An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gelsemium elegans (G. elegans) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/n-hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine (1, 12.1 mg), sempervirine (2, 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine (3, 10.1 mg), koumine (4, 50.5 mg), gelsemine (5, 32.2 mg), gelselvirine (6, 50.5 mg), and 11-hydroxyhumanmantenine (7, 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans, holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.

A high-resolution mass spectrometric approach to a qualitative and quantitative comparative metabolism of the humantenine-type alkaloid rankinidine.

RATIONALE: Rankinidine belongs to the humantenine-type alkaloids isolated from Gelsemium. Currently, the mechanism behind the toxicity differences of rankinidine has not been explained. In this study, our purpose was to elucidate the major in vitro metabolic pathways of rankinidine and to compare the formation of metabolites of rankinidine in human (HLMs), rat (RLMs), goat (GLMs) and pig (PLMs) liver microsomes. METHODS: This is the first study to compare the in vitro metabolism of rankinidine with high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF). The MS/MS data and LC/MS peak area acquired in positive ion mode were used to analyze metabolite structures and compare metabolism. RESULTS: We identified 11 metabolites (M1-M11) in total and found five main metabolic pathways, consisting of demethylation (M1), reduction (M2), oxidation at different positions (M3-M5), oxidation and reduction (M6-M10) and demethylation and oxidation (M11). The metabolism of rankinidine has qualitative and quantitative species-specific differences in vitro. In PLMs and GLMs, the main metabolic pathway of rankinidine was oxidation. Notably, among the four species, the oxidation ability of rankinidine was highest in pigs and goats, and the demethylation and reduction abilities of rankinidine were highest in humans and rats. CONCLUSIONS: The interspecific metabolic differences of rankinidine in HLMs, PLMs, GLMs and RLMs were compared and studied for the first time using LC/QTOF. These findings will certainly support future studies of rankinidine metabolism in vivo and will contribute to elucidating the cause of species-specific differences behind Gelsemium toxicity.

Excretion, Metabolism, and Tissue Distribution of Gelsemium elegans (Gardn. & Champ.) Benth in Pigs.

Gelsemium elegans (Gardn. & Champ.) Benth is a toxic flowering plant in the family Loganiaceae used to treat skin diseases, neuralgia and acute pain. The high toxicity of G. elegans restricts its development and clinical applications, but in veterinary applications, G. elegans has been fed to pigs as a feed additive without poisoning. However, until now, the in vivo processes of the multiple components of G. elegans have not been studied. This study investigates the excretion, metabolism and tissue distribution of the multiple components of G. elegans after feeding it to pigs in medicated feed. Pigs were fed 2% G. elegans powder in feed for 45 days. The plasma, urine, bile, feces and tissues (heart, liver, lung, spleen, brain, spinal cord, adrenal gland, testis, thigh muscle, abdominal muscle and back muscle) were collected 6 h after the last feeding and analyzed using high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Five natural products in plasma, twelve natural products and five metabolites in urine, and three natural products in feces were characterized, suggesting that multiple components from G. elegans were excreted in the urine. However, ten natural products and four metabolites were detected in bile samples, which suggested that G. elegans is involved in enterohepatic circulation in pigs. A total of seven of these metabolites were characterized, and four metabolites were glucuronidated metabolites. Ten natural products and six metabolites were detected in the tissues, which indicates that G. elegans is widely distributed in tissues and can cross the blood-brain barrier. Among the characterized compounds, a highly toxic gelsedine-type alkaloid from G. elegans was the main compound detected in all biological samples. This is the first study of the excretion, metabolism and tissue distribution of multiple components from G. elegans in pigs. These data can provide an important reference to explain the efficacy and toxicity of G. elegans. Additionally, the results of the tissue distribution of G. elegans are of great value for further residue depletion studies and safety evaluations of products of animals fed G. elegans.

[Mechanism of atractylenolide â ¢ in alleviating H9c2 cell apoptosis through ROS/GRP78/caspase-12 signaling pathway based on molecular docking].

This study aims to investigate the effect of atractylenolide Ⅲ(ATL-Ⅲ) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-Ⅲ to GRP78 was determined by molecular docking.The result showed that ATL-Ⅲ had a good binding activity to GRP78, and the binding activity of ATL-Ⅲ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 µmol·L~(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-Ⅲ(15, 30, and 60 µmol·L~(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca~(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-Ⅲ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-Ⅲ(15, 30, and 60 µmol·L~(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-Ⅲ.In conclusion, ATL-Ⅲ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.

Is hyaluronic acid production transcriptionally regulated? A transcriptional repressor gene deletion study in Streptococcus zooepidemicus.

Hyaluronic acid (HA) is a multiple-function biopolymer that is widely used in food, cosmetic, and biomedical fields. In group C streptococci, the major workhorse of HA production in industry, the HA biosynthetic pathway has been proposed, while how HA synthesis is regulated is unclear. In this study, we identified twenty-five putative transcriptional repressors in S. zooepidemicus and studied whether they regulate HA synthesis or not. The individual gene deletion strain was firstly constructed, and the phenotypic changes of the corresponding deletion strains in stress tolerance and HA production were detected. The hrcA deletion strain is more sensitive to high temperature, and the rex deletion strain is more resistant to the oxidative stress. Three transcriptional repressor deletions resulted significantly decreased transcriptional levels of hasA, among which the scrR deletion strain shows most dramatical decrease in HA production. The regulatory mechanism of how ScrR affects the production of HA was further explored by transcriptional expression analysis of scrA and scrB, two direct target genes of ScrR regulon. Our results indicates that the deficiency of ScrR results in the unbalanced expression of scrA and scrB, which might also partly account for the decreasing production of HA. In agreement with the speculation, overexpression of scrB in ΔscrR genetic background results in 80% improvement in HA production. Taken together, the systemic genetic study of transcriptional repressors expands our understanding for the physiological regulation process of S. zooepidemicus and should help in the development of high-performance industrial strains for the efficient production of HA. KEY POINTS: • Twenty-two transcriptional repressor genes in S. zooepidemicus were deleted individually, and the phenotypes of corresponding mutants on a variety of conditions were characterized. • HrcA deficiency showed inferior cell tolerance to high temperature, and Rex deficiency showed superior cell tolerance to reactive oxygen stress, and four repressors deficiency showed inferior hyaluronic acid synthesis, among which the transcriptional levels of hasA of three mutants decreased significantly. • Optimizing sucrose metabolic flux can enhance hyaluronic acid synthesis significantly.

A Tri-Stable Piezoelectric Vibration Energy Harvester for Composite Shape Beam: Nonlinear Modeling and Analysis.

To reveal the nonlinear mechanism of the tri-stable piezoelectric vibration energy harvester based on composite shape beam (TPEH-C) and its influence on the system response, the nonlinear restoring force and the nonlinear magnetic force are discussed and analyzed in this paper. The nonlinear magnetic model is acquired by using equivalent magnetizing current theory, and the nonlinear resilience model is obtained by fitting experimental data. The corresponding distributed parameter model based on generalized Hamiltonian variation principle has been established. Frequency response functions for the TPEH-C are derived according to harmonic balance expansion, and the influence of different magnet distances and different excitation accelerations on the response amplitude and bandwidth of the TPEH-C are investigated. More importantly, the correctness of the theoretical analysis is verified by experiments. The results reveal that the spectrum of composite beam shows hard characteristic and the depth of potential well is changed, which provides a new way to ameliorate the potential well of the TPEH-C. A suitable magnet distance enables the TPEH-C to improve the response amplitude and the effective frequency range. The results in this paper have a theoretical guiding significance for the optimal design and engineering application of the TPEH-C.

Characterization of in vitro metabolites of three tetrahydroprotoberberine alkaloids in rat liver S9 by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Tetrahydroberberine (THB), tetrahydrocoptisine (THCP) and tetrahydrocolumbamine (THCB) belong to the tetrahydroprotoberberine (THPB) alkaloids. Most of them have been extensively studied because of their pharmacological activities such as anti-hypertension, anti-arrhythmia, antimicrobial activity and antioxidant. However, limited information on the pharmacokinetics and metabolism of the three alkaloids has been reported. The purpose of this study was to investigate the in vitro metabolism of THB, THCP and THCB in rat liver S9 by using a rapid and accurate high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) method. METHODS: The incubation mixture was processed with 15% trichloroacetic acid. Chromatographic separation of the three THPB alkaloids and their metabolites was achieved by HPLC/QqTOF-MS and accurate mass measurements of metabolites were automatically performed through data-dependent acquisition in only a 30-min analysis. The detailed structural elucidations of these metabolites were performed by comparing the changes in their accurate molecular masses, elemental compositions and product ions with those of the parent drug. RESULTS: Five, five and four metabolites of THB, THCP and THCB were identified in rat liver S9, respectively. The results show that O-demethylenation of the 9,10-vicinal methoxyl group was the main metabolic pathway of THB and THCB and that demethylenation of the two methylenedioxy groups was the main metabolic pathway of THCP. In addition, minor oxidation and methylation reactions could occur for these alkaloids in rat liver S9. CONCLUSIONS: This was the first investigation of the in vitro metabolism of THB, THCP and THCB in rat liver S9 by using a sensitive and accurate HPLC/QqTOF-MS method. The tentatively proposed metabolic pathways of the three alkaloids will provide a basis for further studies of the in vivo metabolism of the three compounds in animals and humans.

Characterization of N-methylcanadine and N-methylstylopine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: N-Methylcanadine and N-methylstylopine are two types of isoquinoline alkaloids which are considered to be the main medicinally active constituents of the genus Papaveraceae. However, to date, no metabolism studies of N-methylcanadine and N-methylstylopine have been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of these two alkaloids in rat liver S9. METHODS: N-Methylcanadine or N-methylstylopine was incubated with rat liver S9 for 1 h, and then the incubation mixture was processed with 15% trichloroacetic acid. High-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) as a reliable analytical method was used. The structural characterization of these metabolites was performed by the combination of the accurate MS/MS spectra and the known elemental composition. RESULTS: As a result, a total of four metabolites of N-methylcanadine and five metabolites of N-methylstylopine in rat liver S9 were tentatively identified. The cleavage of the methylenedioxy group of the drugs was the main metabolic pathway of N-methylcanadine and N-methylstylopine. CONCLUSIONS: The present study is the first in vitro metabolic investigation of N-methylcanadine and N-methylstylopine in rat liver S9 using a reliable HPLC/QqTOF-MS method. The metabolic pathways of N-methylcanadine and N-methylstylopine are tentatively proposed. This work lays the foundation for the in vivo metabolism of the two compounds in animals.

An Arc-shaped Piezoelectric Bistable Vibration Energy Harvester: Modeling and Experiments.

In order to improve vibration energy harvesting, this paper designs an arc-shaped piezoelectric bistable vibration energy harvester (ABEH). The bistable configuration is achieved by using magnetic coupling, and the nonlinear magnetic force is calculated. Based on Lagrangian equation, piezoelectric theory, Kirchhoff's law, etc., a complete theoretical model of the presented ABEH is built. The influence of the nonlinear stiffness terms, the electromechanical coupling coefficient, the damping, the distance between magnets, and the load resistance on the dynamic response and the energy harvesting performance of the ABEH is numerically explored. More importantly, experiments are designed to verify the energy harvesting enhancement of the ABEH. Compared with the non-magnet energy harvester, the ABEH has much better energy harvesting performance.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Comparative Analysis of the Gelsemium Alkaloids Metabolism in Human, Pig, Goat, and Rat Liver Microsomes.

AIM: The aim of this study was to investigate the metabolism of Gelsemium elegans in human, pig, goat and rat liver microsomes and to elucidate the metabolic pathways and cleavage patterns of the Gelsemium alkaloids among different species. METHODS: A human, goat, pig and rat liver microsomes were incubated in vitro. After incubating at 37°C for 1 hour and centrifuging, the processed samples were detected by HPLC/Qq-TOFMS was used to detect alcohol extract of Gelsemium elegans and its metabolites. RESULTS: Forty-six natural products were characterized from alcohol extract of Gelsemium elegans and 13 metabolites were identified. These 13 metabolites belong to the gelsemine, koumine, gelsedine, humantenine, yohimbane, and sarpagine classes of alkaloids. The metabolic pathways included oxidation, demethylation and dehydrogenation. After preliminary identification, the metabolites detected in the four species were different. All 13 metabolites were detected in pig and rat microsomes, but no oxidative metabolites of Gelsedine-type alkaloids were detected in goat and human microsomes. CONCLUSION: In this study, Gelsemium elegans metabolic patterns in different species are clarified and the in vitro metabolism of Gelsemium elegans is investigated. It is of great significance for its clinical development and rational application.

Effect of cytochrome P450 3A4 on tissue distribution of humantenmine, koumine, and gelsemine, three alkaloids from the toxic plant Gelsemium.

Humantenmine, koumine, and gelsemine are three indole alkaloids found in the highly toxic plant Gelsemium. Humantenmine was the most toxic, followed by gelsemine and koumine. The aim of this study was to investigate and analyze the effects of these three substances on tissue distribution and toxicity in mice pretreated with the Cytochrome P450 3A4 (CYP3A4) inducer ketoconazole and the inhibitor rifampicin. The in vivo test results showed that the three alkaloids were absorbed rapidly and had the ability to penetrate the blood-brain barrier. At 5 min after intraperitoneal injection, the three alkaloids were widely distributed in various tissues and organs, the spleen and pancreas were the most distributed, and the content of all tissues decreased significantly at 20 min. Induction or inhibition of CYP3A4 in vivo can regulate the distribution and elimination effects of the three alkaloids in various tissues and organs. Additionally, induction of CYP3A4 can reduce the toxicity of humantenmine, and vice versa. Changes in CYP3A4 levels may account for the difference in toxicity of humantenmine. These findings provide a reliable and detailed dataset for drug interactions, tissue distribution, and toxicity studies of Gelsemium alkaloids.

Camrelizumab, apatinib and hepatic artery infusion chemotherapy combined with microwave ablation for advanced hepatocellular carcinoma.

BACKGROUND: Hepatic arterial infusion chemotherapy and camrelizumab plus apatinib (TRIPLET protocol) is promising for advanced hepatocellular carcinoma (Ad-HCC). However, the usefulness of microwave ablation (MWA) after TRIPLET is still controversial. AIM: To compare the efficacy and safety of TRIPLET alone (T-A) vs TRIPLET-MWA (T-M) for Ad-HCC. METHODS: From January 2018 to March 2022, 217 Ad-HCC patients were retrospectively enrolled. Among them, 122 were included in the T-A group, and 95 were included in the T-M group. A propensity score matching (PSM) was applied to balance bias. Overall survival (OS) was compared using the Kaplan-Meier curve with the log-rank test. The overall objective response rate (ORR) and major complications were also assessed. RESULTS: After PSM, 82 patients were included both the T-A group and the T-M group. The ORR (85.4%) in the T-M group was significantly higher than that (65.9%) in the T-A group (P < 0.001). The cumulative 1-, 2-, and 3-year OS rates were 98.7%, 93.4%, and 82.0% in the T-M group and 85.1%, 63.1%, and 55.0% in the T-A group (hazard ratio = 0.22; 95% confidence interval: 0.10-0.49; P < 0.001). The incidence of major complications was 4.9% (6/122) in the T-A group and 5.3% (5/95) in the T-M group, which were not significantly different (P = 1.000). CONCLUSION: T-M can provide better survival outcomes and comparable safety for Ad-HCC than T-A.

Facile and scale-up syntheses of high-performance enzyme@meso-HOF biocatalysts.

Facile immobilization is essential for the wide application of enzymes in large-scale catalytic processes. However, exploration of suitable enzyme supports poses an unmet challenge, particularly in the context of scale-up biocatalyst fabrication. In this study, we present facile and scale-up syntheses of high-performance enzyme biocatalysts via in situ encapsulation of cytochrome c (Cyt-c) as mono-enzyme and glucose oxidase (GOx)-horseradish peroxidase (HRP) as dual-enzyme cascade (GOx&HRP) systems, respectively, into a stable mesoporous hydrogen-bonded organic framework (meso-HOF) matrix. In situ encapsulation reactions occur under ambient conditions, and facilitate scale up (∼3 g per reaction) of enzyme@meso-HOF within a very short period (5-10 min). The resultant biocatalysts not only exhibit high enzyme loading (37.9 wt% for mono-enzyme and 22.8 wt% for dual-enzyme) with minimal leaching, but also demonstrate high catalytic activity, superior reusability, and durability. This study represents an example of scale-up fabrication of enzyme@meso-HOF biocatalysts on the gram level and highlights superior meso-HOFs as suitable host matrices for biomolecular entities.

Hepatic arterial infusion chemotherapy with anti-angiogenesis agents and immune checkpoint inhibitors for unresectable hepatocellular carcinoma and meta-analysis.

BACKGROUND: Hepatic arterial infusion chemotherapy (HAIC) has been proven to be an ideal choice for treating unresectable hepatocellular carcinoma (uHCC). HAIC-based treatment showed great potential for treating uHCC. However, large-scale studies on HAIC-based treatments and meta-analyses of first-line treatments for uHCC are lacking. AIM: To investigate better first-line treatment options for uHCC and to assess the safety and efficacy of HAIC combined with angiogenesis inhibitors, programmed cell death of protein 1 (PD-1) and its ligand (PD-L1) blockers (triple therapy) under real-world conditions. METHODS: Several electronic databases were searched to identify eligible randomized controlled trials for this meta-analysis. Study-level pooled analyses of hazard ratios (HRs) and odds ratios (ORs) were performed. This was a retrospective single-center study involving 442 patients with uHCC who received triple therapy or angiogenesis inhibitors plus PD-1/PD-L1 blockades (AIPB) at Sun Yat-sen University Cancer Center from January 2018 to April 2023. Propensity score matching (PSM) was performed to balance the bias between the groups. The Kaplan-Meier method and cox regression were used to analyse the survival data, and the log-rank test was used to compare the suvival time between the groups. RESULTS: A total of 13 randomized controlled trials were included. HAIC alone and in combination with sorafenib were found to be effective treatments (P values for ORs: HAIC, 0.95; for HRs: HAIC + sorafenib, 0.04). After PSM, 176 HCC patients were included in the analysis. The triple therapy group (n = 88) had a longer median overall survival than the AIPB group (n = 88) (31.6 months vs 14.6 months, P < 0.001) and a greater incidence of adverse events (94.3% vs 75.4%, P < 0.001). CONCLUSION: This meta-analysis suggests that HAIC-based treatments are likely to be the best choice for uHCC. Our findings confirm that triple therapy is more effective for uHCC patients than AIPB.

Potential Use of Extracellular Vesicles in the Treatment of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain, and several studies have evaluated the efficacy of extracellular vesicles (EVs) in the treatment of IVDD. The databases PubMed, Embase, and Cochrane Library were systematically searched from inception to the end of 2022 to identify studies investigating the therapeutic potential of cell-derived EVs for IVDD treatment. The following outcome measures were utilized: magnetic resonance imaging (MRI) Pfirrmann grading system, disc height index (DHI), histological grading, and apoptosis rate. A comprehensive meta-analysis was conducted, including a total of 13 articles comprising 19 studies involving 218 experimental animals. Comparative analysis between normal cell-derived EVs and placebo revealed significant reductions in MRI grade, increased DHI values, decreased nucleus pulposus cell apoptosis rates, and improved tissue grades. These findings collectively demonstrate the effective inhibition of IVDD through the application of EVs derived from cells. In conclusion, this study provides an updated synthesis of evidence supporting the efficacy of EVs as a promising therapeutic approach for IVDD treatment.

Combining Preoperative Clinical and Imaging Characteristics to Predict MVI in Hepatitis B Virus-Related Combined Hepatocellular Carcinoma and Cholangiocarcinoma.

BACKGROUND: Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA) is a rare form of primary liver malignancy. Microvascular invasion (MVI) indicates poor postsurgical prognosis in cHCC-CCA. The objective of this study was to investigate preoperative predictors of MVI in hepatitis B virus (HBV) -related cHCC-CCA patients. METHODS: A total of 69 HBV-infected patients with pathologically confirmed cHCC-CCA who underwent hepatectomy were included. Univariate and multivariate analyses were conducted to determine independent risk factors that were then incorporated into the predictive model associated with MVI. Receiver operating characteristic analysis was used to assess the predictive performance of the new model. RESULTS: For the multivariate analysis, γ-glutamyl transpeptidase (OR, 3.69; p = 0.034), multiple nodules (OR, 4.41; p = 0.042) and peritumoral enhancement (OR, 6.16; p = 0.004) were independently associated with MVI. Active replication of HBV indicated by positive HBeAg showed no differences between MVI-positive and MVI-negative patients. The prediction score using the independent predictors achieved an area under the curve of 0.813 (95% CI 0.717-0.908). A significantly lower recurrence-free survival was observed in the high-risk group with a score of ≥1 (p < 0.001). CONCLUSION: γ-glutamyl transpeptidase, peritumoral enhancement and multiple nodules were independent preoperative predictors of MVI in HBV-related cHCC-CCA patients. The established prediction score demonstrated satisfactory performance in predicting MVI pre-operatively and may facilitate prognostic stratification.

Protocol to characterize target components of Gelsemium using an in-house herb database and high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Currently, the identification of herb metabolites is challenging due to a lack of clear standards. Here, using Gelsemium as an example, we present a protocol for characterizing target components of herbs. This approach utilizes high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry guided by an in-house herb metabolite database based on reported studies and mass spectrometry. We describe steps for creating an in-house database, preparing and detecting samples, processing data, and characterizing compounds. This approach offers a reference for future research on the identification of herb metabolites. For complete details on the use and execution of this protocol, please refer to Liu et al. (2017).1.

Increased Neuron-Specific Enolase Level Predicts Symptomatic Intracranial Hemorrhage in Patients with Ischemic Stroke Treated with Endovascular Treatment.

BACKGROUND: Neuron-specific enolase (NSE), which is a highly specific marker for neurons, could be a predictor for prognosis in patients with symptomatic intracranial hemorrhage (sICH) with acute ischemic stroke who are receiving endovascular treatment (EVT). This study aimed to investigate the relationship between NSE and sICH in patients with acute anterior circulation stroke undergoing EVT. METHODS: A total of 215 consecutive patients with acute stroke treated with EVT were included. Patients with stroke and acute anterior circulation occlusion, receiving EVT treated at our hospital, were enrolled between January 2017 and August 2021. NSE level was measured on arrival at the neurology intensive care unit after EVT. The patients were divided into 2 groups according to whether sICH was present. Univariate and multivariate analyses were performed. NSE level was also incorporated into the TAG score (modified Thrombolysis in Cerebral Infarction score, Alberta Stroke Program Early CT Score, and glucose level), which was developed as a scoring system to predict sICH, and the prediction capability was compared with the TAG score alone. Causal inference was performed using the package DoWhy in Python to evaluate the causal relationship between NSE and sICH. RESULTS: The area under the curve (AUC) value of NSE showed moderate accuracy, with an AUC value of 0.729 (95% confidence interval, 0.655-0.795; P < 0.001). The NSE cutoff value was set at 23.88 ng/mL. When the NSE level ≥23.88 ng/mL, the sensitivity was 58.33% and the specificity was 78.72% (P < 0.001). The AUC for the TAG + NSE score was 0.801 compared with an AUC of 0.632 for the TAG score (Z = 2.034; P = 0.042). A causal inference model using the DoWhy library shows a proportional relationship between NSE and the diagnosis of sICH. CONCLUSIONS: This study is the first to show that increased NSE level is an independent predictor of sICH in patients with acute anterior circulation stroke who are undergoing endovascular treatment.