Crystal structure of the Caenorhabditis elegans apoptosome reveals an octameric assembly of CED-4.

The CED-4 homo-oligomer or apoptosome is required for initiation of programmed cell death in Caenorhabditis elegans by facilitating autocatalytic activation of the CED-3 caspase zymogen. How the CED-4 apoptosome assembles and activates CED-3 remains enigmatic. Here we report the crystal structure of the complete CED-4 apoptosome and show that it consists of eight CED-4 molecules, organized as a tetramer of an asymmetric dimer via a previously unreported interface among AAA(+) ATPases. These eight CED-4 molecules form a funnel-shaped structure. The mature CED-3 protease is monomeric in solution and forms an active holoenzyme with the CED-4 apoptosome, within which the protease activity of CED-3 is markedly stimulated. Unexpectedly, the octameric CED-4 apoptosome appears to bind only two, not eight, molecules of mature CED-3. The structure of the CED-4 apoptosome reveals shared principles for the NB-ARC family of AAA(+) ATPases and suggests a mechanism for the activation of CED-3.

Simultaneous Assay of Oxygen-Dependent Cytotoxicity and Genotoxicity of Anticancer Drugs on an Integrated Microchip.

Oxygen deprivation is a common feature in a variety of cancer tissues and associated with tumor progression, acquisition of antiapoptotic potential, and clinical therapeutic resistance. Thus, great interest has been aroused to develop new platforms or approaches of activity assays to impact on the hypoxic microenvironment and oxygen-dependent drug responses to improve the productivity of new drug discovery. In this study, an integrated microsystem is established to combine the cytotoxic and genotoxic tests together for continuous multiple measurements under mimicking hypoxic tumor microenvironment. We fabricated a double-layer chip device by combining a single-cell-arrayed agarose layer with a microfluidics-based oxygen gradient-generating layer using a PDMS membrane. Using tirapazamine (TPZ) and blemycin (BLM) as model anticancer drugs, we demonstrated its application and performance in single cell loading, cell cultivation, and subsequent drug treatment as well as in situ analysis of oxygen-dependent cytotoxicity and genotoxicity of anticancer drugs. The results demonstrated the opposite oxygen-dependent toxicity of TPZ and BLM, which also indicated that the formation of DNA breaks is related with cell apoptosis. Compared with the traditional assays, this device takes advantage of microfluidic phenomena to generate various oxygen concentrations while exhibiting the combinatorial diversities achieved by the single cell microarray, offering a powerful tool to study single cell behaviors and responses under different oxygen conditions with desired high-content and high-throughput capabilities.

A new method to evaluate the dose-effect relationship of a TCM formula Gegen Qinlian Decoction: "Focus" mode of integrated biomarkers.

It is difficult to accurately evaluate the efficacy of traditional Chinese medicine (TCM), which leads to the uncertainty and complexity of dose-effect analysis. In this study we established the "Focus" mode of biomarkers to characterize the dose-effect relationship of Gegen Qinlian Decoction (GQD), a TCM formula for treating type 2 diabetes mellitus (2-DM). A rat model of 2-DM was established through high fat diet feeding combined with low-dose STZ injection. Rats with 2-DM were administered high, middle or low doses (6.785, 4.071, 1.357 mg·kg-1·d-1, respectively) of GQD extract for 60 d. Metformin (300 mg·kg-1·d-1) was taken as the positive control. Blood samples were collected to assess serum biochemical indexes and metabolic profiling. After "Focus" analysis, the biochemical index triglycerides (TG) and insulin sensitivity (ISI) were identified as focused integrated biomarkers (FIBs), while arachidonic acid and docosatetraenoic acid were the metabolic FIBs. Dose-effect relationship curves of GQD were built based on these types of FIBs. Furthermore, the two dose-effect relationship curves showed similar trends with the middle dosage displaying the greatest efficacy, suggesting that insulin function and arachidonic acid metabolism played important roles in 2-DM and the responses to GQD. The metabolic FIB docosatetraenoic should be further explored for understanding its involvement in the process of 2-DM occurrence and the treatment. This "Focus" mode provides a novel strategy to evaluate the dose-effect relationship of a TCM. The system and concepts established here may also be applicable for assessing the dose-effect relationships of Western medicines.

[Effect of processed Polygonum multiflorum on mRNA expression level of five subtypes of CYP450 enzymes in rat liver].

To observe the effect of processed Polygonum multiflorum on mRNA expression levels of five subtypes of CYP450 enzymes in rat liver. SD rats were randomly divided into the normal control group, processed P. multiflorum high dose and low dose groups (5.40 g•kg⁻¹ and 1.08 g•kg⁻¹). The rats in administration groups were continuously given with processed P. mutiflorum for 7 days by ig administration, and the rats in normal control group were given with the same volume of distilled water. After successive administration of 7 days, the serum biochemical indications were detected, and Real-time quantitative PCR technology was used to detect the mRNA expression levels of five subtypes of CYP450 enzymes in rat liver. Experimental results showed that AST was decreased significantly in both low and high dose groups. ALT was significantly decreased in low dose group and significantly increased in high dose group. The mRNA expression levels of five subtypes of CYP450 enzymes in rat liver were decreased in high dose and low dose groups in a dose-dependent manner. Especially the high dose processed P. multiflorum could significantly inhibit CYP1A2 and CYP2E1 mRNA expression levels in rats. The study showed that high dose P. multiflorum water extract had hepatotoxicity, and the degree of liver damage was increased with the increase of dose. It shall be noted that 5.40 g•kg⁻¹ water extract of P. multiflorum could significantly inhibit CYP1A2 and CYP2E1 mRNA expression levels in the liver of rats.

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents.

Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.

Serum Metabolomic Characterization of Liver Fibrosis in Rats and Anti-Fibrotic Effects of Yin-Chen-Hao-Tang.

Yin-Chen-Hao-Tang (YCHT) is a famous Chinese medicine formula which has long been used in clinical practice for treating various liver diseases, such as liver fibrosis. However, to date, the mechanism for its anti-fibrotic effects remains unclear. In this paper, an ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS)-based metabolomic study was performed to characterize dimethylnitrosamine (DMN)-induced liver fibrosis in rats and evaluate the therapeutic effects of YCHT. Partial least squares-discriminant analysis (PLS-DA) showed that the model group was well separated from the control group, whereas the YCHT-treated group exhibited a tendency to restore to the controls. Seven significantly changed fibrosis-related metabolites, including unsaturated fatty acids and lysophosphatidylcholines (Lyso-PCs), were identified. Moreover, statistical analysis demonstrated that YCHT treatment could reverse the levels of most metabolites close to the normal levels. These results, along with histological and biochemical examinations, indicate that YCHT has anti-fibrotic effects, which may be due to the suppression of oxidative stress and resulting lipid peroxidation involved in hepatic fibrogenesis. This study offers new opportunities to improve our understanding of liver fibrosis and the anti-fibrotic mechanisms of YCHT.

Comparative proteomic analysis using 2DE-LC-MS/MS reveals the mechanism of Fuzhuan brick tea extract against hepatic fat accumulation in rats with nonalcoholic fatty liver disease.

Fuzhuan brick tea has received increasing attention in recent years owing to its benefits for nonalcoholic fatty liver disease (NAFLD) and associated metabolic syndrome. For exploring the ameliorative mechanism, the liver proteomes from three groups of rats fed either a normal control diet (NCD), a high fat diet (HFD), or a HFD supplemented with high-dose Fuzhuan brick tea extract (FTE) (HFD + HFTE) were comprehensively compared by quantitative proteomics using 2DE-LC-MS/MS. This is the first study of the effects of tea aqueous extract on the liver proteome of rats suffering from metabolic syndrome. The results showed that 57 proteins displayed more than 1.5-fold differences in at least one of two comparisons of HFD versus NCD and HFD versus HFD + HFTE due to HFD feeding and FTE treatment, respectively. Of them, over 75% of proteins exhibited a similar tendency of expression in the two comparisons, meaning FTE was able to correct HFD effects on rat livers. By function analyses, an extensive list of proteins was involved in sugar and lipid metabolism. Compared with HFD-fed rats, the reduced lipogenesis and enhanced ß-oxidation, tricarboxylic acid cycle and respiratory chain in HFD + HFTE-fed rats, which mainly contributed to ameliorate hepatic fat accumulation and associated NAFLD. Additionally, some putative drug targets were also revealed such as COX2, PGAM1, ACACB, FAS, and ECHS1.

Identification of NF-κB inhibitors in Qishenyiqi dropping pills for myocardial infarction treatment based on bioactivity-integrated UPLC-Q/TOF MS.

Qishenyiqi dropping pills (QSYQ) are a type of standardized cardiovascular multiherb medicine for the treatment of myocardial infarction (MI). Knowledge concerning the systemic identification of nuclear factor-kappa B (NF-κB) inhibitors of QSYQ is generally lacking. Therefore, it is necessary to establish an effective method to screen the bioactive components of NF-κB inhibition. In the present study, a rat model of coronary artery ligation was used to assess the cardioprotective effects of QSYQ. The electrocardiograms, histopathology of heart tissues and serum biochemical indicators, such as brain natriuretic peptide, cardiac troponin I and inflammatory cytokines, were measured. Subsequently, ultra-performance liquid chromatography quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF MS) combined with the NF-κB luciferase reporter assay system was applied to screen the potential anti-inflammatory compounds in QSYQ. The results revealed that the administration of QSYQ could improve heart function, ameliorate neutrophil infiltration and diminish the levels of inflammatory cytokines in MI rats. Furthermore, 22 compounds were determined to be potential NF-κB inhibitors. In conclusion, NF-κB inactivation and cytokine suppression might be the main mechanisms of QSYQ for MI treatment. The method of UPLC-Q/TOF MS combined with a bioactive human cell functional evaluation system was proved to be a simple and effective strategy for screening bioactive compounds in traditional Chinese medicine prescriptions.

Metabolites software-assisted flavonoid hunting in plants using ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry.

Plant secondary metabolism drives the generation of metabolites used for host plant resistance, as biopesticides and botanicals, even for the discovery of new therapeutics for human diseases. Flavonoids are one of the largest and most studied classes of specialized plant metabolites. To quickly identify the potential bioactive flavonoids in herbs, a metabolites software-assisted flavonoid hunting approach was developed, which mainly included three steps: firstly, utilizing commercial metabolite software, a flavonoids database was established based on the biosynthetic pathways; secondly, mass spectral data of components in herbs were acquired by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS); and finally, the acquired LC-MS data were imported into the database and the compounds in the herbs were automatically identified by comparison of their mass spectra with the theoretical values. As a case study, the flavonoids in Smilax glabra were profiled using this approach. As a result, 104 flavonoids including 27 potential new compounds were identified. To our knowledge, this is the first report on profiling the components in the plants utilizing the plant metabolic principles with the assistance of metabolites software. This approach can be extended to the analysis of flavonoids in other plants.

[The mechanism of rosiglitazone compound based on network pharmacology].

Applications of network pharmacology are increasingly widespread and methods abound in the field of drug development and pharmacological research. In this study, we choose rosiglitazone compound as the object to predict the targets and to discuss the mechanism based on three kinds of prediction methods of network pharmacology. Comparison of the prediction result has identified that the three kinds of prediction methods had their own characteristics: targets and pathways predicted were not in accordance with each other. However, the calcium signaling pathway could be predicted in the three kinds of methods, which associated with diabetes and cognitive impairment caused by diabetes by bioinformatics analysis. The above conclusion indicates that the calcium signaling pathway is important in signal pathway regulation of rosiglitazone compound, which provides a clue to further explain the mechanism of the compound and also provides a reference for the selection and application of methods of network pharmacology in the actual research.

Oil-water biphasic parallel flow for the precise patterning of metals and cells.

Fluidic patterning is a convenient and versatile tool for the patterning of materials, cells and microstructures on surface and in microchannels. However, its performance is usually limited by transverse diffusion between fluid streams. It would blur the boundary and deteriorate the precision of patterns. In this paper, we adopted geometric confinement to generate biphasic parallel flow that is constituted of oil and water. Since there is minimum transverse diffusion in biphasic parallel flow, the performance of fluid patterning is expected to be improved. The results show that the metal (Silver and Chromium) patterns have distinct boundary and well-controlled geometry in comparison with that by conventional laminar flow patterning. Furthermore, the high biocompatibility of oil phase (perfluorodecalin, PFD) enables the precise patterning of viable bacteria inside microchannels. Our work demonstrated a new route of using biphasic parallel flow to patterning, which would serve wide applications in prototyping and research settings.

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry.

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.

Evidence for the involvement of JAK/STAT/SOCS pathway in the mechanism of Tangshen formula-treated diabetic nephropathy.

Diabetic nephropathy is one of the most significant microvascular complications associated with diabetes. Until now, there is no effective treatment and the gene mechanism of diabetic nephropathy is still unclear. Tangshen formula is a traditional Chinese medicine, and has been shown to have good clinical efficacy in diabetic nephropathy treatment. The objective of this study was to investigate the changes of gene expression profiling and explore the molecular mechanism using a db/db mice model treated by Tangshen formula. After administration for 12 weeks, a microarray was applied to detect the gene expression of db/db mice kidney tissues. Quantitative real-time PCR was used to confirm the differential gene expression and carry out a JAK/STAT/SOCS signaling pathway study. Treatment with Tangshen formula reduced the levels of serum glucose and urinary albumin in db/db mice, and the effects of Tangshen formula on db/db mice were significantly different from the positive control (Losartan potassium tablets) on microarray data. It also showed that the JAK/STAT/SOCS signaling pathway played an important role in the treatment process. The expressions of JAK1, JAK2, and STAT3 were upregulated, and STAT4 was downregulated in Tangshen formula-treated db/db mice. SOCS1, 3, and 7 were all activated, while negative feedback regulated other related genes in the JAK/STAT/SOCS pathway. Our study suggested that Tangshen formula has beneficial effects on diabetic nephropathy treatment via regulating the JAK/STAT/SOCS signaling pathway. This study will help to provide evidence-based recommendations for Tangshen formula clinical treatment.

Absorption, metabolism and effect of compatibility on absorption of qishenyiqi dropping pill.

Qishenyiqi dropping pill (QSYQ), is a traditional Chinese medicine (TCM) prescription for treating heart diseases in China. Knowledge concerning the systemic identification of active compounds and metabolic components of QSYQ is generally lacking. Therefore, it is essential to develop a valid method for the analysis of active compounds of the combined prescription and determination of interactions among the herbs. The absorbable compounds and metabolites of QSYQ were profiled using computational chemistry prediction, an improved everted gut sac in vitro experiment, the Caco-2 cell monolayer in vitro test, a rat in vivo experiment and ultra-performance liquid chromatography/diode array detection/quadrupole-time of flight mass spectrum (UPLC/DAD/Q-TOF MS). In total, 42 prototype compounds were recognized as absorbable compounds, and eight metabolites were identified by UPLC/DAD/Q-TOF MS. The absorption rates of phenolic acids and saponins were significantly improved and the absorption of isoflavone was inhibited after compatibility. The volatile oil component had an improved effect on the absorption of other compounds, while its own absorption was inhibited. In conclusion, the present study established a rapid and effective strategy for demonstrating the absorption and metabolism of QSYQ and revealing the compatible relationship among herbs. This investigation can provide a reference for the compatibility of prescriptions and the modernization of TCM.

Identification of nuclear factor-κB inhibitors and ß2 adrenergic receptor agonists in Chinese medicinal preparation Fuzilizhong pills using UPLC with quadrupole time-of-flight MS.

INTRODUCTION: Fuzilizhong Pills (FZLZ), a modified form of a famous traditional Chinese medicine (TCM) Lizhong Wan in Treatise on Febrile Diseases and consisting of Panax ginseng C.A.Mey. (Ren Shen), Aconitum carmichaelii Debx. (Fu Zi, Zhi), Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. (Gan Cao), Atractylodes macrocephala Koidz. (Bai Zhu) and Zingiber offcinale Rosc. (Gan Jiang), show strong clinical therapeutic effects for dyspnea and pulmonary oedema. However, the bioactive compounds are still unclear. In this study, FZLZ was analysed using a rapid detection method to identify its anti-inflammatory and spasmolytic constituents. OBJECTIVE: To develop a simple screening method to detect the anti-inflammatory and spasmolytic constituents of FZLZ. METHODS: Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and ß2 -adrenergic receptor) luciferase reporter assay systems was employed. RESULTS: Two ß2 -adrenergic receptor agonists (salsolinol and higeramine) and three terpenoidal analogues of NF-κB inhibitors such as ginsenosides derivatives, triperpenoids derivatives and diester-diterpenoid aconitum alkaloid derivatives were characterised. Mesaconitine, flaconitine, ginsenosides Rb2, Rf, Rg2, F1 and Ro were considered to be new NF-κB inhibitors. Furthermore, IL-8 detection by enzyme linked immunosorbent assay confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. CONCLUSION: Compared with conventional fingerprints, activity-integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical composition of plant materials. This strategy clearly demonstrated that dual bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.

[Study on inhibitory effects and mechanism of lipophilic components in Salvia miltiorrhiza on angiogenesis in vitro].

In this study, the human umbilical vein endothelial cell model was used to study the regulating effect of lipophilic components in Salvia miltiorrhiza on angiogenesis, and explore its possible mechanism. The cell model was established to determine the effect of lipophilic components in S. miltiorrhiza on the proliferative activity and migration capacity of endothelial cells. Then the realtime fluorescence quantification PCR technology was applied to detect the changes in the gene expressions of angiogenesis-related cytokines VEGF-A, VEGF-C and MMP-9. The results showed that 5 mg x L(-1) lipophilic components in S. miltiorrhiza could inhibit the proliferation and migration of endothelial cells, and reduce the expression of VEGF-A and MMP-9 genes. It indicated that lipophilic components in S. miltiorrhiza may inhibit the proliferation and migration of endothelial cells by inhibiting the expression of VEGF-A and MMP-9 genes, so as to show the inhibitory effect on angiogenesis.

Arctigenin, a potential anti-arrhythmic agent, inhibits aconitine-induced arrhythmia by regulating multi-ion channels.

BACKGROUND/AIMS: Arctigenin possesses biological activities, but its underlying mechanisms at the cellular and ion channel levels are not completely understood. Therefore, the present study was designed to identify the anti-arrhythmia effect of arctigenin in vivo, as well as its cellular targets and mechanisms. METHODS: A rat arrhythmia model was established via continuous aconitine infusion, and the onset times of ventricular premature contraction, ventricular tachycardia and death were recorded. The Action Potential Duration (APD), sodium current (I(Na)), L-type calcium current (I(Ca, L)) and transient outward potassium current (I(to)) were measured and analysed using a patch-clamp recording technique in normal rat cardiomyocytes and myocytes of arrhythmia aconitine-induced by. RESULTS: Arctigenin significantly delayed the arrhythmia onset in the aconitine-induced rat model. The 50% and 90% repolarisations (APD50 and APD90) were shortened by 100 µM arctigenin; the arctigenin dose also inhibited the prolongation of APD50 and APD90 caused by 1 µM aconitine. Arctigenin inhibited I(Na) and I(Ca,L) and attenuated the aconitine-increased I(Na) and I(Ca,L) by accelerating the activation process and delaying the inactivation process. Arctigenin enhanced Ito by facilitating the activation process and delaying the inactivation process, and recoverd the decreased Ito induced by aconitine. CONCLUSIONS: Arctigenin has displayed anti-arrhythmia effects, both in vivo and in vitro. In the context of electrophysiology, I(Na), I(Ca, L), and I(to) may be multiple targets of arctigenin, leading to its antiarrhythmic effect.

Proteomic analysis of the inhibitory effect of epigallocatechin gallate on lipid accumulation in human HepG2 cells.

BACKGROUND: (-)-Epigallocatechin-3-gallate (EGCG), the most abundant catechin found in green tea, effectively reduces body weight and tissue and blood lipid accumulation. To explore the mechanism by which EGCG inhibits cellular lipid accumulation in free fatty acid (FFA) induced HepG2 cell culture, we investigated the proteome change of FFA-induced HepG2 cells exposed to EGCG using two-dimensional gel electrophoresis and mass spectrometry. RESULTS: In this study, 36 protein spots showed a significant change in intensity by more than 1.5-fold from the control group to the FFA group and from the FFA group to the FFA + EGCG group. Among them, 24 spots were excised from gels and identified by LC-MS/MS. In total, 18 proteins were successfully identified. All identified proteins were involved in lipid metabolism, glycometabolism, antioxidant defense, respiration, cytoskeleton organization, signal transduction, DNA repair, mRNA processing, iron storage, or were chaperone proteins. This indicated that these physiological processes may play roles in the mechanism of inhibition of lipid accumulation by EGCG in FFA-induced HepG2 cells. Western blotting analysis was used to verify the expression levels of differentially expressed proteins, which agree with the proteomic results. CONCLUSIONS: From the proteomic analysis, we hypothesized that EGCG reduced cellular lipid accumulation in FFA-induced HepG2 cells through the activation of AMP-activated protein kinase (AMPK) resulting from the generation of reactive oxygen species (ROS). The induction of ROS may be a result of EGCG regulation of the antioxidant defense system. Activation of AMPK shifted some FFA toward oxidation, away from lipid and triglyceride storage, and suppressed hepatic gluconeogenesis. The findings of this study improve our understanding of the molecular mechanisms of inhibition of lipid accumulation by EGCG in HepG2 cells.

Preparation of functionalized alkynyl magnetic microspheres for the selective enrichment of cell glycoproteins based on click chemistry.

Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25-6.31 µm in size and 48.53-73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.

Bioactivity-integrated ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the identification of nuclear factor-κB inhibitors and ß2 adrenergic receptor agonists in Chinese medicinal preparation Chuanbeipipa dropping pills.

A simple and dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with dual-bioactive [nuclear factor-κB (NF-κB) and ß2 -adrenergic receptor] luciferase reporter assay systems was developed to rapidly characterize the chemical structure of various bioactive compounds of TCM preparations. Chuanbeipipa dropping pills, a traditional Chinese medicine preparation used for the clinical therapy of chronic obstructive lung disease and cough caused by bronchial catarrh, was analyzed with this method. Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and ß2 -adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time-of-flight mass spectrometry data. One ß2-adrenergic receptor agonist (ephedrine) and two structural types of NF-κB inhibitors (platycosides derivatives and ursolic acid derivatives) were characterized. Platycodin D3 and E were considered new NF-κB inhibitors. Further cytokine and chemokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. Compared with conventional fingerprints, activity-integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that multiple bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential multi-target lead compounds in complex herbal medicines.