Objective To develop a dual real-time fluorescent RT-PCR method for rapid detection of enterovirus(EV)and en terovirus type 71(EV71).Methods Specific primers and probes were designed and the dual real-time fluorescent RT-PCR reaction system was established.The quantitative standard curve was drawn;its sensitivity and precision were evaluated.Feces and throat swab specimens of 109 clinical patients with hand foot and mouth disease were collected and tested by using this method.Then the obtained results were compared with those detected by commercial EV71 PCR kit.Results The relative coefficient(2)of EV and EV71 standard curve established by the dual real-time fluorescent RT-PCR method were both 0.998.Its sensitivity reached 0.5 TCID50/mL for detecting EV and 0.05 TCID50/mL for detecting EV71.The within-run precision for detecting EV and EV71 was ＜3％ and total precision≤4％.The results showed good specificity for the detection of enterovirus and non-enterovirus.In 109 detected clinical samples,84 cases of EV positive samples were detected,in which 56 cases were EV71 positive with the total positive rate of 51.4 ％,which was consistent with the result of simple fluorescent RT-PCR commercialization kit(P=1.000).Conclusion The established dual real-time fluorescent RT-PCR method has high sensitivity and good stability,which has an important significance for early high throughput rapid diagnosis of hand foot and mouth disease.

OBJECTIVE: To assess the association of cytochrome P450 (CYP450) gene polymorphisms with the occurrence of ischemic stroke (IS). METHODS: From January 2020 to August 2022, 390 IS patients treated at the Zhengzhou Seventh People's Hospital were enrolled as the study group, and 410 healthy individuals undergoing physical examination during the same period were enrolled as the control group. Clinical data of all subjects were collected, which included age, sex, body mass index (BMI), smoking history and results of laboratory tests. Chi-square test and independent sample t test were used for comparing the clinical data. Multivariate logistic regression analysis was used to analyze the non-hereditary independent risk factors for IS. Fasting blood samples of the subjects were collected, and the genotypes of rs4244285, rs4986893, rs12248560 of the CYP2C19 gene and rs776746 of the CYP3A5 gene were determined by Sanger sequencing. The frequency of each genotype was calculated by using SNPStats online software. The association between the genotype and IS under the dominant, recessive and additive models was analyzed. RESULTS: The levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C), apolipoprotein B (Apo-B) and homocysteine (Hcy) of the case group were significantly higher than those of the control group, whilst the levels of high density lipoprotein (HDL-C) and Apo-A1 (APO-A1) were significantly lower (P < 0.05). Multivariate Logistic regression analysis showed that TC (95%CI = 1.13-1.92, P = 0.02), LD-C (95%CI = 1.03-2.25, P = 0.03), Apo-A1 (95%CI = 1.05-2.08, P = 0.04), Apo-B (95%CI = 1.7-4.22, P < 0.01) and Hcy (95%CI = 1.12-1.83, P = 0.04) were non-genetic independent risk factors for the occurrence of IS. Analysis of the association between the genetic polymorphisms and the risk of IS showed that the AA genotype at rs4244285 of the CYP2C19 gene, the AG genotype and A allele at rs4986893 of the CYP2C19 gene, and the GG genotype and G allele at rs776746 of the CYP3A5 gene were significantly associated with IS. Under the recessive/additive model, dominant model and dominant/additive model, polymorphisms of the rs4244285, rs4986893 and rs776746 loci were also significantly associated with the IS. CONCLUSION TC, LDL-C, Apo-A1, Apo-B and Hcy can all affect the occurrence of IS, and CYP2C19 and CYP3A5 gene polymorphisms are closely associated with the IS. Above finding has confirmed that the CYP450 gene polymorphisms can increase the risk of IS, which may provide a reference for the clinical diagnosis.
Humans ; Cytochrome P-450 CYP3A/genetics* ; Cytochrome P-450 CYP2C19/genetics* ; Ischemic Stroke ; Cholesterol, LDL/genetics* ; Polymorphism, Single Nucleotide ; Genotype ; Apolipoproteins B/genetics* ; Gene Frequency

Objective: As cancer stem cells (CSCs) are considered as the origin of tumor development, recurrence, and drug resistance, we aimed to explore the mechanism related to modulating stemness in CSCs, thus facilitating to search for new therapeutic strategy for ovarian cancer. Methods: In this study, ovarian cancer stem cells (OCSCs) induced from cell line 3AO and A2780 were enriched in serum-free medium (SFM). The effect of SURF4 on CSC-like properties was evaluated by sphere-forming assays, re-differentiation assays, quantitative real-time polymerase chain reaction, flow cytometry, Western blotting, cell viability assays and in vivo xenograft experiments. The downstream molecule participating in SURF4 maintaining stemness was screened by RNA-sequencing and identified by the experiments of gene function. Results: SURF4 was upregulated expressed in OCSCs. Knockdown of SURF4 reduced the expression of the related stem markers (SOX2 and c-MYC), inhibited self-renewal ability, and improved the sensitivity to chemotherapeutic drugs (paclitaxel and cisplatin) in OCSCs.SURF4 knockdown also inhibited tumorigenesis in nonobese diabetic/severe combined immunodeficiency mice. BIRC3 expression was controlled by SURF4, and BIRC3 showed the similar effect as SURF4 did, and BIRC3 overexpression partially recovered stem-like properties abolished by SURF4 knockdown. Conclusion Our findings suggest that SURF4 possesses the ability to maintain stemness of OCSCs via BIRC3, and may serve as a potential target in stem cell-targeted therapy for ovarian cancer.

OBJECTIVE To study the protective mechanism of Bawei chenxiang powder containing serum on H9c2 cells injured by oxygen-glucose deprivation （OGD）. METHODS H9c2 cells were divided into blank group， model group and Bawei chenxiang powder low-dose， medium-dose and high-dose groups （the dose of drug containing serum 2.5， 8， 12 g/kg）. H9c2 cells were cultured in vitro to establish OGD model. After intervention with drug-containing serum， survival rate of cell was detected. The cell morphology was observed； the levels of lactate dehydrogenase （LDH）， creatine kinase （CK）， superoxide dismutase （SOD）， catalase （CAT）， respiratory chain complexⅠ （ComplexⅠ）， glutathione peroxidase （GSH-Px） and malondialdehyde （MDA） were detected. The contents of reactive oxygen species （ROS）， mitochondrial membrane potential and apoptosis were also detected. The expressions of oxidative stress-related proteins [Kelch ECH association protein 1 （Keap1）， nuclear factor erythroid 2- related factor 2 （Nrf2）， heme oxygenase 1 （HO-1）， NADH oxidoreductase coenzyme 10 （Ndufa10）， thioredoxin （Trx）] and apoptosis-related proteins [B-cell lymphoma 2 （Bcl-2）， Bcl-2 associated X protein （Bax）， Caspase-3 and cytochrome C （Cytc）] were detected. RESULTS Compared with blank group， the cell morphology of model group was damaged； the levels of LDH， CK and MDA were significantly increased （P＜0.01）， while the levels of CAT， ComplexⅠ， SOD and GSH-Px and mitochondrial membrane potential were significantly decreased （P＜0.01）. The content of intracellular ROS and apoptotic rate were significantly increased （P＜0.01）； the expressions of oxidative stress-related proteins （Keap1， Nrf2， HO-1， Ndufa10 and Trx） and pro- apoptosis proteins （Bax， Caspase-3 and Cytc） were significantly increased （P＜0.05）， while the expression of anti-apoptotic protein Bcl-2 was significantly decreased （P＜0.05）. After administration of Bawei chenxiang powder containing serum， the cell morphology improved， and most of the above indexes were significantly reversed （P＜0.05 or P＜0.01）.CONCLUSIONS Bawei chenxiang powder containing serum E-mail：345783110@qq.com has a good protective effect on H9c2 cells damaged by OGD，the mechanism of which is related to the reduction of oxidative damage and inhibition of cell apoptosis.

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K _D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.