Integrating Anti-Influenza Virus Activity and Chemical Pattern Recognition to Explore the Quality Evaluation Method of Lonicerae Japonicae Flos.

Lonicerae japonicae flos (LJF, Lonicera japonica Thunb.) is adopted as a core herb for preventing and treating influenza. However, the anti-influenza virus components of LJF and the impact of quality-affecting factors on the anti-influenza activity of LJF have not been systematically investigated. In this study, a strategy integrating anti-influenza virus activity, ultrahigh-performance liquid chromatography fingerprint and chemical pattern recognition was proposed for the efficacy and quality evaluation of LJF. As a result, six bioactive compounds were screened out and identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 4,5-Di-O-caffeoylquinic acid, sweroside and secoxyloganin. Based on the bioactive compounds, chemical pattern recognition models of LJF were established by a linear discriminant analysis (LDA). The results of the LDA models and anti-influenza virus activity demonstrated that cultivation pattern significantly affected the anti-influenza effect of LJF and that the neuraminidase inhibition rate of wild LJF was significantly higher than that of cultivated LJF. Moreover, the quality of LJF samples with different processing methods and geographical origins showed no obvious difference. Overall, the proposed strategy in the current study revealed the anti-influenza virus components of LJF and provided a feasible method for thequality evaluation of LJF, which has great importance for assuring the clinical effect against influenza of LJF.

Ginsenoside Rg5 allosterically interacts with P2RY12 and ameliorates deep venous thrombosis by counteracting neutrophil NETosis and inflammatory response.

Background: Deep venous thrombosis (DVT) highly occurs in patients with severe COVID-19 and probably accounted for their high mortality. DVT formation is a time-dependent inflammatory process in which NETosis plays an important role. However, whether ginsenoside Rg5 from species of Panax genus could alleviate DVT and its underlying mechanism has not been elucidated. Methods: The interaction between Rg5 and P2RY12 was studied by molecular docking, molecular dynamics, surface plasmon resonance (SPR), and molecular biology assays. The preventive effect of Rg5 on DVT was evaluated in inferior vena cava stasis-induced mice, and immunocytochemistry, Western blot, and calcium flux assay were performed in neutrophils from bone marrow to explore the mechanism of Rg5 in NETosis via P2RY12. Results: Rg5 allosterically interacted with P2RY12, formed stable complex, and antagonized its activity via residue E188 and R265. Rg5 ameliorated the formation of thrombus in DVT mice; accompanied by decreased release of Interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α in plasma; and suppressed neutrophil infiltration and neutrophil extracellular trap (NET) release. In lipopolysaccharide- and platelet-activating factor-induced neutrophils, Rg5 reduced inflammatory responses via inhibiting the activation of ERK/NF-κB signaling pathway while decreasing cellular Ca2+ concentration, thus reducing the activity and expression of peptidyl arginine deiminase 4 to prevent NETosis. The inhibitory effect on neutrophil activity was dependent on P2RY12. Conclusions: Rg5 could attenuate experimental DVT by counteracting NETosis and inflammatory response in neutrophils via P2RY12, which may pave the road for its clinical application in the prevention of DVT-related disorders.

Discrimination of Lonicerae Japonicae Flos according to species, growth mode, processing method, and geographical origin with ultra-high performance liquid chromatography analysis and chemical pattern recognition.

Lonicerae japonicae flos (LJF, Lonicera japonica Thunb.) is often confused and/or adulterated with Lonicerae flos (LF, Lonicera macrantha (D.Don) Spreng.). Ecological conditions and processing methods strongly influenced the safety and efficacy of LJF. For the strict quality control of LJF, a rapid and feasible strategy for identification and classification of LJF by species, growth mode, processing method and geographical origin, based on chromatographic profiles and pattern recognition analysis, in 119 batches of Lonicera samples was systematically established. Firstly, comprehensive analysis of the chemical compositions of LJF was achieved using ultra-high performance liquid chromatography (UHPLC). Next, unsupervised principal component analysis showed that the influence of species, growth mode, processing method and geographical origin displayed a decreasing trend. Subsequently, classification models for authentication of LJF samples were established by linear discriminant analysis (LDA) with good classification abilities. Finally, sweroside and secoxyloganin could be considered as markers associated of cultivated and wild LJF, respectively, while 3-O-caffeoylquinic acid and 3,5-Di-O-caffeoylquinic acid could be regarded as markers for LF. Consequently, the findings suggest that UHPLC profiles combined with pattern recognition analysis is precise and feasible strategy for the discrimination and quality control of LJF.

The role of histone methylase and demethylase in antitumor immunity: A new direction for immunotherapy.

Epigenetic modifications may alter the proliferation and differentiation of normal cells, leading to malignant transformation. They can also affect normal stimulation, activation, and abnormal function of immune cells in the tissue microenvironment. Histone methylation, coordinated by histone methylase and histone demethylase to stabilize transcription levels in the promoter area, is one of the most common types of epigenetic alteration, which gained increasing interest. It can modify gene transcription through chromatin structure and affect cell fate, at the transcriptome or protein level. According to recent research, histone methylation modification can regulate tumor and immune cells affecting anti-tumor immune response. Consequently, it is critical to have a thorough grasp of the role of methylation function in cancer treatment. In this review, we discussed recent data on the mechanisms of histone methylation on factors associated with immune resistance of tumor cells and regulation of immune cell function.

Application of UHPLC Fingerprints Combined with Chemical Pattern Recognition Analysis in the Differentiation of Six Rhodiola Species.

Rhodiola, especially Rhodiola crenulate and Rhodiola rosea, is an increasingly widely used traditional medicine or dietary supplement in Asian and western countries. Because of the phytochemical diversity and difference of therapeutic efficacy among Rhodiola species, it is crucial to accurately identify them. In this study, a simple and efficient method of the classification of Rhodiola crenulate, Rhodiola rosea, and their confusable species (Rhodiola serrata, Rhodiola yunnanensis, Rhodiola kirilowii and Rhodiola fastigiate) was established by UHPLC fingerprints combined with chemical pattern recognition analysis. The results showed that similarity analysis and principal component analysis (PCA) could not achieve accurate classification among the six Rhodiola species. Linear discriminant analysis (LDA) combined with stepwise feature selection exhibited effective discrimination. Seven characteristic peaks that are responsible for accurate classification were selected, and their distinguishing ability was successfully verified by partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA), respectively. Finally, the components of these seven characteristic peaks were identified as 1-(2-Hydroxy-2-methylbutanoate) ß-D-glucopyranose, 4-O-glucosyl-p-coumaric acid, salidroside, epigallocatechin, 1,2,3,4,6-pentagalloyglucose, epigallocatechin gallate, and (+)-isolarisiresinol-4'-O-ß-D-glucopyranoside or (+)-isolarisiresinol-4-O-ß-D-glucopyranoside, respectively. The results obtained in our study provided useful information for authenticity identification and classification of Rhodiola species.

Chemical Pattern Recognition for Quality Analysis of Lonicerae Japonicae Flos and Lonicerae Flos Based on Ultra-High Performance Liquid Chromatography and Anti-SARS-CoV2 Main Protease Activity.

Lonicerae japonicae flos (L. japonicae flos, Lonicera japonica Thunb.) is one of the most commonly prescribed botanical drugs in the treatment or prevention of corona virus disease 2019. However, L. japonicae flos is often confused or adulterated with Lonicerae flos (L. flos, Lonicera macrantha (D.Don) Spreng., Shanyinhua in Chinese). The anti-SARS-CoV2 activity and related differentiation method of L. japonicae flos and L. flos have not been documented. In this study, we established a chemical pattern recognition model for quality analysis of L. japonicae flos and L. flos based on ultra-high performance liquid chromatography (UHPLC) and anti-SARS-CoV2 activity. Firstly, chemical data of 59 batches of L. japonicae flos and L. flos were obtained by UHPLC, and partial least squares-discriminant analysis was applied to extract the components that lead to classification. Next, anti-SARS-CoV2 activity was measured and bioactive components were acquired by spectrum-effect relationship analysis. Finally, characteristic components were explored by overlapping feature extracted components and bioactive components. Accordingly, eleven characteristic components were successfully selected, identified, quantified and could be recommended as quality control marker. In addition, chemical pattern recognition model based on these eleven components was established to effectively discriminate L. japonicae flos and L. flos. In sum, the demonstrated strategy provided effective and highly feasible tool for quality assessment of natural products, and offer reference for the quality standard setting.

[Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF].

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.