A modified dye test with microplate was to be established to detect Toxoplasma antibodies with cell-cultured Toxoplasma gondii. Numbers of stained and unstained tachyzoites were estimated in every 100 tachyzoites in each well after dyeing with methylene blue. The dilution with 50% tachyzoites stained was used as final dilution. Better results of the microplate dye test has been received when the concentration of tachyzoites in suspension reaches 109/ml with 1% sodium citrate as accessory factor.

Objective: To investigate chronic kidney disease (CKD) and its risk factors in people receiving physical examination. Methods: hTis retrospective study included people over 20 years old who had physical examination in the Health Management Center of Third Xiangya Hospital from Janurary 2008 to June 2011. CKD and its risk factors as well as questionnaire were recorded. hTe risk factors were analyzed by multivariate logistic analysis. CKD was deifned by kidney damage (microalbuminuria≥30 mg/L) and/or hematuria and/or reduced kidney function [evaluate glomerular ifltration rate (eGFR)<60mL/(min.1.73 m2)]. We counted eGFR according to the modiifcation of diet in renal disease (MDRD). Results: A total of 5 708 physical examination reports were included. The detection rate of albuminuria, reduced renal function and hematuria was 25.0%, 1.7% and 1.1%. hTe detection rate of CKD was 25.6%, and detection rate of CKD stage 1-5 was 17.8%, 6.7%, 1.1%, 0 and 0, respectively. Multivariate logistic analysis indicated that diabetes mellitus, hypertension, hypercholesterolemia, male, age, and smoking were the risk factors for CKD. Increasing physical activity was the protective factor against CKD. Conclusion: High prevalence of CKD in people receiving physical examination is found in Changsha, especially stage 1 and 2 CKD. Physical examination is important to screen CKD. Stopping smoking, control of blood glucose, blood pressure, blood lipids and increasing physical activity may help reduce the prevalence of CKD.

Objective To understand the status of oipA gene of Helicobacter pylori (H.pylori) and to evaluate the relationship between oipA gene and digestive disease, Methods Biopsy gastric mucosa were obtained from 360 patients with digestive disease under gastroscopy. H.pylori was isolated from urease positive and some negative samples, and then identified by microscope, urease test and 16 S rRNA PCR. oipA and cagA genes of the isolates were amplified by PCR and the status of oipA signal region was analyzed after sequencing. The relationship between functional oipA gene of H. pylori and digestive disease was investigated. Results One hundred and six isolats were obtained. Seventy-two strains were positive for cagA gene and 87 were positive for oipA gene in signal region. Eighty strains were on open status in signal region called functioal oipA gene and 1 was on off status. The status of other 6 strains were uncertain. The frequency of functional oipA in ulcer and atrophic gastritis were higher than that of cagA(100% and 78.26% vs 58.33% and 39.13%).Conclusion Functional oipA gene is closely related with ulcer and atrophic gastritis.

Objective:To express Helicobacter pylori oipA gene with different fragments and determine the protein and its peptides with finest antigenicity.Methods:oipA gene was obtained from international standard Hp NCTC 11637 and cloned into vector pGEM-T then was identified by PCR and sequencing.Six different fragments of oipA gene were amplified by PCR with the template pGEM-T/oipA.The gene fragments and expressing vector pET-42a were ligated.The recombinant vector were transformed into BL-21.The correct recombinants were identified by PCR,enzyme digestion and sequencing.Recombination protein and the peptides were detected by Western Breeze chemiluminescent after induced by IPTG.Optimization of the time and IPTG concentration for induction were conducted.The expressed products were affinity purified by Ni-NTA of His?Bind.The antigenicity of the recombination protein was determined with Western blot indicated by goat anti-Hp polyclonal antibody,and the recombination protein with finest antigenicity was selected with indirect ELISA.Results:The six oipA gene fragments could all expressed and the products were recognized by goat-anti Hp polyclonal antibody.The finest peptide with antigenicity was the smallest peptide.Conclusion:OipA gene fragments could be expressed in prokaryotic system and the smallest peptide had the finest antigenicity.It may be used as the reagent to detect corresponding antibody in serum.

Thirteen compounds were isolated from the ethyl acetate fraction of Crepis crocea by column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. The structures were elucidated on the basis of spectral analysis as tectorone I (1), 8β- (2-methyl- 2-hydroxy-3-oxobutanoyloxy) -glucozaluzanin C (2), tectoroside (3), luteolin-7-O-glucoside (4), cosmosiin (5), esculetin (6), 3,4-dihydroxybenzaldehyde (7), trans-4-hydroxycinnamic acid (8), Caffeic acid (9), methyl p-hydroxyphenyllactate (10), ethylp- hydroxyphenyllactate (11), cis-3,4-dihydroxy-β-ionion (12). All the compounds, except for compounds 4 and 9, were isolated from this plant for the first time, and tectorone I (1) is a new natural product.
Crepis ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure

Objective To compare the results of TEOAE and DPOAE in the same population of normal newborns, to provide information on choosing appropriate screening tools.Methods A two-steps protocol was taken with the first screening during the first 48 to 72 hours of birth and rescreened from one to two months old if the newborns failed the first screening.For each step of screening, TEOAE and DPOAE were performed simultaneously using AccuScreen hearing screening instrument (Madsen-GN Otometrics, Taastrup, Denmark).A total of 1 062 normal newborns (F/M=508/554) delivered in Peking Union Medical College Hospital were enrolled in this research for the first screening.Infants who failed either TEOAE or DPOAE screening in the first screening were referred to a second screening.Among them, 135 performed both DPOAE and TEOAE in the second step.The newborns who failed the second screening would receive ABR when they were 3 months old.Results In the first screening,the failure rate for TEOAE was 11.0% (117/1 062) and 13.7% (145/1 062) for DPOAE.In the second screening step, the failure rates were 17.8% (24/135) and 20.7% (28/135) for TEOAE and DPOAE, respectively.Chi-square and Fisher's test showed that the failure rates of DPOAE were significant higher than TEOAE for both steps (P<0.001).The agreements between TEOAE and DPOAE were 96.0% and 95.6% for the first and second steps respectively, and the kappa values were 0.817 and 0.857.As to the average time taken to accomplish the screening for one ear, TEOAE was 24±25 s and DPOAE was 40±34 s during the first screening;in the rescreening, TEOAE was 52±41 s and DPOAE was 73±62 s.Paired-t tests showed that the differences between DPOAE and TEOAE testing time were statistically significant (P=0.000) in both screening steps.Finally, 7 newborns (10 ears) were diagnosed conductive hearing loss(except 1 ear was sensorineural hearing loss).Conclusion As a screening tool, TEOAE got lower refer rates and took less time than DPOAE implicating TEOAE a better screening tool for normal neonates.

Objective To investigate the effect on incidence of ventilator associated pneumonia(VAP),the cost of hospitalization with closed endotracheal suctioning and vital sign as well in postoperative cardiac patients.Methods 304 postoperative cardiac patients supporting by ventilation were enrolled in this cohort study during January,2012-November,2013 in The Second affiliated Hospital& Yuying Children Hospital of Wenzhou Medical University.All the subjects were randomly divided into observational group and control group by coin side.Closed endotracheal suctioning system was applied in observation group and opened mode was applied in control group.Compare the vital sign(heart rate,blood pressure,saturation) at the moment of aspiration,suction time,incidence of VAP,duration of ventilation,mortality,the cost of suction,hospital stays and hospitalization expense.Results The baseline is no significant difference between two groups.The fluctuation of blood pressure and heart rate is lower in observational group at 30 second since completed the suction(P ＜0.05),but saturation is higher at 30 second and 60 second since completed the suction respectively(P ＜ 0.05).There is no significant difference of incidence of unexpected tube displacement and pneumothorax between two groups.Average time of each suction of experimental groups is shorter than Control groups[(156 ± 6) s vs (225 ± 8) s,t =-84.86,P ＜ 0.01].VAP incidence is lower in experimental group (12.0％ vs.18.6％,x2 =4.37,P ＜ 0.05).Duration of ventilation is lower in experimental group[(72 ± 33) h vs.(98 ± 38) h,t =-6.35,P ＜ 0.05].The cost of suction is higher in observational group [(346 ± 15) RMB vs.(178 ± 26) RMB,t =69.00,P ＜ 0.01],but the hospitalization expense is lower in experimental group [(32 011 ± 2 525) yuan vs.(35 264 ± 3 846)yuan,t =-8.72,P ＜ 0.05].There is no significant difference in mortality between two groups (x2 =0.08,P ＞ 0.05).Conclusion Application of closed endotracheal suction system can result in reduction vital sign fluctuation and incidence of cross infection and reducing the workload of nurses and decreasing the complication of suction,shorting the duration of ventilation and hospitalization and saving the expense of hospitalization in postoperative cardiac patients comparing with open mode.It is worthy to be populized in cardiac care unit.

Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma.Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan,so as to predict the miRNA targeting CCL18 gene.Three kinds of C CL18 3'UTR dual-luciferase reporter vectors,including mutant 3'UTR vector (mutant 3'UTR group),wildtype 3'UTR vector (wild-type 3'UTR group) and empty vector (blank control group),as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis,and then were used to co-transfect 293T cells.After 48-hour treatment,the cells were lysed for detection of luciferase activity.Real-time fluorescence-based quantitative PCR was performed to measure the expression of CCL 18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens (control tissues),and their correlations were analyzed.Results Online software analysis showed that some miRNAs were identified to target the 3'UTR of CCL18 gene,including miR-183,miR-128 and miR-33a.Luciferase reporter vectors and miRNA vectors were constructed successfully.As luciferase activity assay showed,when miR-183 and miR-128 were bound to the CCL18 3'UTR,the luciferase activities were significantly higher in their mutant 3'UTR groups (11.63 ± 0.42;8.80 ± 0.49) than in their wild-type 3'UTR groups (4.86 ± 0.39;5.01 ± 0.54;both P ＜ 0.05) and blank control groups (2.41 ± 0.13;2.39 ± 0.05;both P ＜ 0.01),while there were no significant differences between miR-33a-hinding mutant 3'UTR group (6.41 ± 0.47) and miR-33a-binding wild-type 3'UTR group (6.16 ± 0.22,P ＞ 0.05).Real-time fluorescence-based quantitative PCR revealed higher mRNA expression of the CCL18 gene (3.52 ± 1.68),but lower expression of miR-183 (0.49 ± 0.32),miR-128 (0.30 ± 0.20) and miR-33a (0.46 ± 0.40) in the malignant melanoma tissues compared with the control tissues.The mRNA expression of the CCL18 gene was negatively correlated with the expression of miR-128 (rs =-0548,P ＜ 0.05),but showed no significant correlations with the expression of miR-183 and miR-33a (both P ＞ 0.05).Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18.

Adult ; Aged ; Capsid Proteins ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology ; Uterine Cervicitis ; metabolism ; pathology ; Young Adult

Objective To screen microRNAs(miRNAs)related to early mycosis fungoides(MF). Methods A high?throughput miRNA PCR array was used to determine miRNA expression profiles in skin lesions of 6 patients with early MF (early MF group) and 6 patients with lichen planus (control group), followed by screening of differentially expressed miRNAs between the two groups. Then, real?time fluorescence?based quantitative PCR(RT?qPCR)was performed to verify the differentially expressed miRNAs in lesional specimens from 13 patients with early MF and 13 patients with eczema or lichen planus, as well as in Myla cells and normal human T?lymphocytes. Results The high?throughput miRNA PCR array showed that the expressions of hsa?miR?378a?5p, hsa?miR?107 and hsa?miR?302c?3p were significantly higher in the early MF group than in the control group(all P<0.05). For skin lesions, the results from RT?qPCR were similar to those from the miRNA array assay. Compared with normal human peripheral blood T?lymphocytes, Myla cells showed significantly increased expressions of hsa?miR?378a?5p and hsa?miR?107, which was consistent with the results from the miRNA array assay. However, no significant difference was observed in the expression of hsa?miR?302c?3p between the two kinds of cells. Conclusion MiRNA expression profiles in early MF are different from those in inflammatory skin diseases.