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

Objective To develop a rapid,sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus. Methods The 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. Results The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly,and the sensitivity was 1 copy/μl.The coefficient of variation of intra-assay and inter-assay wasless 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 bronehiolitis ( 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. Conclusion It is demonstrated that real time reverse transcription PCR is a reliable,accurate and feasible assay for human metapneumovirus,whieh has become one of the most important pathogens induced acute respiratory infections in pediatric patients.

Objective To investigate the clinical and angiographic characteristics of left coronaroventricular microfistula. Methods In his retrospective review, clinical, electrocardiogram, echocardiography and coronary angiography data were analyzed for patients with left coronaroventricular microfistula. Results Left coronarovcntricular microfistula was identified in 9 out of 8300 patients underwent coronary angiagraphies 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. Mierofistula 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. Conclusion Microfistula between coronary arteries and left ventricle is a rare disease, often originates from two coronary vessels and diagonal artery is involved in most cases.

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

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.

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.

The epidemiology and etiology of three suspected cases of imported skin diphtheria infection in Fujian Province were investigated.Secretion samples of patients with skin damage were collected for isolation and culture of Corynebacterium diphtheriae.Biochemical identification and mass spectrum analysis of pure cultures of suspected C.diphtheriae were conduc-ted,the virulence-related genes,including diphtheria toxin reporter(dtxR),toxin A(toxA),and toxin B(toxB)were detec-ted,and multilocus sequence typing(MLST)was performed.All three cases had typical clinical manifestations of cutaneous diphtheria,and C.diphtheriae was isolated from the damaged skin.The virulence genes of two C.diphtheriae strains isolated from two cases were identified as dtxR(+),toxA(-),and toxB(-),and the MLST type was ST-703.The virulence genes of C.diphtheriae isolated from one case were identified as dtxR(+),toxA(+),toxB(+),and the MLST type was ST-248.There is an increased risk of diphtheria in Fujian Province.C.diphtheriae without diphtheria toxin genes can also cause skin diphtheria.

ObjectiveTo explore Mycoplasma genitalium (MG) infection in the urogenital tract of infertile men and its influence on semen quality.

METHODSSemen samples were collected from 352 infertile males in the Center of Reproductive Medicine of Nanjing General Hospital from March to July 2015. MG infection was detected by real-time fluorescence simultaneous amplification and testing and semen analyses were conducted according to the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed) on the semen pH value, semen volume, total sperm count, sperm concentration, total sperm motility, percentages of progressively motile sperm (PMS) and immotile sperm (IMS), and sperm DNA fragmentation index (DFI). The data obtained were subjected to statistical analysis by t-test and non-parametric test (Wilcoxon test).

RESULTSMG infection was found in 3.4% (12/352) of the infertile patients. Compared with the MG-positive cases, the MG-negative ones showed a significantly higher semen volume (［2.85 ± 0.14］ vs ［3.84 ± 0.12］ ml, P = 0.008) and percentage of PMS (［15.86±1.72］ vs ［60.95 ± 5.63］ %, P = 0.032) but a lower DFI (［30.73 ±2.24］ vs ［20.71 ± 1.55］%, P = 0.014). However, no statistically significant differences were observed between the two groups in the semen pH value (7.38 ±0.02 vs 7.39 ± 0.01, P = 0.774), sperm concentration (［52.96 ± 15.78］ vs ［60.05 ± 4.29］×10⁶/ml, P = 0.683), sperm count (［154.15 ± 46.37］ vs ［221.56 ± 15.43］×106, P = 0.236), total sperm motility (［29.04 ± 3.11］ vs ［33.52 ± 1.51］ %, P = 0.626), or percentage of IMS (［23.57 ± 0.99］ vs ［62.34 ± 1.69］ %, P = 0.691).

CONCLUSIONSUrogenital MG infection is common in infertile males and potentially affects the semen quality, especially sperm vitality of the patient.

DNA Fragmentation ; Humans ; Infertility, Male ; microbiology ; physiopathology ; Male ; Male Urogenital Diseases ; microbiology ; Mycoplasma Infections ; complications ; Mycoplasma genitalium ; Semen ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa ; physiology