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Objectives: Triterpenoid saponins of Ilex pubescens (IPTS), the main active components of Ilex pubescens, has a therapeutic effect on atherosclerosis (AS). The ingredients in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs) may play an essential role in AS. This study attempted to explore its mechanism from the perspectives of HUVECs' inflammation, apoptosis, and autophagy. Methods: By using a tumour necrosis factor-α (TNF-α)-induced HUVECs injury model, cell viability and the expression of intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9), cleave-caspase-3 and cleave-caspase-9, in combination with the results of flow cytometry, JC-1 and Hoechst 33258 staining were investigated to evaluate the anti-inflammatory and anti-apoptotic impact effects of IPTS on HUVECs. Afterwards, the expression of microtubule-associated proteins light chain 3II (LC3II) and sequestosome 1 (p62) was determined to test the effect of IPTS on autophagy. Finally, by adding an autophagy inhibitor 3-methyladenine (3-MA), we investigated whether IPTS exerts anti-inflammatory and anti-apoptotic effects through the autophagy pathway. Key findings: We firstly demonstrated that pretreatment with IPTS could increase the cell viability, maintain the cell morphology and reduce TNF-α-induced inflammation and apoptosis of HUVECs. Moreover, IPTS pretreatment was proved to raise the expression of LC3II /LC3I while decreasing the expression of p62, which indicated that IPTS could activate HUVECs' autophagy. IPTS has been shown for the first time to exert anti-inflammatory and anti-apoptotic effects through autophagy and thereby resisting TNF-α-induced inflammatory injury of HUVECs. Conclusions: This study preliminarily confirmed that IPTS ameliorated HUVECs' inflammation and apoptosis by increasing autophagy. [ABSTRACT FROM AUTHOR]

Ilex pubescens is a famous Chinese herbal medicine, frequently used to treat cardiovascular disease in South China. In this study, we aim to explore the absorption properties of ilexgenin A (C1) and ilexsaponin B1 (C3) in vascular endothelial cells after administration of the total triterpenoid saponins from Ilex pubescens (IPTS) and clarify the possible transport mechanisms. A UPLC–qTOF–MS/MS system was used to identify the components in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs). Afterwards, a rapid, highly selective and sensitive method was established to simultaneously quantify the concentration of C1 and C3 in HUVECs after administration of IPTS. The results demonstrate that pretreatment with IPTS could promote the survival of HUVECs and reduce the damage caused by TNF‐α to HUVECs. Among the main 11 components in IPTS, eight components could be absorbed by HUVECs, including seven triterpenoids and one phenolic acid. The uptake of C1 and C3 by HUVECs occurred in a time‐, temperature‐ and concentration‐dependent manner, indicating the participation of passive diffusion and active transportation mechanisms. The two triterpenoid saponins all exhibited rapid absorption and a bimodal phenomenon in their concentration–time profiles, and equilibrium could be achieved after 6 h. Furthermore, C1 and C3 intracellular transportation was regulated by serum proteins, sodium‐dependent glucose transporter 1 and P‐glycoprotein. The current research for the first time demonstrates the in vitro pharmacokinetics characteristics of C1 and C3 in HUVECs lines, which could supply a new way of understanding the treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

In this study, the pathogenic fungus of stem spot disease of pitaya (Hylocereus spp.) stems was identified by isolation and purification, pathogenicity test, morphological characteristics, and analysis of rDNA-ITS sequences. The results turned out that the rDNA-ITS sequences of the H1 strain showed 100% of identity with Botryosphaeria dothidea, indicating that the pathogenic fungus of stem spot disease of pitaya stems was Botryosphaeria dothidea. Meanwhile, the H1 strain was then used as a reference strain to screen some commercial fungicides. The bioassay test results indicated that prochloraz had an obvious inhibitory effect on Botryosphaeria dothidea with the EC50 value of 0.0798 μg/mL. Our study could provide a theoretical basis for the effective control method of stem spot disease of pitaya. [ABSTRACT FROM AUTHOR]

The Drosophila wing was proposed to be a taste organ more than 35 years ago, but there has been remarkably little study of its role in chemoreception. We carry out a differential RNA-seq analysis of a row of sensilla on the anterior wing margin and find expression of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, we devised a dye-transfer paradigm and found that large, hydrophobic molecules comparable to pheromones can be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. Unlike other chemoreceptors that act in males to inhibit male–male interactions and promote male–female interactions, IR52a acts in both males and females, and can promote male–male as well as male–female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly. [ABSTRACT FROM AUTHOR]

Context:Baicalin (BL) and baicalein (B) as the major flavonoids ofScutellaria baicalensisGeorgi (Lamiaceae) have been investigated intensively, and shown to possess a multitude of pharmacological activities. Objective:This study systematically evaluates the stability of BL and B in monomer and total flavonoid fraction (FSR) formin vitro, and further studies whether the protective measures are effective to make B and BL stable enough to meet the requirement of quantitative analysis in various biological samples. Materials and methods:The stability of BL and B was evaluated by investigating the influence factors such as pH (2.0, 3.0, 4.5, 6.8, 7.4 and 9.0), temperature (4, 25 and 40 °C), antioxidant (vitamin C and Na2SO3) and sunlight. After the protective measures were taken, stability of BL and B in plasma, urine and tissue homogenates was evaluated through post-preparative stability (stored at 4 °C for 24 h), three freeze–thaw cycles stability and long-term stability test (stored in refrigerator at −20 °C for 15 days). In addition, by comparing the degradation parameters of BL and B obtained from the monomer administration group with those of the FSR administration group, drug–drug interaction of coexistent components in FSR on the stability of BL and B was discussed. Results:The degradation of BL and B was both pH- and temperature-dependent with their correlation coefficents for first-order kinetics equation larger than 0.99, and acidic environment (pH 2–4.5), lower temperature (<4 °C) and acidic antioxidant (e.g. vitamin C) were conducive to stabilize B and BL. Furthermore, coexistent components in FSR were proved to have function on inhibiting the degradation of BL and B in our study for the first time, which was characteristic of prolonging their biological half-life (t1/2) significantly, e.g., from 2.89 h to indefinite for BL (pH 6.8, 25 °C), from 2.63 h to 4.48 h for B (pH 6.8, 25 °C) and so on. Antioxidant of Na2SO3could inhibit the degradation of BL witht1/2increasing from 1.8 h to 3.5 h, but aggravate the bio-transformation of B witht1/2decreasing from 0.92 h to 0.29 h. Our research proved that BL monomer, and BL and B in FSR form could be stabilized enough to meet the requirement of biological quantitative analysis under the protection of coexistent components in FSR. Discussion and conclusion:The results obtained indicated that the stability of BL and B was affected not only by its environmental parameters, but also by the coexistent components in the effective total flavonoids fractions. [ABSTRACT FROM PUBLISHER]

Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721–1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that vitamin D (VD) has a therapeutic effect on non-alcoholic fatty liver disease (NAFLD). Pyroptosis and gut microbiota have been recognized as critical factors of the progression of NAFLD. However, the effect of VD on the pyroptosis and gut microbiota in NAFLD remains inconclusive. Herein, rats were fed high fat diet (HFD) for 12 weeks and concurrently treated with 5 μ g/kg 1,25(OH) 2 D 3 twice a week. BRL-3A cells were stimulated with 0.4 mmol/L palmitic acid (PA) and 1 μ g/ml lipopolysaccharide (LPS) for 16 h and treated with 10−6 mol/L 1,25(OH) 2 D 3. Effect of VD on the hepatic injury, lipid accumulation, activation of NLRP3 inflammasome and pyroptosis was determined in vivo and in vitro. Next, gasdermin D N -terminal (GSDMD-N) fragment was overexpressed in BRL-3A cells to investigate the role of pyroptosis in the therapeutic effect of VD on NAFLD. In addition, gut microbiota in NAFLD rats was also analyzed. Results showed that VD attenuated HFD-induced hepatic injury in vivo and PA-LPS-induced impairment of cell viability in vitro , and inhibited lipid accumulation, activation of NLRP3 inflammasome and pyroptosis in vivo and in vitro. GSDMD-N fragment overexpression suppressed the protective effect of VD on PA-LPS-induced activation of NLRP3 inflammasome, impairment of cell viability and lipid accumulation, indicating that VD might attenuate NAFLD through inhibiting pyroptosis. Additionally, VD also restored HFD-induced gut microbiota dysbiosis by increasing the relative abundance of Lactobacillus and reducing that of Acetatifactor , Oscillibacter and Flavonifractor. This study provides a novel mechanism underlying VD therapy against NAFLD. • Vitamin D inhibited activation of NLRP3 inflammasome and pyroptosis in NAFLD rat. • Vitamin D might attenuate HFD-induced NAFLD by inhibiting pyroptosis in NAFLD rat. • Vitamin D restored HFD-induced gut microbiota dysbiosis in NAFLD rat. [ABSTRACT FROM AUTHOR]

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples. [ABSTRACT FROM AUTHOR]

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT–mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT–mTOR signaling pathway to modulate cell proliferation. [ABSTRACT FROM AUTHOR]

The quantitative analysis of multiple components with a single marker (QAMS) method was firstly established for simultaneous determination of 18 active components in Ilex kudingcha C. J. Tseng by HPLC. Using rutin, isochlorogenic acid A and kudinoside A as internal refererence substances (IRS), compatibility results showed that the relative correction factors (RCFs) of all compounds showed good reproducibility under different chromatographic conditions. On the basis of previous studies, the accuracy of the QAMS method was systematically evaluated by investigating the influences of curve intercept, analytes and IRS concentration. The results showed that the concentration (especially at low level) of analytes and curve intercept were the major influencing parameters for the LRG-QAMS method (LRG = linear regression), whereas the influence of IRS concentration seemed more apparent in terms of the AVG-QAMS method (AVG = average). The two approaches were complementary with each other. In addition, hierarchical clustering analysis (HCA), principal components analysis (PCA) and similarity analysis (SA) were performed to differentiate and classify the samples based on the contents of 18 marker compounds. The results of the different chemometric analyses were completely consistent with each other, and could be supported by the quantification results. [ABSTRACT FROM AUTHOR]

The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches. [ABSTRACT FROM AUTHOR]