Jian-Pi-Yi-Shen formula ameliorates renal fibrosis-induced anemia in rats with chronic kidney disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Jian-Pi-Yi-Shen (JPYS) formula is an effective herbal therapy against renal injury, and JPYS has been clinically applied to ameliorate chronic kidney disease (CKD) and CKD-associated anemia. Increasing evidence supports the link between renal fibrosis and anemia in CKD. JPYS possessed anti-fibrosis effects in experimental CKD. Nevertheless, research on the mechanisms of JPYS in ameliorating renal anemia (RA) through suppressing renal fibrosis remains to be clarified. AIM OF THE STUDY: Our study here was carried out to investigate the mechanisms of JPYS in protecting against RA. MATERIALS AND METHODS: An adenine-induced anemia model in rats with CKD at three different time points was established, and bio-samples taken from each group were analyzed. Biochemical analysis was employed to detect kidney function and hematological parameters. Masson staining was used to evaluate renal fibrosis of rats. Western blot and immunohistochemistry were utilized to evaluate the expressions of fibrotic markers, erythropoietin (EPO) and hypoxia inducible factor-2α (HIF-2α) in the kidneys of rats. Subsequently, transcriptomic analysis was conducted to disclose the possible mechanisms of JPYS in treating RA. Finally, the expression levels of key targets were analyzed and validated by using Western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: JPYS treatment improved kidney function, suppressed renal fibrosis and enhanced hematological parameters in CKD rats. Moreover, JPYS treatment restored the increased expression levels of fibrotic markers and the declined EPO with time dependence. In parallel, data indicated JPYS treatment stimulated the translocation of HIF-2α into nucleus in the renal interstitium and thus promoted the expression of EPO. Transcriptomic profiling disclosed that activations of both nuclear factor kappa B (NF-κB) and transforming growth factor-ß (TGF-ß)/Smad pathways were closely associated with RA. Ultimately, experimental validation results presented that the increased expressions of target proteins from the above-mentioned two pathways in the kidneys were decreased significantly after JPYS treatment. CONCLUSION: Our findings suggest that JPYS may improve RA by alleviating renal fibrosis, and the mechanisms of which involve in inhibiting the NF-κB and TGF-ß/Smad pathways.

Comprehensively analysis of IL33 in hepatocellular carcinoma prognosis, immune microenvironment and biological role.

IL33 plays an important role in cancer. However, the role of liver cancer remains unclear. Open-accessed data was obtained from the Cancer Genome Atlas, Xena, and TISCH databases. Different algorithms and R packages are used to perform various analyses. Here, in our comprehensive study on IL33 in HCC, we observed its differential expression across cancers, implicating its role in cancer development. The single-cell analysis highlighted its primary expression in endothelial cells, unveiling correlations within the HCC microenvironment. Also, the expression level of IL33 was correlated with patients survival, emphasizing its potential prognostic value. Biological enrichment analyses revealed associations with stem cell division, angiogenesis, and inflammatory response. IL33's impact on the immune microenvironment showcased correlations with diverse immune cells. Genomic features and drug sensitivity analyses provided insights into IL33's broader implications. In a pan-cancer context, IL33 emerged as a potential tumour-inhibitor, influencing immune-related molecules. This study significantly advances our understanding of IL33 in cancer biology. IL33 exhibited differential expression across cancers, particularly in endothelial cells within the HCC microenvironment. IL33 is correlated with the survival of HCC patients, indicating potential prognostic value and highlighting its broader implications in cancer biology.

Transcriptomics and UHPLC-QQQ-MS analyses reveal the dysregulation of branched chain amino acids metabolism in renal fibrotic rats.

The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB, the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.

Bafi A1 inhibits nano-copper oxide-induced mitochondrial damage by reducing the release of copper from lysosomes.

This study demonstrated that both copper oxide nanoparticles (CuO-NPs) and copper nanoparticles (Cu-NPs) can cause swelling, inflammation, and cause damage to the mitochondria of alveolar type II epithelial cells in mice. Cellular examinations indicated that both CuO-NPs and Cu-NPs can reduce cell viability and harm the mitochondria of human bronchial epithelial cells, particularly Beas-2B cells. However, it is clear that CuO-NPs exhibit a more pronounced detrimental effect compared with Cu-NPs. Using bafilomycin A1 (Bafi A1), an inhibitor of lysosomal acidification, was found to enhance cell viability and alleviate mitochondrial damage caused by CuO-NPs. Additionally, Bafi A1 also reduces the accumulation of dihydrolipoamide S-acetyltransferase (DLAT), a marker for mitochondrial protein toxicity, induced by CuO-NPs. This observation suggests that the toxicity of CuO-NPs depends on the distribution of copper particles within cells, a process facilitated by the acidic environment of lysosomes. The release of copper ions is thought to be triggered by the acidic conditions within lysosomes, which aligns with the lysosomal Trojan horse mechanism. However, this association does not seem to be evident with Cu-NPs.

Unveiling the role of the KLF4/Lnc18q22.2/ULBP3 axis in the tumorigenesis and immune escape of hepatocellular carcinoma under hypoxic condition.

Hepatocellular carcinoma (HCC) represents a significant global health burden, necessitating an in-depth exploration of its molecular underpinnings to facilitate the development of effective therapeutic strategies. This investigation delves into the complex role of long non-coding RNAs (lncRNAs) in the modulation of hypoxia-induced HCC progression, with a specific emphasis on delineating and functionally characterizing the novel KLF4/Lnc18q22.2/ULBP3 axis. To elucidate the effects of hypoxic conditions on HCC cells, we established in vitro models under both normoxic and hypoxic environments, followed by lncRNA microarray analyses. Among the lncRNAs identified, Lnc18q22.2 was found to be significantly upregulated in HCC cells subjected to hypoxia. Subsequent investigations affirmed the oncogenic role of Lnc18q22.2, highlighting its critical function in augmenting HCC cell proliferation and migration. Further examination disclosed that Kruppel-like factor 4 (KLF4) transcriptionally governs Lnc18q22.2 expression in HCC cells, particularly under hypoxic stress. KLF4 subsequently enhances the tumorigenic capabilities of HCC cells through the modulation of Lnc18q22.2 expression. Advancing downstream in the molecular cascade, our study elucidates a novel interaction between Lnc18q22.2 and UL16-binding protein 3 (ULBP3), culminating in the stabilization of ULBP3 protein expression. Notably, ULBP3 was identified as a pivotal element, exerting dual functions by facilitating HCC tumorigenesis and mitigating immune evasion in hypoxia-exposed HCC cells. The comprehensive insights gained from our research delineate a hitherto unidentified KLF4/Lnc18q22.2/ULBP3 axis integral to the understanding of HCC tumorigenesis and immune escape under hypoxic conditions. This newly unveiled molecular pathway not only enriches our understanding of hypoxia-induced HCC progression but also presents novel avenues for therapeutic intervention.

Dynamic graph transformer network via dual-view connectivity for autism spectrum disorder identification.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that requires objective and accurate identification methods for effective early intervention. Previous population-based methods via functional connectivity (FC) analysis ignore the differences between positive and negative FCs, which provide the potential information complementarity. And they also require additional information to construct a pre-defined graph. Meanwhile, two challenging demand attentions are the imbalance of performance caused by the class distribution and the inherent heterogeneity of multi-site data. In this paper, we propose a novel dynamic graph Transformer network based on dual-view connectivity for ASD Identification. It is based on the Autoencoders, which regard the input feature as individual feature and without any inductive bias. First, a dual-view feature extractor is designed to extract individual and complementary information from positive and negative connectivity. Then Graph Transformer network is innovated with a hot plugging K-Nearest Neighbor (KNN) algorithm module which constructs a dynamic population graph without any additional information. Additionally, we introduce the PolyLoss function and the Vrex method to address the class imbalance and improve the model's generalizability. The evaluation experiment on 1102 subjects from the ABIDE I dataset demonstrates our method can achieve superior performance over several state-of-the-art methods and satisfying generalizability for ASD identification.

Chemical characterization and metabolic profiling of Xiao-Er-An-Shen Decoction by UPLC-QTOF/MS.

Background: Xiao-Er-An-Shen decoction (XEASD), a TCM formula composed of sixteen Chinese medicinal herbs, has been used to alleviate tic disorders (TD) in clinical practice for many years. However, the chemical basis underlying the therapeutic effects of XEASD in the treatment of TD remains unknown. Purpose: The present study aimed to determine the major chemical components of XEASD and its prototype compounds and metabolites in mice biological samples. Methods: The chemical constituents in XEASD were identified using ultra-high Performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Following this, XEASD was orally administered to mice, and samples of plasma, urine, feces, bile, and tissue were collected in order to identify effective compounds for the prevention or treatment of TD. Result: Of the total 184 compounds identified to be discriminated in the XEASD, comprising 44 flavonoids, 26 phenylpropanoids, 16 coumarins, 16 triterpenoids, 14 amino acids, 13 organic acids, 13 alkaloids, 13 ketones, 10 cyclic enol ether terpenes, 7 citrullines, 3 steroids, and 5 anthraquinones, and others. Furthermore, we summarized 54 prototype components and 78 metabolic products of XEASD, measured with biological samples, by estimating metabolic principal components, with four prototype compounds detected in plasma, 58 prototypes discriminated in urine, and 40 prototypes identified in feces. These results indicate that the Oroxylin A glucuronide from Citri reticulatae pericarpium (CRP) is a major compound with potential therapeutic effects identified in brain, while operating positive effect in inhibiting oxidative stress in vitro. Conclusion: In summary, our work delineates the chemical basis underlying the complexity of XEASD, providing insights into the therapeutic and metabolic pathways for TD. Various types of chemicals were explored in XEASD, including flavonoids, phenylpropanoids, coumarins, organic acids, triterpenoid saponins, and so on. This study can promote the further pharmacokinetic and pharmacological evaluation of XEASD.

Black phosphorus quantum dots induced ferroptosis in lung cell via increasing lipid peroxidation and iron accumulation.

Black Phosphorus Quantum Dots (BP-QDs) have potential applications in biomedicine. BP-QDs may enter the body through the respiratory tract during grinding and crushing production and processing, causing respiratory toxicity. Ferroptosis is an oxidative, iron-dependent form of cell death. Here, respiratory toxicity of BP-QDs has been validated in mice and human bronchial epithelial cells. After 24 h of exposure to different doses (4-32 µg/mL) of BP-QDs, intracellular lipid peroxidation and iron overload occurred in Beas-2B cells. After 4 times exposures by noninvasive tracheal instillation at four doses [0, 0.25, 0.5 and 1 (mg/kg/48h)], all animals were sacrificed, organs were removed, processed for pathological examination and molecular analysis. Iron overload, glutathione (GSH) depletion and lipid peroxidation in the lung tissue of mice in the exposure group. Furthermore, based on the ferroptosis-associated protein and mRNA expression, it was hypothesized that BP-QDs induced ferroptosis through increasing intracellular free iron and polyunsaturated fatty acid synthesis. By comparing with previous studies, we speculate that primary cells generally are more sensitive to BP-QDs-induced damage than cancer cells. In summary, findings in the present study confirmed that BP-QDs induce ferroptosis via increasing lipid peroxidation and iron accumulation in vitro and in vivo.

CCNF is a potential pancancer biomarker and immunotherapy target.

Background: CCNF catalyzes the transfer of ubiquitin molecules from E2 ubiquitin-conjugating enzymes to target proteins, thereby regulating the G1/S or G2/M transition of tumor cells. Thus far, CCNF expression and its potential as a pancancer biomarker and immunotherapy target have not been reported. Methods: TCGA datasets and the R language were used to analyze the pancancer gene expression, protein expression, and methylation levels of CCNF; the relationship of CCNF expression with overall survival (OS), recurrence-free survival (RFS), immune matrix scores, sex and race; and the mechanisms for posttranscriptional regulation of CCNF. Results: CCNF expression analysis showed that CCNF mRNA expression was higher in cancer tissues than in normal tissues in the BRCA, CHOL, COAD, ESCA, HNSC, LUAD, LUSC, READ, STAD, and UCEC; CCNF protein expression was also high in many cancer tissues, indicating that it could be an important predictive factor for OS and RFS. CCNF overexpression may be caused by CCNF hypomethylation. CCNF expression was also found to be significantly different between patients grouped based on sex and race. Overexpression of CCNF reduces immune and stromal cell infiltration in many cancers. Posttranscriptional regulation analysis showed that miR-98-5p negatively regulates the expression of the CCNF gene. Conclusion: CCNF is overexpressed across cancers and is an adverse prognostic factor in terms of OS and RFS in many cancers; this phenomenon may be related to hypomethylation of the CCNF gene, which could lead to cancer progression and worsen prognosis. In addition, CCNF expression patterns were significantly different among patients grouped by sex and race. Its overexpression reduces immune and stromal cell infiltration. miR-98-5p negatively regulates CCNF gene expression. Hence, CCNF is a potential pancancer biomarker and immunotherapy target.

Black phosphorus quantum dots induce autophagy and apoptosis of human bronchial epithelial cells via endoplasmic reticulum stress.

PURPOSE: The board application of black phosphorus quantum dots (BP-QDs) increases the risk of inhalation exposure in the manufacturing process. The aim of this study is to explore the toxic effect of BP-QDs on human bronchial epithelial cells (Beas-2B) and lung tissue of Balb/c mice. METHODS: The BP-QDs were characterized using transmission electron microscopy (TEM) and a Malvern laser particle size analyzer. Cell Counting Kit-8 (CCK-8) and TEM were used to detect cytotoxicity and organelle injury. Damage to the endoplasmic reticulum (ER) was detected by using the ER-Tracker molecular probe. Rates of apoptosis were detected by AnnexinV/PI staining. Phagocytic acid vesicles were detected using AO staining. Western blotting and immunohistochemistry were used to examine the molecular mechanisms. RESULTS: After treatment with different concentrations of BP-QDs for 24 h, the cell viability decreased, as well as activation of the ER stress and autophagy. Furthermore, the rate of apoptosis was increased. Inhibition of ER stress caused by 4-phenyl butyric acid (4-PBA) was shown to significantly inhibit both apoptosis and autophagy, suggesting that ER stress could be an upstream mediator of both autophagy and apoptosis. BP-QD-induced autophagy can also inhibit the occurrence of apoptosis using molecules related to autophagy including rapamycin (Rapa), 3-methyladenine (3-MA), and bafilomycin A1 (Bafi A1). In general, BP-QDs activate ER stress in Beas-2B cells, which further induces autophagy and apoptosis, and autophagy may be activated as a factor that protects against apoptosis. We also observed strong staining of related proteins of ER stress, autophagy, and apoptosis proteins in mouse lung tissue following intracheal instillation over the course of a week. CONCLUSION: BP-QD-induced ER stress facilitates autophagy and apoptosis in Beas-2B cells and autophagy may be activated as a protective factor against apoptosis. Under conditions of ER stress induced by BP-QDs, The interplay between autophagy and apoptosis determines cell fate.

ST-segment elevation predicts the occurrence of malignant ventricular arrhythmia events in patients with acute ST-segment elevation myocardial infarction.

BACKGROUND: ST-segment elevation (STE) represents a repolarization dispersion marker underlying arrhythmogenesis in ST-segment elevation myocardial infarction (STEMI); however, its value for predicting malignant ventricular arrhythmia events (MVAEs) remains uncertain. METHODS: In total, 285 patients with STEMI and those with or without MVAEs who presented within 6 h of symptom onset were enrolled. The relationships between STE and clinical characteristics of MVAEs (defined as ventricular tachycardia or ventricular fibrillation) were analyzed using t-test, chi-square test, binary multivariate logistic regression, and receiver operating characteristic curve analysis. RESULTS: Patients with STEMI and MVAEs had a shorter time from symptom onset to balloon time (p = 0.0285) and greater STE (p < 0.01) than those without MVAEs. The symptom-to-balloon time, age, and STE were associated with MVAEs after stepwise regression analysis in all cases. Only STE was significantly associated with the occurrence of MVAEs (all, p < 0.01). The area under the curve (AUC) of STE for predicting MVAEs was 0.905, and the cut-off value was 4.5 mV. When only infarct-related arteries were included in the analysis, the AUC of the left anterior descending artery was 0.925 with a cut-off value of 4.5 mV, that of the right coronary artery was 0.915 with a cut-off value of 4.5 mV, and that of the left circumflex artery was 0.929 with a cut-off value of 4.0 mV. CONCLUSIONS: In patients with STEMI presenting within 6 h of symptom onset, age, symptom-to-balloon time, and STE were the main predictors for MVAEs. However, among these, STE was the strongest predictor for MVAEs and was an index for repolarization dispersion of cardiomyocytes in infarcted and non-infarcted areas.

MC-UNet: Multimodule Concatenation Based on U-Shape Network for Retinal Blood Vessels Segmentation.

Accurate retinal blood vessels segmentation is an important step in the clinical diagnosis of ophthalmic diseases. Many deep learning frameworks have come up for retinal blood vessels segmentation tasks. However, the complex vascular structure and uncertain pathological features make blood vessel segmentation still very challenging. This paper proposes a novel multimodule concatenation via a U-shaped network for retinal vessels segmentation, which is based on atrous convolution and multikernel pooling. The proposed network structure retains three layers of the essential structure of U-Net, in which the atrous convolution combining the multikernel pooling blocks are designed to obtain more contextual information. The spatial attention module is concatenated with the dense atrous convolution module and the multikernel pooling module to form a multimodule concatenation. And different dilation rates are selected by cascading to acquire a larger receptive field in atrous convolution. Adequate comparative experiments are conducted on these public retinal datasets: DRIVE, STARE, and CHASE_DB1. The results show that the proposed method is effective, especially for microvessels. The code will be released at https://github.com/rocklijun/MC-UNet.

UCR-Net: U-shaped context residual network for medical image segmentation.

Medical image segmentation prerequisite for numerous clinical needs is a critical step in biomedical image analysis. The U-Net framework is one of the most popular deep networks in this field. However, U-Net's successive pooling and downsampling operations result in some loss of spatial information. In this paper, we propose a U-shaped context residual network, called UCR-Net, to capture more context and high-level information for medical image segmentation. The proposed UCR-Net is an encoder-decoder framework comprising a feature encoder module and a feature decoder module. The feature decoder module contains four newly proposed context attention exploration(CAE) modules, a newly proposed global and spatial attention (GSA) module, and four decoder blocks. We use the proposed CAE module to capture more multi-scale context features from the encoder. The proposed GSA module further explores global context features and semantically enhanced deep-level features. The proposed UCR-Net can recover more high-level semantic features and fuse context attention information from CAE and global and spatial attention information from GSA module. Experiments on the retinal vessel, femoropopliteal artery stent, and polyp datasets demonstrate that the proposed UCR-Net performs favorably against the original U-Net and other advanced methods.

"We don't normally require that in other contexts, do we": Interpersonal meanings of tag questions in British university seminars based on the BASE corpus.

This study investigates the interpersonal meanings expressed by English tag questions in the context of British university seminars from two dimensions: evidential modification of tags and conduciveness of responses. The data for the study derive from seminars in the British Academic Spoken English (BASE) Corpus, which is herein both quantitative and qualitative analyzed. The findings reveal that: (1) three-quarters of tag questions in the data are utilized by teachers, and the unmarked form of tag questions in university seminars are generally positive-negative forms, with a few examples of other varieties; (2) regarding evidential modification of tags, depending on the degree of epistemic certainty of the speakers on the proposition of the anchor, the default function of teachers' tag questions in the data is to convey emphasis, followed by confirmatory function, with only a few cases of informational function; and (3) regarding conduciveness of responses, over 70% of tag questions are followed by no verbal response, while less than 30% are followed by explicit responses. Accordingly, these findings raise the question of whether tag questions can really provide sufficient scope for interactions in classroom. It is hoped that this nuanced, corpus-based analysis of tag question utilization within the context of British university seminars would empirically reveal the interpersonal relations between teachers and students and thereby shed light on more efficient seminar discussions.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

Obesity-Related Glomerulopathy: From Mechanism to Therapeutic Target.

Obesity-related glomerulopathy (ORG) is a secondary glomerular disease caused by obesity, with clinical manifestations such as proteinuria and glomerulomegaly. Currently, the high incidence of obesity brings a change in the spectrum of kidney diseases across the globe, including China. ORG has become another important secondary nephropathy leading to end-stage renal disease (ESRD), and its incidence has increased significantly. This trend is bound to bring about a serious socioeconomic burden. Therefore, it is urgent to study its pathogenesis and intervention measures. Currently, the occurrence and development mechanisms in ORG are complicated by many factors, which are still unclear. In the past 20 years, with the continuous intensive research on mechanisms such as hypoxia in the metabolic process, immune inflammation, and pyroptosis, there have been new advances in the mechanism of ORG, especially the important role of inflammation in podocyte injury and its impact on the progress of ORG. Here, we briefly review the possible pathogenic role of the inflammasome in the podocyte damage in ORG and summarize the possible therapeutical strategies targeting inflammasome.

Qualitative Analysis and Componential Differences of Chemical Constituents in Taxilli Herba from Different Hosts by UFLC-Triple TOF-MS/MS.

Taxilli Herba (TH) is a well-known traditional Chinese medicine (TCM) with a wide range of clinical application. However, there is a lack of comprehensive research on its chemical composition in recent years. At the same time, Taxillus chinensis (DC) Danser is a semi parasitic plant with abundant hosts, and its chemical constituents varies due to hosts. In this study, the characterization of chemical constituents in TH was analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS). Moreover, partial least squares discriminant analysis (PLS-DA) was applied to reveal the differential constituents in TH from different hosts based on the qualitative information of the chemical constituents. Results showed that 73 constituents in TH were identified or tentatively presumed, including flavonoids, phenolic acids and glycosides, and others; meanwhile, the fragmentation pathways of different types of compounds were preliminarily deduced by the fragmentation behavior of the major constituents. In addition, 23 differential characteristic constituents were screened based on variable importance in projection (VIP) and p-value. Among them, quercetin 3-O-ß-D-glucuronide, quercitrin and hyperoside were common differential constituents. Our research will contribute to comprehensive evaluation and intrinsic quality control of TH, and provide a scientific basis for the variety identification of medicinal materials from different hosts.

Metabolite Profiling and Transcriptome Analysis Explains Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) Under Salt Stress.

Salinity stress significantly affects the contents of bioactive constituents in licorice Glycyrrhiza uralensis. To elucidate the molecular mechanism underlying the difference in the accumulation of these constituents under sodium chloride (NaCl, salt) stress, licorice seedlings were treated with NaCl and then subjected to an integrated transcriptomic and metabolite profiling analysis. The transcriptomic analysis results identified 3,664 differentially expressed genes (DEGs) including transcription factor family MYB and basic helix-loop-helix (bHLH). Most DEGs were involved in flavonoid and terpenoid biosynthesis pathways. In addition, 121 compounds including a triterpenoid and five classes of flavonoids (isoflavone, flavone, flavanone, isoflavan, and chalcone) were identified, and their relative levels were compared between the stressed and control groups using data from the ultrafast liquid chromatography (UFLC)-triple quadrupole-time of flight-tandem mass spectrometry (TOF-MS/MS) analysis. Putative biosynthesis networks of the flavonoids and triterpenoids were created and combined with structural DEGs such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase [4CL], cinnamate 4-hydroxylase [C4H], chalcone synthase [CHS], chalcone-flavanone isomerase [CHI], and flavonoid-3',5' hydroxylase (F3',5'H) for flavonoids, and CYP88D6 and CYP72A154 for glycyrrhizin biosynthesis. Notably, significant upregulation of UDP-glycosyltransferase genes (UGT) in salt-stressed licorice indicated that postmodification of glycosyltransferase may participate in downstream biosynthesis of flavonoid glycosides and triterpenoid saponins. Accordingly, the expression trend of the DEGs is positively correlated with the accumulation of glycosides. Our study findings indicate that key DEGs and crucial UGT genes co-regulate flavonoid and saponin biosynthesis in licorice under salt stress.

[Simultaneous determination of multiple bioactive constituents in Abelmoschi Corolla by UFLC-QTRAP-MS/MS].

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 38 active components in Abelmoschi Corolla, including flavonoids, organic acids, nucleosides and amino acids, so as to investigate the effects of different harvesting and processing methods on multi-active components in Abelmoschi Corolla. The chromatographic separation was performed on a XBridg®C_(18) column(4.6 mm×100 mm, 3.5 µm) with(0.1% formic acid water) methanol-acetonitrile(1∶1) as the mobile phase for gradient elution at 30 ℃. The flow rate was 0.5 mL·min~(-1). The components were detected in a multiple-reaction monitoring(MRM) mode. The gray relational analysis(GRA) was used to comprehensively evaluate the multiple active components of Abelmoschi Corolla at different harvesting times and drying temperatures. The results showed that 38 components had a good linearity with correlation coefficients all above 0.999 0. The method featured a good precision, repeatability and stability with the relative stan-dard deviations(RSDs) of less than 5.0%. Recoveries ranged from 98.06% to 104.4% with RSD between 0.22% and 4.9%. The results of GRA indicated that a better quality in the samples collected on September 9 th. Samples dried at 90 ℃ had a better quality. The established method is accurate and reliable, and can be used to assess the internal quality of Abelmoschi Corolla. This study can provide basic materials for determining appropriate harvesting time and processing method of Abelmoschi Corolla.

Comparison of Multiple Bioactive Constituents in the Corolla and Other Parts of Abelmoschus manihot.

Abelmoschus manihot (L.) Medic (AM), called Huangshukui in Chinese, is a widely used medicinal plant. Each part of AM has medicinal value, including Abelmoschi Radix (AR), Abelmoschi Herba (AH), Abelmoschi Folium (AF), Abelmoschi Corolla (AC), and Abelmoschi Semen (AS). However, only AC is documented in the Chinese Pharmacopoeia. In order to investigate whether there is any difference between AC and the other parts of AM, an analytical method based on ultra-fast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) was established for the simultaneous determination of 35 constituents in different parts of AM. Moreover, principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied to classify and evaluate the different parts of AM based on the content of the 35 constituents. The total contents of the 35 constituents in AC were significantly higher than in the other parts of AM and the results revealed significant differences between AC and the other parts of AM. Eight constituents were remarkably related to the sample classifications. This research does not just provide the basic information for revealing the distribution patterns in different parts of AM from the same origin, but also complements some of the scientific data for the comprehensive quality evaluation of AC.