With the improvement of people's living standard and changes of their lifestyle, the incidence of coronary artery disease (CAD) has increased quite significantly, which has attracted great concern. Examination methods for CAD are more and more advanced, while the cost of inspection is rising. As a traditional test, the exercise treadmill test (ETT) is convenient, safe, and cheap. ETT can be conducted in almost every hospital as it doesn't need advanced equipments. The values of some new evaluation indexes for diagnosis and prognosis of CAD are reviewed in this article.

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Objective To evaluate the efficacy and safety of high-frequency electric dissection for colorectal cysts.Methods The personal information,clinical data,operation methods and postoperative complications of patients who were diagnosed as having colorectal cysts and underwent high-frequency electric dissection in Nanfang hospital and Zhujiang Hospital of Southern Medical University between January 1st,2005 and July 1st,2015 were analysed.All patients enrolled in the study were followed up to obtain their resuits of colonoscopy.Results A total of 63 patients were enrolled into our study,9 lesions located in the ileocecus,17 in the ascending colon,19 in the transverse colon,10 in the descending colon,7 in the sigmoid colon and 1 in the rectum.The maximum diameter of the cysts was 20.2+7.5 mm (5-40 mm).All patients underwent high-frequency electric dissection to remove the cysts completely or part of the cyst wall for drainage.Hemorrhage occurred in only one patient and bleeding stopped after being clipped by Titanium clip.Forty-five patients were followed up and there were no delayed complications or recurrence during a postoperative follow-up of 24.1 + 14.3 months (6-87 months).Conclusion High-frequency electric dissection is a safe and effective procedure for the treatment of colorectal cysts.

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein- mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-tansfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors,encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

Objective To explore the diagnosis and differential diagnosis of tricholemmal carcinoma and the significance of expression of cytokeratin(CK)and oncogene protein(c-erbB-2)in it.Method Twenty-two cases of tricholemmal carcinoma were studied with histologic and immunohistochemical tech-niques,and compared with those in squamous cell carcinoma.The neoplastic cells were stained with cytok-eratin-H,cytokeratin-L and c-erbB-2immunohistochemically(S-P method).Results The morphological characteristics of the tricholemmal carcinoma were the lobular structure and sudden keratinization in the cen-ter of the lobule,which were the key points to be differentiated with squamous cell carcinoma.The positive rate of cytokeratin-H of tricholemmal carcinoma was81.8%(18/22),and the positive cells distributed in the middle zone between the keratinized lobular center and tumor cells in the peripheral area,which differed from squamous cell carcinoma.Cytokeratin-L expression was not found in tricholemmal carcinoma.The posi-tive rate of c-erbB-2was50%(11/22)in tricholemmal carcinoma,with positive cells distributed to the mar-gin of the carcinoma cell nests,and increased with the poor differentiation.Conclusions The particular dis-tribution of cytokeratin-H suggests the tricholemmal origin of tricholemmal carcinoma.C-erbB-2may take part in the activation and differentiation of this tumor.

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.

Objective To investigate whether there has any difference of gastric and small bowel transit time and completion rates between two capsule endoscopes with different size and weight. Methods Clinical data of patients who had undergone OMOM or MiroCam (smaller and lighter than OMOM) capsule endoscopy were retrospectively studied. Comparison of gastric and small bowel transit time and completion rates were made between the two kinds of capsule endoscopy. Results 1, 448 patients (628 in OMOM group and 820 in MiroCam group) were finally includ-ed. In patients with Crohn's disease or suspected Crohn's disease, gastric transit time of OMOM was significantly longer than that of MiroCam [(53.4 ± 52.6) minutes vs (41.1 ± 47.9) minutes, = 0.022]. In patients with gastroin-testinal bleeding, gastric transit time in OMOM was significantly shorter than that in MiroCam [(42.1 ± 44.8) minutes vs (62.0 ± 78.6) minutes, = 0.016). No significant difference in small bowel transit time or completion rate was found. Conclusions We conclude that the differences of gastric transit time, small bowel transit time and completion rates between the two kinds of capsule endoscopy with different size and weight are not significantly. Whereas, in patients with Crohn's disease or suspected Crohn's disease, gastric transit time of smaller and lighter capsule en-doscopy is shorter in patients with gastrointestinal bleeding, but longer of gastric transit time in smaller and lighter capsule endoscopy.

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.