BACKGROUNDPromoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.

METHODSHuman embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.

RESULTSThe sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.

CONCLUSIONSTo detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

Cells, Cultured ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Luciferases ; genetics ; Plasmids ; Promoter Regions, Genetic ; Transfection

OBJECTIVETo investigate the clinical and angiographic characteristics of left coronaroventricular microfistula.

METHODSIn his retrospective review, clinical, electrocardiogram, echocardiography and coronary angiography data were analyzed for patients with left coronaroventricular microfistula.

RESULTSLeft coronaroventricular microfistula was identified in 9 out of 8300 patients underwent coronary angiographies from 1998 to 2008 in our center. Seven patients were female (77.8%) and the average age was 71.5 years. All 9 patients had presenting symptoms of chest distress or dyspnea, coronary artery disease was documented in 5 (55.6%), hypertension in 2 (22.2%), valve disease in 1 (11.1%)and cardiomyopathy in 1 (11.1%) patient. Microfistula originated from one single coronary artery was seen in 1 patient (11.1%), from two coronary arteries in 6 patients (66.7%), from three coronary arteries in 2 patients (22.2%). The diagonal artery was involved in all patients. The characteristic sign of microfistula from CAG was intracavitary staining.

CONCLUSIONMicrofistula between coronary arteries and left ventricle is a rare disease, often originates from two coronary vessels and diagonal artery is involved in most cases.

Aged ; Aged, 80 and over ; Coronary Angiography ; Coronary Artery Disease ; diagnostic imaging ; epidemiology ; Coronary Vessels ; pathology ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Sex Distribution ; Vascular Fistula ; diagnostic imaging ; epidemiology

OBJECTIVETo develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus.

METHODSThe Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens.

RESULTSThe human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years.

CONCLUSIONIt is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.

Child, Preschool ; Feasibility Studies ; Humans ; Metapneumovirus ; genetics ; isolation & purification ; Respiratory Tract Infections ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Reverse Transcription ; Sensitivity and Specificity

Objective To observe the brain cerebrovascular function and blood flow changes in patients with brain diseases during hyperbaric oxygen therapy of different states to provide evidence for their clinical treatment.Methods Forty-nine patients with brain diseases,admitted to our hospital from November 2010 to May 2011,were chosen in our study; their rheoencephalography data captured during a complete hyperbaric oxygen therapy of 4 different states were collected and compared; these 4 states were:state Ⅰ,before pressure raising,which could also be the beginning of the therapy; state Ⅱ,before oxygen intake while pressure coming to a stable level; state Ⅲ,40 min after oxygen uptake; and state Ⅳ,while the pressure inside the chamber coming back to normal level.Results Carotid artery and vertebrobasilar system enjoyed likely changes.Parameters of rheoencephalography showing cerebral blood flow changes,including impedance,raising time,total retraction time and high turn ratio,did not show any differences among the four states (P＞0.05),those showing vascular tension and elasticity,including elasticity index,angle of increasing,top angle of peak value,filling time and amplitude,showed statistically significant difference between each 2 states (P＜0.05),and those showing cerebral vascular resistance,such as resistance index,rising time and high turn ratio,showed significant difference between each 2 states (P＜0.05).Conclusion The hyperbaric oxygen therapy has little effect on changing the brain blood flow; it mainly functions as controlling the impedance and extending the contraction of brain vascular.

OBJECTIVETo compare the difference of effects on SiO(2)-induced alveolitis and early fibrosis between bone marrow-derived mesenchymal-like stem cells (BM-MSCs) and BM-MSCs transfected by pcDNA3.1-HGF and to explore the mechanism of this effects.

METHODSThe Primary BM-MSCs from Wistar male young rats were cultured and labeled by 4, 6-diamidino-2-phenylindole (DAPI). Fifty Wistar rats were randomly divided into 3 groups:model group (10 rats),which was administered with SiO(2) by the trache, the next day,injected PBS via the tail vein; BM-MSCs group (20 rats),which was administered with SiO(2) by the trache, the next day,injected with 1 ml suspension of BM-MSCs via the tail vein; pcDNA3.1-HGF plus BM-MSC group (20 rats),which was administered with SiO(2) by the trache, the next day,injected with 1 ml suspension of BM-MSCs transfected by pcDNA3.1-HGF via the tail vein. On the 14th and 28th days after treatment, half of the animals were sacrificed, respectively, and the lungs were harvested for frozen section to observe the cell marked by DAPI. HE staining under a fluorescent microscope, and to observe the pulmonary alveolitis and fibrosis by HE and Masson staining under a light microscope. Western blot assay was used to detect the expression of HGF in rat lungs. The expression levels of tumor necrosis factor-α (TNF-α) in pulmonary tissues were analyzed quantitatively by ELISA. The contents of HYP in pulmonary tissues were analyzed quantitatively by sample hydrolysis method.

RESULTSOn the 14th and 28th days after treatment, the scores of pulmonary alveolitis and early fibrosis in pcDNA3.1-HGF plus BM-MSCs group were 2.36 ± 0.17, 2.8 ± 0.14 and 0.1 ± 0.11, 1.16 ± 0.13, which were significantly lower than those (1.68 ± 0.17, 1.58 ± 0.31 and 0.54 ± 0.15, 1.36 ± 0.13) in BM-MSCs group, also which were significantly lower those (2.36 ± 0.17, 2.80 ± 0.14 and 0.64 ± 0.09, 1.84 ± 0.17) in model group (P < 0.05); On the 14th and 28th days after treatment, the TNF-α contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 280.4 ± 23.11 and 249.78 ± 22.33 pg/mg, which were significantly lower than those (341.58 ± 35.34, 442.29 ± 36.76 pg/mg and 319.51 ± 17.84, 348.53 ± 33.95 pg/mg) in BM-MSCs and model groups (P < 0.05); On the 14th and 28th days after treatment, the HYP contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 0.46 ± 0.04 and 0.65 ± 0.05 µg/mg, which were significantly lower than those (0.63 ± 0.04, 1.04 ± 0.07 µg/mg and 0.72 ± 0.60, 1.39 ± 0.60 µg/mg) in BM-MSCs and model groups (P < 0.05).

CONCLUSIONThe effects of BM-MSCs transfected by pcDNA3.1-HGF on suppressing pulmonary alveolitis and early fibrosis induced by SiO2 were better than those of BM-MSCs. The mechanism may be associated with the reduced pulmonary inflammation.

Animals ; Bone Marrow Cells ; cytology ; Hepatocyte Growth Factor ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; metabolism ; Pulmonary Fibrosis ; chemically induced ; prevention & control ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Silicosis ; prevention & control ; Transfection

BACKGROUNDIt is unclear whether edge segments have different responses to paclitaxel eluting stent (PES) and sirolimus eluting stent (SES) implantation in patients with unstable angina. This study aimed to compare the different vascular edge responses in patients with unstable angina and single de novo coronary lesion treated with SES and PES.

METHODSTwo hundred and fifty-five patients with unstable angina and single de novo lesion were randomly assigned to PES and SES groups. Serial volumetric intravascular ultrasound (IVUS) images were taken immediately after stenting and at an eight-month follow-up. Five-mm edge segments proximal and distal to the stents were analyzed.

RESULTSBaseline characteristics were comparable between the two groups. At proximal-edge segment, the vessel area decreased and the plaque area increased significantly in the PES group as compared with the SES group. A significant net loss of lumen area was found in the PES group (from (11.10 +/- 3.12) mm(2) at baseline to (9.92 +/- 3.59) mm(2) at the follow-up, P < 0.001). At the distal-edge segment, the net loss of lumen area in the PES group (from (7.71 +/- 2.81) mm(2) at baseline to (6.66 +/- 2.29) mm(2) at the follow-up, P < 0.001) was attributed to a significant increase of plaque area. Proximal-edge stenosis was commonly seen in the PES group (20.0%) as compared with the SES group (5.0%, P = 0.001). This correlated with the higher incidence of target lesion revascularization in the PES group (P = 0.03). Subsegmentally, the smallest Delta lumen area was located at 2 mm proximally in both groups, at 0 mm distally in the PES group, and at 1 mm distally in the SES group.

CONCLUSIONSThe two groups demonstrated negative remodeling of edge segments. PES was less effective than SES in inhibiting the growth of plaque within the first 1-mm length proximal to the stent.

Aged ; Aged, 80 and over ; Angina, Unstable ; diagnostic imaging ; drug therapy ; therapy ; Coronary Angiography ; Drug-Eluting Stents ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Paclitaxel ; therapeutic use ; Sirolimus ; therapeutic use ; Treatment Outcome ; Ultrasonography

The stiffness can respond to the function of muscle and tendon. Resistance training leads to the change of muscle-tendon stiffness, and various with the patterns and strength of contraction. This paper reviewed the impacts of mechanical load on stiffness of muscle and tendon, to explore the adaptation of muscle and tendon to mechanical load.

Objective Throush identify biochemical characteristics and virulence factors of 2 strains suspected Yersinia pestis(Y.pestis)isolated from the dead Marmota himalayana(M.himalayana)to confirm the nature epidemic focus in Dege County,Sichuan Province.Methods Y.pestis was analyzed by specific staining and shape,culturing characteristics,splitting-test by bacteriophage,test of biochemical characteristics and glycolysis ability,virulence factors,virulence,nutritional requirement,plasmid,genetic test and genetic type. Results The tested strains were Gram staining bacilus.The main biochemical characteristics were Arabinose(+)、 Rhamnose(-),Maltose(+),Melibiose(-),Glycerol(+),Denitrification(+).The virulence factors with FI+.VW+, Pgm+,Pst I+;and with the common 6.0×106,45.0×106,65.0×106 plasmids,also with the virulence-relative plasmid gene.Both their absolutely lethal dose(LD100)in mice were 50 bacteria.The nutritional requirement appeared which were depended on Phenylalanine and Methionine.With the Genomovar 5 genotype characteristics of M.himalayana plague foci of Qinghai-Tibet plateau.The difference between tested strains and Yersinia pseudotubercuosis on the 3 different culture medium was obvious.The tested strains had a Y.pestis' specific 3a fragment,Pst I and FI-Ag,at 22 ℃,the strains could be split by bacteriophage completely.Conclusions According to the diagnostic criteria of plague in China,the 2 suspected strains isolated from Dege County,Sichuan Province ale confirmed as Y.pestis.both with powerful virulenceand with the characteristics of the Y.pestis of M.himahtyana in Qinghai-Tibet plateau plague natural focus.

Objective: To investigate the value of real-time RNA simultaneous amplification and testing (SAT) in the detection of Ureaplasma urealyticum (UU) in the semen of infertile males and its clinical significance. METHODS: We collected semen samples from 542 infertility patients and 120 normal fertile men as controls in the Andrology Clinic of Nanjing General Hospital from March to September 2015. We detected UU infection in the samples using the culture method and SAT technology, respectively. RESULTS: All the UU positive cases (except 4 false positive cases) detected by the culture method were also shown to be positive in SAT. The UU detection rate of SAT was significantly higher than that of the culture method both in the infertility patients (54.1 vs 19.7%, P<0.05) and in the normal controls (42.5 vs 12.5%, P<0.05). CONCLUSIONS SAT is a rapid and accurate method for detecting UU infection in semen samples, with a higher sensitivity and accuracy than the culture method, and it can also be used to evaluate the therapeutic effects. However, the culture method has its own advantages, such as low requirement of technical equipment, easy operation, and possibility of drug sensitivity test at the same time. Therefore, SAT and the culture method can be used alternatively according to the clinical need.
Andrology ; Humans ; Infertility, Male ; microbiology ; Male ; Nucleic Acid Amplification Techniques ; RNA, Bacterial ; analysis ; Semen ; chemistry ; microbiology ; Semen Analysis ; Ureaplasma Infections ; diagnosis ; Ureaplasma urealyticum ; genetics ; isolation & purification

Asymptomatic Infections ; Betacoronavirus ; Carrier State ; Coronavirus Infections ; transmission ; Female ; Humans ; Middle Aged ; Pandemics ; Pneumonia, Viral ; transmission