ObjectiveTo investigate the effects of thyme herbal tea （BLX） on the proliferation of human coronavirus OC43 （HCoV-OC43） in vitro and in vivo. MethodThe chemical composition of BLX was analyzed by UPLC-MS. The cytotoxicity of BLX in HRT-18 cells and the effect of BLX treatment on the proliferation of HCoV-OC43 in cells were analyzed. Copies of viral gene were detected by real-time PCR. The effect of BLX treatment on the life cycle of HCoV-OC43 was detected by time-of-addition assay. The maximum tolerated dose of BLX and the influences of BLX on the body weight and survival time of suckling mice infected with HCoV-OC43 were determined. The expression of viral protein in the brain and lung tissue was analyzed by immunohistochemistry. ResultThere were 11 chemical components identified in BLX by UPLC-MS. BLX showed the 50% cytotoxic concentration （CC₅₀） of （13 859.56±319） mg·L^-1， the median inhibitory concentration （IC₅₀） of （1 439.09±200） mg·L^-1， and the selection index of 8.26-11.44 for HCoV-OC43 in HRT-18 cells. Compared with the cells infected with HCoV-OC43， BLX at the concentrations of 1 500， 1 000， 500 mg·L^-1 inhibited the proliferation of this virus （P<0.05， P<0.01）. BLX exhibited antiviral effect in the early stage of virus infection， and the inhibition role in the attachment stage was more significant than that in the entry stage （P<0.05）. In the suckling mice infected with HCoV-OC43， BLX at 1200 and 600 mg·kg^-1·d^-1 alleviated the symptoms， prolonged the survival period， reduced the death rate， and down-regulated the mRNA level of nucleocapsid protein in the mice. Moreover， BLX at 1 200 mg·kg^-1·d^-1 down-regulated the expression of nucleocapsid protein in the brain （P<0.01） and the lung （P<0.01）. ConclusionBLX contained multiple antiviral ingredients. It inhibited the proliferation of HCoV-OC43 both in vitro and in vivo by interference with viral attachment. This study provides theoretical reference for the treatment of acute respiratory tract infection with HCoV-OC43 and for further development and application of BLX.

AlM: The Chinese medicinal herb Hanfangji is dried roots of Stephania tetrandra S. Moore (Family, Menispermaceae). Tetrandrine and fangchinoline are two major constituents of Hanfangji and these bisbenzyltetrahydroisoquinoline alkaloids possess anti - cancer and other pharmacological activities. To facilitate further pharmacodynamic investigation of these compounds, a pharmacokinetic investigation was performed in rats and in vitro. METHODS: Pharmacokinetics of tetrandrine and fangchinoline were characterized in rats p.o. or i.v. dosing an aqueous extract of Hanfangji or the individual compound. Unbound levels of systemic exposure to these two alkaloids were assessed using in vitro studies of plasma protein binding, blood-plasma partition, and lysosomal trapping. All the study samples were analyzed by liquid chromatography/mass spectrometry.RESULTS: We found two pharmacokinetic features of tetrandrine and fangchinoline. First, the two compounds had blood levels of systemic exposure substantially higher than the respective plasma levels of systemic exposure. Second, the two compounds exhibited significantly higher systemic exposure levels after p.o. dosing an aqueous extract of Hanfangji than the respective exposure levels after p.o. dosing the individual compound, at the same compound dose levels and under the same conditions for analytical measurement and the same conditions for animal study. Unbound fractions of tetrandrine and fangchinoline in rat plasma were 2%-5% and the concentrations of the alkaloids in rat erythrocytes were 5-times higher than those in rat plasma. Lysosomal inhibitor could block their trapping in lysosomes and significantly reduce their concentrations in HEK-293 cells. CONCLUSlON: The following pharmacokinetic aspects should be noted in pharmacodynamic investigation of tetrandrine and fangchinoline: extensive binding with plasma proteins, extensive binding with erythrocytes, and trapping by lysosomes of tissue cells substantially reduce the levels of unbound tetrandrine and fangchinoline in the systemic circulation.

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.
Alcoholic Beverages ; Alcohols/analysis* ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

This article summarizes the experience in the teaching model of flipped classroom combined with group learning in the course of biostatistics in Duke University and discusses the application of this teaching model in universities and colleges in China.The study results show that this teaching model introduces group learning in flipped classroom to develop continuous peer-assisted learning.Meanwhile,this teaching model follows the process of knowledge internalization and designs four successive steps of individual readiness assessment,team readiness assessment,mini-lectures,and application activity,in order to strengthen the understanding and practical application of key points.This teaching model also establishes a transparent mechanism for individual and curriculum evaluation and feedback collection to find and solve the issues in this course.These teaching experiences provide a good reference for the application of flipped classroom in large-class teaching in China.

Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Objective To investigate the characteristics of epidemic and genotype/subtype distribution of hepatitis C virus (HCV) among entry travelers at Tengchong port,to provide references for HCV prophylaxis and treatment.Methods A total of 54 serum samples were collected from anti-HCV positive travelers at Tengchong port from June 2009 to June 2016.HCV NS5B gene was amplified using reverse transcription polyonerase chain reation (RT-PCR) and subsequently sequenced.Based on the obtained sequences and retrieved reference sequences,phylogenetic analysis was conducted to determine HCV genotype/subtype.Results HCV infection rate among entry travelers at Tengchong ports was 0.45 ％ (54/12 059).Forty five samples were successfully genotyped.Phylogenetically,HCV genotype 3b was revealed to be the predominant subtype (28.89 ％,13/45) in this population,followed by genotype 6n (20.0％,9/45),genotype 1b (17.78％,8/45),genotype 3a (13.33％,6/45),genotype 2a (11.11％,5/45),genotype 1a (2.22％,1/45) and genotype 6a (2.22％,1/45).The major genotype in Myanmar travelers was genotype 6,while in Chinese population,genotype 1 predominated.Genotype 6 in the population showed close phylogenetic relationship with strains prevalent in China and Southeast Asia.Genotype 3 was closely clustered with strains prevalent in China.Conclusions The distribution of HCV genotypes among entry travelers at Tengchong port is impacted by HCV epidemic strains both in Yunnan province and neighboring regions.This population serves as a transmitting media which may influence the epidemiological characteristics of HCV in Tengchong and neighboring areas.

AIM:To establish and characterize the patient-derived esophageal squamous-cell carcinoma xeno-graft (PDECX) in mice.METHODS:The samples of human esophageal cancer were grafted into severe combined immu-nodeficient ( SCID) mice.The xenografts were transferred to SCID mice when the first passage of xenografts grew up .The growth of tumors in the first, second and third passages was observed .HE staining was performed.The expression of CK5/6, p63 and p40 in the patient samples , and the first and third passages of the xenografts were detected by immunohisto-chemical analysis.The expression of mTOR, p-mTOR, p70S6K, p-p70S6K, Akt1, p-Akt (Ser473), Erk1/2 and p-Erk1/2 were determined by Western blot .RESULTS:The PDECX was successfully established .The positive expression of CK5/6, p63 and p40 in the xenografts was consistent with that in the patients ’ samples.The levels of phosphorylated and total proteins of proliferation-related signaling pathways were different in the xenografts from different patients .CONCLU-SION:The PDECX model adequately reflects the tumal heterogeneity that is observed in the patients .

OBJECTIVETo investigate the prevalence of serum antibodies against Japanese encephalitis virus (JEV) in bats.

METHODSBlood samples from the heart were obtained from bats captured in Guangdong and Hainan Provinces in 2013. The anti-JEV antibodies in bat sera were tested using indirect ELISA and virus neutralization test.

RESULTSA total of 201 bat serum samples were tested, in which the total positivity rate of anti-JEV antibodies was 46.27% (93/201). The positive rate of anti-JEV antibodies in bats from Hainan and Guangdong Provinces was 88.89% (48/54) and 30.61% (45/147), respectively. All the samples from Rousettus leschenaultia, Miniopterus schreibersii, Pipistrellus abramus, and Rhinolophus macrotis were positive for anti-JEV antibodies, and up to 95.56% (43/45) of the samples from Miniopterus schreibersii (from Hainan Province) yielded positive results. Of the 28 samples with positive results by indirect ELISA, 15 showed positive results in virus neutralization test (53.57%) with neutralization antibody titers ranging from 1:10 to 1:28.22.

CONCLUSIONBats from different regions and of different species can be naturally infected with JEV and have a high prevalence of anti-JEV antibodies in their sera. The role of bats in the natural cycle of JEV awaits further study.

Animals ; Antibodies, Viral ; blood ; China ; Chiroptera ; immunology ; virology ; Encephalitis Virus, Japanese ; Enzyme-Linked Immunosorbent Assay ; Neutralization Tests

Objective To investigate the prevalence of serum antibodies against Japanese encephalitis virus (JEV) in bats. Methods Blood samples from the heart were obtained from bats captured in Guangdong and Hainan Provinces in 2013. The anti-JEV antibodies in bat sera were tested using indirect ELISA and virus neutralization test. Results A total of 201 bat serum samples were tested, in which the total positivity rate of anti-JEV antibodies was 46.27%(93/201). The positive rate of anti-JEV antibodies in bats from Hainan and Guangdong Provinces was 88.89% (48/54) and 30.61% (45/147), respectively. All the samples from Rousettus leschenaultia, Miniopterus schreibersii, Pipistrellus abramus, and Rhinolophus macrotis were positive for anti-JEV antibodies, and up to 95.56%(43/45) of the samples from Miniopterus schreibersii (from Hainan Province) yielded positive results. Of the 28 samples with positive results by indirect ELISA, 15 showed positive results in virus neutralization test (53.57%) with neutralization antibody titers ranging from 1∶10 to 1∶28.22. Conclusion Bats from different regions and of different species can be naturally infected with JEV and have a high prevalence of anti-JEV antibodies in their sera. The role of bats in the natural cycle of JEV awaits further study.