Adult ; Female ; Humans ; Myocardial Infarction ; Puerperal Disorders

Hematoma ; diagnosis ; diagnostic imaging ; etiology ; Humans ; Kidney Diseases ; complications ; diagnostic imaging ; physiopathology ; Male ; Middle Aged ; Ultrasonography

OBJECTIVETo investigate the difference of soluble P-selectin levels in different subtype of coronary heart disease and the relationship between soluble P-selectin levels with the severity of coronary artery lesions.

METHODSEnzyme linked immuoserbent assay (ELISA) was used to measure the plasma soluble P-selectin levels in 69 patients with angiocardiography documented coronary heart disease and 19 normal coronary arteries persons without angiocardiography detectable coronary artery disease (control group). The coronary artery lesions score was recorded according to single, double and triple-vessel lesions while the American College of Cardiology and the American Heart Association proposed type A, B, C lesion and Gensini scoring system. The relationships between plasma soluble P-selectin levels and the coronary artery score (the severity of coronary heart disease) were assessed.

RESULTS(1) The level of plasma soluble P-selectin was obviously higher in the coronary heart disease group than in the control group (180.6 +/- 60.5 ng/L vs. 145.3 +/- 21.7 ng/L, P<0.05). (2) The level of plasma soluble P-selectin was significantly higher in the acute coronary syndrome group (191.4 +/- 63.7 ng/L) than in the control group (145.3 +/- 21.7 ng/L, P< 0.01) and in the stable angina pectoris group (141.3 +/- 17.9 ng/L, P<0.01). (3) The level of plasma soluble P-selectin was high in multi-vessel coronary artery lesions group than in single-vessel group (190.1 +/- 64.2 ng/L vs. 157.2 +/- 43.4 ng/L, P < 0.05). The level of plasma soluble P-selectin was positively correlated with the Gensini score (r = 0.391, P = 0.001); the numbers of vessels lesions (rs = 0.349, P = 0.003); Type A, B and C lesions (rs = 0.358, P = 0.002).

CONCLUSIONThe positive correlation between the level of soluble P-selectin and the coronary artery score may indicate that soluble P-selectin levels might reflect the severity of coronary heart disease. The elevated soluble P-selectin level in acute coronary syndrome suggested the possible relation of P-selectin to the pathogenesis of acute coronary syndrome, which may save as a potential marker of plaque unstability.

Case-Control Studies ; Coronary Disease ; blood ; physiopathology ; Coronary Vessels ; pathology ; Female ; Humans ; Male ; Middle Aged ; P-Selectin ; blood ; chemistry ; Solubility

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.
Animals ; Cells, Cultured ; Chlorides ; metabolism ; Codon ; genetics ; Cricetinae ; Cystic Fibrosis ; therapy ; Cystic Fibrosis Transmembrane Conductance Regulator ; genetics ; metabolism ; DNA, Complementary ; genetics ; Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Inteins ; physiology ; Kidney ; cytology ; Protein Processing, Post-Translational ; Recombinant Proteins ; genetics ; metabolism ; Trans-Splicing ; Transfection

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.
Animals ; COS Cells ; Cercopithecus aethiops ; Cysteine ; genetics ; metabolism ; Disulfides ; metabolism ; Factor VIII ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; Mutation ; Peptide Fragments ; genetics ; metabolism ; Protein Splicing ; Transfection

Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIII gene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediated protein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally spliced fVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcine fVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. A pair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfected into COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatant of cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of spliced hfVIII secreted from the cells co-transfected with human fVIII [(184+/-34 ng/mL) vs (48+/-12) ng/mL, P<0.01; (1.18+/-0.22) IU/mL vs (0.31+/-0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of intein-spliced HP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separately transfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independent of cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularly spliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. These results provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIII gene in animal models.
Animals ; COS Cells ; Cercopithecus aethiops ; Dependovirus ; genetics ; metabolism ; Factor VIIIa ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Inteins ; Protein Splicing ; Recombinant Fusion Proteins ; genetics ; Swine ; Trans-Splicing

OBJECTIVETo prepare a lymph node-targeted ultrasound/fluorescence bi-functional imaging contrast agents, and observe its effectiveness both on contrast-enhanced ultrasound (CEUS) and vivo near infrared fluorescence (NIR) imaging through animal experiments.

METHODSThe chimeric lymph node-targeted ligand (phosphatidylserine) and near-infrared fluorescent substance were assembled to form bi-functional contrast microbubbles. The morphology and size distribution were detected by optical microscope and Malvern potential tests. Five normal New Zealand white rabbits were subcutaneously injected with the prepared contrast agent in bilateral footpads, and the imaging effectiveness of lymph nodes and lymphatic vessel were observed by CEUS and NIR technique. Then blue dye was subcutaneously injected at the same site, and the rabbits were sacrificed for lymph nodes pathological examination.

RESULTSLipid ultrasound microbubbles,with a mean size of 3-5 Μm in diameter, appeared to be uniform in distribution and regular in configuration. The images of inflow lymphatic vessel and relevant lymph node were quickly showed up after the subcutaneous injection by CEUS, which was identical to the result detected by NIR. Biopsy confirmed that all the blue-stained lymph nodes could be displayed by NIR.

CONCLUSIONSThe self-made bi-functional contrast agent has a good imaging ability in CEUS and NIR imaging. It may be a better agent as lymph node tracer.

Animals ; Contrast Media ; chemistry ; Fluoresceins ; chemistry ; Lymph Nodes ; anatomy & histology ; diagnostic imaging ; Lymphatic Metastasis ; pathology ; Male ; Rabbits ; Ultrasonography

OBJECTIVETo explore the risk factors and prevention strategies of post-operative complications in elderly patients with colorectal cancer.

METHODSData of 107 elderly patients (≥75 years) undergoing surgery for colorectal cancer were collected from January 2006 to December 2009 in the Department of Gastrointestinal Surgery, Peking University People's Hospital. POSSUM and E-POSSUM scoring systems were used to predict post-operative complications. ROC curve and observe/expect(O/E) were used to assess the validity of scoring systems. Logistic regression was used to evaluate the independent risk factors associated with post-operative complications of elderly patients with colorectal cancer.

RESULTSThe predictive complication rates of E-POSSUM and POSSUM in elderly patients with colorectal cancer were 13.9%-86.6%(average, 32.7%) and 19.1%-99.1% (average, 55.5%). The predictive validity of E-POSSUM was better than POSSUM(AUC of ROC: 0.862 vs. 0.576, O/E: 0.771 vs. 0.454), the former was closer to the actual complication rate(25.2%, 27/107). Concurrent diabetes mellitus(P=0.019) and rectal lesion(P=0.005) were independent risk factors associated with surgery-related post-operative complications. Anastomotic leakage was the most common surgery-related post-operative complications. Chronic obstructive pulmonary disease(P=0.026), ASA score(P=0.025), intestinal obstruction(P=0.037) and perforation(P=0.001) were independent risk factors associated with non-surgery-related post-operative complications. Pulmonary infection was the most common non-surgery-related post-operative complication.

CONCLUSIONSThe application of E-POSSUM scoring system can provide more accurate prediction of post-operative complications in elderly patients undergoing surgery for colorectal cancer. Positive interventions should be taken for high-risk patients to prevent post-operative complications.

Aged ; Colorectal Neoplasms ; surgery ; Female ; Humans ; Male ; Postoperative Complications ; etiology ; prevention & control ; Risk Factors

Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.
Cell Line ; Dependovirus ; Factor VIII ; metabolism ; Genetic Vectors ; Humans ; Inteins ; Protein Splicing

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.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection