Background:Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are important for both the integrated diagnosis and the prognosis of diffuse gliomas.The p.R132H mutation of IDH1 is the most frequently observed IDH mutation,while IDH2 mutations were relatively rarely studied.The aim of the study was to determine the pathological and genetic characteristics of lowergrade gliomas that carry IDH2 mutations.Methods:Data from 238 adult patients with lower-grade gliomas were retrospectively analyzed.The status of IDH1/2 gene mutations,telomerase reverse transcriptase (TERT) promoter mutations,O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation,1p/19q co-deletion and the expressions of IDH1 R132H,alpha-thalassemia X-linked mental retardation,and p53 were evaluated.Progression-free survival (PFS) and overall survival (OS) were calculated via Kaplan-Meier estimation using the log-rank test.Results:Totally,71％ (169/238) of patients were positive for IDH mutations,including 12 patients harboring mutations in IDH2.Among the 12 patients with IDH2 mutations,ten patients harbored the R172K mutation,one patient harbored the R172S mutation and one harbored the R172W mutation.Of these,11 tumors occurred in the frontal lobe and showed morphology typical of oligodendroglioma.The proportion of grade Ⅱ tumors was higher than that of grade Ⅲ tumors in IDH2 mutant-gliomas.IDH2 mutations were frequently associated with TERT promoter mutations,1p/19q co-deletion and MGMT promoter methylation.IDH2 mutations were associated with better outcomes compared with IDH wild-type gliomas (P ＜ 0.05).However,the PFS and OS did not differ from that of IDH1 mutant patients (P =0.95 and P =0.60,respectively).Conclusions:IDH2 mutations are more frequent in oligodendrogliomas and associated with a better prognosis.IDH2 mutations may segregate in distinct clinico-pathological and genetic subtypes of gliomas,and therefore may merit routine investigation.

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.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

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.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

OBJECTIVETo evaluate three dimensional dynamic contrast-enhanced magnetic resonance angiography (3D-DCE MRA) in diagnosis of cavernous transformation of portal vein (CTPV).

METHODSTwenty-four patients with CTPV underwent 3D-DCE MRA examinations and the reconstructed images were retrospectively analyzed. A series of clinical, laboratory and imaging studies were performed on all these cases. Among all cases 14 underwent operations and 2 with hepatocellular carcinoma complicated portal thrombosis received transhepatic artery chemoembolization.

RESULTThe CTPA was located in the main trunk in 10 cases, in both the main trunk and left/right branches in 8, and in left or right branches of the portal vein in 4. In the remaining 2 cases CTPA was located at the level of superior mesenteric vein. MRA revealed multiple circuitous collateral veins striding over obstruction to extend into the liver in 9 cases,and in 7 it simultaneously showed streaky or dot-like low signal intensities representing thrombi in the extensively dilated network of portal system. MRA did not clearly demonstrate the structure of the portal vein but only showed multiple sinuous network of venous collaterals strangling together in 6 cases. In 15 cases it also showed the route and distribution of multiple hepatofugal venous collaterals.

CONCLUSION3D-DCE MRA can provide adequate information about the site and severity of CTPA.

Adult ; Aged ; Aged, 80 and over ; Contrast Media ; Female ; Hemangioma, Cavernous ; diagnosis ; etiology ; pathology ; Humans ; Image Processing, Computer-Assisted ; Imaging, Three-Dimensional ; Liver Neoplasms ; complications ; Magnetic Resonance Angiography ; methods ; Male ; Middle Aged ; Portal Vein ; pathology ; Retrospective Studies ; Venous Thrombosis ; diagnosis ; etiology ; pathology

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

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.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology

The purpose of this study was to clarify the characteristics of the pacemaker cells in the left ventricular outflow tract (aortic vestibule) and compare them with those of the cells in the sinoatrial node (SAN). By using conventional intracellular microelectrode technique to record their action potentials, some ionic channel blockers were used to observe their electrophysiological effects on the two types of pacemaker cells in the rabbit, especially on the ionic movement during phase 0 and phase 4. The results obtained are as follows. (1) Perfusion with 1 micromol/L verapamil (VER) resulted in a significant reduction in the amplitude of action potential (APA), maximal rate of depolarization (V(max)), absolute value of the maximal diastolic potential (MDP), velocity of diastolic depolarization (VDD) and rate of pacemaker firing (RPF), and also a prolongation of the 90% of the duration of action potential (APD(90)) in the pacemaker cells of the SAN and aortic vestibule (P<0.05). (2) Perfusion with 180 micromol/L nickel chloride (NiCl2) resulted in a decrease in VDD in the two types of the pacemaker cells (P<0.01). APA, V(max) and RPF fell notably, and the APD(90) prolonged in the sinoatrial node cells (P<0.05). (3) 2 mmol/L 4-aminopyridine (4-AP) led to a increase in VDD in both types of pacemaker cells (P<0.01). At the same time the absolute values of MDP, APA and V(max) decreased significantly, and APD(90) prolonged notably (P<0.05). During the perfusion, RPF in SAN increased markedly, while RPF in aortic vestibule exhibited no significant change. (4) 2 mmol/L cesium chloride (CsCl) led to a decrease in VDD and RPF in the two types of the pacemaker cells (P<0.05).These results suggested: (1) the ion currents in phase 0 and phase 4 of depolarization and repolarization of slow-response activity in aortic vestibule are similar to those in dominant pacemaker cells of sinoatrial node; (2) for the pacemaker cells in the left ventricular outflow tract, Ca(2+) current is the main depolarizing ion current of the phase 0, K(+) current is the main factor responsible for the repolarization. Attenuation of K(+) current is responsible for the phase 4 spontaneous depolarization. In addition, it seems that I(Ca-T), I(Ca-L) and I(f ) play some role in the pacemaker currents.
4-Aminopyridine ; pharmacology ; Action Potentials ; drug effects ; Animals ; Aorta, Thoracic ; cytology ; physiology ; Female ; Male ; Nickel ; pharmacology ; Periodicity ; Rabbits ; Sinoatrial Node ; cytology ; physiology ; Verapamil ; pharmacology

OBJECTIVETo review the development of tissue culture on Rehmannia glutinosa.

METHODDocumentaries at home and abroad were consulted.

RESULT AND CONCLUSIONThe tissue culture conditions of R. glutinosa were summarized and the problems of tissue culture in R. glutinosa were also pointed out.

Culture Media ; Lighting ; Plant Diseases ; Plant Leaves ; growth & development ; Plant Roots ; growth & development ; Plant Stems ; growth & development ; Plants, Medicinal ; growth & development ; Pollen ; growth & development ; Rehmannia ; growth & development ; Tissue Culture Techniques ; Tissue Preservation

The risk factors of high trait anger of juvenile offenders were explored through question naire study in a youth correctional facility of Hubei province,China.A total of 1090 juvenile offenders in Hubei province were investigated by self-compiled social-demographic questionnaire,Childhood Trauma Questionnaire (CTQ),and State-Trait Anger Expression Inventory-Ⅱ (STAXI-Ⅱ).The risk factors were analyzed by chi-square tests,correlation analysis,and binary logistic regression analysis with SPSS 19.0.A total of 1082 copies of valid questionnaires were collected.High trait anger group (n=316) was defined as those who scored in the upper 27th percentile of STAXI-Ⅱ trait anger scale (TAS),and the rest were defined as low trait anger group (n=766).The risk factors associated with high level of trait anger included:childhood emotional abuse,childhood sexual abuse,step family,frequent drug abuse,and frequent internet using (P＜0.05 or P＜0.01).Birth sequence,number of sibling,ranking in the family,identity of the main care-taker,the education level of care-taker,educational style of care-taker,family income,relationship between parents,social atmosphere of local area,frequent drinking,and frequent smoking did not predict to high level of trait anger (P＞0.05).It was suggested that traumatic experience in childhood and unhealthy life style may significantly increase the level of trait anger in adulthood.The risk factors of high trait anger and their effects should be taken into consideration seriously.

The clinical tumor therapy was greatly challenged due to the complex characteristics of tumor microenvironment, however, which also provide arena for novel therapeutic strategies. In this study, poly(2-ethyl-2-oxazoline)-poly(lactic acid)-SS-poly(β-amino ester (PEOz-PLA-SS-PBAE) triblock copolymers with pH and GSH double response were synthesized, polymer micelles were prepared by thin film hydration method for loading of silybin to improve its antitumor activity. The critical micelle concentration was determined by pyrene fluorescence method as 1.8 μg·mL^-1. The particle size was 155.30 ± 1.80 nm as determined by dynamic light scattering, with polydispersity index of 0.168 ± 0.004. The drug loading and entrapment efficiency of the micelles were determined by HPLC as (5.48 ± 0.04)% and (68.52 ± 0.48)%, respectively. The in vitro drug release profiles showed that the micelles have low pH sensitivity and high GSH responsiveness, and exhibited sustained release profiles. The good biocompatibility of the material was proved by measuring the hemolysis rate and cytotoxicity of the blank micelle. The cytotoxicity and apoptosis rate of tumor cells showed that the drug loaded PEOz-PLA-SS-PBAE micelles had significant inhibitory effect and apoptosis-inducing effect on MDA-MB-231 cells. The results of wounding healing assay and Transwell invasion test showed that the drug loaded PEOz-PLA-SS-PBAE micelles could significantly inhibit the metastasis of MDA-MB-231 cells. The PEOz-PLA-SS-PBAE drug-loaded micelles prepared in this study have good inhibitory effect on tumor growth and anti-tumor metastasis in vitro, which lays the foundation for the further application of silybin.