Objective To optimize the concentration of polyethylene glycol (PEG) for the growth of Chlamydia trachomatis (Ct) reference strains D-UW-5/Cx and E-UW-5/Cx,and to evaluate the effects of PEG on the sensitivity of Ct to 4 common antibiotics.Methods After the inoculation of Ct standard strains (D-UW-5/Cx and E-UW-5/Cx) into McCoy cell monolayer,different concentrations of PEG were added into the culture medium followed by centrifugation.After 2 hours of incubation at 37 ℃,the inoculum was removed and a complete culture medium containing 1 μg/mL cycloheximide was added followed by another 48-hour culture.Subsequently,the culture was fixed and subjected to iodine staining for the calculation of Ct inclusions and optimization of PEG for the growth of Ct.Some Ct standard strains were used to infect McCoy cells,with PEG (0.7％,wt/vol) added to the culture medium after inoculation and before centrifugation process,and when the infection rate reached higher than 90％,a microdilution method was utilized to evaluate the minimal inhibitory concentration (MIC) of 4 antibiotics,including azithromycin,minocyline,moxifloxacin and doxycycline.Thirty-one clinical specimens,which had been confirmed to be positive for Ct serovar D or E strain,were inoculated into McCoy cell monolayer for the passage of Ct with or without the presence of 0.7％ PEG.Results The optimal concentration of PEG was 0.7％ for the growth of Ct,and this concentration of PEG could increase the number of inclusion bodies of Ct serovar E by 3.44 folds,and that of Ct serovar D by 3.56 folds.In vitro,the MICs of the 4 antibiotics were consistent between PEG-treated and untreated Ct reference strains.Moreover,PEG notably increased the quantity of inclusion bodies of Ct serovar E or D from clinical specimens after passages.Conclusions PEG (0.7％) can enhance the growth of Ct serovar D and E,but has no obvious influence on antimicrobial susceptibility of Ct.

ObjectiveTo detect the changes of heat shock protein 60 (HSP60) during the subcultures of standard and clinical strains of Chlamydia trachomatis(Ct),and to determine if the absence of chlamydial inclusions is associated with the persistent infection of Ct.MethodsA total of 40 Ct strains isolated by cell culture from patients were included in this study and classified into 2 groups according to whether inclusions appeared after initial culture.Reverse transcription PCR was conducted to quantify the levels of HSP60 in specimens containing Ct after 1-4 subcuhures.Chi-squared test was performed to analyze the relationship between the levels of HSP60 and treatment outcomes of patients.ResultsThe levels of HSP60 in clinical specimens containing Ct serovar E were significantly higher in subcultures prior to the appearance of inclusions than in subcultures with the appearance of inclusions(P ＜ 0.05).The ratio of HSP60:16S rRNA mRNA expression after the first,second,third,fourth passage was 0.38 ± 0.06,0.39 ± 0.03,0.38 ± 0.04 and 0.39 ±0.03 respectively in 18 specimens with inclusions appearing after the initial culture,1.18 ± 0.10,0.28 ± 0.06,0.30 ± 0.03 and 0.29 ± 0.05 respectively in 12 specimens with inclusions appearing after the second culture,1.20 ± 0.04,1.20 ± 0.04,0.28 ± 0.04 and 0.28 ± 0.05 in 10 specimens with inclusions appearing after the third culture.Whether inclusions appeared after the initial culture was associated with the treatment outcome of patients.Inclusions were undetected after the initial culture in 16 of 20 specimens from patients with poor response to treatment,but observed in 14 of 20 specimens from patients who tested negative for Ct after one course of treatment.Conclusions It is implicated that no inclusions form after the initial culture in 80％ of specimens from patients experiencing treatment failure.The Ct strains whose inclusions do not form after passages may be in a persistent state,and the expression of HSP60 is high in these strains.Specimens should be subjected to at least 3 blind passages to avoid missed diagnosis of Ct infection.

Objective To explore the effect of recovery sleep on the executive function after 36 h of total sleep deprivation by event related potential technology.Methods Thirteen healthy male college students participated in two trials. At the first trial normal sleep as control was investigated. At the second trial participants experienced 36 h of sleep deprivation and then accepted 8 h recovery sleep. In each trial six Go/Nogo tests were employed to test the executive control function and the ERP data were recorded. Results There was no statistical difference in behavior and ERP results at each time point as the subjects had normal sleep. After 36 h of sleep deprivation, the behavior results were statistically significant when compared to the baseline. The amplitude and latency of Nogo-N2, Nogo-P3 on Fz electrode, the amplitude and latency of Nogo-P3 on Cz electrode showed statistical significance when compared to the baseline. After 8 h recovery sleep, the average correct reaction time and the Go correct reaction rate had statistical significance compared to 36 h value. The amplitude of Nogo-N2 and Nogo-P3 had no statistical significance compared to the baseline.However,it was of statistical significance[(-6.80 3.95)vs(-3.37 2.63)μV,(10.63±6.62)vs(5.63±5.45)μV,(9.49±7.37)vs(6.08±6.56)μV] compared to 36 h value. The latency of the recovery value of Nogo-N2 and Nogo-P3 was statistically significant[(254.14±15.55)vs(243.08±13.97)ms(382.14±41.07)vs(349.17±30.36)ms,(369.86±26.48)vs(347.48±29.24)ms]compared to the baseline.Conclusion As the time of sleep deprivation is prolonged, the executive function is impaired and the executive function is not completely recovered after 8 h recovery sleep.

OBJECTIVETo evaluate visual inspection with Lugol's iodine (VILI) in cervical cancer screening program and to provide evidence for designing a cervical cancer screening algorithm in high risk areas of existing low-resource settings to reduce the incidence and mortality of cervical cancer.

METHODSWomen in Yangcheng county, Shanxi province were screened with VILI, colposcopy, liquid-based cytology test and human papilloma virus (HPV) DNA test. The efficacy of different screening tests was compared by Youden's index based on the pathology as the gold standard.

RESULTSIn the population being screened, the mean age was 40.80 +/- 10.75 years old. Based on pathological findings, 4.35% (32/735) of the subjects had >or= CIN (cervical intraepithelial neoplasia) II. The sensitivity and specificity for the VILI test (>or= positive) were 53.13 and 82.19, while 56.25 and 79.09 were for colposcopy (>or= low grade dysplasia) respectively. Comparing by the Youden's indexs, there was no statistically significant difference (P > 0.05) between VILI and colposcopy. However, statistical significant difference (P < 0.05) was found between VILI and liquid-based cytology test and HPV DNA test. In addition, there was no statistically significant difference (P > 0.05) found between the experienced doctors and the newly-trained doctors working in the field station.

CONCLUSIONWith low sensitivity when using microscope but low cost of equipments, VILI can be one of the primary screening tests in China's rural area with low-resource settings if the screening frequency is to be increased.

Adult ; Cervical Intraepithelial Neoplasia ; diagnosis ; epidemiology ; China ; epidemiology ; Early Detection of Cancer ; economics ; methods ; Female ; Health Resources ; Humans ; Incidence ; Iodides ; Program Evaluation ; Rural Health ; Sensitivity and Specificity ; Socioeconomic Factors ; Uterine Cervical Neoplasms ; diagnosis ; epidemiology

OBJECTIVETo identify the candidate auto-antigen of rheumatic heart disease as a molecular marker for this disease.

METHODSThe total RNA of the heart tissue of patients with rheumatic heart disease was extracted and reverse-transcribed into long cDNA to construct the phage expression library. The library was screened using the serum from patients with active rheumatic fever, and the positive clone was identified and analyzed by bioinformatics and expressed in vitro. The expressed products were evaluated with Western blotting and its cross-reactivity was assessed.

RESULTSThe phage expression library of the heart tissue of patients with rheumatic heart disease was constructed, with the titer of the primary library of 3.3×10(6) pfu/ml, recombinant rate of 99%, and 81% of the inserted segments were larger than 1 kb. An auto-antigen RHDAG1 was identified by screening, which was homologous to keratin 18. RHDAG1 was detected in the serum of patients with active rheumatic fever and of those with rheumatic heart disease, but not in the serum of healthy subjects.

CONCLUSIONPhage display library can be an effective strategy to screen the auto-antigens of rheumatic heart disease. The auto-antigen RHDAG1 can be a candidate molecular biomarker of rheumatic heart disease and/or rheumatic fever.

Autoantibodies ; blood ; immunology ; Autoantigens ; immunology ; isolation & purification ; Autoimmune Diseases ; blood ; immunology ; Humans ; Peptide Library ; Rheumatic Heart Disease ; immunology

OBJECTIVEPrader-Willi Sydrome (PWS) is a human disorder related to genomic imprinting defect on 15q11-13. It is characterized by a series of classic features such as hypotonia, hyperphagia, obesity, osteoporosis, typical facial and body dysmorphosis, hypogonadism, mental and behaviour disorders. Our study was designed to precisely detect the microdeletions, which accounts for 65%-70% of the PWS.

METHODSPhysical and laboratory examinations were firstly performed to diagnose PWS clinically, and to discover novel clinical features. Then the patient was screened with bisulfite-specific sequencing and precisely delineated through high-density array CGH.

RESULTSWith the bisulfite-specific sequencing, the detected CpG island in the PWS critical region was found homozygously hypermethylated. Then with array CGH, a 2.22 Mb type II microdeletion was detected, covering a region from MKRN3, MAGEL2, NDN, PWRN2, PWRN1, C12orf2, SNURF-SNRPN, C/D snoRNAs, to distal of UBE3A.

CONCLUSIONSArray CGH, after the fast screening of Bisulfite-specific sequencing, is a feasible and precise method to detect microdeletions in PWS patients. A novel feature of metacarpophalangeal joint rigidity was also presented, which is the first time reported in PWS.

Base Sequence ; Chromosome Deletion ; DNA Primers ; Female ; Humans ; Infant, Newborn ; Nucleic Acid Hybridization ; Prader-Willi Syndrome ; genetics

Adult ; Electroencephalography ; Evoked Potentials ; Humans ; Memory, Short-Term ; Reaction Time ; Sleep Deprivation ; Young Adult

UNLABELLEDOBJECTICE: To evaluate the application of high-throughput sequencing to sequence the FMS-like Tyrosine Kinase 3 internal tandem duplication (FLT3-ITD) in de novo acute myeloid leukemia (AML) patients with lower allelic ratio FLT3-ITD mutation or more than one ITD, and to analyze the feature of ITD.

METHODSThe genomic DNA of 23 AML patients with positive FLT3-ITD was amplified by PCR, capillary electrophoresis was used to detect the ITD mutation. Then, the FLT3 gene was amplified using primer with different barcode, and the product was analyzed by illumina Miseq, and the results were compared with UCSC database.

RESULTSOut of 23 AML patients, 17 had a single ITD, and 3 had 2 ITDs, and the remaining 3 had 3 ITD detected by capillary electrophoresis. The high-throughput sequencing showed that 17 ITD were the complate duplications of wild-type FLT3, and the remaining 16 ITD were partial duplications in the all 33 ITDs. The same length ITD mutation contained 2 different ITD sequences in one patient with more than one ITD, and the other patient with 2 ITD had the same ITD insertion position. The ITD occurred in the regions from p. Y572 to p. L602 of the FLT3 protein, and all the patient ITD covered one or more amino acid between p. V592 and p. E598.

CONCLUSIONIllumina Miseq can analyze the sequence of ITDs precisely and accurately. ITD mutation varies widely, but the hotspots are concentrated.

Alleles ; DNA Mutational Analysis ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; fms-Like Tyrosine Kinase 3 ; genetics

Objective::To investigate the antihypertensive effect of Tianmu Jiangya powder and its related antihypertensive mechanism by using SHR rats as a model, and protein expressions provide an experimental basis for the clinical application of Tianmu Jiangya powder in the treatment of hypertension. Method::Sixty male SHR rats were randomly divided into six groups according to body weight after one week of adaptive feeding: model group, valsartan group (12 mg·kg^-1), captopril group (9 mg·kg^-1), hydrochlorothiazide group (6 mg·kg^-1), Tianmu Jiangya powder low and high-dose group (0.36, 1.44 g·kg^-1), WKY rats were used as the normal group, and the intragastric administration lasted for 16 weeks. Softron BP-2010A intelligent non-invasive blood pressure meter was used to measure the systolic blood pressure (SBP)and heart rate (HR) of rat tail arteries. Adobe Photoshop CS5 software was used to analyze the left auricle and claw fixed selected areas to evaluate the effect on blood stasis syndrome. Vevo 2100 small animal ultrasound imaging system detects left ventricular ejection fraction (LVEF), left ventricular shortening (FS), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systole dimension (LVIDs), left ventricular end-diastole dimension (LVIDd), interventricular septum end-systolic depth (IVSs), and interventricular septum end-diastolic depth (IVSd). Then the rats were sacrificed and the materials were taken (blood, heart, aorta, liver, kidney, tibia), and the weight of heart, liver, kidney and tibia length were measured and recorded. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of the heart and thoracic aorta. Separation of serum and plasma, and determination of nitric oxide (NO) in serum by nitrate reductase method. Radioimmunoassay was used to detect plasma adrenaline/3 methoxyadrenaline (MN), urea (UREA), and uric acid (UA) contents. The expression of nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) protein in thoracic aorta of each group was detected and analyzed by immunohistochemical method. Result::Compared with normal group, the SBP and HR of the rats in model group were significantly increased (P<0.05). The r value of the claw was significantly reduced and the g value was significantly increased at 8 and 16 weeks (P<0.05). LVEF and FS significantly decreased, LVESV, LVIDs, IVSd increased significantly (P<0.05). Heart weight, heart weight /tibia length, liver weight and liver weight /tibia length, plasma of MN, UREA, and UA contents significantly increased, and promoted the expression of iNOS and VEGF proteins in the aortic (P<0.05). Compared with the model group, the Tianmu Jiangya powder administration group could continuously reduce SBP in SHR rats, maintain HR stability (P<0.05), significantly increase the claw of r value, lower the claw of g value(P<0.05). LVEF, FS significantly increased, LVEDV, LVESV, LVIDd and LVIDs significantly decreased (P<0.05), significantly increased serum NO content, decreased liver weight, liver weight/tibia length, plasma MN, UREA, UA content (P<0.05), and down-regulated the expression of iNOS and VEGF protein in the aorta(P<0.05). Conclusion::Tianmu Jiangya powder has a certain antihypertensive effect, and its mechanism may be mainly related to protecting heart function, improving vascular endothelial function, reducing catecholamines and sedative analgesia.