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.

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.

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.

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

OBJECTIVETo investigate the clinical outcomes of the posterior C1,2 screw-rod combined with C2 unilateral translaminar screw and contralateral pedicle screw fixation and autogenous bicortical iliac crest graft fusion in treating upper cervical instability with vertebral artery variations.

METHODSFrom June 2008 to December 2012, the clinical data of 12 patients with upper cervical instability underwent C1 lateral mass screws-C2 unilateral laminar and contralateral pedicle screws fixation combined with autogenous bicortical iliac crest graft fusion were analyzed retrospectively. There were 8 males and 4 females with a mean age of 47.5 years (ranged, 16 to 77 years). Patients suffered from occipitocervical activity limitation of motion with pain or not, VAS was 0-7 points with an average of (3.50 +/- 2.71) points. Unilateral vertebral artery hypoplasia was demonstrated by vertebral arteriography (VAG) or CTA in all patients. Cervical X-ray and CT scans were done within 7 days after surgery in order to confirm internal fixation position. Internal fixation loosening and breakage, reduction losing, bone fusion ratio were observed during follow-up.

RESULTSNo nerves and vertebral artery injuries occurred during operation. Cervical pain obviously decreased and VAS was (0.92 +/- 0.90) points. Cervical alignment of 12 patients had well-recovered by X-ray while Atlantoaxial ventral lamina cortex of 1 case was encroached by CT scan without neurological symptom. All patients were followed up for 6 months to 3 years, no internal fixation loosening and breakage, reduction losing were found. All patients obtained bone fusion in 6-12 months after operation.

CONCLUSIONPosterior C1 lateral mass screws-C2 unilateral laminar and contralateral pedicle screws fixation combined with autogenous bicortical iliac crest graft fusion can achieve biomechanical stability and raise the successful rate of bone fusion, while avoiding the risk of vertebral artery injury and overcoming the insufficient of bone fusion during bilateral laminar screws placement as well. Posterior C1 lateral mass screws fixation is a safe and effective additional method in treating upper cervical instability with vertebral artery variations.

Adolescent ; Adult ; Aged ; Bone Screws ; Cervical Vertebrae ; surgery ; Female ; Humans ; Internal Fixators ; Joint Instability ; surgery ; Male ; Middle Aged ; Tomography, X-Ray Computed ; Vertebral Artery ; pathology

Objective To discuss the relationships between qi-arrival and mind-control, qi-arrival and qi-regulation, and to define qi-arrival by tracing the relevant records in Nei Jing (Classic of Internal Medicine) and Nan Jing (Classic of Difficult Issues). Method Thirty-seven relevant ancient books of the two classics were reviewed and the notes by WANG Bing, MA Shi, and YANG Shang-shan were referred. Result Mind-control requires the spirits of the practitioner, patient and environment, as well as observing and waiting for the arrival of qi. Regarding qi-regulation, theories involve that qi-arrival achieves same reinforcing and reducing effect as qi-regulation, qi-regulation is elucidated from the correct location of acupoint, proper needling depth and direction, and to regulate qi by observing the arrival of qi. Besides, the changes of internal and external environments also influence qi-arrival via qi-regulation and mind-control. Conclusion Qi-arrival is closely related to mind-control and qi-regulation in obtaining, differentiation, and adjustment. Mind-control and qi-regulation assist each other through the whole acupuncture process, which is the core of qi-arrival.

BACKGROUND: Previous studies have shown that HLA-identical sibling allogeneic peripheral blood hematopoietic stemcelltransplantation (allo-HSCT) provides higher disease-free and overall survival rates for patients with intermediatecytogenetic risk acute myeloid leukemia (AML) in complete remission (CR). But prognosis factors have not been fullydefined.OBJECTIVE: To evaluate the outcome of patients with intermediate cytogenetic risk AML undergoing HLA-matchedallo-HSCT in CR, and to analyze the prognostic factors.METHODS: Fifty cases of intermediate cytogenetic risk AML in CR receiving HLA-matched allo-HSCT from January2009 to January 2015 were retrospectively analyzed. Primary outcome measures of the study included overall survival(OS), relapse rate and non-relapse mortality.RESULTS AND CONCLUSION: The 4-year OS of the study population reached to 64%, and the relapse rate and NRMreached to 18% and 20% respectively. Incidence of acute graft-versus-host disease was 26%. Different prognosis wasobserved between female donor/male recipient (FDMR) combination transplant and control (4-year OS: 50% vs. 71.9%,P=0.041), between patients requiring more than one course of induction chemotherapy to achieve CR and control(4-year OS: 40% vs. 70%, P=0.038), between older age (≥ 40 years) and control (4-year OS: 44.4% vs. 68.3%,P=0.056). The 4-year OS for matched sibling donor and matched unrelated donor was 63.2% and 66.7% (P=0.427),respectively. Further analysis revealed significantly high non-relapse mortality in FDMR combination transplant (P=0.024)and older age (≥ 40 years; P=0.043). Multivariate analysis revealed three negative prognostic factors: FDMRcombination (P=0.031, RR=1.38, 95% CI: 1.03-1.95), requiring more than one course of induction chemotherapy toachieve CR (P=0.016, RR=1.46, 95% CI: 1.10-1.98) and older age (≥ 40 years; P=0.024, RR=1.63, 95% CI: 1.32-2.12).To conclude, HLA-matched allo-HSCT is a choice for the intermediate cytogenetic risk AML case in CR. FDMRcombination, requiring more than one course of induction chemotherapy to achieve CR and older age (≥ 40 years) areconfirmed as risk factors of poor prognosis for HLA-matched allo-HSCT patients with intermediate cytogenetic risk AMLin CR. To these cases, the donor-recipient sex combination is more important than the donor type in donor selection.

Objective:To analyze the clinical manifestations, radiographic features, and pathological classification of the juvenile ossifying fibroma (JOF) of the jaws and discuss its clinical management and prognosis. Methods: From January 2005 to December 2014, 15 patients with JOF who underwent surgery were retrospectively investigated with regard to clinical and radiologic data. On the basis of the standards of the World Health Organization in 2005, JOF was divided into juvenile psammomatoid ossifying fibroma (JPOF) and juvenile trabecular ossifying fibroma (JTOF). Results:Among the 15 patients, 10 were female and 5 were male. Patient age ranged from 7 years old to 18 years old with a mean of 10.93 years old. Nine cases were located in the mandible and 6 in the maxilla. The clinical manifestation was painless swelling of the jaw, but 20%of the cases showed jaw swelling with pain. Various JOF radiolog-ic appearances, such as radiolucent, mixed radiopaque-radiolucent, or ground-glass pattern, were observed. Ten of the 15 patients were JTOF and 5 were JPOF With regard to treatment, 4 patients underwent conservative surgery, 3 patients lived with tumors, and 11 pa-tients underwent radical surgery during the follow-up period; no lesion recurrence occurred. Nine patients underwent reconstruction, that is, 5 cases with fibula flap graft, 3 cases with free iliac graft, and 1 case with costal cartilage graft. Conclusion:JOF is a rare form of benign fibro-osseous lesions and occurs in adolescents. Mandible and maxilla are two of the most common locations. Early diagnosis and treatment and strict clinical and radiological follow-up is important in the clinic because of the aggressiveness and high recurrence rate of JOF. Operation time and treatment options should be selected according to the patients' specific situation.