Hybrid Bayesian Optimization-Based Graphical Discovery for Methylation Sites Prediction.

Protein methylation is one of the most important reversible post-translational modifications (PTMs), playing a vital role in the regulation of gene expression. Protein methylation sites serve as biomarkers in cardiovascular and pulmonary diseases, influencing various aspects of normal cell biology and pathogenesis. Nonetheless, the majority of existing computational methods for predicting protein methylation sites (PMSP) have been constructed based on protein sequences, with few methods leveraging the topological information of proteins. To address this issue, we propose an innovative framework for predicting Methylation Sites using Graphs (GraphMethySite) that employs graph convolution network in conjunction with Bayesian Optimization (BO) to automatically discover the graphical structure surrounding a candidate site and improve the predictive accuracy. In order to extract the most optimal subgraphs associated with methylation sites, we extend GraphMethySite by coupling it with a hybrid Bayesian optimization (together named GraphMethySite +) to determine and visualize the topological relevance among amino-acid residues. We evaluated our framework on two extended protein methylation datasets, and empirical results demonstrate that it outperforms existing state-of-the-art methylation prediction methods.

Fufang Fanshiliu Decoction Revealed the Antidiabetic Effect through Modulating Inflammatory Response and Gut Microbiota Composition.

Background: Diabetes mellitus brings serious threats and financial burdens to human beings worldwide. Fufang Fanshiliu decoction (FFSLD), a traditional Chinese medicine formula showing great antidiabetic effects, has been used in clinics for many years. Objective: This study aims to explore the underlying therapeutic mechanisms of FFSLD in Type II diabetes mellitus (T2DM). Methods: Sprague-Dawley rats induced by high-fat diet feeding combined with streptozotocin injection were used to establish the T2DM model. All rats were randomly divided into 6 groups: control, model, metformin, high dosage, middle dosage, and low dosage of FFSLD. After 4 weeks of treatment, serum, intestinal mucosa, and fecal samples were collected for further analysis. ELISA was used to detect the diabetic-related serum indicators and proinflammation cytokines. Gene or protein expressions of mitogen-activated protein kinase (MAPK), interleukin 1 beta (IL-1ß), transforming growth factor-beta (TGF-ß), and tumor necrosis factor-alpha (TNF-α) in intestinal mucosa were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) or western blot. 16s rRNA gene sequencing was used to detect the changes of gut microbiome in these groups. Intestinal gut microbiota (GM) composition was further analyzed according to the sequencing libraries. Results: FFSLD effectively recovered the diabetic-related biochemical indexes by reducing fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), insulin, and increasing high-density lipoprotein cholesterol (HDL-C). Furthermore, FFSLD significantly ameliorated the abnormal levels of proinflammation cytokines including IL-1ß, IL-6, TNF-α, and TGF-ß. In addition, the GM compositions of rats in control, model, and FFSLD treated groups were different. FFSLD significantly increased the relative abundance of Lactobacillus, Akkermansia, and Proteus, and reduced the relative abundance of Alistipes, Desulfovibrio, and Helicobacter. Moreover, these changed bacteria were closely related to the diabetic-related serum indicators and proinflammatory cytokines. Conclusion: These results suggest that FFSLD alleviates diabetic symptoms in T2DM rats through regulating GM composition and inhibiting inflammatory response, which clarify the therapeutic mechanism of FFSLD on T2DM and provide a theoretical basis for its further clinical application.

Comparison of two Polygonum chinense varieties used in Chinese cool tea in terms of chemical profiles and antioxidant/anti-inflammatory activities.

Despite the extensive use of Polygonum chinense (PC) as a detoxifying ingredient of Chinese cool tea, the efficacy of different PC varieties remains underexplored. Herein, we compare the chemical profiles and antioxidant/anti-inflammatory activities of the aqueous extracts of two PC varieties, namely P. chinense var. chinense (PCC) and P. chinense var. hispidum (PCH). Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MSMS) and multivariate analysis are used to rapidly identify extract components, while DPPH radical scavenging and xylene-induced mice ear edema assays are used to evaluate antioxidant and anti-inflammatory activities, respectively. Correlation analysis reveals that ellagic acid and quercitrin contents are positively correlated with the magnitude of the anti-inflammatory effect, and the adopted technique is concluded to allow for the rapid discrimination of PC varieties used in Chinese cool tea formulations.

Pharmacokinetics Comparison, Intestinal Absorption and Acute Toxicity Assessment of a Novel Water-Soluble Astragaloside IV Derivative (Astragalosidic Acid, LS-102).

BACKGROUND AND OBJECTIVES: Astragaloside IV (AGS IV) is the most important bioactive constituent of Radix Astragali. However, its disappointing clinical application is mainly caused by its very low solubility in biologic fluids, resulting in poor bioavailability after oral administration. We recently obtained a novel water-soluble derivative of AGS IV (astragalosidic acid, LS-102) that displayed significant cardioprotective potential against hypoxia-induced injury. The objective of this study was to investigate the intestinal absorption, main pharmacokinetic parameters and acute toxicity of LS-102 in rodents compared with AGS IV. METHODS: An oral dose of LS-102 and AGS IV (20 mg/kg) was administered to Sprague-Dawley (SD) rats, and blood samples were collected at predetermined time points. The plasma concentrations were detected by a validated UHPLC-MS/MS method, and pharmacokinetic parameters were calculated using a compartmental model. In the intestinal permeability study, the transport of LS-102 across Caco-2 cell monolayers was investigated at six concentrations from 6.25 to 250 µM. Moreover, the acute toxicity of LS-102 (40-5000 mg/kg) via a single oral administration was investigated in BALB/c mice. RESULTS: LS-102 was rapidly absorbed, attaining a maximum concentration of 248.7 ± 22.0 ng/ml at 1.0 ± 0.5 h after oral administration. The relative bioavailability of LS-102 was twice that of AGS IV. LS-102 had a Papp (mean) of 15.72-25.50 × 10-6 cm/s, which was almost 500-fold higher than that of AGS IV, showing that LS-102 had better transepithelial permeability and could be better absorbed in the intestinal tract. The acute toxicity study showed no abnormal changes or mortality in mice treated with LS-102 even at the single high dose of 5000 mg/kg body weight. CONCLUSIONS: Oral LS-102 produced a pharmacokinetic profile different from AGS IV with higher bioavailability, while the toxic tolerance was similar to previous estimates. Thus, we speculated that LS-102 might provide better clinical efficacy and be a potential candidate for the new drug development of Radix Astragali.

Discovery of markers for discriminating the age of cultivated ginseng by using UHPLC-QTOF/MS coupled with OPLS-DA.

BACKGROUND: Ginseng (Ginseng Radix et Rhizoma, Panax ginseng C.A. Meyer) is gaining more publicity in modern society due to its health benefit and huge value in market. In the practice of grading and pricing of ginseng, the age is one of the major factor influencing the price and grade of ginseng. Therefore, the age discrimination is an important task for the quality control of ginseng. However, the traditional morphological methods are too subjective to be reproductive in discrimination. PURPOSE: To establish a method that can discriminate the ginseng samples with different cultivation years. STUDY DESIGN: To analyze the correlation between chemical compositions and cultivation years of cultivated ginseng samples of different age and thus discover potential quality marker (Q-marker) for discriminating the age of cultivated ginseng. METHODS: In the present study, the ultra-high performance liquid chromatography coupled with the quadrupole-time of flight mass spectrometry (UHPLC-QTOF/MS) were utilized for the age discrimination and marker discovery. A statistical data processing procedure was established to screen markers and reduce the false positive rate. RESULTS: The results showed that the ginseng samples from 2- to 6-year-old could be well separated in the orthogonal projections on the latent structure - discrimination analysis (OPLS-DA) using the markers screened by the established statistical procedure, which could reduce approximately 20% of the insignificant markers and false positive discoveries. Ultimately, more than 50 compounds contributing to the age discrimination were identified including one new compound (malonylginsenoside). One negative marker (1038.4825@8.98) was discovered for the 2-year-old ginseng, and an equation was established to effectively predict the age of 3- to 6-year-old of ginseng. CONCLUSION: The constructed method can discriminate the ginseng samples with different cultivation years and is a complement to the traditional discrimination method of ginseng age.

An integrated strategy for rapid discovery and identification of quality markers in Guanxin Kangtai preparation using UHPLC-TOF/MS and multivariate statistical analysis.

BACKGROUND: Guanxin Kangtai preparation (GXKT), consisting of Panax ginseng, Panax notoginseng and Ilex pubescens, is a new proprietary Chinese medicines under development for treating coronary heart disease. Like other Chinese medicines, the components of GXKT were complex and the bioactive compounds remained unclear. PURPOSE: To discover bioactive compounds as quality markers (Q-markers) for better quality control of GXKT. STUDY DESIGN: Chinese medicines was separated into fractions. The correlation between chemical information and bioactivity of these fractions were analyzed with multivariate statistical methods to discover bioactive compounds responsible for the actions of Chinese medicine. METHOD: GXKT was separated into fractions by using high-performance liquid chromatography (HPLC). Ultra HPLC coupled with time-of-flight mass spectrometer (UHPLC-TOF/MS) was applied to detect compound information from these fractions to form a chemical database. The bioactivity of these fractions in protecting cardiomyocytes from ischemia/reperfusion injury was examined in H9c2 cells that were exposed to hypoxia followed by reoxygenation (H/R). Then, partial least square model and orthogonal projections to latent structures discriminant analysis were employed to discover bioactive compounds from the chemical database that were positively correlated with the bioactivity of GXKT fractions. Finally, the bioactivity of these compounds was confirmed by bioassay in H9c2 cells. RESULTS: The chemical information of 120 fractions separated from GXKT was detected and extracted by UHPLC-TOF/MS, and a chemical database including 61 high abundance compounds were formed from all fractions. These fractions produced different extent of protective effect to H9c2 cell underwent H/R treatment with cell viability ranging from 33.43% to 74.91%, demonstrating the separation of bioactive compounds among different fractions. The multivariate analysis discovered 16 compounds from GXKT positively correlated with the bioactivity of GXKT. Of these compounds, 6 compounds, i.e.: ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were chemical identified and also confirmed for their responsibility to the action of GXKT by bioassay. CONCLUSION: Ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were bioactive compounds and qualified as Q-markers for quality control of GXKT. This research provided a useful reference for the quality research of Chinese medicines.

Suppressing mPGES-1 expression by sinomenine ameliorates inflammation and arthritis.

Recently, microsomal prostaglandin E synthase 1 (mPGES-1) has attracted much attention from pharmacologists as a promising strategy and an attractive target for treating various types of diseases including rheumatoid arthritis (RA), which could preserve the anti-inflammatory effect while reducing the adverse effects often occur during administration of non-steroidal anti-inflammatory drugs (NSAIDs). Here, we report that sinomenine (SIN) decreased prostaglandin (PG)E2 levels without affecting prostacyclin (PG)I2 and thromboxane (TX)A2 synthesis via selective inhibiting mPGES-1 expression, a possible reason of low risk of cardiovascular event compared with NSAIDs. In addition, mPGES-1 protein expression was down-regulated by SIN treatment in the inflamed paw tissues both in carrageenan-induced edema model in rats and the collagen-II induced arthritis (CIA) model in DBA mice. More interestingly, SIN suppressed the last step of mPGES-1 gene expression by decreasing the DNA binding ability of NF-κB, paving a new way for drug discovery.

Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance.

Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and are some of the best-selling natural products in the world. The accurate quantification of ginsenoside Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1 chemical reference substance (CRS) is often measured with high-performance chromatography coupled with an ultraviolet detector (HPLC-UV), which is a selective detector with unequal responses to different compounds; thus, this detector introduces probable error to purity assessments. In the present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in methanol-d4 at 4.37ppm was selected to avoid interfering signals, enabling accurate quantitative analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data acquisition. For post-processing, the Lorentz/Gauss deconvolution method was employed to increase the signal accuracy by separating the impurities and noise in the integrated region of the quantitative proton. The method validation showed that the developed method has acceptable sensitivity, linearity, precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed in this research was 90.34±0.21%, which was obviously lower than that reported by the manufacturer (>98.0%, HPLC-UV). The cross-method validation shows that the commonly used HPLC-UV, HPLC-ELSD (evaporative light scattering detector) and even LC-MS (mass spectrometry) methods provide significantly higher purity values of Rg1 CRS compared with the qNMR method, and the accuracy of these LC-based methods largely depend on the amount of the sample that was loaded and the properties of the impurities.

Rg1 prevents myocardial hypoxia/reoxygenation injury by regulating mitochondrial dynamics imbalance via modulation of glutamate dehydrogenase and mitofusin 2.

PURPOSE: Mitochondrial dysfunction is a prominent feature of ischemia heart disease but the underlying mechanism of dynamics (fusion/fission) is still unclear. Here we investigated a novel function and underlying mechanism of Rg1 on an in vitro cardiomyocyte model of hypoxia/reoxygenation (H/R). METHODS: Cellular cytotoxicity was evaluated by MTT, mitochondrial viable staining, and cardiac marker detection. Mitochondrial function was evaluated by ATP content measurement, MMP determination, ROS, OCR and ECAR assay. Mitochondrial dynamics was investigated by Live-cell imaging with time-lapse fluorescence microscopy and morphological features were evaluated by the high-content image analysis. Mitochondrial fusion and fission-related proteins, GDH were determined by Western blot, RT-PCR and immunofluorescence. RESULTS: Rg1 moderated GDH dysregulation and then protected against H/R-induced cellular damage and mitochondrial dysfunction in a dose-dependent manner. Rg1 significantly increased mitochondrial length, reduced the number of cells with fragmented mitochondria and up-regulated the MFN2 expression finally leading to preventing the imbalance of mitochondrial dynamics following H/R. Knock-down of MFN2 by specific siRNA completely abolished the ability of Rg1 to cell survival by H/R. CONCLUSION: Rg1 through modulation of GDH and MFN2 maintained mitochondrial dynamics that resulted in protection against H/R-induced cardiomyocyte injury. All these results put forward a new protective mechanism of Rg1 on the therapeutic potential in cardiac I/R disorders.

An ultra-high performance liquid chromatographic-tandem mass spectrometric method for the determination of sinomenine in human plasma after transdermal delivery of the zhengqing fengtongning injection.

A sensitive, precise and selective ultra-high performance liquid chromatography method coupled with triple-quadrupole mass spectrometry was developed and validated for the determination of trace amounts of sinomenine (ng/mL) in minute volumes of human plasma. Fifty microliter plasma samples were precipitated using methanol to extract sinomenine. Separation was carried out on a C18 column with a water and acetonitrile mobile phase gradient with formic acid as an additive. The mass spectrometry data were obtained in the positive ion mode, and the transition of multiple reactions was monitored at m/z 330.2→181.0 for sinomenine quantification. The working assay range for sinomenine was linear from 0.1173 to 15.02 ng/mL with the lower limit of quantification of 0.1173 ng/mL. The precision and accuracy of the method was less than 15% in intra-day and inter-day experiments with a matrix effect of less than 6.5%. After validation, the quantitative method was applied to analyze sinomenine levels in human plasma after transdermal delivery of the Zhengqing Fengtongning Injection. The results showed that some samples contained sinomenine within the concentration range 0.4131-4.407 ng/mL.