OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

Factor VII Deficiency ; diagnosis ; genetics ; Female ; Humans ; Middle Aged ; Pedigree ; Phenotype

OBJECTIVETo evaluate the diagnosis and management of adrenal myelolipoma.

METHODSThe clinical data of 26 cases were analyzed retrospectively and the selected articles were reviewed. There were no specific clinical symptom and endocrine abnormality, except increasing catecholamine in 2 cases. All cases but two were diagnosed by B-model ultrasound scanning (B-US), CT or magnetic resonance imaging (MRI).

RESULTSTwenty-six cases were surgically treated, the diameter of the tumor was 5 - 10 cm, simple tumor resection was performed in 16 cases, and complete adrenal resection was performed in 10 cases. All the operated cases were proved by pathologists. The duration of follow-up was from 6 - 28 months after surgery. No recurrence was observed.

CONCLUSIONSThe diagnosis of adrenal myelolipoma could be established based on B-US, CT or MRI. The tumors whose diameter is larger than 5 cm in size should be removed.

Adrenal Gland Neoplasms ; diagnosis ; surgery ; Adrenalectomy ; Adult ; Female ; Follow-Up Studies ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Myelolipoma ; diagnosis ; surgery ; Retrospective Studies ; Tomography, X-Ray Computed

OBJECTIVETo evaluate the various operative details of strictures of the posterior urethra that are essential for a successful result.

METHODSThe clinical data of 191 patients with posterior urethral strictures or distraction defects from January 1990 to January 2006 were analyzed retrospectively. All patients underwent a retrograde and voiding urethrogram, 62 patients had urethral ultrasonography, 48 patients had urethroscopy, 3 patients had MRI. Repair was performed with a simple anastomosis after urethral mobilization in 66 patients, separation of the corporeal bodies in 48 patients, separation of the corporeal bodies and inferior pubectomy in 30 patients, transpubic anastomosis in 18 patients, pull-through operation in 3 patients, and optical urethrotomy in 26 patients. Followup ranged from 6 to 48 months.

RESULTSThe mean stricture length was 3.6 cm (range from 1.5 to 8.0 cm). Posterior urethral strictures is in 31 (16%), posterior distraction defects is in 160 (84%), of which the length of the distraction defects < 3 cm is in 102 (53.4%), and the the length of the distraction defects > 3 cm is in 58 (30.6%). The overall successful results (Qmax > 15 ml/s) after operation was 84.3%. Optical urethrotomy was 69%, the successful results with anastomotic urethroplasty were 97% with a simple anastomosis; 79% with separation of the corporeal bodies; 80% with separation of the corporeal bodies and inferior pubectomy; 83% with transpubic anastomosis; and 67% with pull-through operation.

CONCLUSIONSThe anastomotic urethroplasty is better than the optical urethrotomy, the length of the strictures or distraction defect which is lower than 3 cm is much more successfully corrected.

Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; Urethral Stricture ; surgery ; Urologic Surgical Procedures, Male ; methods

OBJECTIVETo evaluate the selection of different procedures and the feasibility for the treatment of long segment urethral stricture.

METHODSSeventy-six patients with complex urethral stricture greater than 8 cm long underwent different procedures of urethroplasty. Of them various mucosa grafts urethral reconstruction were adopted in 42 cases (colonic mucosal graft, n = 26; buccal mucosal graft, n = 10; bladder mucosal graft, n = 6); One-stage pedicle flaps urethroplasty in 20; two-stage urethroplasty of Johanson procedure in 12; and penile urethra-prostatic urethra anastomosis, three-stage urethroplasty in 2.

RESULTSIn early followed up (within 6 months postoperatively), 67 patients (88%) voided well and complications developed in 10. Among the 70 patients who lasted more than 1 year after operation, 51 cases were followed up. Forty-four patients voided well, and complications developed in 8. Of the 8 cases urethral restructure developed in 2 (18%) for pedicle flaps urethroplasty, 2 for colonic mucosal urethroplasty (9%), 1 for buccal mucosal graft (1/7), 1 for bladder mucosal graft (1/3); penile chordee in 2 (2/5), and one of them was accompanied by hair bearing neourethra for two-stage urethroplasty of Johanson procedure.

CONCLUSIONSColonic mucosal and buccal mucosal grafts urethroplasty are feasible procedures for the treatment of long segment urethral stricture, and Colonic mucosal graft urethroplasty may be considered when more conventional procedures fail or complicated urethral strictures greater than 10 cm long.

Adolescent ; Adult ; Aged ; Follow-Up Studies ; Humans ; Intestinal Mucosa ; surgery ; Male ; Middle Aged ; Mouth Mucosa ; surgery ; Surgically-Created Structures ; Treatment Outcome ; Urethral Stricture ; pathology ; surgery ; Urologic Surgical Procedures, Male ; methods

Animals ; Colon ; pathology ; Dogs ; Female ; Graft Survival ; Intestinal Mucosa ; pathology ; transplantation ; Mouth Mucosa ; pathology ; transplantation ; Reconstructive Surgical Procedures ; Urethra ; surgery

Objective To study the clinical features and curative effect of hepatitis associated aplastic anemia (HAAA) in children of China.Methods Patients' records in China Biological Medicine Database (CBM-disc 1980-2011) and Wan Fang Database were reviewed.The clinical data of the children with newly diagnosed HAAA were respectively studied,including clinical manifestations,blood routine,bone marrow examination and viral serology results,as well as the treatment and clinical effect.Results A total of 50 children were confirmed as HAAA.There were 41 boys and 9 girls.The median age was 8.2 years(range 0.8 to 15.0 years) on diagnosis.The causes of hepatitis could not be identified.The median interval between hepatitis occurrence to blood cell reduction was 10 weeks.Twenty-two cases were diagnosed as severe aplastic anemia and 13 cases as very severe aplastic anemia.Fifteen children died within 1 month after diagnosis.In the immunosuppression treatment group,the percentage of overall responders was 69.2％,which was higher than that of the non-immunosuppression treatment group (18.5％) (x2 =9.920,P ＜ 0.01).Conclusions Severe HAAA is very common in school children,especially in boys.The children with HAAA have a higher early death rate.Immunosuppression therapy is effective if combined with androgenic hormone with an earlier diagnosis.

OBJECTIVETo assess the value of (99m)Tc-N-NOET ((99m)Tc-N-ethoxy-N-ethyl dithiocarbamato-nitrito) myocardial perfusion SPECT for the diagnosis of coronary artery disease.

METHODSA total of 42 patients [mean age (54 +/- 9) years, 35 men] with suspected chest pain were included in this study. 740 MBq of (99m)Tc-N-NOET was injected intravenously during bicycle exercise when the heart rate attained reached more than 85% of the expected maximum, or in cases of angina pectoris, severe arrhythmias and ischemic ST segment changes. (99m)Tc-N-NOET 740 MBq, SPECT myocardial imaging acquisitions were obtained at 15 minutes and 2 hours after (99m)Tc-N-NOET injection. Coronary angiography was performed in all patients.

RESULTSCoronary artery stenosis was detected in 26 patients and normal coronary angiography was shown in 16 patients. (99m)Tc-N-NOET myocardial perfusion imaging was abnormal in twenty-one patients out of the 26 patients with significant coronary artery stenosis (sensitivity, 81%); 14 out of 16 patients with normal angiography had a normal myocardial perfusion imaging (specificity, 88%). The positive predictive value, negative predictive value and predictive accuracy of (99m)Tc-N-NOET myocardial perfusion imaging for detection of CAD was 91%, 74% and 83%, respectively. The sensitivity of the imaging for detecting single vessel, double vessels and triple vessels disease were 60% (6/10), 86% (6/7) and 100% (9/9), respectively. There was mild (99m)Tc-N-NOET lung uptake in patients with coronary artery stenosis 15 minutes post (99m)Tc-N-NOET injection.

CONCLUSIONSPECT myocardial perfusion imaging with (99m)Tc-N-NOET supplied an important diagnostic tool for detecting coronary artery disease. Lung uptake with stress (99m)Tc-N-NOET might be related to coronary artery disease.

Adult ; Aged ; Coronary Artery Disease ; diagnostic imaging ; Exercise Test ; Female ; Humans ; Male ; Middle Aged ; Organotechnetium Compounds ; Thiocarbamates ; Tomography, Emission-Computed, Single-Photon

Objective:To investigate the long-term safety of digoxin in patients with coronary artery disease (CAD) and atrial fibrillation (AF).Methods:This was a prospective study, in which 25 512 AF patients were enrolled from China Atrial Fibrillation Registry Study. After exclusion of patients receiving ablation therapy at the enrollment, 1 810 CAD patients [age: (71.5±9.3)years] with AF were included. The subjects were grouped into the digoxin group and non-digoxin group, and were followed up for a period of 80 months. Long-term outcomes were compared between the groups and an adjusted Cox regression analysis was applied to evaluate the risk of digoxin on the long-term outcomes. The primary endpoint was all-cause mortality.Results:The patients were followed up for a median period of 3.05 years. After multivariable adjustment, the Cox regression analysis showed that digoxin significantly increased the risk of all-cause mortality ( HR=1.28, 95% CI 1.01-1.61, P=0.038), cardiovascular mortality ( HR=1.48,95% CI 1.10-2.00, P=0.010), cardiovascular hospitalization ( HR=1.67,95% CI 1.35-2.07, P=0.008) and the composite endpoints ( HR=2.02,95% CI 1.71-2.38, P<0.001). In the subgroup of patients with heart failure (HF), digoxin was not associated with the risk of all-cause mortality, but was still associated with the increased risk of cardiovascular mortality ( HR=1.44,95% CI 1.05-1.98, P=0.025), cardiovascular hospitalization ( HR=1.44,95% CI 1.09-1.90, P=0.010) and the composite endpoints ( HR=1.37, 95% CI 1.01-1.70, P=0.004). However, in the subgroup of patients without HF, digoxin was only associated with all-cause mortality ( HR=2.56,95% CI 1.44-4.54, P=0.001). Conclusion:Digoxin significantly increased the risk of all-cause mortality in CAD patients with AF, especially in patients without HF.

OBJECTIVE: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation. METHODS: Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status. RESULTS: In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0. CONCLUSION The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.
Amelogenin ; genetics ; Blastomeres ; cytology ; metabolism ; Chromosomes, Human, X ; genetics ; Chromosomes, Human, Y ; genetics ; Cytogenetic Analysis ; methods ; Exons ; genetics ; Female ; Gene Deletion ; Humans ; Lymphocytes ; cytology ; metabolism ; Male ; Muscular Dystrophy, Duchenne ; blood ; diagnosis ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Preimplantation Diagnosis ; methods