Objective To investigate the role of Wnt/β-catenin pathway in regulating allergic airway inflammation in asthmatic mice.Methods We induced dendritic cells (DCs) from bone marrow of BALB/c mice,and then treated the cells with LiCl and PKF118-310,separately.We observed the morphological features of DCs under light microscope.Mixed lymphocyte reaction (MLR) was used to observe the functional changes of DCs.Western blot was used to detect the expressions of GSK-3β and β-catenin at the protein level.We established a mouse asthma model by using ovalbumin (OVA),and then treated these mice with LiCl and PKF118-310.The total number of cells and eosinophil percentage in BALF were determined.The lungs of mice were observed by HE staining to evaluate the degree of allergic inflammation.The cytokines in BALF and spleen cells supernatant were assayed by enzyme-linked immunoassay (ELISA),and the total IgE in the serum was also measured by ELISA.The protein expression levels of GSK-3β and β-catenin in lung tissue were assayed by Western blot.Results ① The DCs treated with LiCl promoted the proliferation of allogeneic T lymphocytes in MLR more weakly than those treated with PKF118-310 (P＜0.01).② The GSK-3β protein expression level of DCs treated with LiCl was significantly lower than DCs treated with PKF118-310.In contrast,the β-catenin protein expression of DCs treated with LiCl was higher than that of DCs treated with PKF118-310 (P ＜ 0.01).③ The total number of cells and eosinophil percentage in BALF were significantly increased in the experimental group compared with those in the control group (P＜0.01).There was also a significant difference between LiCl group and PKF118-310 group (P＜0.01).④ In the three experimental groups,the severity of inflammation in the lungs of LiCl group was weaker than that in PKF118-310 group (P＜0.05).⑤ Compared with that in the normal control group,IL-4 in BALF and spleen cell culture supernatant of the experimental group was significantly higher while IFN-γ was the opposite (P＜0.01).LiCl group had the lowest level of IL-4 and the highest level of IFN-γ;PKF groups was the opposite (P＜0.05).⑥ The total IgE in serum was significantly increased in the experimental group compared with the control group (P＜0.01).There was also a significant difference between LiCl group and PKF118-310 group (P＜0.05).⑦ GSK-3β protein expression was significantly lower in LiCl group than in PKF118-310 group (P＜0.05),while β-catenin protein expression was significantly higher in LiCl group than in PKF118-310 group (P＜0.05).Conclusion LiCl and PKF118-310 can affect the severity of asthma by regulating Wnt/β-catenin signal pathway and the expressions of GSK-3β andβ-catenin protein,which provides a new direction for asthma treatment.

BACKGROUNDTo study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line.

METHODS801D cell line with p53 deletion and mutation at 248 codon was selected as a parent cell line. An 1.8 kb human p53 full length cDNA was inserted into a mammalian expression vector PEGFP to construct a p53 antisense RNA recombined plasmid PEGFP-p53(AS) and GFP gene at plasmid was a report gene to monitor extraneous gene expression. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohitochemical stain of p53 monoclonal antibody. The inhibition growth efficacy of extraneous p53 in vitro was determined by clonogenic survival assay. Sensitivity of cells to cisplatin was examined with MTT assay. FCM analysis was performed to measure the effect of p53 antisense RNA on cell cycle.

RESULTSTwo cell lines, PEGFP-p53(AS)-801D and PEGFP-801D, were established after transfection of 801-D cells by lipofection and selection. Presence of extraneous p53 gene in PEGFP-p53(AS)-801D was proved by PCR and expression of extraneous p53 was estimated when green fluorescence in those cells was found out under the fluorescent microscopy. Mutated p53 protein in parent cell line 801D was positive and in PEGFP-p53(AS)-801D was negative with immunochemical stain. The inhibition rate of colony formation was 61% for PEGFP-p53(AS)-801D (P < 0.001). The sensitivity of PEGFP-p53(AS)-801D cells to cisplatin was increased. FCM analysis showed that the cell line was arrested at G1 phase.

CONCLUSIONSp53 mutation at 248 code plays an important role on malignant growth and resistance to cisplatin of human lung cancer cell line 801D. Malignant growth of cells with p53 deletion and mutation at 248 codon can be inhibited by extraneous p53 antisense RNA, and simultaneously the sensitivity to cisplatin is also increased.

BACKGROUND AND OBJECTIVEThe p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot.

METHODSUsing pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E. coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infected-virus was detected by FCM scatter plot.

RESULTSp53 recombinant adenoviruses named Ad-p53(wtp), Ad-p53(del) and Ad-(empty carrier) were produced. Results of sequences indicate that the Ad-p53(del) was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53(wtp) no loss of any p53 base, the Ad-(empty carrier) no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity.

CONCLUSIONThe preparation of recombinant adenovirus, Ad-p53(del), pA-p53(wtp) and Ad-(empty carrier). The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells.

Adenoviridae ; genetics ; Animals ; Flow Cytometry ; methods ; Genes, p53 ; Green Fluorescent Proteins ; genetics ; Humans ; Mice ; Recombination, Genetic

BACKGROUNDTo study the inhibition effects of both extraneous right sense and antisense p53 on malignant phenotype of human lung cancer cell line.

METHODSThe named 801D cell line with p53 deletion and mutation at 248 code was selected as a model in vitro. The recombined plasmid pEGFP-p53(RS) and pEGFP-p53(AS) were constructed. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohistochemical stain of p53 antibody. The inhibition effect of extraneous p53 on tumor growth in vitro were determined by clonogenic survival assay. FCM analysis was carried out in cells. The inhibition effect on malignant growth of extraneous p53 in vivo was observed by heteroplastic transplant on nude mouse.

RESULTSThe transfected cell lines, pEGFP-p53(AS)-801D, pEGFP-p53(RS)-801D and pEGFP-801D were established. Presence of extraneous p53 and neo genes in pEGFP-p53(AS)-801D and pEGFP-p53(RS)-801D was proved by PCR and green fluorescence was found out in those cells under the microscope. Mutant protein in pEGFP-p53(AS)-801D was negative by immunohistochemical stain. The malignant growth of these transfected cell lines was inhibited comparing with parents in vivo and in vitro. Inhibition rate of colony formation was 62.0% for pEGFP-p53(AS)-801D and 80.8% for pEGFP-p53(RS)-801D. The tumorigenicity in nude mice was suppressed. Inhibition effects of extraneous right sense p53 on malignant growth of 801D was more distinct. FCM analysis showed that pEGFP-p53(AS)-801D cells were arrested at G1 phase.

CONCLUSIONSThe transfected cell lines with extraneous right sense and antisense p53 appear that malignant growth can be inhibited in vivo and in vitro.

Electrocardiogram (ECG) signal is an important basis for the diagnosis of arrhythmia and myocardial infarction. In order to further improve the classification effect of arrhythmia and myocardial infarction, an ECG classification algorithm based on Convolutional vision Transformer (CvT) and multimodal image fusion was proposed. Through Gramian summation angular field (GASF), Gramian difference angular field (GADF) and recurrence plot (RP), the one-dimensional ECG signal was converted into three different modes of two-dimensional images, and fused into a multimodal fusion image containing more features. The CvT-13 model could take into account local and global information when processing the fused image, thus effectively improving the classification performance. On the MIT-BIH arrhythmia dataset and the PTB myocardial infarction dataset, the algorithm achieved a combined accuracy of 99.9% for the classification of five arrhythmias and 99.8% for the classification of myocardial infarction. The experiments show that the high-precision computer-assisted intelligent classification method is superior and can effectively improve the diagnostic efficiency of arrhythmia as well as myocardial infarction and other cardiac diseases.
Humans ; Electrocardiography ; Heart Diseases ; Myocardial Infarction/diagnostic imaging* ; Algorithms ; Electric Power Supplies

Objective : To investigate the expression characteristics of mu opioid receptor ( MOR) in rat colon smooth muscle cells by cultured rat primary colonic smooth muscle cells . Methods : colonic smooth muscle cells were isolated , cultured and identified ; immunofluorescence double labeling method was used to observe the distribution characteristics of MOR , Endomorphin⁃2 (EM2) , and calmodulin (CaM) in colonic smooth muscle cells ; Western Blot method was used to detect the expression of MOR and EM2 in smooth muscle cells of the colon . After the intervention measures Acetylcholine (ACh) ( 1 × 10 - 3 mol/L) and EM2 (2 μmol/L) were applied , the changes of CaM protein expression were observed ; The calciumion imaging method was used to detect the changes of calciumi on concentration in smooth muscle cells. Results: The colonic smooth muscle cells were cultured and identified . The positive cells labeled with α ⁃smooth muscle actin ( α ⁃SMA) accounted for more than 95% of the total number of cells . Immunofluorescence double labeling showed that there were MOR and EM2 distributions in colonic smooth muscle cells , and all MOR and EM2 positive cells coexisted with α ⁃SMA . Western Blot results showed that there were MOR and EM2 expressions in colonic smooth muscle cells . CaM in the ACh group significantly increased at 10 minutes (P < 0. 05) , CaM in the EM2 group significantly decreased ( P < 0. 05) ; The calciumion imaging results showed that alone applied ACh , the calciumion concentration in smooth muscle cells significantly increased ( P <0. 05) ; Alone applied EM2 , the calciumion concentration in colonic smooth muscle cells was down⁃regulated (P <0. 05) ; Applied ACh and EM2 sequentially , EM2 significantly reduced the increase of the calciumion concentration in smooth muscle cells induced by ACh (P < 0. 05) . Conclusion MOR and EM2 are expressed in colonic smooth muscle cells , and EM2 may inhibit the expression of CaM and reduce the concentration of calcium ions through MOR .

Objectives: To understand the status of studies about influenza economic burden in mainland China and summarize their major results. Methods: The words of influenza, flu, cost, economic, burden, effectiveness, benefit, utility, China, and Chinese, were used as search keywords. Journal papers published during 2000-2018 were searched from Chinese electronic databases (CNKI and Wanfang) and English electronic databases (PubMed, Web of science, EconLit and Cochrane Library). The language of literature was restricted to Chinese and English. A total of 23 effective documents were included, and the descriptive characteristics, research indexes and methods included in the literature were analyzed. The monetary unit used in this review is Chinese Yuan (CNY). Results: The 23 study sites were mainly in the relatively developed and populous regions. The total cost per capita of laboratory-confirmed influenza,of all age-group was reported in 6 literatures, and only 4 literatures reported it in out-patients (range: 768.0-999.9 CNY), Only one study reported this indicator in inpatients (9 832.0 CNY). One literature reported the total cost per capita of influenza-like illness,, which was 205.1 CNY. And one literature reported that the direct medical cost of inpatients per capita in children under 5 years of age was 6 072.0 CNY while two literature reported this index for the elderly over 60 years of age, ranging from 14 250.0 to 19 349.1 CNY. Four articles reported the economic burden of influenza in urban and rural areas, one of which showed that the related expenses of urban influenza inpatients accounted for 31% of the average annual income, while which for the rural flow was 113%. Conclusion The average economic burden of lab-confirmed influenza case is higher than that of influenza-like illness, and there are differences in outpatient indirect expenses and inpatients direct medical expenses. The direct medical burden for the hospitalized 60-years-and-beyond influenza case group is heavier thar other age group. By region, the influenza associated individual economic burden in rural area is higher than that of urban area..

The oral microbiota is associated with oral diseases and digestive systemic diseases. Nevertheless, the causal relationship between them has not been completely elucidated, and colonisation of the gut by oral bacteria is not clear due to the limitations of existing research models. The aim of this study was to develop a human oral microbiota-associated (HOMA) mouse model and to investigate the ecological invasion into the gut. By transplanting human saliva into germ-free (GF) mice, a HOMA mouse model was first constructed. 16S rRNA gene sequencing was used to reveal the biogeography of oral bacteria along the cephalocaudal axis of the digestive tract. In the HOMA mice, 84.78% of the detected genus-level taxa were specific to the donor. Principal component analysis (PCA) revealed that the donor oral microbiota clustered with those of the HOMA mice and were distinct from those of specific pathogen-free (SPF) mice. In HOMA mice, OTU counts decreased from the stomach and small intestine to the distal gut. The distal gut was dominated by Streptococcus, Veillonella, Haemophilus, Fusobacterium, Trichococcus and Actinomyces. HOMA mice and human microbiota-associated (HMA) mice along with the GF mice were then cohoused. Microbial communities of cohoused mice clustered together and were significantly separated from those of HOMA mice and HMA mice. The Source Tracker analysis and network analysis revealed more significant ecological invasion from oral bacteria in the small intestines, compared to the distal gut, of cohoused mice. In conclusion, a HOMA mouse model was successfully established. By overcoming the physical and microbial barrier, oral bacteria colonised the gut and profiled the gut microbiota, especially in the small intestine.
Animals ; Bacteria ; Gastrointestinal Microbiome ; Germ-Free Life ; Humans ; Mice ; Microbiota ; RNA, Ribosomal, 16S

Objective: To systematically review the mortality burden study of influenza in mainland China. Method: "influenza", "flu", "H1N1", "pandemic", "mortality", "death", "fatality", "burden", "China" and "Chinese" were used as keywords, and a systematic literature search was conducted to identify articles in three English databases (PubMed, Web of Science and Embase) and three Chinese database (CNKI, WanFang and VIP) during 1990-2018 (excluding Hong Kong, Macao and Taiwan). The language of literature was restricted to Chinese and English. The inclusion criteria were human-oriented researches with method based on population, and research indexes included mortality and excess mortality. The exclusion criteria were non-primary research materials, predictive research and research on the burden of avian influenza related deaths. A total of 17 literatures were included, and the basic information to descriptive characteristics, methodology of modeling and the corresponding results were extracted. Results: All the 17 studies adopted indirect statistical models, with 14 of which adopted the regression model, and all the research index was excess mortality. All causes (16 studies), respiratory and circulatory diseases (14 studies) and pneumonia and influenza (10 studies) were the main causes of death associated with influenza. Influenza associated mortality burden in the elderly was higher, with the lowest excess mortality rates of all causes, respiratory and circulatory diseases, pneumonia and influenza being 49.57, 30.80 and 0.69 per 100 000 people, and the highest rates being 228.16, 170.20 and 30.35 per 100 000 people, respectively. In the non-elderly, the corresponding lowest rates were -0.27, -0.08 and 0.04 per 100 000 people respectively, and the highest rates were 3.63, 2.6 and 0.91 per 100 000 people, respectively. The influenza-related excess mortality was higher in the north, with a minimum of 7.8 per 100 000 and a maximum of 18.0 per 100 000, and slightly lower in the south, with a minimum of 6.11 per 100 000 and a maximum of 18.7 per 100 000. There were also differences in deaths caused by different influenza virus subtypes, with influenza A(H3N2) and influenza B virus possibly posing a heavier mortality burden. Conclusions Studies on influenza mortality burden is mainly based on indirect model and urban level in China. The mortality burden of influenza in the elderly, the northern and subtype A(H3N2) and B were more severe.

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate,