There is still a serious challenge of the measurement of critical quality attributes (CQAs) related to clinical efficacy for Chinese materia medica manufacturing. To overcome this challenge, an integrated strategy of biosensor and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was proposed using Tongren niuhuang qingxin pills as a trial. Firstly, an original biosensor was created using a semiconductor chip material high electron mobility transistor (HEMT) as the transducer and the macrophage migration inhibitory factor (MIF) as the identification element. By this MIF-HEMT biosensor, the efficacy on stoke of different components from Tongren niuhuang qingxin pills was measured. It was clear that all three components of Tongren niuhuang qingxin pills had strong therapeutic effects on stroke, especially the section A, the K_D of which reached to 8.722×10^-10 g·mL^-1. Furthermore, MIF-HEMT biosensor integrated UPLC-MS/MS was introduced to identify the efficacy CQAs of different components of Tongren niuhuang qingxin pills. As a result, 19 potential CQAs, such as albiforin, paeoniflorin, and prim-O-glucosylcimifugin, were measured as the efficacy CQAs of Tongren niuhuang qingxin pills on stroke treatment by MIF. These results provided vital measurement techniques and methodological guidance for the CQAs study of Tongren niuhuang qingxin pills intervention in MIF-induced stroke treatment. This also provided an essential guideline for the efficient utilization and quality control measurement of high-quality classical recipes.

Xanthine oxidoreductase (XOR), the key enzyme catalyzing purine to produce uric acid, including two subtypes, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), respectively, in vivo. Usually, XDH and XO can transform to each other. In this study, based on the principle that the subtype XO or XDH uses different electron acceptors, the methods for the measuring the activities of bovine milk XOR (pure enzyme) and its subtypes were established. The optimal concentrations of substrate xanthine (50 μmol·L^-1) and electron acceptor NAD⁺ (50 μmol·L^-1), pH value (7.80) were investigated. The ranges of the XOR, XO, XDH activity which could be determined were 0.97-17.5 U·L^-1, 1-9 U·L^-1, and 66-1 191 mU·L^-1, respectively. Furthermore, the methods for determining the activities of XOR and its subtypes in mouse liver were established. The preparation of liver samples, the optimal concentrations of xanthine (100 μmol·L^-1) and NAD⁺ (100 μmol·L^-1) were researched. And the activity ranges of XOR, XO and XDH in mouse liver which could be determined were 0.67-3.98, 0.19-1.08, and 0.52-3.55 U·gprot^-1, respectively. With the methods above, the effects of classic XOR inhibitor allopurinal (Allo) on XOR, XO and XDH from both milk and mouse liver were determined. All animal experiments have been approved by the Animal Experimental Center, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College (00003346). This study established new methods for the determination of XOR and its subtypes activity in pure enzyme system and in mouse liver, respectively, which were accurate and convenient. It laid the experimental foundation for exploring the different pathophysiological effects of XOR in the body and developing new XOR inhibitors.

Bacterial biofilm widely exists in all kinds of bacteria, and is related to about 80 percent of bacterial infections. It is one of the main reasons for bacterial tolerance and resistance to existing antibiotics. Therefore, there is unmet clinical need for new anti-biofilm drugs. At present, there are three kinds of anti-biofilm agents under research, including biofilm inhibitors, biofilm dispersal agents and biofilm eradication agents. Among them, the biofilm eradication agent is unique, which can not only kill bacteria in biofilm but also eliminate biofilm as a monotherapy. Based on modifications of natural products with antibacterial activity, a variety of compounds with biofilm eradicating activities have been obtained, such as, acyldepsipeptides, pyrrolomycins, halogenated phenazines and halogenated 8-hydroxyquinolines. In this review, we summarize several major biofilm eradication agents above according to their structures and mechanisms.

A quantitative analysis of multi-components by single marker method (QAMS) was established for simultaneous determination of gallic acid, protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin in Cynomorium songaricum Rupr. The analysis was performed on a ChromCore Polae C₁₈ column (250 mm×4.6 mm, 5 μm), with a mobile phase consisting of acetonitrile-0.3% phosphoric acid aqueous solution for gradient elution. The volume flow rate, column temperature and sample injection volume were set at 1.0 mL·min^-1, 25 ℃, and 40 µL, respectively. The relative correction factors of gallic acid and protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin were calculated and the durability was also investigated. The contents of these seven compounds in fourteen batches of Cynomorium songaricum Rupr. from different producing areas or batches were determined by external standard method (ESM) and quantitative analysis of multi-components with a single-marker method (QAMS), respectively. SPSS and Origin Pro software were employed for principal components assay, similarity evaluation and cluster analysis. The specificity, precision, repeatability, stability and linear range (R² > 0.999 0) of the seven components were all good. The average recovery was 96.89%-103.16% and RSD was 0.55%-2.76%. Then gallic acid was chosen as internal reference for calculation the correction factors for the other six components, the average relative correction factors of protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin were 1.141 5, 0.200 5, 0.208 0, 2.361 9, 1.867 7, 0.204 6, respectively. Student's test results showed that there was no significant difference between the data analyzed by ESM and the data obtained from QAMS method. Through data visualization analysis, the contents of gallic acid, protocatechuic acid, catechin and epicatechin in different samples were significantly different, indicating that these four components might be the main quality markers of Cynomorium songaricum Rupr. for gaving more contributes to the principal components. The cluster analysis showed that samples from Xinjiang and samples from Inner Mongolia were clustered in significantly different categories, meaning that the quality of Cynomorium songaricum Rupr. had great relation with producing areas. The method of QAMS established in this study is a simple, economical and practical method with scientific and applicable charactistics for evaluating the quality of Cynomorium songaricum Rupr. efficiently and scientifically.

Asparaginyl endopeptidases (AEPs) in plants belong to the family of cysteine protease that undergo self-activation in the form of zymogen in acidic vacuole and play important physiological roles in maturation of seed storage proteins, protein degradation, programmed cell death and host defense. Bioprocessing enzymes (peptidyl Asx-specific ligases, PALs) that promote the maturation of cyclotides have recently been isolated and identified from several cyclotide-rich plants. PALs derived from AEPs can site-specifically catalyze the formation of asparagine or aspartate peptide bonds. Due to the advantages of relatively traceless peptide bonds and broad substrate spectrum and high catalytic efficiency, they have been playing important roles in the cyclization and modification of peptides and proteins, and are powerful tools for improving the stability of peptide drugs. This review describes the physiological functions of AEPs in plants and summarizes the discoveries, structural characteristics, catalytic mechanism and protein engineering of PALs, as well as the limitation of their applications and future trends. In addition, the applications of PALs in cyclotides biosynthesis and the development of macrocyclic peptides are highlighted, with the aim of providing a new idea for the biocatalytic synthesis of cyclic peptides.

As an important component of nucleosomes on the chromatin of eukaryotic cells, histones play an important role in the development and progression of tumour diseases by regulating epigenetic post-translational modifications such as acetylation and methylation. In addition, development of inhibitors targeting methyltransferase and deacetylase provides novel therapeutic strategies for cancer treatment. Mass spectrometry-based proteomics can reveal the global changes of histone modifications under the action of drugs during disease progression, which in turn provides important support for revealing drug action mechanism, drug resistance mechanism, and investigating novel drug combination strategies. This article focuses on the progress and status of proteomic research on a variety of histone modifying enzyme inhibitors, including methyltransferase inhibitors and histone deacetylase inhibitors, which will help to understand the current and further utilization of proteomics in studying histone modifications.

Attenuated Salmonella typhimurium VNP20009 is a widely used natural oncolytic bacterium, which has great application potential given its unique characteristics, including clinical safety, tumor targeting specificity, and explicit genome sequence. Here, we show that tumor progression can be effectively reduced by intraperitoneal administration with VNP20009 in a mouse model of melanoma (all animal experiments were conducted in accordance with the Animal Ethics Committee of China Pharmaceutical University); co-culture experiment in vitro demonstrated that VNP20009 can induce the polarization of macrophage M1, accompanied by expression of inflammation-related factors; flow cytometry analysis showed that VNP20009 induced the increase of immune cell infiltration in tumor. Further analysis showed that T cells infiltration in tumor-draining lymph node (TDLN) increased, and VNP20009 induced the activation of CD4⁺ T cells and CD8⁺ T cells in tumor. Our results demonstrate that VNP20009 treatment significantly inhibited melanoma tumors by remodeling tumor-associated macrophages to an M1-like phenotype, as well as recruiting and activating cytotoxic T cells, combined with its own antigenic activity to exert anti-tumor immunity.

The morbidity and mortality of gastrointestinal malignancies are the highest in the world. For patients with poor response to conventional chemotherapy, new treatment methods are urgently needed. In recent years, under the background of precision medicine, antibody-drug conjugates (ADCs) with high tumor specificity and potent toxicity have become a hot research spot in the field of biomedicine. However, due to the complex structure and mechanism of ADCs, its pharmacokinetic research is facing great challenges which are the biggest resistance to the development of ADCs at present. In this case, it is of great significance to understand the pharmacokinetic properties of ADCs and make use of it to improve the efficacy of ADCs in the treatment of gastrointestinal malignancies. Based on the basic composition and mechanism of ADCs, this review summarizes the pharmacokinetic properties of ADCs, discusses its recent advances in the treatment of gastrointestinal malignancies, in order to provide more references for follow-up research on ADCs.

α-Conotoxin ArIB[V11L,V16D] is currently the most optimal selective inhibitor of α7 nicotinic acetylcholine receptor (nAChR) known. In order to explore chemical modification methods and enrich its application in targeting nAChR, this study utilized the linker to covalently connect camptothecin and 7-amino-4-methylcoumarin to the [2,4] disulfide bond of ArIB[V11L,V16D]. Therefore, two peptide-drug conjugates (PDCs), ArIB[V11L,V16D]-5 and ArIB[V11L,V16D]-6, and one fluorescent-labeled peptide, ArIB[V11L,V16D]-7 were constructed. Cytotoxicity evaluation showed that the IC₅₀ values against non-small cell lung cancer cell line A549 of the two PDCs were respectively 1.3 and 4.1 times of camptothecin, indicating slight reduction in activity at the cellular level which was related to the linker structure. Fluorescence spectrum scanning revealed that the excitation and emission wavelength of the fluorescent-labeled peptide were 340 nm and 403 nm respectively, and the fluorescence features of 7-amino-4-methylcoumarin as a marker were retained without fluorescence quenching. This modification strategy laid a solid foundation for the further application of α-conotoxin ArIB[V11L,V16D] in PDCs and fluorescent probes.

Autophagy is an important physiological process that can degrade cell components and maintain cell homeostasis, divided into three types including macroautophagy, microautophagy and chaperon-mediated autophagy generally, and macroautophagy is the most common form. Autophagy can affect the progression of a variety of diseases, such as cancer, neurodegenerative diseases, heart-related diseases, and autoimmune diseases, etc. However, autophagy can promote or inhibit diseases in different circumstances because of the dual roles of autophagy. Therefore, targeted regulating autophagy may be a potential treatment plan for diseases in specific stages of disease development. Now, with the development of traditional Chinese medicine (TCM) resources and the deepening of researches on the modern utilization of TCM, many active compounds from TCM have been discovered that can target autophagy to exert pharmacological activity. Most of the natural compounds activate or inhibit autophagy by affecting the classical PI3K/AKT/mTOR autophagy pathway. In addition, some compounds can also affect autophagy through MAPKs signaling pathways such as MEK/ERK, JNK and p38MAPK. These active compounds exert various biological activities by regulating autophagy, including anti-tumor, inhibiting neurodegenerative diseases, protecting cardiomyocytes, and relief of inflammatory response. In this review, we summarized the active compounds in TCM that affect autophagy by targeting different signaling pathways and their mechanisms of regulating autophagy, also introduced the effects of active compounds on diseases after affecting autophagy. Finally, this paper summarized and prospected the development of targeted autophagy for the treatment of diseases by TCM compounds, hoping to provide clues for subsequent exploration and research.