Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey . Methods To detect the expression of P21 gene in COS-7 cells ( derived from the kidney of African green monkey , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern blot assay .Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA.Their silencing rate was (91.82 ±3.21)%, (82.47 ±2.48)%, (81.31 ±2.69 )% and ( 87.35 ±4.59 )%, and the protein expression was ( 11.97 ±0.70 )%, ( 20.22 ±0.65 )%, ( 23.21 ± 0.63)%and (14.42 ±0.86)%, respectively.Conclusions Four effective silencing target sequences are screened at cel-lular level , which can be used in gene silencing research of rhesus monkeys .

BACKGROUND:In the earliest stages of embryonic development and organ formation, fibroblast growth factor family members function as mediating the growth, differentiation, survival, and morphology of progenitor cels. Fibroblast growth factor mediates metabolic function, tissue repair and regeneration in mature tissues by reactivation of signal pathways.
OBJECTIVE:To summarize and explore the role of the fibroblast growth factor signaling pathway in tissues and organs.
METHODS:A computer-based online search was conducted in CNKI and PubMed databases by using the key words of “fibroblast growth factor, signaling pathway” from 2010 to 2016 and 2000 to 2016, respectively to screen the relevant literatures. The language was limited to both Chinese and English. Research progress in the fibroblast growth factor signaling pathway was summarized.
RESULTS AND CONCLUSION:A total of 47 literatures were included. Mammalian fibroblast growth factor family is composed of 18 secreted signal proteins which interact with 4 tyrosine kinase signal fibroblast growth factor receptors. Interaction of fibroblast growth factor ligand with the receptor is regulated by a protein or cofactor binding proteoglycans and extracelular proteins. Activation of fibroblast growth factor receptor mediates interaction with cytoplasmic adapter protein, RAS-MAPK, and PI3K-AKT, phospholipase Cγand STAT signaling pathway by phosphorylation on a specific tyrosine residue. Four structuraly related intracelular non-signaling fibroblast growth factors regulate the voltage-gated sodium ion channels by their interactions. Fibroblast growth factors exist in almost al tissues and organs, and developmental defects and abnormal activity of this pathway (destruction of organogenesis) is associated with damage response to injury, metabolic disorders and cancer.

Background: The purpose of this study was to extract the major areas of interest in health insurance research in Korea, and infer policy agendas related to health insurance by analyzing research keywords. Methods: For this study, 2,590 articles were selected from among 7,459 academic papers related to health insurance published between January 1987 and December 2018, which were looked up using the Research Information Sharing Service (RISS). Keyword extraction and keyword network analysis were performed using the KrKwic, KrTitle, and UCINET software. Results: First, the number of studies in the area of health insurance continued to increase in all government terms, and it was not until after the 2000s that the subjects of health insurance researches were diversified. Second, degree centrality showed that ‘medical expenditure’ and ‘medical utilization’ were consistently high–ranking keywords regardless of the government in power. Aging and long-term care insurance-related keywords were ranked higher in the Lee Myung-bak government, Park Geun-hye government, and Moon Jae-in government. Third, betweenness centrality showed the same high ranking in key topics such as medical expenditure and medical utilization, while the ranking of key keywords differed depending on the interests and characteristics of each government policy. Conclusion We confirm that health insurance as a research topic has been the main theme in Korean health care research fields. Research keywords extracted from articles also corresponded to the main health policies promoted during each government period. Efforts to systematically investigate policy megatrends are needed to plan adaptive future policies.

Objective To establish an indirect immunofluorescence assay for detection of murine norovirus ( MNV) .Methods Mouse leukaemic monocyte macrophage cell line RAW 264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells , and to make antigen glass slides .BALB/c mice were gavaged with MNV-1 (107 TCID50) and infected sera were collected as positive control .The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay ( IFA ) .80 serum samples were tested using the two methods , IFA and ELISA, and the discrepant samples were validated by Western blotting .Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred.IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection , reaching a maximum antibody concentration at 4 weeks after infection , and maintained a stable level later .The mouse serum at four weeks after MNV-1infection was used as positive quality control . Among the 80 serum samples , 27 positive and 53 negative cases were detected by IFA method , and 32 positive and 48 negative cases were detected by ELISA .The five discrepant samples were verified by Western blotting , resulted in 3 positive and 2 negative cases . The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally .IFA and ELISA detection can be selected as initial screening measures , and use Western blot assay to verify the discrepant samples .

Objective In order to establish a rhesus monkey model of p53 gene silencing, firstly we screened and determined the effective silencing targets of p53 gene at the cellular level in rhesus monkey.Methods The expression of p53 gene was detected in COS-7 cells ( derived from the kidney of the African Green Monkey, Cercopithecus aethiops).Three small hairpin RNA ( shRNA) sequences targeting rhesus monkey p53 gene were designed, analysed by bioinformatics, and inserted into lentivirus-based gene silencing constructs FUGW-TDT.The plasmids of p53-RNAi and control vector were transfected into the COS-7 cells, respectively.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western blot assay.Results p53 gene expression was detected in COS-7 cells.Bioinformatics analysis showed that three gene-silencing sequences were screened which lied in the open reading frame ( ORF) region and targeted 238 -258bp, 681 -701bp, 169 -189bp of the rhesus monkey p53 mRNA.At 48 hrs after transfection of the three silencing constructs, p53 mRNA was suppressed by(87.17 ±4.03)%, ( 72.62 ±4.11)% and(76.22 ±0.98 )%, and p53 protein was suppressed by ( 84.44 ±2.18 )%, ( 71.04 ±1.18)% and ( 74.17 ±0.95 )%, respectively. Conclusions We obtained three effective target sequences showing high efficiency in p53silencing, which can be used in further studies on gene silencing in rhesus monkey.

Chronic cough is one of the common symptoms of childhood respiratory diseases, which cause serious impact to children and parents in the growth and development of children, life, learning and other aspects.The top three causes of childhood chronic cough are cough variant asthma(CVA), upper airway cough syndrome(UACS), post-infectious cough(PIC). CVA is the most common.However, in recent years, the literature reports about the first cause of UACS higher than CVA gradually appear and show an increasing trend.This paper summarizes the literature related to the etiological composition of chronic cough in Chinese children, and analyzes the etiological changes and causes of chronic cough, so as to help clinicians recognize the etiological trends of chronic cough, and timely make correct diagnosis and corresponding treatment programs.

Objective To investigate the role of nicorandil pretreatment on protecting myocardium after coronary microembolization (CME) and on the PDCD4/NF-κB/TNF-α signaling pathway in miniature pigs. Methods Fifteen Bama miniature pigs were randomly(random number) divided into the sham operation group (sham group), microembolization group (CME group) and CME plus nicorandil group, with 5 pigs in each group. The CME model was constructed by injecting polyethylene microspheres via microcatheter into the left anterior descending artery, and pigs in the sham group were injected with the same amount of saline. Pigs in the CME plus nicorandil group were injected intravenously with nicorandil (150 μg/kg) via ear vein 30 min before CME. Cardiac function indexes were measured using cardiac ultrasonography. The expression of PDCD4 and TNF-α mRNA in myocardium were detected by fluorescence quantitative PCR, and the protein expression of PDCD4 and TNF-α in myocardium were detected by Western blotting. NF-κB activation was evaluated by electrophoretic mobility shift assay. Results (1) Cardiac function was significantly lower and the level of serum cTnI was significantly higher in the CME group compared with the sham group. CME reduced myocardial systolic dysfunction and left ventricular dilatation. The CME plus nicorandil group showed improved CME-induced cardiac function and reduced serum cTnI level when compared with the CME Group (P < 0.05). (2) Compared with the CME group, the CME plus nicorandil group showed lower PDCD4 and TNF-α expression and NF-κB activity as well as improved cardiac function (P < 0.05). Conclusions The pretreatment of nicorandil effectively reduced the myocardial damage caused by CME, mainly through inhibiting the PDCD4/NF-κB/TNF-α pathway in cardiomyocytes.

Objective:To evaluate the related factors of gallstones related gallbladder intraepithelial neoplasia(GBIN) and establish the prediction models for gallstones related GBIN.Methods:The clinicopathological data of 750 patients who underwent cholecystectomy for gallstones at Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi′an Jiaotong University from January 2013 to December 2018 and the postoperative pathological examination showed chronic cholecystitis or GBIN were analyzed retrospectively,including 150 cases of gallstones with GBIN and 600 cases of gallstones with chronic cholecystitis.There were 264 males and 486 females with age of (51.3±14.5) years (range: 18 to 90 years).The related factors for gallstones related GBIN were screened by χ 2 test and Logistic regression model,and the prediction models were established based on independent related factors and internal validation was conducted.The original data were randomly divided into a training cohort(526 cases) and a validation cohort(224 cases) at a ratio of 7∶3,and the nomogram and tree augmented na?ve Bayes were conducted to establish the prediction model for gallstones related GBIN.The consistency index(C-index),calibration chart,area under the receiver operating characteristic curve(AUC) and confusion matrix were used to evaluate the prediction performance of the two models. Results:Univariate analysis showed that age,gallstones history(years),gallbladder size,whether the gallbladder mucosa smooth or not,whether the gallbladder wall thickened or not,gallstones diameter,and number of gallstones were related factors for the occurrence of gallstones related GBIN (χ2=19.957,8.599,9.724,9.301,8.341,15.288,9.169,all P<0.05).Multivariate analysis showed that age ( OR=2.23,95% CI:1.50-3.31, P<0.01),gallbladder size ( OR=2.11,95% CI:1.17-3.80, P=0.013),whether the gallbladder mucosa smooth or not ( OR=1.80,95% CI:1.13-2.88, P=0.014),gallstones diameter( OR=2.98,95% CI:1.71-5.21, P<0.01),and number of gallstones ( OR=2.14,95% CI:1.34-3.42, P<0.01) were independent related factors for the occurrence of gallstones related GBIN; the C-index of the nomogram in training cohort and validation cohort were 0.708 and 0.696,respectively.The AUC of the two models in training cohort were 70.60% and 70.73%,and in validation cohort were 68.14% and 67.47%,respectively.The accuracy of the two models in training cohort were 69.96% and 70.72%,and in validation cohort were 66.96% and 67.41%,respectively. Conclusion:Age,gallbladder size,whether the gallbladder mucosa smooth or not,gallstones diameter and number of gallstones are independent related factors for the occurrence of gallstones related GBIN,and the nomogram and tree augmented na?ve Bayes prediction models based on the above factors can be used to predict the occurrence of GBIN.

Objective:A subunit vaccine against SARS-CoV-2 BA.2 variant was prepared by prokaryotic expression system and its immunogenicity was evaluated.Methods:The recombinant plasmid of BA.2 variant RBD and the tandem of RBD and TT-P2 epitopes was constructed by molecular cloning technology, and recombinant proteins So and Sot were obtained by protein purification technology. Mice were immunized by intramuscular injection after mixing protein So/Sot as immunogens with Al(OH) 3 adjuvant to evaluate the effect of cellular and humoral immunity. Results:High purity soluble protein were obtained by dialysis and renaturation after expressed by prokaryotic system. The number of antigen-specific T lymphocytes secreting IFN-γ and IL-4 (213.7±0.6 and 311.7±1.5) in the Sot group induced by mice was significantly higher than that in the So group (94.3±16.8 and 185.7±4.2) ( P<0.001). The serum antibody level induced by Sot group was higher than that in the So group, the geometric mean titer (GMT) of neutralizing antibodies against BA.2 strain were 588 and 337, respectively ( P<0.05). Sot protein induced Th1/Th2 mixed type immune response with the predominance of Th2 type. Conclusions:The protein subunit vaccine expressed by the prokaryotic system have excellent cellular and humoral immunogenicity, which provides a strong theoretical basis for the development of the protein subunit vaccines of the SARS-CoV-2 Omicron variant.

Objective:To evaluate the related factors of gallstones related gallbladder intraepithelial neoplasia(GBIN) and establish the prediction models for gallstones related GBIN.Methods:The clinicopathological data of 750 patients who underwent cholecystectomy for gallstones at Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi′an Jiaotong University from January 2013 to December 2018 and the postoperative pathological examination showed chronic cholecystitis or GBIN were analyzed retrospectively,including 150 cases of gallstones with GBIN and 600 cases of gallstones with chronic cholecystitis.There were 264 males and 486 females with age of (51.3±14.5) years (range: 18 to 90 years).The related factors for gallstones related GBIN were screened by χ 2 test and Logistic regression model,and the prediction models were established based on independent related factors and internal validation was conducted.The original data were randomly divided into a training cohort(526 cases) and a validation cohort(224 cases) at a ratio of 7∶3,and the nomogram and tree augmented na?ve Bayes were conducted to establish the prediction model for gallstones related GBIN.The consistency index(C-index),calibration chart,area under the receiver operating characteristic curve(AUC) and confusion matrix were used to evaluate the prediction performance of the two models. Results:Univariate analysis showed that age,gallstones history(years),gallbladder size,whether the gallbladder mucosa smooth or not,whether the gallbladder wall thickened or not,gallstones diameter,and number of gallstones were related factors for the occurrence of gallstones related GBIN (χ2=19.957,8.599,9.724,9.301,8.341,15.288,9.169,all P<0.05).Multivariate analysis showed that age ( OR=2.23,95% CI:1.50-3.31, P<0.01),gallbladder size ( OR=2.11,95% CI:1.17-3.80, P=0.013),whether the gallbladder mucosa smooth or not ( OR=1.80,95% CI:1.13-2.88, P=0.014),gallstones diameter( OR=2.98,95% CI:1.71-5.21, P<0.01),and number of gallstones ( OR=2.14,95% CI:1.34-3.42, P<0.01) were independent related factors for the occurrence of gallstones related GBIN; the C-index of the nomogram in training cohort and validation cohort were 0.708 and 0.696,respectively.The AUC of the two models in training cohort were 70.60% and 70.73%,and in validation cohort were 68.14% and 67.47%,respectively.The accuracy of the two models in training cohort were 69.96% and 70.72%,and in validation cohort were 66.96% and 67.41%,respectively. Conclusion:Age,gallbladder size,whether the gallbladder mucosa smooth or not,gallstones diameter and number of gallstones are independent related factors for the occurrence of gallstones related GBIN,and the nomogram and tree augmented na?ve Bayes prediction models based on the above factors can be used to predict the occurrence of GBIN.