The 1.23 kb DNA fragment encoding the early protein EP0 of pseudorabies virus (PRV) Ea strain was amplified by PCR technique and cloned into pBluescriptII sk+.Three sequencing plasmids containing the partial fragment of the EP0 gene were constructed and the sequences were obtained by Sanger's sequencing technique. Compared with PRV InFh strain, there were multipile site-mutations and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity of amino acid residues also existed.Then, the EP0 gene was inserted into an expression vector, pET-28a, fused into the downstream of the 6ΧHis-Tag in frame, to yield the expression plasmid pETEP0. After induction by IPTG, a high expression of fusion protein was obtained, SDS-PAGE analysis and Western blotting showed that the fusion protein was 62kD and the protein was specific to antisera against PRV Ea strain. This indicated that the EP0 gene be expressed in BL21(DE3) and the expression products have immuno-genicity.

Autopsy ; Coxsackievirus Infections ; Enterovirus ; isolation & purification ; Enterovirus B, Human ; isolation & purification ; Enterovirus Infections ; Hand, Foot and Mouth Disease ; pathology ; virology ; Humans ; Infant ; Male

BACKGROUND: It is quite difficult to produce a decellularized lung scaffold, in which cells are removed and the extracellular matrix components (ECM) are preserved effectively. Perfusion of detergent-enzymes is an effective method with wide applications for decellularized lung scaffolds. OBJECTIVE: To investigate the effects of two detergents (sodium deoxycholate, SDC and sodium dodecyl sulfate, SDS) on the preparation of decellularized lung scaffolds. METHODS: Twenty-four male Sprague-Dawley rats were randomized into three groups: control group with no intervention, SDC group and SDS group. Decellularized lung scaffolds were prepared by perfusion of SDC or SDS combined with enzymes. The rat lung tissues in the three groups were taken for histological staining, immunofluorescent staining and DNA quantification. A549 cells were cultured and seeded onto the decellularized lung scaffolds for 7 days followed by hematoxylin-eosin staining. The decellularized lung scaffolds prepared by perfusion of SDC or SDS were subcutaneously implanted into the rat back, and the implants were retrieved and assessed by Masson staining after 2 weeks. RESULTS AND CONCLUSION: In the control group, there were abundant cells in the lung tissues. In the other two groups, the decellularized lung scaffolds were nearly transparent, and the morphology of the SDC scaffold was more close to the native lung. There were no residual cells and nuclei on the two scaffolds, and the DNA content in the SDS and SDC groups was significantly lower than that in the control group (P＜ 0.01). At 7 days of culture, A549 cells cultured on the SDS and SDC scaffolds migrated from the edge to the center of the scaffold. Comparatively speaking, the migration ability of A549 cells on the SDC scaffolds was stronger, and there was obvious cell invasion and growth in the middle part of the lung. After 2 weeks of scaffold transplantation, the SDC implants poorly fused with the surrounding tissues, with a clear boundary, a large number of infiltrating cells distributed evenly, and intravascular blood cells were clearly visible; the number of new blood vessels with larger diameter in the SDC scaffold was significantly higher than that in the SDS scaffold. These findings indicate that the SDC scaffold has better biocompatibility than the SDS scaffold, which can fuse with the surrounding tissues faster and produce more infiltrating cells and new blood vessels.

No abstract available.
Aged, 80 and over ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics ; China ; Drug Resistance, Multiple, Bacterial ; Humans ; Male ; Methyltransferases/*genetics ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/genetics/metabolism ; Serratia marcescens/drug effects/*enzymology/*genetics/isolation & purification ; Urinary Tract Infections/diagnosis/microbiology

Objective To describe a simple and efficient method to obtain large quantities of higllly purified Schwann cells(SCs).Methods SCs were isolated from the sciatic and brachial nerves of 3-to 5-day-old newborn SD rats by collagenase digestion.The isolated SCs were plated at the density of3x105/mL for primary culture,several rounds oftrypsin digestion were performed within 72 h to purify SCs. Results Compared with the purification using 1.25 g/L trypsin digestion in serial differential detachment procedures, Our protocol allowed easier and more efficient separation of the SCs from the fibroblasts.Immunocytochemical staining showed that the purity of the SCs exceeded 95%.Conclusion The purification protocol of the SCs we established can be easily carried out and yields well reproducible results to obtain large quantities ofhighly purified SCs for transplantation studies.

OBJECTIVETo compare the efficacy and safety of tolterodine and oxybutynin in the treatment of idiopathic overactive bladder in children.

METHODSA total of 204 children with idiopathic overactive bladder were randomly divided into three groups (n=68 each): placebo, tolterodine-treated and oxybutynin-treated. The efficacy and safety were evaluated two weeks after treatment.

RESULTSThe effective rate was 25% in the placebo group, 89% in the tolterodine-treated group, and 92% in the oxybutynin-treated group. The effective rate in the two treatment groups was significantly higher than that in the placebo group (P<0.05). There was a similar efficacy between the two treatment groups. The incidence of adverse events in the tolterodine-treated group (28%) was significantly lower than that in the oxybutnin-treated group (57%) (P<0.05).

CONCLUSIONSTolterodine has a similar efficacy to oxybutynin in the treatment of idiopathic overactive bladder in children, with better safety in pharmacotherapy.

Adolescent ; Benzhydryl Compounds ; adverse effects ; therapeutic use ; Child ; Child, Preschool ; Cresols ; adverse effects ; therapeutic use ; Female ; Humans ; Male ; Mandelic Acids ; adverse effects ; therapeutic use ; Muscarinic Antagonists ; therapeutic use ; Phenylpropanolamine ; adverse effects ; therapeutic use ; Tolterodine Tartrate ; Urinary Bladder, Overactive ; drug therapy

Objective To explore the diagnosis and treatment of eosinophilic cystitis (EC) in children.Methods Six patients who experienced EC in Nanjing Children's Hospital Affiliated to Nanjing Medical University from Mar.2007 to Feb.2012 were analyzed and the relevant literature was reviewed.This group included 5 male and 1 female,with mean age of 5.3 years.Their major complains included urinary frequency in 5 cases,odynuria in 3 cases,dysuria in 2 cases,hematuria in 2 cases,proteinuria in 2 cases,urinary dribbling in 1 case,and nocturia in 1 case.Physical examination and urine culture was positive in 1 case,eosinophil proportion of peripheral blood increased in 3 cases,imaging studies suggested the bladder wall thickening in 5 cases and space-occupying lesions in 2 cases.Cystoscopy in 4 cases and biopsy in 3 cases showed EC.Results Three patients were cured after receiving anti-infection and urine alkalinization,and another 3 patients were cured after adding Prednisone and Ketotifen,or Oxybutynin or Loratadine.Five patients were followed up for 1-60 months,and clinical symptoms,blood,urine,and B-ultrasound in these patients were normal.The information of 1 case was lost.Conclusions The incidence of EC in children tends to increase in recent years,the diagnosis can be confirmed combined with the clinical manifestations,laboratory tests,imaging studies and histological examination.Conservative treatment is preferred.

OBJECTIVETo investigate the association of polymorphisms of CDT1 and GMNN gene, two important genes participating in DNA replication, with the risk of sporadic breast cancer.

METHODSUsing polymerase chain reaction-restriction fragment length polymorphism (PCR - RFLP) and the primer-introduced restriction analysis (PIRA)-PCR assay to genotype the CDT1 838G/A and GMNN 387C/A polymorphisms in a case-control study of 427 breast cancer cases and 477 cancer-free controls in a Chinese population.

RESULTSNo significant association of the CDT1 838G/A and GMNN 387C/A polymorphisms with the risk of breast cancer was found (adjusted OR:1.16, 95% CI:0.88-1.54 for CDT1 GA+AA genotypes and adjusted OR:0.90, 95% CI:0.67-1.21 for GMNN CA+AA genotypes). However, in the stratified analyses, a significant association of CDT1 GA+AA genotypes with breast cancer risk among subjects with family history of cancer was found (adjusted OR:2.21, 95% CI:1.20-4.09).

CONCLUSIONThese findings suggest that the CDT1 838G/A and GMNN 387C/A polymorphisms may not play a major role in the etiology of breast cancer, but CDT1 variant may have a potential role only in genetically susceptible women.

Adult ; Asian Continental Ancestry Group ; genetics ; Breast Neoplasms ; ethnology ; genetics ; Case-Control Studies ; Cell Cycle Proteins ; genetics ; China ; Female ; Geminin ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Polymorphism, Restriction Fragment Length

Objective: To investigate the current status of antithrombotic strategy for elderly patients with atrial fibrillation (AF) and acute coronary syndrome (ACS) after stent implantation in Beijing area and to study the safety and efficacy of different therapeutic strategy. Methods: A total of 467 relevant patients were enrolled by re-travelling electronic medical records from 12 hospitals in Beijing area. The patients' mean age was (78.70±3.32) years and they were divided into 2 groups by antithrombotic therapy condition: Triple therapy group, n=17 (3.64％), Double therapy group, n=450 (96.36％). The incidence of major adverse cardiac and cerebral events (MACCE) including all-caused death, non-fatal myocardial infarction, stent thrombosis, target vessel revascularization (TVR), stoke and bleeding was compared between Triple therapy group and Double therapy group.Results: The medication in Double therapy group included aspirin+ticagrelor, aspirin+clopidogrel, clopidogrel+warfarin and cilostazol+clopidogrel; in Triple therapy group was aspirin+clopidogrel+warfarin. Patient with HAS-BLED score≥3 was defined as high risk of bleeding and they were all treated by double therapy; HAS-BLED<3 was defined as low risk of bleeding, only 5.03％ patients were treated by triple therapy. 3 patients in Triple therapy group and 33 in Double therapy group suffered from gastrointestinal bleeding, P=0.338; 6 patients in Triple therapy group and 128 in Double therapy group had MACCE, P=0.589; 3 and 80 patients died in Triple therapy group and Double therapy group, P=0.766. Conclusion: Triple therapy was rarely used in elderly AF and ACS patients after stent implantation, double therapy was the main strategy; the incidence of MACCE and mortality were similar between triple and double therapies; patients with triple therapy had the higher incidence of gastrointestinal bleeding.

OBJECTIVES: To observe the changes of the formation time of venous thrombus in rats, and to provide new ideas and methods for the estimation on thrombus formation time of the forensic cases died from thrombosis. METHODS: Totally 80 rats were randomly divided into 10 groups （0 h, 3 h, 6 h, 12 h, 1 d, 3 d, 1 week, 2 weeks, 3 weeks and 4 weeks after operation）. A vein thrombosis model was established by the "narrow" method. The processes of thrombosis, organization, recanalization and the features of change on hemosiderin and calcium salt were observed by HE stain, Perls stain and Von Kossa stain. The expression changes of CD61, α-SMA and CD34 were observed by immunohistochemical staining technique. RESULTS: Platelets adhered to the exposed blood vessel intima 3 h after operation, and platelet trabeculae were formed by the repeated accumulation of platelets 1 d after operation. The thrombus organization formed through the fibroblasts from vessel wall that grew into the interior of the thrombus 3 d after operation. Endothelial cells covered the surface of thrombus and then the new blood vessels were reformed, and the vessels were reconstructed. The expression of CD61 upregulated at the stages of the thrombus formation （3 h） and thrombus reformation （4 weeks）, and reached the peak 1 d after thrombus formation. The release of hemosiderin and the initial expression of α-SMA were detected 3 d later. Calcium deposit and expression of CD34 were observed 1 week later. CONCLUSIONS The hemosiderin, calcium salt, CD61, α-SMA and CD34 show time-dependent changing characteristics, which is expected to provide a reference for the estimation on thrombus formation time of the forensic cases died from thrombosis.
Animals ; Antigens, CD34/analysis* ; Hemosiderin/metabolism* ; Rats ; Venous Thrombosis/pathology*