Objective To develop a sensitive, specific, rapid and easy method for detecting E. coli O157 : H7 and other Shiga toxin-producing E. coli in food and clinical specimens. Methods A circular probe and capture probe specific for Shiga toxin-2 (stx2) gene had been synthesized and was used for determining the sensitivity of ramification amplification method (RAM). Different serotypes which contained stx2 gene, including an E. coli O157 : H7, an E. coli O46 : H38, an E. coli O111 : NM, an E. coli O22 : H8 and E. coli ATCC23716 (stx2 gene negative) were used for determining the specificity. All strains were detected by RAM to determine whether they contained stx2 gene. Real-time RAM was further studied to detect stx2 gene. Results The lowest number targets detected by RAM assay was 10 copies of stx2, indicating that RAM assay was as sensitive as conventional PCR. The result of specificity showed that different serotypes of strains were all positive for stx2 gene, while nonpathogenic E. coli ATCC23716 was negative. Among 32 isolates, 28 STEC isolates containing stx2 gene were positive by RAM assay, while others were negative. The RAM results were 100% in concordance with that of PCR. The real-time RAM results also showed that as many as 10 bacteria can be detected and the time of appearance of detectable signal was depended on the target concentration. Conclusion RAM assay can offer an alternative method for PCR to detect E. coli O157 : H7 and STEC in all types of specimens because of its simplicity and isothermal amplification.

AIM: To observe the changes of serum nitric oxide and the production level of IL-1 in different period of coxsackievirus B 4 (CB 4V)-induced insulin-dependent diabetic mice.METHODS: The insulin-dependent diabetes mellitus (IDDM) animal model induced by CB 4V infection was established. Serum nitric oxide level was estimated by nitrate reductase method after infection 72 h,1 week, 3 weeks,6 weeks,8 weeks, respectively. At the same time, level of IL-1 produced by peritoneal M? was measued.RESULTS: (1) Changes of serum nitric oxide: serum nitric oxide level in control group remained normal level. The serum nitric oxide level in diabetic group increased significantly at 72 h after infection(P0.05). (2) IL-1 activities: IL-1 activities were increased obviously from 72 h to 3 weeks after virus infection, but decreased to normal level after 6 weeks.CONCLUSION: Nitric oxide may be one of the important factors in the development of CB 4V-induced IDDM.

Objective To investigate current situation of otoscope use by practitioners at community health-care service facilities in Hainan and Shenzhen and explore its importance and feasibility of spread of its use in community health-care service facilities in China.Methods Group discussion, telephone personal interview, otoscope market survey and literature research all were used among 112 practitioners at 67 community health-care service facilities in Hainan, 47 practitioners at 23 community health-care service facilities in Shenzhen, Guangdong, and 11 representatives involved in general practice training from 11 districts.Results In Hainan, only one community health-care service facility ( 1.5% ) was equipped with otoscopes and only one practitioner (0.9% ) could use it.In Shenzhen, two community health-care service facilities (8.7%) were equipped with it, but which had never be used.All the practitioners at community health-care service facilities surveyed have not been trained for its use, so most practitioners there did not have basic skills to use otoscope.Conclusions Otoscope is not equipped at most community health-care service facilities in Hainan and Shenzhen, and it is little used there even equipped with it.Otoscopy should be used as an adequate and feasible technique at community health-care service and be spread among general practitioners in China.

Objective:To prepare the immunoglobulin Y(IgY) against Avain Influenza Virus (H5N1) and to investgate its inhibitory activity to Influenza virus A (FM1).Methods:Iimmunized by Avain Influenza vaccine,IgY against Avain Influenza Virus was purified from the yolk and then the antibody was enriched by the means:caprylate acid-two-step salt precipitation-gel chromatograph.TD0 of IgY and the inhibory effectiveness were showed by nhibiting cytopathic effect (CPE) thst in MDCK infected with Influenza virus A(FM1).Results:IgY against Avain Influenza Virus was obtained.The TD0 value of IgY against Avain Influenza to MDCK cell line was 1.764 mg/ml,while at the dosage as low as 0.082 8 mg/ml the lnfluenza virus A(FM1) was still inhibited in vitro.Connchusion:Caprylate acid-two-step salt precipitation-gel chromatograph can prepare IgY successfully and the inhibitory effectiveness of IgY is good for inhibition of Influenza virus A(FM1).

Background: Insomnia often responds to the orexin receptor antagonist suvorexant. This study aimed to evaluate the efficacy and adverse events of suvorexant for Chinese patients with primary insomnia over 6 months. Methods: A total of 120 patients with primary insomnia were assigned randomly to two groups that received placebo or suvorexant (40 mg) for 6 months. The primary outcomes were the total sleep time (sTST), time to sleep onset (sTSO), and sleep quality (sQUAL). The secondary outcomes were the Insomnia Severity Index (ISI) score and adverse events. Results: A total of 111 patients completed the study and all of them were included in the final analysis. Suvorexant showed greater efficacy than the placebo in enhancing sTST, sTSO, sQUAL and ISI score at months 1 and 6. Serious adverse events were documented in 2 patients (3.3%) in the suvorexant group and 1 patients (1.7%) in the placebo group. The most common adverse event was somnolence, which occurred in 7 patients (11.7%) in the suvorexant group and 2 patients (3.3%) in the placebo group. No death related to suvorexant treatment was recorded. Conclusions: Suvorexant was efficacious and well-tolerated in a group of Chinese patients with primary insomnia over 6 months.
Sleep Initiation and Maintenance Disorders

BACKGROUND: Mesenchymal stem cells (MSCs) are an important component of the in vivo microenvironment and act on multiple biological behaviors of tumor cells. The potential clinical value of MSCs has become an issue of concern in recent years.OBJECTIVE: To investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with umbilical cord-derived MSCs (UC-MSCs) using cDNA microarray.METHODS: In vitro co-culture system was constructed, and then cellular proliferation, apoptosis and differentiation status of NB4 cells treated with UC-MSCs were evaluated. Two cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without UC-MSCs. The probes were labeled with fluorescence dyes individually, hybridized with cDNA microarray, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in two gene expression profiles.RESULTS AND CONCLUSION: UC-MSCs promoted the proliferation and differentiation, while reduced the apoptosis of NB4 cells. The analysis of gene expression profiles indicated that after co-culture with UC-MSCs, 530 genes were up-regulated and 53 genes were down-regulated. Accordingly, specific gene function and pathway signaling related were also regulated to some extent. Overall, UC-MSCs influence can major biological behaviors of NB4 cells by changing expression of a large amount of genes, gene-related function and multiple intracellular signaling pathways.

Objective　To establish a dual droplet digital PCR (ddPCR) assay for herpes simplex virus type I (HSV-1) and varicella-zoster virus (VZV). Methods The specific primers and probes were derived based on the conserved regions of HSV-1 and VZV genome. The primer-probe combinations were screened, and the annealing temperatures and primer-probe concentration ratios of the dual-droplet digital PCR reaction were optimized to establish a dual-droplet digital PCR reaction system for HSV-1 and VZV, which was tested for other viruses and validated for clinical samples. The sensitivity, specificity, and reproducibility of the established dual microtiter digital PCR method were analyzed. Results The optimal concentrations of primers and probes for the dual ddPCR detection method of HSV-I and VZV were determined to be 800 nmol/L and 250 nmol/L, respectively, with an optimal annealing temperature of 56 ℃. The correlation coefficient (R2) of the standard curve of the dual ddPCR assay was 0.99, showing a clear linear relationship. The method showed high sensitivity, with the lowest detection limit of herpes simplex virus type I being 2.97 copies/μL, and for VZV being 2.73 copies/μL. The repeatability was high with a small coefficient of variation and stable detection results; the specificity was excellent, and no cross-reaction was found with herpes simplex virus type Ⅱ, Epstein-Barr virus, Adenovirus, Coxsackievirus (CA6/CA10/CA16), Cytomegalovirus, Human Cytomegalovirus, Human enterovirus 71, Japanese Encephalitis virus, West Nile virus, Measles virus, Mumps virus, and human nucleic acids. Conclusions The dual droplet digital PCR assay for herpes simplex virus type I and varicella-zoster virus established in this experiment has strong sensitivity, specificity, and high repeatability, and can provide a solution for rapid quantitative detection of the two viruses in different scenarios.

Objective To investigate the pathogenesis of the production of anti-neutrophil cytoplasmic antibodies (ANCA) in the rat models of chronic bronchitis (CB) with recurrent infections. Methods The CB models were made by double element of smoking and lipopolysaccharide (LPS) stimulation. The rats were divided into four groups, including normal control group (n=5), phorbol-12-myristate-13-acetate (PMA)-treated healthy rats control group (n=5), CB rats group (n=5) and PMA-treated CB rats group (n=6). Renal function of rats was detected. The histopathological lung and kidney tissues were observed by HE staining of paraffin section. Immunological markers, including myeloperoxidase anti-neutrophil cytoplasmic antibodies (MPO-ANCA), proteinase 3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA) and citrullinated histone H3 (CitH3), were measured by enzyme-linked immune-sorbent assay (ELISA) at different time points. Correlation between CitH3 and MPO-ANCA was analyzed by the Spearman rank correlation. NETs components were further detected in lung and kidney tissue by confocal immunofluorescence and colocalization analysis. Results (1) The serum levels of CitH3 and MPO-ANCA in CB+PMA group showed an increased trend. Compared with those in the normal control group and CB rats group, the serum levels of CitH3 and MPO-ANCA in CB+PMA group increased significantly at the sixth week (both P＜0.05). Serum CitH3 levels in rats were positively correlated with serum MPO-ANCA levels (rs=0.490,P=0.024). (2) There were pathological manifestations of CB in the lung tissues of rats in CB group and CB+PMA group, and no obvious abnormalities in the lung tissues of rats in the normal control group and control group. In the rat kidney tissue of CB+PMA group, there were inflammatory cells infiltrated in the glomerular and around the renal tubules, but glomerular necrosis was not found. No obvious abnormalities were observed in the kidney tissues of rats in the normal control group, PMA-treated healthy rats control group and CB group. (3) In the lung and kidney tissues of CB+PMA group NETs could be detected by confocal immunofluorescence analysis. Conclusion CB rats with the recurrent infections can release large amounts of NETs, in which the exposure of MPO antigen will break the immune tolerance and result in the production of MPO-ANCA.

Tibetan medicine Lamiophlomis rotata(Benth.)Kudo is a plant belonging to the genus L.rotata(Benth.)in Labiatae,which is a commonly used bulk Tibetan medicine.As a first-class endangered Tibetan medicine,its sustainable utilization of resources is representative,and it is particularly important to master its species characteristics and adaptation mechanism under high Plateau adversity.This paper sorts out the habitat characteristics,morphological characteristics,population ecological characteristics,reproductive characteristics and other aspects of L.rotata(Benth.),analyzes the growth characteristics and adverse ecological factors of L.rotata(Benth.)in the Qinghai Tibet Plateau grassland,and analyzes its adaptation mechanism in drought,cold and different degraded grass from two aspects of biological characteristics and physiological characteristics.The author believes that it is necessary to study its stress resistance mechanism from many aspects,such as gene and molecular biology,and explore the suitable planting mode of monoculture,so as to provide a theoretical basis for introduction and domestication,cultivation management,resource protection and sustainable utilization.

Objective The HPLC fingerprints of different varieties of Tibetan medicine"Bangjian"were investigated to compare and analyse the differences in the chemical composition of different varieties and to further classify them,so as to provide reference for their quality control and safe clinical use.Methods HPLC method was used to establish the fingerprints of the different species of"Bangjian",and UPLC-Q-TOF-MS method was used to analyse the similarities and differences of the chemical components of the mainstream species and to identify the characteristic peaks.Chemometrics methods were used for the analysis including cluster analysis(HCA),principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Results A total of 11 chromatographic peaks were identified from 93 batches of"Bangjian"samples.The HCA and PCA methods were used to classify the 93 batches into 2 categories,and then OPLS-DA was used to classify them into 3 categories in more detail,while 4 main components were selected according to the principle of VIP>1.The results of UPLC-Q-TOF-MS and chemometric showed that the Tibetan medicine"Bangjian"could be divided into iridoids with benzoyl fragments represented by Gentiana szechenyii Kanitz and monocyclic iridoids represented by Gentiana veitchiorum Hemsl.according to their chemotypes.The latter could be divided into alpine gentian group subtype and multi-branch group ornate subtype according to the content of components.The results of the chemotypic classification proved that the traditional classification of the"Bangjian"has a material basis in science but were also flawed.Conclusion The present study demonstrates that the established HPLC fingerprint can be used to classify the"Bangjian"of the complex base elements effectively,which is expected to provide an effective reference for the improvement of quality standards of"Bangjian"and clinical safety medication.