Dysregulation of microRNA-181b and TIMP3 is functionally involved in the pathogenesis of diabetic nephropathy.

This study aimed to study the roleof microRNA (miR)-181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real-time polymerase chain reaction (RT-PCR) was adopted to compare the miR-181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR-181b. Real-time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR-181b inhibitors, miR-181b mimics, and TIMP3 siRNA. And the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR-181b on cell survival and apoptosis. miR-181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR-181b target. The luciferase activity of cells transfected with wild-type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR-181b expression with a correlation efficient of -0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR-181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR-181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR-181b promoted the survival of cells and inhibited their apoptosis. The miR-181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM.

USP10 regulates Musashi-2 stability via deubiquitination and promotes tumour proliferation in colon cancer.

Recent studies have demonstrated that ubiquitin-specific protease 10 (USP10) plays a catalytic role in tumour suppression mainly by deubiquitinating its target proteins to enhance their stabilities. However, we found that USP10 could interact with and regulate the expression of oncogenic factor Musashi-2 (MSI2). We investigated whether USP10 positively regulates the expression of MSI2 by deubiquitination and confirmed the type of polyubiquitin chain that is linked to MSI2. We also explored the role of USP10 in regulating the proliferation of colon cancer through different experiments. This study provides a completely new perspective in understanding the role of USP10 in deubiquitination. In the future, USP10 may serve as a target for colon cancer treatment.

Aquaporin 11 rs2276415 variant and progression of chronic kidney disease.

BACKGROUND: The Aquaporin 11 (AQP11) rs2276415 variant has been implicated in kidney disease in Type 2 diabetes. Association of the AQP11 variant with chronic kidney disease (CKD) beyond diabetic nephropathy is unknown, with no studies reported in the Chinese population. We explored the risk of CKD progression associated with the AQP11 rs2276415 variant in a population-based study in China. METHODS: We conducted a prospective cohort study of 620 participants with CKD (Stages 2-5 and who were not receiving dialysis) at the Nephrology Center of First Affiliated Hospital of Jiaxing University between July 2011 and December 2014 and followed up for 3 years. Incident CKD progression, defined as an increase in creatinine levels of at least 0.4 mg/dL (35 µmol/L) above baseline or maintenance dialysis initiation or transplantation, was examined by AQP11 genotypes. RESULTS: During the follow-up period, CKD progression developed in 170 individuals. Cumulative events-free survival was significantly dependent on AQP11 genotypes with an apparent gene-dose effect (log-rank P < 0.001). Adjusting for sex, age and major CKD risk factors, the A allele of AQP11 gene (GA + AA) increased the risk of CKD progression by 1.92 (95% confidence interval 1.31- 2.84). CONCLUSIONS: The AQP11 rs2276415 variant predicts CKD progression in the Chinese population, independent of traditional risk factors. Exploring the pathways mediating the association may shed light on novel therapeutic targets in the pathophysiology of CKD.

Low serum levels of vitamin D are associated with anxiety in children and adolescents with dialysis.

Anxiety is a frequent and serious complication of children and adolescents receiving dialysis. Low serum vitamin D levels have been associated with anxiety in non-pediatric patients. This study sought to examine the possible association between serum vitamin D levels and the presence of anxiety in children and adolescents with dialysis in China. A total of 156 pediatric patients who were on hemodialysis or peritoneal dialysis and 100 healthy controls were included in the current study. Serum 25-hydroxyvitamin D [25(OH)D] levels were measured by using a competitive protein-binding assay. Anxiety was assessed by using the Chinese version of the Screen for Child Anxiety Related Emotional Disorders (SCARED, = 25 as cutoff). Among 156 patients, 110 had a current anxiety (70.5%) and 46 did not (29.5%). Serum levels of 25(OH)D were significantly lower in patients with anxiety than in normal controls (19.4 ± 10.3 vs. 38.6 ± 15.5 ng/ml, P < 0.001). Serum 25(OH)D levels (≤15.0 ng/ml) were independently associated with the existent of anxiety in children and adolescents receiving dialysis (OR 4.650, 95% CI 1.663-13.001, P = 0.003). Our research demonstrates that low serum levels of vitamin D are independently associated with anxiety among children and adolescents on dialysis, which needs to be confirmed in future experimental and clinical studies.

Cartilage oligomeric matrix protein is a prognostic factor and biomarker of colon cancer and promotes cell proliferation by activating the Akt pathway.

PURPOSE: Recent studies have determined that cartilage oligomeric matrix protein (COMP) plays a vital role in carcinogenesis. We sought to clarify the role of COMP in colon cancer. METHODS: We investigated gene expression data from The Cancer Genome Atlas (TCGA) dataset. Tissue microarrays (TMA) containing paired samples from 253 patients with colon cancer were subjected to immunostaining. COMP levels in serum of colon cancer patients and healthy donors were measured with ELISA. We established COMP-knockout cells using the CRISPR/Cas9 system and COMP-overexpressing cells using lentiviral vectors to detect the effects of COMP on colon cancer cells using Cell Counting Kit-8 (CCK8), colony formation, apoptosis detection kit, and tumorigenesis assays in nude mice. RESULTS: The analysis of TCGA dataset and the results of the TMA suggested that COMP expression levels were significantly higher in cancer tissues than in adjacent normal tissues. Moreover, high COMP expression was correlated with the poor outcome of colon cancer patients. COMP levels in the sera of preoperative patients with colon cancer were much higher than those in healthy donors and were significantly reduced after colectomy. Colon cancer cells without COMP were defective with respect to the ability to proliferate, colony formation, the ability to resist 5-Fluorouracil-induced apoptosis and the growth of xenograft tumors in mice. Contrasting results were observed in COMP overexpressed cells. COMP promoted colon cancer cell proliferation partially through the activation of PI3K/ Akt/ mTOR/ p70S6K pathway. CONCLUSIONS: COMP may be a novel prognostic indicator and biomarker and also a potential therapeutic target for colon cancer.

Cambogin suppresses dextran sulphate sodium-induced colitis by enhancing Treg cell stability and function.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract, and an impaired immune response plays a critical role in IBD. The current drugs and therapies for IBD treatment are of limited use, therefore, there is a need to find novel drugs or therapies for this disease. We investigated the effect of cambogin in a mouse model of dextran sulphate sodium (DSS)-induced colitis and whether cambogin attenuates inflammation via a Treg-cell-mediated effect on the immune response. EXPERIMENTAL APPROACH: Chronic colitis was established in mice using 2% DSS, and cambogin (10 mg·kg-1 , p.o.) was administered for 10 days. Body weight, colon length and colon histology were assessed. Cytokine production was measured using elisa and quantitative real-time PCR. To evaluate the mechanism of cambogin, human CD4+ CD25hi CD127lo Treg cells were isolated from peripheral blood mononuclear cells. Major signalling profiles involved in Treg cell stability were measured. KEY RESULTS: Cambogin attenuated diarrhoea, colon shortening and colon histological injury and IL-6, IFN-γ and TNF-α production in DSS-treated mice. Cambogin also up-regulated Treg cell numbers in both the spleen and mesenteric lymph nodes. Furthermore, cambogin (10 µM) prevented Foxp3 loss in human primary Treg cells in vitro, and promoted USP7-mediated Foxp3 deubiquitination and increased Foxp3 protein expression in LPS-treated cells. CONCLUSIONS AND IMPLICATIONS: The effect of cambogin on DSS-induced colitis is expedited by a Treg-cell-mediated modification of the immune response, suggesting that cambogin could be applied as a novel agent for treating colitis and other Treg cell-related diseases.

Protective effect of regular physical activity on major depressive episodes in patients with early stages of chronic kidney disease.

Previous studies have demonstrated an association between physical activity (PA) and depression in diverse population. The purpose of our study is to examine if PA within the recommended level over time is associated with major depressive episode (MDE) in patients with early stages of chronic kidney disease (CKD) in Mainland China. Patients with stages 2-5 CKD not receiving dialysis were enrolled from a nephrology outpatient clinic between May 2014 and February 2016. Based on the patterns of PA over time, all patients were divided into four groups: persistently active, from inactive to active, from active to inactive, and persistently inactive. An MDE was diagnosed by using the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition)-based the Mini International Neuropsychiatric Interview. Among 150 patients, 34 had a current MDE (22.7%) and 116 did not (77.3%). After multivariable adjustment, patients being persistently active had significantly lower odds of having an MDE (odds ratio 0.102, 95% confidence interval, 0.022-0.467, p = .003) compared with those who were persistently inactive. Additionally, patients with diabetes mellitus had significantly higher odds of having an MDE (odds ratio 4.287, 95% confidence interval, 1.473-12.483, p = .008) compared with those without diabetes mellitus. Our results suggest a protective effect of regular PA on MDE in patients with early stages of CKD in Mainland China.

Association between Serum Vitamin D Levels and Sleep Disturbance in Hemodialysis Patients.

Sleep disturbance is a frequent and serious complication of hemodialysis (HD). Low serum vitamin D levels have been associated with sleep quality in non-HD subjects. Our aim was to examine the possible association between serum vitamin D levels and the presence of sleep disturbance in HD patients. We recruited 141 HD patients at the HD center of the First Affiliated Hospital of Jiaxing University during 2014-2015. Serum levels of 25-hydroxyvitamin D (25(OH)D) were determined by the competitive protein-binding assay. Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI). Demographic, clinical and laboratory data were recorded. Meanwhile, 117 healthy control subjects were also recruited and underwent measurement of 25(OH)D. Eighty-eight patients (62.4%) had sleep disturbance (PSQI scores ≥ 5). Patients with sleep disturbance showed lower levels of 25(OH)D as compared to those without sleep disturbance (85.6 ± 37.4 vs. 39.1 ± 29.1 nmol/L, p < 0.001). In multivariate analyses, serum levels of 25(OH)D (≤48.0 nmol/L) were independently associated with sleep disturbance in HD patients (OR 9.897, 95% CI 3.356-29.187, p < 0.001) after adjustment for possible variables. Our study demonstrates that low serum levels of vitamin D are independently associated with sleep disturbance in HD patients, but the finding needs to be confirmed in future experimental and clinical studies.

Serum levels of copeptin are associated with type 2 diabetes and diabetic complications in Chinese population.

PURPOSE: The aim of this study was to investigate copeptin levels in serum, and assess their associations with type 2 diabetes (T2DM) and diabetic complications. METHODS: In this post hoc analysis, serum levels of copeptin were tested in 306 patients with T2DM. Clinical information including diabetic retinopathy (DR) and diabetic nephropathy (DN) were collected. The relation of serum copeptin with DR and DN were investigated with the use of logistic regression models according to equal quartiles of the distributions of serum copeptin. RESULTS: We found that serum copeptin levels were significantly higher in diabetes as compared to normal controls [9.4(IQR, 7.4-12.5) pmol/L vs. 4.1(IQR, 2.5-6.2) pmol/L; P<0.0001]. In multivariate analysis, there was an increased risk of T2DM associated with copeptin levels (OR 1.312, 95% CI: 1.204-1.403; P<0.0001) after adjusting for possible confounders. After adjustment for possible confounders, serum copeptin levels were positively associated with the DR (odds ratio [OR], 1.117; 95% confidence interval [CI], 1.072-1.241; P<0.001) and DN (OR, 1.259; 95% CI, 1.198-1.323; P<0.001). Compared with the first quartile of serum copeptin levels, the ORs for DR and DN were as follows: second quartile, 1.19 (95% CI, 0.94-1.51, P=0.12) and 1.37 (95% CI, 0.78-2.37, P=0.28); third quartile, 1.61 (95% CI, 1.18-2.43, P=0.005) and 2.12 (95% CI, 1.32-3.27, P=0.003); fourth quartile, 2.83 (95% CI, 2.04-4.93; P<0.001) and 3.48 (95% CI, 1.77-7.03; P<0.001), respectively. CONCLUSIONS: Using a post-hoc analysis our data show that elevated serum levels of copeptin are associated with type 2 diabetes and diabetic complications in Chinese population, suggesting a potential role of the AVP system (copeptin) in the pathophysiology of diabetes.

[Trans-splicing of Cys mutated coagulation factor VIII].

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.

[Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system].

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.

[Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells].

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.

[Glycosylation modifications improve secretion and activity of intein spliced coagulation factor VIII].

OBJECTIVE: to investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FVIII (BDD-FVIII). METHODS: a total of 226 amino acid residues of FVIII B domain with six potential asparagines-linked glycosylation sites (N6) were incorporated into heavy chain of BDD-FVIII. By dual-vector co-transfer of heavy and light chain genes with fused intein (N6HCIntN and IntCLC) into cultured 293 cells, the amounts of spliced BDD-FVIII protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively. RESULTS: the amounts of spliced BDD-FVIII protein and activity were up to (123 ± 18) ng/ml and (0.94 ± 0.11) U/ml in supernatant from cells co-transfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy (HCIntN) and light chain (IntCLC) genes [(86 ± 12) ng/ml and (0.65 ± 0.07) U/ml, both P < 0.05]. Spliced BDD-FVIII protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [(18 ± 6) ng/ml and (0.15 ± 0.05) U/ml]. CONCLUSION: it demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FVIII and the protein splicing can occur independent of cellular mechanism.

[vWF improves secretion and activity of intein spliced BDD-FVIII].

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.

[Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII].

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.

[Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII].

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.

[Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion].

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.

[Post-translational ligation and function of dual-vector transferred split CFTR gene].

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.

[Enhancing effect of AR-3 on protein splicing-based eukaryotic cell gene transfer of full-length coagulation factor VIII].

OBJECTIVE: To study the effect of an acidic region-3 (AR-3), capable of improving the secretion of heavy chain of coagulation factor VIII (fVIII), on the secretion of protein spicing ligated full-length fVIII. METHODS: A pair of vectors was used to deliver intein fused heavy and light chain genes of a full-length fVIII gene with an additional AR-3 incorporated on the end of heavy chain into cultured 293 cells. The intracellular protein splicing was observed by Western blot. And the secretion of spliced fVIII and activity in culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) and Coatest assay respectively. RESULTS: A noticeable spliced fVIII protein band was observed from the gene co-transfected cells. The culture supernatant displayed a spliced fVIII of (112 ± 18) ng/ml with an activity of (0.76 ± 0.13) U/ml greater than that of cells co-transfected with AR-3-free heavy chain and light chain genes [(64 ± 11) ng/ml and (0.37 ± 0.05) U/ml]. And a spliced fVIII of (27 ± 7) ng/ml with an activity of (0.16 ± 0.05) U/ml was detected in the culture supernatant from combined cells separately transfected with AR-3-fused heavy chain gene and light gene. CONCLUSION: AR-3 can enhance the fVIII gene transfer by improving the secretion of intein spliced full-length fVIII.

[Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

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.