LC-MS/MS method for the quantification of cortisol of hepatocellular carcinoma.

The imbalance of steroid hormones is closely related to the occurrence and development of hepatocellular carcinoma (HCC). However, most research has focused on steroid hormone receptors, and reports about the relationship between the serum concentration of cortisol and the development of HCC are rare. The aim of this research was to establish a simple, specific, sensitive and reliable liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the quantitation of cortisol in human serum and to compare the level of cortisol in serum between 221 HCC patients and 183 healthy volunteers. The results showed that the correlation coefficients of the linear regression with a weighing factor of 1/x2 ranged from 0.9933 to 0.9984 over the range of 2-1,000 ng/ml. The inter- and intra-day precision and accuracy were <10%. The matrix effect and recovery of cortisol were 94.9-102.5% and 96.3-99.8%, respectively. The concentration of cortisol in HCC patients was significantly higher than that in healthy volunteers (p < 0.05) and was not affected by sex, age, menopause or α-fetoprotein (AFP) level. The present study reveals that elevated cortisol might promote the progression of HCC.

Exploring the Mechanism of Hepatotoxicity Induced by Dictamnus dasycarpus Based on Network Pharmacology, Molecular Docking and Experimental Pharmacology.

The root bark of Dictamnus dasycarpus Turcz is a traditional Chinese medicine, Dictamni Cortex (DC), which is mainly used in the clinical treatment of skin inflammation, eczema, rubella, rheumatism, and gynecological inflammation. Unexpectedly, there are some cases of liver injury after the administration of DC. However, the mechanism of hepatotoxicity remains ambiguous. The aim of this study was to explore the mechanism and substance bases of DC hepatotoxicity based on network pharmacology and molecular docking, verified through pharmacological experiments. Partial prototype components and metabolites in vivo of quinoline alkaloids from DC were selected as candidate compounds, whose targets were collected from databases. Network pharmacology was applied to study the potential hepatotoxic mechanism after correlating the targets of candidate compounds with the targets of hepatotoxicity. Molecular docking was simulated to uncover the molecular mechanism. Furthermore, the hepatotoxicity of the extract and its constituents from DC was evaluated in vivo and in vitro. We constructed the "potential toxic components-toxic target-toxic pathway" network. Our results showed that the targets of DC included CYP1A2 and GSR, participating in heterologous steroid metabolism, REDOX metabolism, drug metabolism, heterocyclic metabolic processes, the synthesis of steroid hormone, cytochrome P450 metabolism, chemical carcinogens and bile secretion pathways. In vitro and in vivo experiments displayed that DC could result in a decrease in GSH-Px and oxidative stress, simultaneously inhibiting the expression of CYP1A2 and inducing hepatotoxicity. These results further indicated the mechanism of hepatotoxicity induced by Dictamnus dasycarpus, providing a basic theory to explore and prevent hepatotoxicity in the clinical usage of Dictamnus dasycarpus.

Comparative study on the main active components of Baoyuan decoction in plasma and urine of normal and heart failure rats.

The global morbidity and mortality of heart failure has been increasing in recent years. Traditional Chinese medicine (TCM) was increasingly used to treat cardiovascular diseases. Baoyuan decoction (BYD) was a famous classical prescription in China. Modern pharmacological studies showed that it had obvious therapeutic effects on cardiovascular diseases, but its pathological pharmacokinetic studies were unclear. In this research, the absorption of 16 bioactive components in plasma and the excretion of 9 representative components in urine of control rats and isoproterenol (ISO)-induced heart failure rats were studied using the large-volume direct-injection LC-MS method established by our research group. The results indicated that flavonoid constituents exhibited quicker absorption and elimination than saponin constituents after oral administration of BYD. The half-life period of some bioactive compounds in the model group was increased, which contributed to the longer therapeutic effect. The cumulative excretion rate of major flavonoid components of BYD decreased significantly in the ISO-induced heart failure rats.

The Synergistic Mechanism of Total Saponins and Flavonoids in Notoginseng-Safflower against Myocardial Infarction Using a Comprehensive Metabolomics Strategy.

Notoginseng and safflower are commonly used traditional Chinese medicines for benefiting qi and activating blood circulation. A previous study by our group showed that the compatibility of the effective components of total saponins of notoginseng (NS) and total flavonoids of safflower (SF), named NS-SF, had a preventive effect on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. However, the therapeutic effect on MI and the synergistic mechanism of NS-SF are still unclear. Therefore, integrated metabolomics, combined with immunohistochemistry and other pharmacological methods, was used to systematically research the therapeutic effect of NS-SF on MI rats and the synergistic mechanism of NS and SF. Compared to NS and SF, the results demonstrated that NS-SF exhibited a significantly better role in ameliorating myocardial damage, apoptosis, easing oxidative stress and anti-inflammation. NS-SF showed a more significant regulatory effect on metabolites involved in sphingolipid metabolism, glycine, serine, and threonine metabolism, primary bile acid biosynthesis, aminoacyl-tRNA biosynthesis, and tricarboxylic acid cycle, such as sphingosine, lysophosphatidylcholine (18:0), lysophosphatidylethanolamine (22:5/0:0), chenodeoxycholic acid, L-valine, glycine, and succinate, than NS or SF alone, indicating that NS and SF produced a synergistic effect on the treatment of MI. This study will provide a theoretical basis for the clinical development of NS-SF.

Evaluation of the effects of notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF (CNS) on the activities of cytochrome P450 enzymes using a cocktail method in rats.

Notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF, namely CNS, are used for the treatment of cardiovascular diseases in clinic. This study developed a cocktail assay involving seven cytochrome P450 (CYP) enzymes to elucidate the effect of NS, SF, and CNS on CYP enzymes and to explore the synergistic effect of CNS in terms of CYP enzymes. Ultra-performance liquid chromatography-MS and reverse-transcription polymerase chain reaction were applied to detect the activities and mRNA expression levels of CYP enzymes. SF exhibited inhibitory effects on CYP1A2, 2B1, 2E1, and 2C11 and induction effects on CYP2C19 and 2D4. NS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C11, 2C19, and 2D4. CNS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C19, and 2D4 and inhibitory effects on CYP3A1 in vivo. Moreover, mRNA expression results were consistent with pharmacokinetic results. Potential herb-drug interactions should be studied closely when SF, NS, or CNS with clinical drugs are metabolized by CYP1A2, 2B1, 2E1, 2C11, 2C19, 2D4, and 3A1. CNS could change the inhibition or induction effects of CYP compared to the NS group, which might be one of the causes for the synergistic effects of the combination of NS and SF.

Integrated Array Chip for High-Throughput Screening of Species Differences in Metabolism.

Species differences in metabolism may produce failure prediction of drug efficacy/toxicity in humans. Integration of metabolic competence and cellular effect assays in vitro can provide insight into the species differences in metabolism; however, a co-culture platform with features of high throughput, operational simplicity, low sample consumption, and independent layouts is required for potential usage in industrial test settings. Herein, we developed an integrated array chip (IAC) to evaluate the species differences in metabolism through metabolism-induced anticancer bioactivity as a case. The IAC consisted of two functional parts: a micropillar chip for immobilization of liver microsomes and a microwell chip for three-dimensional (3D) tumor cell culture. First, optimized parameters of the micropillar chip for microsomal encapsulation were obtained by cross-shaped protrusions and a 2.5 µL volume of 3D agarose spots. Next, we examined factors influencing metabolism-induced anticancer bioactivity. Feasibility of the IAC was validated by four model prodrugs using image-based bioactivity detection and mass spectrometry (MS)-based metabolite analysis. Finally, a species-specific IAC was used for selection of animal species that best resembles metabolism-induced drug response to humans at throughputs. Overall, the IAC provides a promising co-culture platform for identifying species differences in metabolism and selection of animal models to accelerate drug discovery.

Pharmacokinetics study of 16 active ingredients from Tabson-2 decoction in normal and d-galactose induced osteoporosis rats by liquid chromatography-tandem mass spectrometry.

Tabson-2 decoction is the traditional Mongolian formula for anti-osteoporosis, and the ambiguous of active ingredient is an important factor in restricting its modernization and globalization. Although pharmacokinetic profiles research is a viable approach to find the components being responsible for formula efficacy, the pharmacokinetics study of Tabson-2 decoction has not been elucidated yet. Owing to the existence of isomers, low bioavailability of some small molecule and interference of endogenous, the pharmacokinetics study of Tabson-2 decoction are more difficult than that of chemical drugs. In our experiment, a specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of 16 active ingredients in Tabson-2 decoction, which could fulfill the requirements of multi-compounds pharmacokinetic study of Tabson-2 decoction. Additionally, the ingredients with significant distributions in rats were gentianic acid, chlorogenic acid, and aucubin, which could be the main potential active components in Tabson-2 decoction. The components with a significant bioavailability difference between normal and d-galactose induced osteoporosis rats were achieved as well. These data offer useful information for screening the active ingredients in Tabson-2 decoction, and assessing the bioavailability of these active ingredients in different physiological status, which might provide a possible mechanism of anti-osteoporosis efficacy of Tabson-2 decoction.

Development and validation of an LC-MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU-48 in rat plasma and its application to a pharmacokinetic study.

A specific, sensitive and stable high-performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of methyl 3-amino-6-methoxythieno [2,3-b]quinoline-2-carboxylate (PU-48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU-48 was achieved with a reversed-phase C18 column (100 × 2.1 mm, 3 µm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU-48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass-to-charge ratio (m/z) 289.1 → 229.2 for PU-48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU-48 was linear over the concentration range of 0.1-1000 ng/mL (r2 > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU-48 in rats.

Effects of crystalline state and self-nanoemulsifying drug delivery system (SNEDDS) on oral bioavailability of the novel anti-HIV compound 6-benzyl-1-benzyloxymethyl-5-iodouracil in rats.

The objective of this study was to investigate the effect of crystalline state and a formulation of self-nanoemulsifying drug delivery system (SNEDDS) on oral bioavailability of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor, in rats. The crystalline states of W-1 were characterized by scanning electron microscope (SEM), differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). The SNEDDS was formulated by medium-chain lipids, characterized by droplet particle size. The plasma concentrations of W-1 were measured by high performance liquid chromatography (HPLC). The results indicated that W-1 compound were presented as crystalline forms, A and B, the degree of crystallization in form B was higher than that in form A. The SNEDDS of W-1 displayed a significant increase in the dissolution rate than W-1 powder. Furthermore, after oral administration of W-1 (100 mg/kg), the pharmacokinetic parameters of form A, form B, and W-1 SNEDDS were as follows: AUC0-t 526.4 ± 123.5, 305.1 ± 58.5 and 2297 ± 451 ng h/mL (p < .05, when W-1 SNEDDS were compared with either form A or form B), respectively. With SNEDDS formulation, the relative bioavailabilities were enhanced by 4.36-fold and 7.53-fold over the form A and form B of W-1, respectively. In conclusion, the present results suggested that the crystalline states of W-1 might lead to the lower oral bioavailability, and SNEDDS formulation is a promising strategy of improving bioavailability, in spite of that crystalline states usually carry small lot-to-lot variability.

Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLint were 2.3 µM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.

Development and validation of a high performance liquid chromatography method for determination of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor and its application to a pharmacokinetic study in rats.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for determination of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor in rat plasma, was developed and validated. Chromatographic separation of W-1 and megestrol acetate (internal standard) was achieved on a reversed-phase C18 column at 25°C. The mobile phase was consisted of acetonitrile-water (60:40, v/v) and pumped at a flow rate of 1.0 mL/min. The ultraviolet (UV) detector was set at the absorption wavelength of 284 nm. The calibration curve for W-1 was linear over the concentration range of 0.01-8 µg/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precision and accuracy were <8.9 and 5.3%, respectively. The extraction recoveries ranged from 97.9 to 101.6%. The validated HPLC method was successfully applied to a pharmacokinetic study of W-1 in rats.

Development of an integrated strategy for comprehensive characterization of Sinomenii Caulis extract and metabolites in rats based on UPLC/Q-TOF-MS.

Sinomenii Caulis (SC), a commonly used traditional Chinese medicine for its therapeutic effects on rheumatoid arthritis, contains rich chemical components. At present, most studies mainly focus on sinomenine, with little research on other alkaloids. In this study, a comprehensive profile of compounds in SC extract, and biological samples of rats (including bile, urine, feces, and plasma) after oral administration of SC extract was conducted via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The fragmentation patterns and potential biotransformation pathways of six main types of alkaloids in SC were summarized, and the corresponding characteristic product ions, relative ion intensity, and neutral losses were obtained to achieve rapid classification and identification of complex components of SC from in vitro to in vivo. As a result, a total of 114 alkaloid compounds were identified, including 12 benzyl alkaloids, 4 isoquinolone alkaloids, 32 aporphine alkaloids, 28 protoberberine alkaloids, 34 morphinan alkaloids and 4 organic amine alkaloids. After administration of SC extract to rats, a total of 324 prototypes and metabolites were identified from rat plasma, urine, feces and bile, including 81 aporphines, 95 protoberberines, 117 morphinans and 31 benzylisoquinolines. The main types of metabolites were demethylation, hydrogenation, dehydrogenation, aldehydation, oxidation, methylation, sulfate esterification, glucuronidation, glucose conjugation, glycine conjugation, acetylation, and dihydroxylation. In summary, this integrated strategy provides an additional approach for the incomplete identification caused by compound diversity and low abundance, laying the foundation for the discovery of new bioactive compounds of SC against rheumatoid arthritis.

A pseudo-targeted metabolomics for discovery of potential biomarkers of cardiac hypertrophy in rats.

Cardiac hypertrophy (CH) is one of the stages in the occurrence and development of severe cardiovascular diseases, and exploring its biomarkers is beneficial for delaying the progression of severe cardiovascular diseases. In this research, we established a comprehensive and highly efficient pseudotargeted metabolomics method, which demonstrated a superior capacity to identify differential metabolites when compared to traditionaluntargeted metabolomics. The intra/inter-day precision and reproducibility results proved the method is reliable and precise. The established method was then applied to seek the potential differentiated metabolic biomarkers of cardiac hypertrophy (CH) rats, and oxylipins, phosphorylcholine (PC), lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), Krebs cycle intermediates, carnitines, amino acids, and bile acids were disclosed to be the possible differentiate components. Their metabolic pathway analysis revealed that the potential metabolic alterations in CH rats were mainly associated with phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, citrate cycle, glyoxylate and dicarboxylate metabolism, and tyrosine metabolism. In sum, this research provided a comprehensiveand reliable LC-MS/MS MRM platform for pseudo-targeted metabolomics investigation of disease condition, and some interesting potential biomarkers were disclosed for CH, which merit further exploration in the future.

Palmitoylation of PKCδ by ZDHHC5 in hypothalamic microglia presents as a therapeutic target for fatty liver disease.

The hypothalamus plays a fundamental role in controlling lipid metabolism through neuroendocrine signals. However, there are currently no available drug targets in the hypothalamus that can effectively improve human lipid metabolism. In this study, we found that the antimalarial drug artemether (ART) significantly improved lipid metabolism by specifically inhibiting microglial activation in the hypothalamus of high-fat diet-induced mice. Mechanically, ART protects the thyrotropin-releasing hormone (TRH) neurons surrounding microglial cells from inflammatory damage and promotes the release of TRH into the peripheral circulation. As a result, TRH stimulates the synthesis of thyroid hormone (TH), leading to a significant improvement in hepatic lipid disorders. Subsequently, we employed a biotin-labeled ART chemical probe to identify the direct cellular target in microglial cells as protein kinase Cδ (PKCδ). Importantly, ART directly targeted PKCδ to inhibit its palmitoylation modification by blocking the binding of zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5), which resulted in the inhibition of downstream neuroinflammation signaling. In vivo, hypothalamic microglia-specific PKCδ knockdown markedly impaired ART-dependent neuroendocrine regulation and lipid metabolism improvement in mice. Furthermore, single-cell transcriptomics analysis in human brain tissues revealed that the level of PKCδ in microglia positively correlated with individuals who had hyperlipemia, thereby highlighting a clinical translational value. Collectively, these data suggest that the palmitoylation of microglial PKCδ in the hypothalamus plays a role in modulating peripheral lipid metabolism through hypothalamus-liver communication, and provides a promising therapeutic target for fatty liver diseases.

Efficient degradation of hexabromocyclododecane using montmorillonite supported nano-zero-valent iron and Citrobacter sp. Y3.

The coupling of modified nanoscale zero-valent iron (nZVI) with organohalide-degrading bacteria provides a promising solution for the remediation of hexabromocyclododecane (HBCD)-contaminated environments. However, the interactions between modified nZVI and dehalogenase bacteria are intricate, and the mechanisms of synergistic action and electron transfer are not clear, and requires further specific investigation. In this study, HBCD was used as a model pollutant, and stable isotope analysis revealed that organic montmorillonite (OMt)-supported nZVI coupled with the degrading bacterial strain Citrobacter sp. Y3 (nZVI/OMt-Y3) can use [13C]HBCD as the sole carbon source and degrade or even mineralise it into 13CO2 with a maximum conversion rate of 100% within approximately 5 days. Analysis of the intermediates showed that the degradation of HBCD mainly involves three different pathways: dehydrobromination, hydroxylation, and debromination. The proteomics results showed that nZVI introduction promoted the transport of electrons and debromination. Combining the results from XPS, FTIR, and Raman spectroscopy with the analysis results of proteinomics and biodegradation products, we verified the process of electron transport and proposed a metabolic mechanism of HBCD degradation by the nZVI/OMt-Y3. Moreover, this study provides insightful avenues and models for the further remediation of HBCD and other similar pollutants in the environment.

An HPLC-MS/MS Method for Pharmacokinetic Study of Y-99: A Novel Diuretic Agent Targeting Urea Transporters.

Y-99, a promising first-in-class diuretic, is a novel urea transporter inhibitor with oral diuretic activity. However, little is known about the pharmacokinetic profiles of Y-99 in experimental animals. In this study, a method of quantitative determination of Y-99 in rat plasma based on high-performance liquid chromatography-tandem mass spectrometry was developed and validated in selectivity, linearity, recovery and matrix effect, accuracy and precision, stability, carry-over and dilution integrity. Chromatographic separation was conducted on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with gradient elution at a 0.3 mL/min flow rate after protein precipitation. Mass spectrometry was performed by a positive electrospray ionization mass spectrometer in multiple reaction monitoring mode. The method showed standard-compliant linearity (1-1,000 ng/mL, r = 0.9991). The intra-day and inter-day accuracy (relative error < 11.2%) and precision (coefficient of variation <8.4%) were within acceptable criteria. The recovery and matrix effects were 97.3-110.7% and 103.7-107.5%, respectively. The stability, dilution integrity and carry-over of the method were also within the acceptable criteria. Pharmacokinetic profiles of Y-99 in rats were first investigated using this method, which was vital for developing novel diuretics without electrolyte imbalance targeting urea transporters.

Citrobacter sp. Y3 harbouring novel gene HBCD-hd-1 mineralizes hexabromocyclododecane via new metabolic pathways according to multi-omics characterization.

Hexabromocyclododecane (HBCD) is a typical persistent organic pollutant that is widely detected in the environment. Despite the significant efforts put into its mineralisation, there is still a lack of microorganism resources that can completely mineralise HBCD. Stable isotope analysis revealed that the Citrobacter sp. Y3 can use [13C]HBCD as its sole carbon source and degrade or even mineralise it into 13CO2, with a maximum conversion rate of 100% in approximately 14 days. Strain Y3 could completely mineralise HBCD, which it used as its only carbon source, and six debromination enzymes related to HBCD degradation were found in Y3, including haloalkane dehalogenase (DhaA), haloacid dehalogenase (HAD), etc. A functional gene named HBCD-hd-1, encoding a HAD, was found to be upregulated during HBCD degradation and heterologously expressed in Escherichia coli. Recombinant E. coli with the HBCD-hd-1 gene transformed the typical intermediate 4-bromobutyric acid to 4-hydroxybutanoic acid and showed excellent degradation performance on HBCD, accompanied by nearly 100% bromine (Br) ion generation. The expression of HBCD-hd-1 in Y3 rapidly accelerated the biodegradation of HBCD. With HBCD as its sole carbon source, strain Y3 could potentially degrade HBCD, especially in a low-nutrient environment.

Metabolomics and network analysis uncovered profound inflammation-associated alterations in hepatitis B virus-related cirrhosis patients with early hepatocellular carcinoma.

Patients with hepatitis B virus (HBV)-related liver cirrhosis (LC) are at high risk for hepatocellular carcinoma (HCC). Limitations in the early detection of HCC give rise to poor survival in this high-risk population. Here, we performed comprehensive metabolomics on health individuals and HBV-related LC patients with and without early HCC. Compared to non-HCC patients (N = 108) and health controls (N = 80), we found that patients with early HCC (N = 224) exhibited a specific plasma metabolome map dominated by lipid alterations, including lysophosphatidylcholines, lysophosphatidic acids and bile acids. Pathway and function network analyses indicated that these metabolite alterations were closely associated with inflammation responses. Using multivariate regression and machine learning approaches, we identified a five-metabolite combination that showed significant performances in differentiating early-HCC from non-HCC than α-fetoprotein (area under the curve values, 0.981 versus 0.613). At metabolomic levels, this work provides additional insights of metabolic dysfunction related to HCC progressions and demonstrates the plasma metabolites might be measured to identify early HCC in patients with HBV-related LC.

Metabolic signatures in pericardial fluid and serum are associated with new-onset atrial fibrillation after isolated coronary artery bypass grafting.

Postoperative atrial fibrillation (POAF) is a common complication of coronary artery bypass grafting (CABG) procedures. However, the molecular mechanism of POAF remains poorly understood, hence the absence of effective prevention strategies. Here we used targeted metabolomics on pericardial fluid and serum samples from CABG patients to investigate POAF-associated metabolic alterations and related risk prediction of new-onset AF. Nine differential metabolites in various metabolic pathways were found in both pericardial fluid and serum samples from patients with POAF and without POAF. By using machine learning algorithms and regression models, a 4-metabolite (aceglutamide, ornithine, methionine, and arginine) risk prediction model was constructed and showed accurate performance in predicting POAF in both discovery and validation sets. This work extends the metabolic insights of the cardiac microenvironment and blood in patients with POAF and paves the way for the use of targeted metabolomics for predicting POAF in patients with CABG surgery.

The roles of ubiquitin-proteasome system and regulator of G protein signaling 4 in behavioral sensitization induced by a single morphine exposure.

AIMS: Opioid addiction is a major public health issue, yet its underlying mechanism is still unknown. The aim of this study was to explore the roles of ubiquitin-proteasome system (UPS) and regulator of G protein signaling 4 (RGS4) in morphine-induced behavioral sensitization, a well-recognized animal model of opioid addiction. METHODS: We explored the characteristics of RGS4 protein expression and polyubiquitination in the development of behavioral sensitization induced by a single morphine exposure in rats, and the effect of a selective proteasome inhibitor, lactacystin (LAC), on behavioral sensitization. RESULTS: Polyubiquitination expression was increased in time-dependent and dose-related fashions during the development of behavioral sensitization, while RGS4 protein expression was not significantly changed during this phase. Stereotaxic administration of LAC into nucleus accumbens (NAc) core inhibited the establishment of behavioral sensitization. CONCLUSION: UPS in NAc core is positively involved in behavioral sensitization induced by a single morphine exposure in rats. Polyubiquitination was observed during the development phase of behavioral sensitization, while RGS4 protein expression was not significantly changed, indicating that other members of RGS family might be substrate proteins in UPS-mediated behavioral sensitization.