS Determination of 88 Psychotropic Drugs in 1,865 Hair Samples from Addicts in Drug Abstinence.

The emergence of novel drugs and the continuous expansion of the scope of the types of drugs under control have greatly increased requests for screening of a range of drugs in hair. Here, a multi-analyte method for the detection and quantification of 88 psychotropic drugs in the hair of addicts in drug abstinence was developed and fully validated using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples (25 mg) were washed, cut into pieces, cryogenically ground and extracted in methanol. The extracted analytes were separated on an Allure PFP Propyl column (100 × 2.1 mm, 5 mm inside diameter, Restek, USA) and analyzed by LC-MS-MS in multiple reaction monitoring modes. The limits of detection and the limits of quantification ranged from 0.1 to 20 pg/mg and 0.2 to 50 pg/mg, respectively. The intra- and inter-assay precisions (relative standard deviation (RSD)) of all analyses ranged from 0.9% to 14.9% and 1.9% to 15.9%, respectively. Accuracy values were 100 ± 20%. The extraction recovery of quality control samples ranged from 50.9% to 99.6% for all analytes. The matrix effects for all analytes ranged from 46.8% to 99.7%. The method was successfully used to analyze 1,865 hair samples from addicts in drug rehabilitation at their own communities. Among the samples, 129 cases were positive; the majority of positive cases were from males (78.29%), 92.25% of whom were >35 years old. Traditional drugs, like methamphetamine and opioids, accounted for most positive cases, and 27 of the abstinence cases with a use history of methamphetamine were still positive. In addition to abused drugs, like methamphetamine, morphine and cocaine, the sedative-hypnotic and psychotherapeutic drugs, including clonazepam, alprazolam, estazolam, zolpidem and quetiapine, were detected in 26% of the hair samples, suggesting that these addicts may have insomnia and mental problems such as depression and psychosis, probably due to the long-term effects of drugs and withdrawal reactions. Three synthetic cannabinoids were also detected in four (2.7%) cases. A total of 37 cases were positive for methadone, tramadol and dextromethorphan, reflecting a new trend of alternative drug use when traditional drugs were not easy to obtain during the coronavirus disease 2019 outbreak.

Chiral analysis of dextromethorphan and levomethorphan in human hair by liquid chromatography-tandem mass spectrometry.

PURPOSE: Methorphan exists in two enantiomeric forms including dextromethorphan and levomethorphan. Dextromethorphan is an over-the-counter antitussive drug, whereas levomethorphan is strictly controlled as a narcotic drug. Chiral analysis of methorphan could, therefore, assist clinicians and forensic experts in differentiating between illicit and therapeutic use and in tracing the source of the drug. METHODS: A method for enantiomeric separation and quantification of levomethorphan and dextromethorphan in human hair was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hair was extracted in hydrochloric acid/methanol (1:20, v/v). The supernatant were separated using a Supelco Astec Chirobiotic™ V2 column (250 × 2.1 mm, i.d., 5 µm particle size) and analyzed on a triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. RESULTS: The limits of detection for dextromethorphan and levomethorphan were 2 and 1 pg/mg, respectively; the lower limit of quantification was 2 pg/mg for both drugs. Good linearity (r > 0.995) was observed for both analytes over the linear range. Precision values were below 10% for both analytes; accuracy values ranged from 87.5 to 101%. The extraction recoveries were 78.3-98.4%, and matrix effects were 70.5-88.6%. This method was applied to human hair samples from 120 people suspected of methorphan use to further distinguish the drug chirality. Dextromethorphan was detected in all 120 samples at a concentration range of 2.7-19,100 pg/mg, whereas levomethorphan was not detected in any sample. CONCLUSIONS: A sensitive quantitative method was established for the enantiomeric separation of dextromethorphan and levomethorphan in hair. This is the first study to achieve chiral analysis of methorphan in human hair.

Establishment of a genetic transformation system for Codonopsis pilosula callus.

Codonopsis pilosula, a traditional Chinese medicinal and edible plant, contains several bioactive components. However, the biosynthetic mechanism is unclear because of the difficulties associated with functional gene analysis. Therefore, it is important to establish an efficient genetic transformation system for gene function analysis. In this study, we established a highly efficient Agrobacterium-mediated callus genetic transformation system for C. pilosula using stems as explants. After being pre-cultured for 3 days, the explants were infected with Agrobacterium tumefaciens strain GV3101 harboring pCAMBIA1381-35S::GUS at an OD600 value of 0.3 for 15 min, followed by co-cultivation on MS induction medium for 1 day and delayed cultivation on medium supplemented with 250 mg l-1 cefotaxime sodium for 12 days. The transformed calli were selected on screening medium supplemented with 250 mg l-1 cefotaxime sodium and 2.0 mg l-1 hygromycin and further confirmed by PCR amplification of the GUS gene and histochemical GUS assay. Based on the optimal protocol, the induction and transformation efficiency of calli reached a maximum of 91.07%. The establishment of a genetic transformation system for C. pilosula calli lays the foundation for the functional analysis of genes related to bioactive components through genetic engineering technology.

The Distribution of Quetiapine and 7-Hydroxyquetiapine in Guinea Pig Hair Roots and Shafts after Repeated Administration: Exploration of the Mechanism of Drug Entry and Retention in Hair.

This study investigated the distribution of quetiapine and 7-hydroxyquetiapine in guinea pig hair roots and shafts after five repeated intragastric administrations at three doses (5, 10 and 25 mg/kg) by segmental analysis to explore the mechanism of drug entry and retention in hair. Hair root samples were collected after 7, 10, 14, 21, 28 and 35 d in area A after the first dose, and a hair shaft was plucked 35 d after the first dose. The maximum concentrations of quetiapine in hair roots in the low-, medium- and high-dose groups occurred at 50, 74 and 98 h after the first administration, and the maximum concentrations were 0.71 ng/mg (range: 0.54-0.84 ng/mg), 6.72 ng/mg (range: 4.59-9.75 ng/mg) and 12.72 ng/mg (range: 10.74-15.76 ng/mg), respectively. The maximum concentrations of 7-hydroxyquetiapine in the low-, medium- and high-dose groups were 0.67 ng/mg (0.23-1.15 ng/mg), 1.07 ng/mg (0.44-1.19 ng/mg) and 3.92 ng/mg (0.656.14 ng/mg), respectively, at 26 h. The maximum concentrations of quetiapine and 7-hydroxyquetiapine in hair roots were significantly positively correlated with the dose (n = 18; r2 = 0.84; P < 0.0001 for quetiapine and n = 18; r2 = 0.61; P = 0.0001 for 7-hydroxyquetiapine). The concentrations of quetiapine and 7-hydroxyquetiapine in hair roots were higher than those in hair shafts 10 d after administration, indicating drug and metabolite entry into the hair through the roots in the first few days after administration. The highest concentrations of quetiapine in the hair shaft in the low-, medium- and high-dose groups were found at the hair ends, and 7-hydroxyquetiapine in the hair shaft showed no obvious peak concentration. Combined with previous studies, we think, by analyzing the drug concentrations in the hair roots and shaft, that the most important way for drugs to enter into and be retained in hair is that the drug enters the hair through the blood circulation from hair root, then spreads and redistributes as the hair grows.

Characteristics of quetiapine and 7-hydroxyquetiapine in hair roots and blood after a single dose of quetiapine.

This study investigated the kinetics of quetiapine and its metabolite 7-hydroxyquetiapine in guinea pig blood and hair roots during the whole time course of absorption and elimination after intragastric administration of three dosages (25 mg/kg, 50 mg/kg, 100 mg/kg). The mean maximum concentration (Cmax) values of quetiapine in the blood of the low-, medium- and high-dose groups were 334.4, 849.0, and 2751.1 ng/mL, respectively, and those of 7-hydroxyquetiapine were 75.6, 175.5, and 173.7 ng/mL, respectively. The corresponding mean Cmax values of quetiapine in hair roots were 2.0, 5.9, and 14.7 ng/mg, and those of 7-hydroxyquetiapine were 1.0, 1.8, and 6.4 ng/mg. The mean half-lives of quetiapine at the three dosages in blood were 3.8 h, 5.0 h, and 6.0 h, and those in hair roots were 48.2 h, 41.5 h, and 162.3 h; for 7-hydroxyquetiapine, the values were 2.9 h, 4.1 h, and 4.2 h in blood and 77.1 h, 103.6 h, and 385.9 h in hair roots. The levels of quetiapine in blood and hair roots were higher than those of 7-hydroxyquetiapine, and there were significant positive correlations (p<0.05) between the concentrations of quetiapine and 7-hydroxyquetiapine in hair roots and the respective doses within 24 h and 48 h. Quetiapine and 7-hydroxyquetiapine could still be detected in some guinea pigs even after 28 days, which means that drugs remain in the hair roots longer than in the blood. This finding shows that hair roots could be a good alternative or supplemental matrix to common biological samples such as blood and urine, as hair roots substantially extend the detection window from days to months. Moreover, quetiapine and 7-hydroxyquetiapine were detected within 15min after administration in hair roots, which also suggests that the drug enters the hair roots quickly. Therefore, hair root analysis may be a good choice to detect acute poisoning and single-dose administration if other matrices are unavailable or to provide complementary information for other matrices.

An LC-MS/MS method for the simultaneous determination of 12 psychotropic drugs and metabolites in hair: Identification of acute quetiapine poisoning using hair root.

A rapid, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of 12 psychotropic drugs and metabolites in hair was developed and validated. After freeze grinding with methanol, the supernatant was determined by LC-MS/MS using an Allure PFPPropyl column (100 × 2.1 mm, 5 µm) with a gradient elution of acetonitrile and 10 mmol/L ammonium acetate with 0.1% formic acid, and in the subsequent analysis using multiple reaction monitoring (MRM) mode, two ion transitions were monitored for each analyte. The limits of detection ranged from 0.002 to 0.05 ng/mg, and the limits of quantitation were in the range of 0.005-0.1 ng/mg. Good linearity (r > 0.995) was observed for all analytes over the linear range. Acceptable intraday and interday precision (RSD ≤ 20%) and accuracy (85.3%-112.9%) were achieved. This method of detection was applied to the analysis of guinea pig hair roots after a single dose of quetiapine. Quetiapine and 7-hydroxyquetiapine were both detected in guinea pig hair roots from 5 min post administration. The concentration of quetiapine (10.3-1733.8 ng/mg) was much higher than that of 7-hydroxyquetiapine (0.1-40.6 ng/mg) in the hair roots of guinea pigs, and higher concentrations of quetiapine and 7-hydroxyquetiapine occurred in black hair root than in that of white and brown. The animal experiment demonstrated that hair roots may be a good specimen for proving acute quetiapine poisoning when other biological matrices are not available.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

[Clone and expression of CpGAPDH gene in Codonopsis pilosula].

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.

Tapetum-specific expression of a cytoplasmic orf507 gene causes semi-male sterility in transgenic peppers.

Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death (PCD) occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature PCD caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.

Cloning and expression analysis of CaPIP1-1 gene in pepper (Capsicum annuum L.).

Plant aquaporins are responsible for water transmembrane transport, which play an important role on abiotic and biotic stresses. A novel plasma membrane intrinsic protein of CaPIP1-1 was isolated from the pepper P70 according to transcriptome databases of Phytophthora capsici inoculation and chilling stress library. CaPIP1-1, which is 1155 bp in length with an open reading frame of 861 bp, encoded 286 amino acids. Three introns, exhibited CT/AC splice junctions, were observed in CaPIP1-1. The numbers and location of introns in CaPIP1-1 were the same as observed in tomato and potato. CaPIP1-1 was abundantly expressed in pepper fruit. Increased transcription levels of CaPIP1-1 were found in the different stresses, including chilling stress, salt stress, mannitol stress, salicylic acid, ABA treatment and Phytophthora capsici infection. The expression of CaPIP1-1 was downregulated by 50 µM HgCl2 and 100 µM fluridone. The pepper plants silenced CaPIP1-1 in cv. Qiemen showed growth inhibition and decreased tolerance to salt and mannitol stresses using detached leaf method.