[Identification of chemical components in Qinggu San reference sample of classial prescription based on UHPLC-Q/Orbitrap HRMS combined with molecular network technology].

The method of ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UHPLC-Q/Orbitrap HRMS)combined with molecular network was developed in this study for rapidly analyzing the chemical components of the Qinggu San reference sample of classical prescription. Firstly, an ACQUITY UPLC BEH Shield RP_(18) column(2.1 mm×100 mm, 1.7 µm)was used, and acetonitrile and 0.1% formic acid were taken as the mobile phases for gradient elution. The flow rate was 0.4 mL·min~(-1), and the column temperature was 30 ℃. Under these conditions, the mass spectrum data were collected in both positive and negative ion modes of the heated electrospray ionization source. Subsequently, the mass spectrum data of the Qinggu San reference sample were uploaded to the Global Natural Products Social Molecular Network(GNPS)platform for calculation and analysis, and a visual molecular network was built with Cytoscape 3.8.2 software. On this basis, the chemical components of the Qinggu San reference sample were identified by fragmentation regularity of standard compounds, retention time, accurate relative molecular weight of HR-MS, characteristic fragment ions information, literature, and databases. Finally, a total of 105 chemical components were identified and speculated in the Qinggu San reference sample, including 19 iridoid glycosides, 23 flavonoids, 15 phenylpropanoids, 11 triterpene saponins, and 37 other components. Meanwhile, two of these components are potential new compounds. The method used in this study not only achieved rapid and accurate identification of chemical components in the Qinggu San reference sample and provided a scie-ntific basis for the study of pharmacological substances and quality control of Qinggu San compound preparations but also provided a refe-rence for the rapid identification of chemical components in traditional Chinese medicine compound preparations.

Developing a High-Umami, Low-Salt Soy Sauce through Accelerated Moromi Fermentation with Corynebacterium and Lactiplantibacillus Strains.

The traditional fermentation process of soy sauce employs a hyperhaline model and has a long fermentation period. A hyperhaline model can improve fermentation speed, but easily leads to the contamination of miscellaneous bacteria and fermentation failure. In this study, after the conventional koji and moromi fermentation, the fermentation broth was pasteurized and diluted, and then inoculated with three selected microorganisms including Corynebacterium glutamicum, Corynebacterium ammoniagenes, and Lactiplantibacillus plantarum for secondary fermentation. During this ten-day fermentation, the pH, free amino acids, organic acids, nucleotide acids, fatty acids, and volatile compounds were analyzed. The fermentation group inoculated with C. glutamicum accumulated the high content of amino acid nitrogen of 0.92 g/100 mL and glutamic acid of 509.4 mg/100 mL. The C. ammoniagenes group and L. plantarum group were rich in nucleotide and organic acid, respectively. The fermentation group inoculated with three microorganisms exhibited the best sensory attributes, showing the potential to develop a suitable fermentation method. The brewing speed of the proposed process in this study was faster than that of the traditional method, and the umami substances could be significantly accumulated in this low-salt fermented model (7% w/v NaCl). This study provides a reference for the low-salt and rapid fermentation of seasoning.

[Health Impacts of Air Pollution in Tianjin].

Ambient air pollution is a dominant determinant of health. The health effects and economic losses due to air pollution are very important for decision-making. Since the implementation of the "Air Pollution Prevention and Control Action Plan" and "blue sky defense war" policies, the air quality of Tianjin has changed significantly. Here, the health effects and economic losses attributable to ambient air pollution in Tianjin from 2013 to 2020 wereestimated. For the particulate matter which has complex components, we assessed the inhalation health risks of heavy metals and polycyclic aromatic hydrocarbons (PAHs) in PM2.5. The variation in the concentration of the main components of PM2.5 was also analyzed. The results showed that improved air quality had positive health benefits. The health benefits from SO2 were the highest among the six air pollutants, and 3786 deaths were avoided in 2020 compared to in 2013 due to lower SO2 concentration. The economic losses caused by air pollutants ranged from several billion to ten billion yuan. Among the six air pollutants, particulate matter and ozone had higher health losses in recent years. The health risks of heavy metals and PAHs in PM2.5 showed a decreasing trend. However, Cr(Ⅵ), As, Cd, and Ni in PM2.5in the winter of 2020 still had respiratorysystem carcinogenic risk, whereas there was no health risk of PAHs in PM2.5in 2019-2020. The concentrations of main components of PM2.5 have decreased significantly. In the future, the reduction of health loss caused by air pollution depends on synergy governance of particulate matter and ozone and further research on health effects.

Circular RNA circFBXO7 attenuates non-small cell lung cancer tumorigenesis by sponging miR-296-3p to facilitate KLF15-mediated transcriptional activation of CDKN1A.

BACKGROUND: Accumulating evidence indicates that circular RNAs (circRNAs) play important roles in various cancers. Hsa_circ_0008832 (circFBXO7) is a circRNA generated from the second exon of the human F-box only protein 7 (FBXO7). Mouse circFbxo7 is a circRNA generated from the second exon of mouse F-box only protein 7 (Fbxo7). The role of human circFBXO7 and mouse circFbxo7 in non-small cell lung cancer (NSCLC) has not been reported. METHODS: The expression of circFBXO7 was measured by quantitative real-time PCR. Survival analysis was performed to explore the association between the expression of circFBXO7 and the prognosis of patients with NSCLC. Lung cancer cell lines were transfected with plasmids. Cell proliferation, cell cycle, and tumorigenesis were evaluated to assess the effects of circFBXO7. Fluorescence in situ hybridization assay was used to identify the location of circFBXO7 and circFbxo7 in human and mouse lung cancer cells. Luciferase reporter assay was conducted to confirm the relationship between circFBXO7 and microRNA. RESULTS: In this study, we found that circFBXO7 was downregulated in NSCLC tissues and cell lines. NSCLC patients with high circFBXO7 expression had prolonged overall survival. Overexpression of circFBXO7 inhibited cell proliferation both in vitro and in vivo. Mechanistically, we demonstrated that circFBXO7 upregulated the expression of miR-296-3p target gene Krüppel-like factor 15 (KLF15) and KLF15 transactivated the expression of CDKN1A. CONCLUSIONS: CircFBXO7 acts as a tumor suppressor by a novel circFBXO7/miR-296-3p/KLF15/CDKN1A axis, which may serve as a potential biomarker and therapeutic target for NSCLC.

Altered phenotypic and metabolic characteristics of FOXP3+CD3+CD56+ natural killer T (NKT)-like cells in human malignant pleural effusion.

Malignant pleural effusion (MPE) is a functional 'cold' tumor microenvironment in which the antitumor activity of CD8+ T cells and natural killer T (NKT)-like cells is suppressed and the function of regulatory T (Treg) cells is enhanced. Using flow cytometry and immunofluorescence staining, we detected a distinct subset of NKT-like cells expressing FOXP3 in MPE. Through single-cell RNA sequencing (scRNA-seq) analysis, we found that the glycolysis pathway and pyruvate metabolism were highly activated in FOXP3+ NKT-like cells. Similar to Treg cells, FOXP3+ NKT-like cells highly expressed monocarboxylate transporter 1 (MCT1) and lactate dehydrogenase B to uptake and utilize lactate, thereby maintaining their immunosuppressive function and hyperlactylation in MPE. Furthermore, we found that MCT1 small molecule inhibitor 7ACC2 significantly reduced FOXP3 expression and histone lactylation levels in NKT-like cells in vitro. In conclusion, we reveal for the first time the altered phenotypic and metabolic features of FOXP3+ NKT-like cells in human MPE.

Concomitant oxidation of fatty acids other than DHA and EPA plays a role in the characteristic off-odor of fish oil.

The aim of the present research was to explore the development of off-odors in fish oil from the perspective of fatty acid oxidation. It was found that the off-odors elicited by the two major ω-3 PUFAs in fish oil, i.e. DHA and EPA, were different from those by fish oil. Results showed that simultaneous oxidation of fatty acids other than DHA and EPA can be involved. The off-odors of fish oil was successfully simulated by combining oxidized samples of DHA, EPA and sunflower oil. Therefore, oxidation of oleic and linoleic acids also contributed to the off-odors in fish oil. A novel analytical approach that consisted in the combination of gas chromatography-ion mobility spectrometry (GC-IMS) and orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to identify differences in the volatile components between the recombinant oil and the fish oil.

A chemometric study on the identification of 5-methylfurfural and 2-acetylfuran as particular volatile compounds of oxidized fish oil based on SHS-GC-IMS.

Fish oil develops particular off-odors, mainly fishy odor, from the oxidation of its characteristic fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA). Anchovy oil (AO) was taken as representative of fish oils. This was compared to three vegetable oils with different fatty acid compositions, i.e. camellia, sunflower and linseed oil, and differential volatile compounds were identified by static-headspace gas-chromatography ion-mobility-spectrometry (SHS-GC-IMS) and orthogonal partial-least-squares discriminant analysis (OPLS-DA) during oxidation at 60 °C. Three groups of differential volatile compounds detected at higher concentrations in the AO were screened out and two compounds, identified as 5-methylfurfural and 2-acetylfuran, were characteristic to the AO and not found in the vegetable oils. They were formed from both EPA and DHA, only present in the AO, and their formation mechanisms were proposed. The contents of 5-methylfurfural and 2-acetylfuran increased linearly with the oxidation time and consequently they could be used as oxidative markers of fish oils.

[Rapid quality evaluation of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle based on color digitization and multi-component determination].

To explore the color value changes after processing and further explore the correlations between color values and internal components, we established a rapid evaluation method for the quality of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle. In this study, the color values of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle were digitized by a spectrophotometer, and the standard ranges of color values of the two herbal medicines were established. Further, a discriminant analysis model was established to quickly and accurately distinguish the two herbal medicines. The content of 9 flavonoids and 1 triterpene in Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle were determined by HPLC, and Pearson correlation analysis was adopted to analyze the correlations between the color values and the content of 10 components. The standard ranges of L~*, a~*, and b~* values were 65.539 6-68.305 8, 7.296 3-8.467 3, and 29.998 8-32.212 8 for Glycyrrhizae Radix et Rhizoma, and 43.654 3-47.166 4, 14.050 0-15.133 8, and 16.424 6-20.984 8 for Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, respectively. Glycyrrhizae Radix et Rhizoma had higher L~* and b~* values and lower a~* value than Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, which indicated that processing with honey decreased the white and yellow values and increased the red value. The original and cross validation of the established discriminant analysis model met the requirements, and the external validation of the model showed the prediction accuracy of 100%. Pearson correlation analysis showed that the a~* value was positively correlated with the content of liquiritin apioside and isoliquiritin apioside(P<0.05), while the L~* and b~* values were negatively correlated with the content of the above two components(P<0.05). After processing with honey, L~* and b~* decreased while a~* increased, and the content of liquiritin apioside and isoliquiritin apioside increased, which was consistent with the content determination results. This study reveals the regularity of the color values of Glycyrrhizae Radix et Rhizoma after processing with honey roasting, as well as the correlations between color values and component content, which provides a basis for the rapid quality evaluation of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle.

Development and Validation of a Prognostic Autophagy-Related Gene Pair Index Related to Tumor-Infiltrating Lymphocytes in Early-Stage Lung Adenocarcinoma.

The role of autophagy in lung cancer is context-dependent and complex. Recent studies have reported the important role of autophagy in tumor immune escape. However, the association between autophagy and tumor-infiltrating lymphocytes (TILs) in early-stage lung adenocarcinoma (LUAD) remains unclear. In this study, we aimed to develop and validate the autophagy-related gene pair index (ATGPI) and autophagy clinical prognostic index (ACPI) in multiple LUAD cohorts, including The Cancer Genome Atlas (TCGA) cohort, Gene Expression Omnibus cohorts, and one cohort from Union Hospital, Wuhan (UH cohort), using a Cox proportional hazards regression model with the least absolute shrinkage and selection operator. Multivariate Cox regression analysis demonstrated that there was a significant difference in overall survival (OS) between patients with high and low ATGPI in the testing [hazard ratio (HR) = 1.97; P < 0.001] and TCGA validation (HR = 2.25; P < 0.001) cohorts. Time-dependent receiver operating characteristic curve analysis was also performed. We found that high ATGPI could accurately identify patients with early-stage LUAD with shorter OS, with the areas under the curve of 0.703 and 0.676 in the testing and TCGA validation cohorts, respectively. Concordance index (C-index) was used to evaluate the efficiency of ATGPI and ACPI. The C-index of ACPI was higher than that of ATGPI in the testing (0.71 vs. 0.66; P < 0.001), TCGA validation (0.69 vs. 0.65; P = 0.028), and UH (0.80 vs. 0.70; P = 0.015) cohorts. TIL analysis demonstrated that the proportions of tumor-infiltrating CD4+ T cells were lower in the high-ATGPI group than in the low-ATGPI group in both the TCGA validation and UH cohorts. These results indicate the potential clinical use of ATG signatures which are associated with TILs, in identifying patients with early-stage LUAD with different OS.

Application of Plackett-Burman Design in Screening of Natural Antioxidants Suitable for Anchovy Oil.

Considering the safety of synthetic antioxidants, more and more natural antioxidants have been developed and utilized in foods. This study aimed to screen out a natural antioxidant combination from many antioxidants, which could significantly affect the oxidation stability of anchovy oil, while Plackett-Burman design (PBD) methodology was employed in this screening. According to the statistical results of this design, sesamol, dihydromyricetin, teapolyphenol, and rosemary acid were four significant parameters on the oxidation stability of anchovy oil. Moreover, dihydromyricetin presented the best antioxidant effect among nine kinds of selected antioxidants when they were used alone in anchovy oil. Meanwhile, a combination including sesamol (0.02%), teapolyphenol (0.02%). and rosemary acid (0.02%) was adopted, and its antioxidant ability was similar to that of tert-butylhydroquinone (TBHQ). Additionally, phytic acid as a synergist was used and combined with sesamol, and the antioxidant ability of this combination was better than that of TBHQ. This study presented a reference for the industrial applications of natural antioxidants and synergists in anchovy oil.

Anticancer activity of recombinant Siva1 protein in human nasopharyngeal carcinoma cell line CNE-2.

AIMS: To clone and express Siva1 protein, and to investigate the role of Siva1 protein in proliferation, apoptosis, invasion, and migration of human nasopharyngeal carcinoma cell line CNE-2 in vitro and in vivo. METHODS: The PCR fragment of Siva1 from human nasopharyngeal carcinoma cell line CNE-2 were double digested with BamHI and SalI and then induced into the pQE30 vector double digested by the same enzymes. The pQE30 vector harboring Siva1 was introduced into M15 competent cells and then induced by isopropyl ß -D-1-thiogalactopyranoside (IPTG). The Siva1 fusion protein was identified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then separated and purified by Ni-affinity chromatography. Subsequently, the effects of recombinant Siva1 protein on proliferation, apoptosis, invasion and migration were assayed in vitro and in vivo. RESULTS: The transformed cells expressed Siva1 fusion protein with a molecular weight of approximately 12 kDa. Cell counting kit-8 (CCK-8) assay showed that the Siva1 protein significantly inhibited the proliferation of the CNE-2 cells at a concentration of 10 µ mol/L. In addition, compared to the control, the Siva1 protein promoted the apoptosis of the cancer cells. And, the Siva1 protein greatly suppressed the invasion and migration of the cancer cells. In vivo, the Siva1 protein significantly inhibited the tumor growth of the tumor-bearing mice. Further, the Siva1 treatment markedly upregulated Bax, caspase-3, and downregulated Bcl-2 protein levels in the transplanted tumor tissue. CONCLUSIONS: The Siva1 protein has a significant anticancer activity on human nasopharyngeal carcinoma cell line CNE-2 including inhibiting proliferation, invasion, migration and promoting apoptosis of the cancer cells.