A global profiling strategy for identification of the total constituents in Chinese herbal medicine based on online comprehensive two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry combined with intelligentized chemical classification guidance.

A comprehensive strategy for effective identification of total constituents in Chinese patent medicine has been advanced applying full scan-preferred parent ions capture-static and active exclusion (FS-PIC-SAE) acquisition coupled with intelligent deep-learning supported mass defect filter (MDF) process, with Naoxintong capsule (NXT) as a case. Online comprehensive two-dimensional liquid chromatography (2DLC) coupled with Q-TOF-MS/MS system was established for obtaining the excellent separation and detection performance of total components, which could exhibit excellent peak capacity with 1052 and orthogonality with 0.69. In addition, a total of 901 unknown compounds could be classified into nine chemical classes rapidly and effectively, based on the intelligent deep-learning algorithm supported MDF model with 96.4% accuracy. Consequently, 276 compounds were successfully identified from NXT, especially including 44 flavonoids, 27 phenolic acids, 25 fatty acids, 17 saponins, 21 phthalocyanines, 20 triterpenes, 10 monoterpenes, 13 diterpenoid ketones, 14 amino acids, and others. It is concluded that the proposed program is an effective and practical strategy enabling the in-depth chemical profiling of complex herbal and biological samples.

Therapeutic effects of columbianadin from Angelicae Pubescentis radix on the progression of collagen-induced rheumatoid arthritis by regulating inflammation and oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was remained unclear. Columbianadin (CBN), one of the main bioactive compounds of APR, has many pharmacological effects including anti-inflammatory and immunosuppression. However, there are few reports on therapeutic effect of CBN on RA. AIM OF THE STUDY: A comprehensive strategy via incorporating pharmacodynamics, microbiomics, metabolomics, and multiple molecular biological methods was adopted to evaluate the therapeutic effects of CBN on collagen-induced arthritis (CIA) mice and explore the potential mechanisms. MATERIALS AND METHODS: A variety of pharmacodynamic methods were used to evaluate the therapeutic effect of CBN on CIA mice. The microbial and metabolic characteristics of CBN anti-RA were obtained by metabolomics and 16S rRNA sequencing technology. The potential mechanism of CBN anti-RA was predicted through bioinformatics network analysis, and verified by a variety of molecular biology methods. RESULTS: CBN can effectively improved symptoms of rheumatoid arthritis in CIA mice, including paw swelling and arthritic scores. The inflammatory and oxidative stress were effectively regulated by the treatment of CBN. The fecal microbial communities and serum and urine metabolic compositions were significantly altered in CIA mice, CBN can ameliorate the CIA-associated gut microbiota dysbiosis, and regulate the disturbance of serum and urine metabolome. The acute toxicity test showed that the LD50 of CBN was greater than 2000 mg kg-1. CONCLUSIONS: CBN exert anti-RA effects from four perspectives: inhibiting inflammatory response, regulating oxidative stress, and improving changes in gut microbiota and metabolites. The JAK1/STAT3, NF-κB and Keap1/Nrf2 pathway may be important mechanism for CBN's inflammatory response and oxidative stress activity. CBN could be considered as a potential anti-RA drug for further study.

A Non-Immunized and BSA-Template Aggregation-Induced Emission Sensor for Noninvasive Detection of Cystatin C in the Clinical Diagnosis of Diabetes Nephropathy.

Diabetes nephropathy (DN) is one of the main causes of death in patients with diabetes. Cystatin C (Cys C) is a reliable indicator of glomerular filtration function. Therefore, it is urgent and meaningful to obtain early warning of DN by noninvasive measurement of Cys C. In this investigation, a novel fluorescence sensor (BSA-AIEgen sensor) was synthesized by cross-linking the aggregation-induced emission (AIE) characteristics of 2-(4-bromophenyl)-3-(4-(4-(diphenylamino) styryl) phenyl) fumaronitrile (TPABDFN) and bovine serum albumin (BSA), which exhibited the "On" state owing to the restriction of the intramolecular motions (RIM) phenomenon of TPABDFN. Intriguingly, a decrease in fluorescence of BSA-AIEgen sensors could be found owing to BSA on the surface of BSA-AIEgen sensor hydrolyzed by papain, but a reverse phenomenon emerged with the increase of Cys C content as the inhibitor of papain. Hence, Cys C was successfully detected by employing the fluorescent differential display and the linear range was from 12.5 ng/mL to 800 ng/mL (R2 = 0.994) with the limit of detection (LOD) of 7.10 ng/mL (S/N = 3). Further, the developed BSA-AIEgen sensor successfully differentiates patients with diabetes nephropathy from volunteers with the advantages of high specificity, low cost, and simple operation. Accordingly, it is expected to become a non-immunized method to monitor Cys C for the early warning, noninvasive diagnosis, and drug efficacy evaluation of diabetes nephropathy.

Simultaneous extraction and determination of alkaloids and organic acids in Uncariae Ramulas Cum Unicis by vortex-assisted matrix solid phase dispersion extraction coupled with UHPLC-MS/MS.

A simple and efficient vortex-assisted matrix solid phase dispersion with a ultra-high-performance liquid chromatography-triple quadrupole mass spectrometer (VA-MSPD-UHPLC-MS/MS) was applied for simultaneous extraction and determination of seven alkaloids and three organic acids from Uncariae Ramulas Cum Unicis. The optimal extraction conditions of the target components were obtained by Box-Behnken design (BBD) combined with response surface methodology (RSM). The results of the method validation showed that this analytical method displayed good linearity with a correlation coefficient (r) no lower than 0.9990. The recoveries of ten active ingredients from Uncariae Ramulas Cum Unicis ranged from 95.9% to 103% (RSD ≤ 2.77%). The RSDs of intra-day and inter-day precisions were all below 2.97%. The present method exhibited not only lower solvent and sample usage, but also shorter sample processing and analysis time. Consequently, the developed VA-MSPD-UHPLC-MS/MS method could be successfully and effectively used for the extraction and analysis of ten active components from Uncariae Ramulas Cum Unicis.

An efficient method for qualitation and quantitation of multi-components of the herbal medicine Qingjin Yiqi Granules.

Qingjin Yiqi Granules (QJYQ) is a Traditional Chinese Medicines (TCMs) prescription for the patients with post-COVID-19 condition. It is essential to carry out the quality evaluation of QJYQ. A comprehensive investigation was conducted by establishing deep-learning assisted mass defect filter (deep-learning MDF) mode for qualitative analysis, ultra-high performance liquid chromatography and scheduled multiple reaction monitoring method (UHPLC-sMRM) for precise quantitation to evaluate the quality of QJYQ. Firstly, a deep-learning MDF was used to classify and characterize the whole phytochemical components of QJYQ based on the mass spectrum (MS) data of ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Secondly, the highly sensitive UHPLC-sMRM data-acquisition method was established to quantify the multi-ingredients of QJYQ. Totally, nine major types of phytochemical compounds in QJYQ were intelligently classified and 163 phytochemicals were initially identified. Furthermore, fifty components were rapidly quantified. The comprehensive evaluation strategy established in this study would provide an effective tool for accurately evaluating the quality of QJYQ as a whole.

Mapping Multi-factor-mediated Chromatin Interactions to Assess Dysregulation of Lung Cancer-related Genes.

Studies on the lung cancer genome are indispensable for developing a cure for lung cancer. Whole-genome resequencing, genome-wide association studies, and transcriptome sequencing have greatly improved our understanding of the cancer genome. However, dysregulation of long-range chromatin interactions in lung cancer remains poorly described. To better understand the three-dimensional (3D) genomic interaction features of the lung cancer genome, we used the A549 cell line as a model system and generated high-resolution chromatin interactions associated with RNA polymerase II (RNAPII), CCCTC-binding factor (CTCF), enhancer of zeste homolog 2 (EZH2), and histone 3 lysine 27 trimethylation (H3K27me3) using long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Analysis showed that EZH2/H3K27me3-mediated interactions further repressed target genes, either through loops or domains, and their distributions along the genome were distinct from and complementary to those associated with RNAPII. Cancer-related genes were highly enriched with chromatin interactions, and chromatin interactions specific to the A549 cell line were associated with oncogenes and tumor suppressor genes, such as additional repressive interactions on FOXO4 and promoter-promoter interactions between NF1 and RNF135. Knockout of an anchor associated with chromatin interactions reversed the dysregulation of cancer-related genes, suggesting that chromatin interactions are essential for proper expression of lung cancer-related genes. These findings demonstrate the 3D landscape and gene regulatory relationships of the lung cancer genome.

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice.

BACKGROUND: The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear. RESULTS: Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete "transcriptional factory" foci in the evening, linking chromatin architecture to coordinated transcription outputs. CONCLUSION: Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Immunosuppressive effect of Columbianadin on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced dendritic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was unclear. Columbianadin (CBN) was one of the main bioactive compounds of APR, and has many pharmacological effects. But the immunosuppressive effect of CBN on DCs and the potential mechanism needed to be explored. AIM OF THE STUDY: The study was aimed to clarify the immunosuppressive effect of CBN on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced DCs. MATERIALS AND METHODS: Bone marrow-derived DCs were obtained and cultured from C57BL/6 mice in accordance with protocol. The phenotypic study (CD11c, CD40, CD80, CD86 and MHC Ⅱ) were measured by flow cytometry. FITC-dextran were uptaked by DCs and the change of endocytosis activity were mediated by acquired mannose receptor. Transwell chambers were used to detect the migration ability of DCs. Mixed leukocyte reaction (MLR) assay was used to detect the allostimulatory ability of CBN on TNF-α stimulated DCs. The secretion of cytokines and chemokines was measured by ELISA Kit. TLRs gene and MAPKs/NF-κB protein expression were checked by qRT-PCR and Western blot. RESULTS: CBN inhibited the maturation of TNF-α-induced DCs while maintaining phagocytosis capabilities. Additionally, CBN inhibited the migration of TNF-α stimulated DCs, which related to reduce the production of chemokines (MCP-1, MIP-1α). Notably, CBN could suppress the proliferation of CD4+T cells by inhibiting DCs maturation, and decrease the proinflammatory cytokines IL-6 production. Furthermore, CBN inhibited mRNA expression of TLR2, TLR7 and TLR9 in TNF-α-activated DCs. Meanwhile, the phosphorylation of p38, JNK1/2 and NF-κB protein were significantly inhibited in CBN treated DCs. CONCLUSIONS: These findings provided novel insights into the pharmacological activity of CBN. They also indicated that inhibition DCs maturation owning to the immunosuppressive effect of CBN. CBN was expected as a potential immunosuppressant and TLRs/MAPKs/NF-κB pathway may be an important mechanism for CBN's immunosuppressive activity.

Traditional herbal medicine: Therapeutic potential in rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease influenced by diverse endogenous and exogenous factors. It is characterized by cartilage and bone destruction. The current conventional allopathic therapy is expensive and carries adverse side effects. Recently, there were some ethnopharmacological studies on RA including anti-RA effects and therapeutic targets of distinct dosage forms of traditional herbal medicines (THMs). AIM OF THE REVIEW: This review provides a brief overview of the current understanding of the potential pharmacological mechanisms of THMs (active constituents, extracts and prescriptions) in RA. This study is intended to provide comprehensive information and reference for exploring new therapeutic strategies of THMs in the RA treatment. MATERIALS AND METHODS: This review captured scientific literatures invivo and vitro experiments on effects of anti-RA THMs published between 2016 and 2021 from journals and electronic databases (e.g. PubMed, Elsevier, Science Direct, Web of Science and Google Scholar). Relevant literatures were searched and analyzed by using keywords such as 'rheumatoid arthritis AND traditional herbal medicines', 'rheumatoid arthritis AND immune cells', 'rheumatoid arthritis AND inflammation', 'rheumatoid arthritis AND miRNA', 'rheumatoid arthritis AND Angiogenesis', 'rheumatoid arthritis AND oxidative stress', 'rheumatoid arthritis AND osteoclasts', 'rheumatoid arthritis AND CIA model', 'rheumatoid arthritis AND AA model' AND 'rheumatoid arthritis herbal prescription'. RESULTS: Experiments in vitro and in vivo jointly demonstrated the potential of THMs in the RA treatment. There are plentiful therapeutic targets in RA. THMs and active ingredients could alleviate RA symptoms through different therapeutic targets, such as immunoregulation, inflammation, fibroblast-like synoviocytes (FLSs), microRNAs (miRNAs), angiogenesis, oxidative stress, osteoclasts and multiple targets interaction. Anti-RA THMs, active ingredients and prescriptions through corresponding therapeutic targets were summarized and classified. CONCLUSIONS: Flavonoids, phenolic acids, alkaloids and triterpenes of THMs are identified as the main components to ameliorate RA. Regulation of different and multiple related therapeutic targets by THMs and their active ingredients were associated with greater therapeutic benefits, among which inflammation is the main therapeutic target. Nonetheless, further studies are required to unravel the complexities and in-depth mechanisms of THMs in alleviating RA.

ChIAMM: A Mixture Model for Statistical Analysis of Long-Range Chromatin Interactions From ChIA-PET Experiments.

Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is an important experimental method for detecting specific protein-mediated chromatin loops genome-wide at high resolution. Here, we proposed a new statistical approach with a mixture model, chromatin interaction analysis using mixture model (ChIAMM), to detect significant chromatin interactions from ChIA-PET data. The statistical model is cast into a Bayesian framework to consider more systematic biases: the genomic distance, local enrichment, mappability, and GC content. Using different ChIA-PET datasets, we evaluated the performance of ChIAMM and compared it with the existing methods, including ChIA-PET Tool, ChiaSig, Mango, ChIA-PET2, and ChIAPoP. The result showed that the new approach performed better than most top existing methods in detecting significant chromatin interactions in ChIA-PET experiments.

Chromatin loops associated with active genes and heterochromatin shape rice genome architecture for transcriptional regulation.

Insight into high-resolution three-dimensional genome organization and its effect on transcription remains largely elusive in plants. Here, using a long-read ChIA-PET approach, we map H3K4me3- and RNA polymerase II (RNAPII)-associated promoter-promoter interactions and H3K9me2-marked heterochromatin interactions at nucleotide/gene resolution in rice. The chromatin architecture is separated into different independent spatial interacting modules with distinct transcriptional potential and covers approximately 82% of the genome. Compared to inactive modules, active modules possess the majority of active loop genes with higher density and contribute to most of the transcriptional activity in rice. In addition, promoter-promoter interacting genes tend to be transcribed cooperatively. In contrast, the heterochromatin-mediated loops form relative stable structure domains in chromatin configuration. Furthermore, we examine the impact of genetic variation on chromatin interactions and transcription and identify a spatial correlation between the genetic regulation of eQTLs and e-traits. Thus, our results reveal hierarchical and modular 3D genome architecture for transcriptional regulation in rice.

Chromatin interaction maps reveal genetic regulation for quantitative traits in maize.

Chromatin loops connect regulatory elements to their target genes. They serve as bridges between transcriptional regulation and phenotypic variation in mammals. However, spatial organization of regulatory elements and its impact on gene expression in plants remain unclear. Here, we characterize epigenetic features of active promoter proximal regions and candidate distal regulatory elements to construct high-resolution chromatin interaction maps for maize via long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The maps indicate that chromatin loops are formed between regulatory elements, and that gene pairs between promoter proximal regions tend to be co-expressed. The maps also demonstrated the topological basis of quantitative trait loci which influence gene expression and phenotype. Many promoter proximal regions are involved in chromatin loops with distal regulatory elements, which regulate important agronomic traits. Collectively, these maps provide a high-resolution view of 3D maize genome architecture, and its role in gene expression and phenotypic variation.

The Epstein-Barr Virus Regulome in Lymphoblastoid Cells.

Epstein-Barr virus (EBV) transforms B cells to continuously proliferating lymphoblastoid cell lines (LCLs), which represent an experimental model for EBV-associated cancers. EBV nuclear antigens (EBNAs) and LMP1 are EBV transcriptional regulators that are essential for LCL establishment, proliferation, and survival. Starting with the 3D genome organization map of LCL, we constructed a comprehensive EBV regulome encompassing 1,992 viral/cellular genes and enhancers. Approximately 30% of genes essential for LCL growth were linked to EBV enhancers. Deleting EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets included MCL1, IRF4, and EBF. MYC ESE looping to the transcriptional stat site of MYC was dependent on EBNAs. Deleting MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study provides a comprehensive view of the spatial organization of chromatin during EBV-driven cellular transformation.