Narcotic plants are strictly regulated worldwide due to their ability to extract drug alkaloids and drug precursor components. Besides the three traditional core species, cannabis, opium poppy, and coca, the misuse of psychoactive plants with addictive properties has become increasingly prevalent globally in recent years, and the establishment of accurate identification methods for such plants has become an urgent need in the field of narcotics control. Within existing identification frameworks, the conventional morphological and chemical analysis methods, despite their long-term application, have demonstrated considerable limitations. In contrast, DNA-based molecular identification techniques have achieved significant advancement in recent years due to their high specificity and stability. This review comprehensively examines current DNA-based identification approaches for narcotic plants through three key dimensions: DNA molecular marker technology, DNA barcoding technology, and emerging molecular biological techniques, and elaborates on the principles, technical characteristics, application scenarios, and research progress of each technology, providing some reference for the scientific selection of DNA identification strategies for narcotic plants in different specific scenarios.

ObjectiveGastrodia elata has evolved ecological types with shortened rhizome internodes and diversified flower and fruit coloration in response to different altitudes. Studying the genetic mechanisms of different ecotype germplasm is significant for guiding variety breeding in different cultivation areas. MethodsThe bHLH gene family was identified based on the whole-genome datasets of G. elata f. elata and G. elata f. glauca. Subsequently， the gene family members were subject to analysis， including gene structure， chromosomal localization， cis-acting elements， gene synteny， and phylogeny. Combined with transcriptome data and quantitative Real-time PCR， the expression patterns of bHLH genes in the stems of the different G. elata ecotype germplasm were analyzed. Finally， correlation analysis was conducted between gene expression patterns and color to obtain the key bHLH genes regulating the color formation of stem. ResultsA total of 63 bHLH genes were identified in both G elata f. elata and G. elata f. glauca， unevenly distributed across 17 chromosomes and clustered into 16 subfamilies， with significant expansion in some family members. Obvious inversions of bHLH genes on the same chromosome and interchromosomal translocations were detected in the two ecotype germplasm. Among these genes， 12 bHLH genes （such as bHLH62-3 and bHLH74） were associated with the bright yellow color of G elata f. elata stem， while 9 bHLH genes （such as PIL13， UNE12， and bHLH130） were correlated with the red color of G. elata f. glauca stem. Compared to G. elata f. glauca， the bHLH48 expression level was significantly higher in flowers and scale leaves of G elata f. elata， and the bHLH62-3 expression level was significantly higher in all organs of G elata f. elata. ConclusionsFunctional pathway divergence of the bHLH family members has occurred across different chromosomes in G elata f. elata and G. elata f. glauca. Through synergism or antagonism with other genes， 21 bHLH genes participate in the coloration metabolic pathway regulation of stems， flowers， and fruits. Specifically， bHLH62-3 is involved in regulating stem color differentiation in the anthocyanin biosynthesis pathway of G. elata， thus relevant to the color formation of stem. Additionally， GebHLH48 positively regulates flowering-related pathways to promote the early-flowering phenotype of G. elata f. elata. These findings have laid the foundation for analyzing the genetic regulatory mechanisms underlying the color formation of the G. elata stem.

Objective To understand the current status of low-density lipoprotein cholesterol (LDL-C) control in patients after coronary artery bypass grafting (CABG). Methods Clinical data of patients who underwent isolated CABG in Beijing Anzhen Hospital in 2023 were collected. All patients returned to our hospital approximately one year after surgery (10-13 months) for a lipid level recheck. We analyzed their LDL-C attainment status and influencing factors. Patients were categorized into two groups based on whether their LDL-C met the target: a LDL-C attainment group and a LDL-C non-attainment group. Results This study included 1456 patients who underwent CABG, including 320 females and 1136 males, with an average age of (61.41±9.12) years. One year post-surgery, 234 patients achieved the LDL-C target, with an attainment rate of 16.07%. The proportion of patients in the LDL-C attainment group who were ultra-high risk (77.35% vs. 92.06%, P＜0.001), female (16.24% vs. 23.08%, P=0.021), and those with comorbid hypertension (55.98% vs. 63.18%, P=0.038) was significantly lower than those in the LDL-C non-attainment group. Additionally, the baseline body mass index (BMI) [(25.37±3.24) kg/m2 vs. (26.03±3.56) kg/m2, P=0.017], total cholesterol levels [(3.30±0.84) mmol/L vs. (4.01±1.03) mmol/L, P＜0.001], LDL-C [(1.62±0.63) mmol/L vs. (2.25±0.85) mmol/L, P＜0.001], and high-density lipoprotein cholesterol [(0.98±0.26) mmol/L vs. (1.02±0.24) mmol/L, P=0.049] upon admission in the attainment group were all lower than those in the non-attainment group. Moreover, the lipid-lowering drug usage rate in the attainment group (100.00% vs. 96.24%, P=0.003) and the proportion using two types of drugs together (25.21% vs. 10.72%, P＜0.001) were both higher than those in the non-attainment group, while the statin monotherapy rate was lower than that in the non-attainment group (74.79% vs. 85.19%, P＜0.001). Logistic regression analysis showed that baseline BMI (OR=0.928, P=0.012) and baseline LDL-C levels (OR=0.207, P<0.001), patient cardiovascular risk stratification (OR=0.155, P<0.001) and lipid-lowering drug treatment regimen (OR=3.758, P<0.001) are significant factors affecting the LDL-C control status. Conclusion The LDL-C compliance rate of patients undergoing CABG is at a relatively low level 1 year after surgery. Patients with very high risk of atherosclerotic cardiovascular disease, high baseline LDL-C levels, and overweight or obesity should be strengthened lipid management. For these patients, the intensity of lipid-lowering drug use or combination medication should be increased upon discharge.

Objective: To investigate the incidence of occult hepatitis B virus infection (OBI) in spouses of asymptomatic hepatitis B virus (HBV) infected individuals in Shenzhen, China, and to analyze their serological and molecular characteristics and possible transmission routes, so as to propose refined strategies for blood safety. Methods: After rapid screening for HBsAg at the blood collection sites, samples from HBsAg-positive blood donors and their concurrently donating spouses were collected. All samples were tested for hepatitis B serological markers by electrochemiluminescence immunoassay (ECLI). Simultaneously, HBV nucleic acid extractiona, nested PCR amplification, gene sequencing of S and BCP/PC regions and qPCR were conducted. Results: A total of 112 samples were collected, including 56 from HBsAg positive donors and 56 from their spouses. All donors were confirmed as HBsAg+/DNA+/anti-HBc+, indicative of asymptomatic chronic hepatitis (CHB) infection. Among their 56 spouses, 11 (19.6%) were identified as HBV DNA+. The prevalence was higher in males (23.1%) than in females (16.7%). Six spouses (10.8%) had OBI, three of whom (5.4%) were negative in routine blood screening tests. The residual risk of HBV were estimated as 1∶127 (95%CI, 1∶356 to 1∶66). Among infected couples, immune escape mutation (E164D) and glycosylation mutations (I126T and T131N/M133T) were identified. Furthermore, sequence analysis suggested partner-to-partner transmission in eight cases. Conclusion: A substantial proportion (19.6%) of spouses of asymptomatic HBV infected donors were HBV-positve, with an OBI prevalence of 10.9%. Among these, 5.4% were negative in routine tests. To ensure blood safety, we recommend that spouses of HBV infected individuals be deferred from blood donation.

ObjectiveGastrodia elata has evolved ecological types with shortened rhizome internodes and diversified flower and fruit coloration in response to different altitudes. Studying the genetic mechanisms of different ecotype germplasm is significant for guiding variety breeding in different cultivation areas. MethodsThe bHLH gene family was identified based on the whole-genome datasets of G. elata f. elata and G. elata f. glauca. Subsequently， the gene family members were subject to analysis， including gene structure， chromosomal localization， cis-acting elements， gene synteny， and phylogeny. Combined with transcriptome data and quantitative Real-time PCR， the expression patterns of bHLH genes in the stems of the different G. elata ecotype germplasm were analyzed. Finally， correlation analysis was conducted between gene expression patterns and color to obtain the key bHLH genes regulating the color formation of stem. ResultsA total of 63 bHLH genes were identified in both G elata f. elata and G. elata f. glauca， unevenly distributed across 17 chromosomes and clustered into 16 subfamilies， with significant expansion in some family members. Obvious inversions of bHLH genes on the same chromosome and interchromosomal translocations were detected in the two ecotype germplasm. Among these genes， 12 bHLH genes （such as bHLH62-3 and bHLH74） were associated with the bright yellow color of G elata f. elata stem， while 9 bHLH genes （such as PIL13， UNE12， and bHLH130） were correlated with the red color of G. elata f. glauca stem. Compared to G. elata f. glauca， the bHLH48 expression level was significantly higher in flowers and scale leaves of G elata f. elata， and the bHLH62-3 expression level was significantly higher in all organs of G elata f. elata. ConclusionsFunctional pathway divergence of the bHLH family members has occurred across different chromosomes in G elata f. elata and G. elata f. glauca. Through synergism or antagonism with other genes， 21 bHLH genes participate in the coloration metabolic pathway regulation of stems， flowers， and fruits. Specifically， bHLH62-3 is involved in regulating stem color differentiation in the anthocyanin biosynthesis pathway of G. elata， thus relevant to the color formation of stem. Additionally， GebHLH48 positively regulates flowering-related pathways to promote the early-flowering phenotype of G. elata f. elata. These findings have laid the foundation for analyzing the genetic regulatory mechanisms underlying the color formation of the G. elata stem.

Objective To explore the predictive value of peripheral blood cells in the efficacy of neoadjuvant immunotherapy combined with chemotherapy for esophageal squamous cell carcinoma. Methods A retrospective study was conducted on patients with esophageal squamous cell carcinoma (clinical stages Ⅱ-Ⅳa) who underwent neoadjuvant immunotherapy combined with chemotherapy at the Department of Thoracic Surgery, Affiliated Hospital of North Sichuan Medical College from April 2020 to November 2023. According to whether the pathology was completely relieved after treatment, patients were divided into a pathological complete remission group and a pathological incomplete remission group. The College of American Pathologists criteria were used to evaluate the tumor pathological regression grade (TRG) after neoadjuvant therapy (TRG=0, 1 defined as a good efficacy group, TRG=2, 3 defined as a poor efficacy group). Results A total of 92 patients with esophageal squamous cell carcinoma were collected, including 72 males and 20 females. The average age was (65.86±7.66) years. The complete remission of pathology was closely related to the number of lymphocytes in the blood before treatment (P=0.019). The area under the curve (AUC) for predicting complete remission of esophageal squamous cell carcinoma after neoadjuvant immunotherapy combined with chemotherapy was 0.678, the maximum Youden index was 0.328, and the optimal cutoff value was 1.845. The incidence of postoperative pulmonary infection in the pathological incomplete remission group was higher than that in the pathological complete remission group (25.0% vs. 5.6%, P=0.030). Using the optimal cutoff value, there were statistically significant differences in pathological N stage and pathological TNM stage between patients with lymphocyte counts <1.845×109/L and ≥1.845×109/L (P<0.05). Treatment response (by TRG) was significantly associated with the pretreatment red blood cell count (P=0.009). The AUC for predicting a good TRG response was 0.669, with a maximum Youden index of 0.385 and an optimal cutoff value of 4.235. Between the good and poor response groups, there were statistically significant differences in postoperative pathological T stage (P<0.001), N stage (P=0.041), and TNM stage (P＜0.001). When stratified by the optimal cutoff value, there were statistically significant differences in age (P＜0.001) and the prevalence of hypertension (P=0.022) between patients with red blood cell counts <4.235×1012/L and ≥4.235×1012/L. Conclusion A pretreatment absolute lymphocyte count ≥1.845×109/L and a red blood cell count <4.235×1012/L are good predictors for pathological complete response and a good pathological response, respectively, following neoadjuvant immunotherapy combined with chemotherapy in patients with esophageal squamous cell carcinoma.

Purpose: Dual anti–human epidermal growth factor receptor 2 (HER2) drugs have become the standard regimen for neoadjuvant systemic treatment (NST) to HER2-positive breast cancer patients. However, the efficacy varies greatly among patients with different HER2 protein expression levels. Materials and Methods: A total of 575 HER2-positive breast cancer patients from multiple centers throughout China from 2013 to 2022 were retrospectively analyzed. We compared clinicopathological features in different HER2 immunohistochemistry classes (HER2 2+/in situ hybridization [ISH] + or HER2 3+), and their difference in response to NST and survival with single or dual anti-HER2 drugs. Drug sensitivity assays were used to evaluate different efficacy of anti-HER2 drugs in vitro. Results: Compared to HER2 3+ subgroup, the HER2 2+/ISH+ group had a higher proportion of hormone receptor–positive status (48.7% vs. 76.1%, p < 0.001), more HER2 protein loss after NST, lower pathological complete response (pCR) rate (46.07% vs. 16.24%, p < 0.001), and tended to have worse disease-free survival (DFS). In HER2 2+/ISH+ patients, treated with pertuzumab and trastuzumab in combination had no significant improvement in pCR (19.12% vs. 12.24%, p=0.287) and DFS (p=0.908) than using alone. Drug sensitivity assay showed poor efficacy with dual anti-HER2 drugs in HER2 2+/ISH+ cell lines; however, fam-trastuzumab deruxtecan drugs had a satisfactory effect. Conclusion Owing to the differences in clinicopathological features and treatment efficacy, we considered the HER2 2+/ISH+ group to be a distinct subtype and defined it as the HER2-moderate–positive subgroup. In this subgroup, dual anti-HER2 drugs did not exert significant improvement in pCR and DFS. Therefore, treatment optimization is warranted, with antibody-drug conjugate drugs as potential options.

OBJECTIVES: To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O₂) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. RESULTS: In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. CONCLUSIONS Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.
Humans ; Reactive Oxygen Species/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Up-Regulation ; Cell Hypoxia ; Cells, Cultured ; Snake Venoms/chemistry* ; Beclin-1

OBJECTIVES: To investigate the active components and possible mechanisms of n-butanol fraction of Periploca forrestii Schltr. ethanol extract for treating Alzheimer's disease (AD). METHODS: The active components of n-butanol fraction of Periploca forrestii Schltr. ethanol extract were analyzed using UPLC-QE-MS technique. In a SD rat model of AD induced by treatment with AlCl₃ and D-gal, the therapeutic effects of low, moderate and high doses of the n-butanol fraction, saline, and donepezil hydrochloride were evaluated using ELISA, HE and Nissl staining, immunohistochemistry and Western blotting. The therapeutic mechanisms of the n-butanol fraction were explored using network pharmacology and molecular docking. RESULTS: Seventeen active components were identified from the n-butanol fraction of Periploca forrestii Schltr. ethanol extract, including phenylpropanoids, flavonoids, anthraquinones, triterpenoids, steroids, and volatile oils. In the rat models of AD, treatment with the n-butanol fraction significantly lowed AChE content in the hippocampus, increased the contents of ACh, SOD, CAT, and GSH-Px, enhanced the expressions of neuronal apoptotic factors Bcl-2, PI3K, Akt, p-PI3K, and p-Akt, and reduced the expressions of Bax and caspase-3 proteins. The treatment also dose-dependently up-regulated hippocampal expressions of Nrf-2, HO-1 and BDNF and down-regulated Keap-1, Aβ and Tau expressions. Bioinformatics analysis identified 14 key intersected targets (including TNF, AKT1 and ESR1) between the n-butanol fraction and AD. CONCLUSIONS The therapeutic effect of n-butanol fraction of Periploca forrestii Schltr. ethanol extract in AD mice is mediated by its multiple active components that regulate multiple targets and pathways.
Animals ; Rats, Sprague-Dawley ; Rats ; 1-Butanol/chemistry* ; Plant Extracts/pharmacology* ; Periploca/chemistry* ; Ethanol/chemistry* ; Alzheimer Disease/drug therapy* ; Male ; Molecular Docking Simulation ; Apoptosis/drug effects*

OBJECTIVES: To investigate the inhibitory effect of Zheng GanDecoction (ZGF) on tumor progression in a rat model of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and explore the possible mechanism. METHODS: Seventy SD rats were subjected to regular intraperitoneal injections of DEN (50 mg/kg) for 12 weeks to induce HCC tumorigenesis, with another 10 rats receiving saline injections as the normal control. After successful modeling, the rats were randomized into 5 groups (n=10) for daily treatment with distilled water ( model group), Huaier Granules (4 g/kg; positive control group), or ZGF at low, medium, and high doses (2, 4, and 8 g/kg, respectively) via gavage for 17 weeks. Body weight changes of the rats were monitored, and after completion of the treatments, the rats were euthanized for measurement of liver, spleen and thymus indices and morphological and histopathological examinations of the liver tissues using HE staining. The expressions of YAP, p-YAP, MST1, LATS1 and p-LATS1 in the liver tissues were detected using immunohistochemistry and Western blotting. RESULTS: Compared with the normal control rats, the rat models with DEN-induced HCC exhibited much poorer general condition with a significantly reduced survival rate, increased body weight and liver and spleen indices, and a lowered thymus index. ZGF treatment obviously reduced liver and spleen indices, increased the thymus index, and improved pathologies of the liver tissues of the rat models. Immunohistochemistry and Western blotting showed a dose-dependent reduction of YAP expression and an increment of p-YAP expression in ZGF-treated rats, which also exhibited significantly upregulated hepatic expressions of MST1, LATS1 and p-LATS1. CONCLUSIONS ZGF inhibits DEN-induced HCC in rats by activating the Hippo/YAP pathway via upregulating MST1 and LATS1 expression, which promotes YAP phosphorylation and degradation to suppress proliferation and induce apoptosis of the tumor cells.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Diethylnitrosamine ; Rats, Sprague-Dawley ; Rats ; Signal Transduction/drug effects* ; Protein Serine-Threonine Kinases/metabolism* ; Carcinoma, Hepatocellular/drug therapy* ; YAP-Signaling Proteins ; Liver Neoplasms/drug therapy* ; Hippo Signaling Pathway ; Male ; Liver Neoplasms, Experimental/metabolism* ; Transcription Factors/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism*