Objective:To clarify the transitional components in the blood of compound Banlangen Granules; To explore the mechanism of drugs in the treatment of epidemic encephalitis B, hepatitis and parotitis.Methods:The transitional components in blood of compound Banlangen Granules were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The regulatory targets and pathways of compound Banlangen Granules in the treatment of epidemic encephalitis B, hepatitis and parotitis were analyzed based on UPLC-MS/MS and network pharmacology.Results:A total of 9 blood components were identified, of which 8 were prototype components, including sucrose, o-aminobenzoic acid, uridine, adenosine, guanosine, indole-3-acetonitrile-2 murine-S-β-D-glucopyranoside and salicylic acid. Through network pharmacological analysis, it was concluded that compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.Conclusion:The 9 blood components of compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.

Objective To investigate the protective effect of Toll-like receptor (TLR)2/TLR9 agonists,Pam2 CSK4(Pam)and CpG ODN (CpG)on mice infected with Acinetobacter baumannii (Ab)in the lungs.Methods Female C57 mice (6～8 weeks old)were randomly divided into PBS,Pam,CpG and Pam+CpG groups.In 24 h after intranasal immunization with different doses of the corresponding agonists,the mice were given a lethal dose of Ab infection in the lungs,and the survival rates of the mice were observed.A sublethal dose lung infection model of Ab was then established,and the bacterial colonization in the blood,lungs,liver,kidneys and spleen was measured respectively in the mice after infection.HE staining was used to observe the pathological damages in the lungs and kidneys.The protective effect of the agonists in the immunized mice against Ab was examined at 1,3 and 7 d after immunization to explore the protective time window.Pam+CpG was used to stimulate A549 cells and RAW264.7 cells to investigate the killing or phagocytic effects on Ab.Results Compared to PBS,Pam+CpG treatment significantly improved the survival rate of the mice after a lethal dose of Ab lung infection (P<0.05,P<0.01 ),reduced bacterial colonization in the blood (P<0.01 ),lungs (P<0.01 ),liver (P<0.01 ),kidneys (P<0.01 )and spleen (P<0.01 )in the mice after sublethal challenge,and alleviated pathological damage caused by infection. Immunization at 1 or 3 d before infection significantly improved the survival rate (P<0.05 ),and the protective effect was the best in 3 d after immunization.Furthermore,compared to single PBS,Pam and CpG immunization,Pam+CpG significantly promoted the killing and phagocytic effects of A549 epithelial cells and RAW264.7 cells,respectively,against Ab (P<0.01 ).Conclusion Combined application of TLR2/TLR9 agonists exerts a significant protective effect on both lethal and sublethal infections of Ab,which might be by its promoting the killing or phagocytic effect of lung epithelial cells and macrophages against Ab.

Objective:To explore the application and practice of "flipped classroom" in the teaching of general surgery interns.Methods:A total of 20 internship groups (3 to 5 people in each group) were randomly selected from the general surgery practice group in the Department of General Surgery of the Second Clinical Medical College of North Sichuan Medical College. They were randomly divided into the flipped group (45 people) and the traditional group (40 people), with 10 subgroups in each group. The flipped group adopted the flipped classroom teaching mode (students' self-study by handing out materials before class, students and teachers' discussion in class, and students and teachers' evaluation after class), while the control group adopted the current conventional teaching mode (students' preview before class, teachers' explanation in class, and teachers' question answering after class). At the end of the teaching, a questionnaire was used to evaluate the participation and completion of each student. The teaching effect was evaluated by medical history collection and case analysis. The participation, completion, and teaching effect between the two groups were compared and analyzed. SPSS 23.0 software was used for t-test and Chi-square test. Results:The participation of the flipped group was better than that of the traditional group [(17.45±1.83) vs. (15.57±1.52)], and the difference was statistically significant ( P < 0.05). There was no statistically significant difference between the flipped group and the traditional group. There was no significant difference in medical history collection scores between the two groups. The case analysis of the flipped group was better than that of the traditional group [(87.30±6.06) vs. (81.50±5.88), P < 0.05]. The questionnaire shows that about 90% of the students think that flipped classroom can improve their interest in learning [96% (43/45)], improve their autonomous learning ability [89% (40/45)], and have better learning effect. At the same time, 78% (35/45) of students think that learning time is too long. Conclusion:The flipped classroom teaching model can improve the teaching participation of general surgery students, improve students' interest in learning, improve their self-learning ability, and improve students' thinking ability of medical record analysis.

Group A Streptococcus (GAS) is a very important pathogen, especially for children.On a global scale, GAS is an important cause of morbidity and mortality.But the burden of disease caused by GAS is still unknown in China and also has not obtained enough attention.For this purpose, the expert consensus is comprehensively described in diagnosis, treatment and prevention of GAS diseases in children, covering related aspects of pneumology, infectiology, immunology, microbiology, cardiology, nephrology, critical care medicine and preventive medicine.Accordingly, the consensus document was intended to improve management strategies of GAS disease in Chinese children.

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serA^T410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serA^T410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA^* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.
Culture Media ; Ergothioneine/metabolism* ; Escherichia coli/metabolism* ; Fermentation ; Histidine/metabolism* ; Metabolic Engineering

Objective:To explore the expressions of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) and clinicopathological characteristics in post-transplant lymphoproliferative disorder (PTLD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children, with the aim of clarifying whether checkpoint inhibition of PD-1/PD-L1 inhibitors may serve as a therapy option.Methods:The clinical data of 13 cases of PTLD after allo-HSCT pathologically confirmed in Shenzhen Children′s Hospital from January 1, 2012 to December 30, 2019 were retrospectively analyzed.The detection was performed by immunohistochemical staining by MaxVision? method, Epstein-Barr virus(EBV) in situ hybridization and lymphoma gene rearrangement.The relationship between the expression of PD-1 and PD-L1 and the clinicopathological characteristics of PTLD were analyzed.Results:The expression of PD-1 was not correlated with gender, age, primary diseases, histopathological types, transplantation mode and the expression of EBV in situ hybridization (all P>0.05). The expression of PD-L1 was correlated with histopathological types ( P<0.05). Furthermore, the expression rate of PD-L1 on severe β-thalassemia was significantly higher than that of severe aplastic anemia [90.0%(9/10 cases) vs. 66.7%(2/3 cases)] and monomorphic PTLD was higher than that of polymorphic PTLD [100.0%(2/2 cases) vs. 83.3%(5/6 cases)]. Moreover, the positive PTLD in EBV was higher than the negative PTLD in EBV [90.9%(10/11 cases) vs. 50.0%(1/2 cases)]. The positive rates of PD-1 and PD-L1 in 13 cases with PTLD were 46.2%(6/13 cases) and 61.5%(8/13 cases) in tumor cells, 92.3% (12/13 cases) and 76.9% (10/13 cases) in microenvironmental cells, and 84.6%(11/13 cases) in EBV, respectively. Conclusions:PD-L1 has a higher positive rate in tumor cells with monomorphic PTLD; and routine staining for PD-1 and PD-L1 can be performed in all types of PTLD when standard immunotherapy and chemotherapy are ineffective.

Objective:To investigate the effect of miR-200c-3p on the proliferation and apoptosis of nephroblastoma SK-NEP-1 cell and its mechanism.Methods:From January 2015 to August 2019, nephroblastoma tissue and peritumoral tissue of 30 patients in Shenzhen Children′s hospital were collected.The experimental group of mimic negative control, miR-200c-3p and miR-200c-3p inhibitor was set up.The expression of miR-200c-3p in 30 paired nephroblastoma tissues and adjacent kidney tissues were detected by real-time fluorescence quantitative PCR.The differential expression of miR-200c-3p was also detected in SK-NEP-1 and 293FT cell lines.The effects of miR-200c and miR-200c-3p inhibitor on the proliferation, cell cycle distribution and apoptosis of SK-NEP-1 cells were detected by cell counting kit-8(CCK-8)and flow cytometry assays, respectively.Xenograft tumors were generated by peri-renal adipose tissue injection to assess the effect of miR-200c-3p on tumor growth in vivo.The pathological morphology of xenograft tumors was observed by HE staining.The proliferation index of Ki-67 were detected by immunohistochemistry.Western blot method was used to detect the B-cell lymphoma 2(Bcl-2), Bcl-2 related X protein (Bax) and cleaved cysteinyl aspartate specific proteinase-3(Caspase-3)expression level on xenograft tumors. Results:The expression level of miR-200c-3p in nephroblastoma(0.420±0.587)was significantly lower than that in matched normal kidney(1.500±0.504)( t=8.613, P<0.001). The expression level of miR-200c-3p in SK-NEP-1 cells (0.363±0.006) was significantly lower than that in human embryonic kidney 293FT cells (0.807±0.186) ( t=4.136, P<0.05). The group and time interaction results of CCK-8 proved that miR-200c-3p could inhibit the proliferation of SK-NEP-1 cells( F=16.81, P<0.001). The flow cytometer test of cell cycle and apoptosis showed that miR-200c-3p could block in G0/G1 and S phase( t=-7.770, P<0.01; t=11.501, P<0.001). Moreover, it increased the early apoptosis rate and decreased the late apoptosis rate ( t=-22.270, P<0.001; t=4.612, P<0.01). A orthotopic transplantation assay was employed to evaluate the effect of miR-200c-3p and miR-200c-3p inhibitor on the proliferation of SK-NEP-1 cells.The final volume of tumor miR-200c-3p group [(0.419±0.16) cm 3]was significantly lower than those in the control group [(2.469±0.914) cm 3, t=0.507, P<0.001]. However, the miR-200c-3p inhibitor group had no significant difference [(1.627±0.189) cm 3; t=2.209, P=0.052]. miR-200c-3p overexpression upregulated expression levels of apoptotic proteins cleaved Caspase-3 and Bax ( t=-47.000, -82.730, all P<0.001), but downregulated the expression level of anti-apoptotic protein Bcl-2( t=53.740, P<0.001). Conclusions:The overexpression of miR-200c-3p can inhibit the proliferation, promote the apoptosis of the SK-NEP-1 cells and partially inhibited tumorigenicity of nude mouse acted as a tumor suppressor gene.

Novel Coronavirus Pneumonia (NCP) is a class B infectious disease, which is prevented and controlled according to class A infectious diseases. Recently, children′s NCP cases have gradually increased, and children′s fever outpatient department has become the first strategic pass to stop the epidemic. Strengthening the management of the fever diagnosis process is very important for early detection of suspected children, early isolation, early treatment and prevention of cross-infection. This article proposes prevention and control strategies for fever diagnosis, optimizes processes, prevents cross-infection, health protection and disinfection of medical staff, based on the relevant diagnosis, treatment, prevention and control programs of the National Health and Health Commission and on the diagnosis and treatment experience of experts in various provinces and cities. The present guidance summarizes current strategies on pre-diagnosis; triage, diagnosis, treatment, and prevention of 2019-nCoV infection in common fever, suspected and confirmed children, which provide practical suggestions on strengthening the management processes of children′s fever in outpatient department during the novel coronavirus pneumonia epidemic period.

ABSTRACT Objective: To investigate the expression and mechanism of microRNA (miRNA)-613 in breast cancer. Methods: A total of 91 specimens of breast cancer tissue were collected from Nanchong Central Hospital between May 2017 and May 2018. Quantitative real-time PCR (qRT-PCR) was used to estimate miRNA-613 expression levels in breast cancer and adjacent tissues and breast cancer ( cells MDA-MB-231, MDA-MB-468, and MCF-7) and normal breast epithelial (HBL-100) cell lines. Based on these data, the relationship between miRNA-613 expression and clinicopathological characteristics and prognosis in breast cancer patients were analyzed using the cancer genome atlas (TCGA) database. A dual-luciferase reporter assay was used to detect the binding of miRNA-613 to the 3'UTR of SOX9. Effects on cell proliferation and cell invasion and migration upon transfection of MDA-MB-231 cells with miRNA-613 mimics were detected by the CCK-8 assay and Transwell invasion and migration assays, respectively. In addition, Western blot was used to estimate the expression levels of SOX9, β-catenin, E-cadherin, and Vimentin in the transfected cells. Results: The expression of miRNA-613 in breast cancer tissues was significantly lower than that in adjacent tissues (P<0.05) and was found to be closely related to TNM stage and lymph node metastasis (P<0.05). TCGA survival data showed that miRNA-613 expression was not related to the overall survival rate of breast cancer patients (P>0.05 ). The expression of miRNA-613 in breast cancer cell lines was significantly lower than that in the normal breast epithelial cell line (P<0.05). Similarly, the expression of miRNA-613 in highly invasive metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-468) was significantly lower than that in the metastatic breast cancer cell line MCF-7 with lower invasion ability (P<0.05). The dual-luciferase reporter assay showed that miRNA-613 could specifically bind to the 3'UTR of SOX9. Upregulation of miRNA-613 expression could inhibit the proliferation, invasion, and migration of MDA-MB-231 cells (P<0.05). This was associated with the downregulated expression of SOX9, β-catenin, and Vimentin (P<0.05) and upregulation of E-Cadherin expression (P<0.05). Conclusions: The expression of miRNA-613 was decreased in breast cancer tissues and cell lines. MiRNA-613 may inhibit the proliferation, invasion, and metastasis of breast cancer cells and epithelial-mesenchymal transition by regulating the SOX9 and Wnt/β-catenin signaling pathways.

OBJECTIVE： To investigate the rationality of TLC identification method （3） of （R，S）-epigoitrin in Isatis indigotica stated in 2015 edition of Chinese Pharmacopeia （partⅠ） （later abbreviated as pharmacopeia）， and make some improvements. METHODS： Three batches I. indigotica were collected and prepared into decoction pieces according to the processing method of I. indigotica in pharmacopoeia. TLC identification of （R，S）-goitrin in I. indigotica decoction piece and medicinal material were conducted according to identification method （3） in pharmacopeia （80％ ethanol as solvent for sample treatment， ultrasound extraction）； the rationality of pharmacopoeia method was investigated. Then the method was improved by changing the extraction solvent and pretreatment method （method one： using water as solvent， ultrasound extraction； method two： soaking in water for 1 h， then adding into methanol， ultrasound extraction； method three： the sample was wetted and then dried， using 80％ methanol as solvent， ultrasound extraction） of samples， and the optimal method was verified. According to the optimal method， the TLC identification of （R，S）-goitrin was detected by using chromatographic plates from different manufacturers， under the conditions of low temperature and low humidity （7 ℃， relative humidity 48％） and high temperature and high humidity （35 ℃， relative humidity 75％） respectively，to investigate the durability of the method. RESULTS： According to the method of pharmacopeia， in the chromatograms of decoction pieces， the same color spots appeared at the corresponding chromatographic positions of reference substance， but no corresponding spots appeared in the medicinal material chromatograms. After the samples were treated by three improvement methods， in medicinal material chromatograms， the same color spots appeared in the corresponding chromatographic positions of reference substances. There were single chromatographic spot after medicinal materials were treated with method one， and there were more spots after medicinal materials were treated with method two and three， and method two consumed less time than method three. The results of validation tests and method durability tests  showed that after the treatment of I. indigotica and its decoction pieces according to method two， the same color spots appeared in the corresponding positions of the decoction pieces and the medicinal materials chromatograms as those of the control. CONCLUSIONS： The improved TLC identification method is effective， the chromatographic spots are clear， and the repeatability is good.