Ring finger protein 31-mediated atypical ubiquitination stabilizes forkhead box P3 and thereby stimulates regulatory T-cell function.

The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.

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.

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.

Regulation of Metabolism Across Different Subsets of T Cells in Cancer.

T cells play a critical role to defend against tumor and maintain immune homeostasis. The diverse functions of T cells require precise regulation of metabolic pathways. Recent studies reveal that metabolic changes are tightly linked to the activation and function of T cells. Given the importance of these cells in tumor progression, it is important to understand how the tumor microenvironment regulates metabolism of T cells and how the metabolic reprogramming of T cells affects tumor growth. Here, we review new findings and discuss how metabolic reprogramming of different types of T cells affects the immune response in tumors.

Adipose Tissue-Resident Regulatory T Cells.

Tissue-resident immune cells play critical roles in regulating tissue function and homeostasis. Obesity-associated visceral adipose tissue inflammation is attributed to the accumulation of M1 macrophages which produce inflammatory cytokines like TNF-α, IL-6, and expansion of effector T cells like Th1 cells, CD8+ cytotoxic T cells which produce interferon-γ to further add to the severity of inflammation in the visceral adipose tissue. Regulatory T cells have been reported to exert key roles in suppressing inflammation, thus maintaining the homeostasis of immune responses, and visceral adipose Tregs exert critical roles in defending against obesity-associated metabolic disorders. They inhibit the infiltration of effector T cells and facilitate the reconstitution of adipose tissue macrophages from M1 to M2 phenotype. What is more, they can take up lipids from the adipocytes through CD36 which is driven by PPARγ. Here we review the recent progress in adipose tissue-resident regulatory T cells (Tregs), a subpopulation of CD4+ T cells which suppress adipose tissue inflammation.

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.

Molecular Characterization of the Complete Genome of Three Basal-BR Isolates of Turnip mosaic virus Infecting Raphanus sativus in China.

Turnip mosaic virus (TuMV) infects crops of plant species in the family Brassicaceae worldwide. TuMV isolates were clustered to five lineages corresponding to basal-B, basal-BR, Asian-BR, world-B and OMs. Here, we determined the complete genome sequences of three TuMV basal-BR isolates infecting radish from Shandong and Jilin Provinces in China. Their genomes were all composed of 9833 nucleotides, excluding the 3'-terminal poly(A) tail. They contained two open reading frames (ORFs), with the large one encoding a polyprotein of 3164 amino acids and the small overlapping ORF encoding a PIPO protein of 61 amino acids, which contained the typically conserved motifs found in members of the genus Potyvirus. In pairwise comparison with 30 other TuMV genome sequences, these three isolates shared their highest identities with isolates from Eurasian countries (Germany, Italy, Turkey and China). Recombination analysis showed that the three isolates in this study had no "clear" recombination. The analyses of conserved amino acids changed between groups showed that the codons in the TuMV out group (OGp) and OMs group were the same at three codon sites (852, 1006, 1548), and the other TuMV groups (basal-B, basal-BR, Asian-BR, world-B) were different. This pattern suggests that the codon in the OMs progenitor did not change but that in the other TuMV groups the progenitor sequence did change at divergence. Genetic diversity analyses indicate that the PIPO gene was under the highest selection pressure and the selection pressure on P3N-PIPO and P3 was almost the same. It suggests that most of the selection pressure on P3 was probably imposed through P3N-PIPO.

Development and Validation of a Nomogram Model for Accurately Predicting Depression in Maintenance Hemodialysis Patients: A Multicenter Cross-Sectional Study in China.

Purpose: Depression is a major concern in maintenance hemodialysis. However, given the elusive nature of its risk factors and the redundant nature of existing assessment forms for judging depression, further research is necessary. Therefore, this study was devoted to exploring the risk factors for depression in maintenance hemodialysis patients and to developing and validating a predictive model for assessing depression in maintenance hemodialysis patients. Patients and Methods: This cross-sectional study was conducted from May 2022 to December 2022, and we recruited maintenance hemodialysis patients from a multicentre hemodialysis centre. Risk factors were identified by Lasso regression analysis and a Nomogram model was developed to predict depressed patients on maintenance hemodialysis. The predictive accuracy of the model was assessed by ROC curves, area under the ROC (AUC), consistency index (C-index), and calibration curves, and its applicability in clinical practice was evaluated using decision curves (DCA). Results: A total of 175 maintenance hemodialysis patients were included in this study, and cases were randomised into a training set of 148 and a validation set of 27 (split ratio 8.5:1.5), with a depression prevalence of 29.1%. Based on age, employment, albumin, and blood uric acid, a predictive map of depression was created, and in the training set, the nomogram had an AUC of 0.7918, a sensitivity of 61.9%, and a specificity of 89.2%. In the validation set, the nomogram had an AUC of 0.810, a sensitivity of 100%, and a specificity of 61.1%. The bootstrap-based internal validation showed a c-index of 0.792, while the calibration curve showed a strong correlation between actual and predicted depression risk. Decision curve analysis (DCA) results indicated that the predictive model was clinically useful. Conclusion: The nomogram constructed in this study can be used to identify depression conditions in vulnerable groups quickly, practically and reliably.

Development and validation of a nomogram model for accurately predicting severe fatigue in maintenance hemodialysis patients: A multicenter cross-sectional study in China.

INTRODUCTION: This study aims to analyze the risk factors for severe fatigue in maintenance hemodialysis (MHD) patients and develop a clinical prediction model to help doctors and patients prevent severe fatigue. METHODS: Multicentre MHD patients were included in this study. The objective was to investigate the risk factors for severe fatigue in MHD patients and develop a prediction model. RESULTS: A total of 243 MHD patients were included in the study, and the incidence of severe fatigue was found to be 20.99%. Using age, body mass index, total cholesterol, and albumin levels, a predictive nomogram for fatigue was constructed. In the training set, the nomogram had an area under the curve of 0.851, sensitivity of 82.86%, specificity of 81.76%, and c-index of 0.851. The nomogram was accurate in calibration and proved to be clinically useful. CONCLUSION: The nomogram developed in this study is a practical and reliable tool for quickly identifying severe fatigue in MHD patients.

Clinical and pathological features of advanced rectal cancer with submesenteric root lymph node metastasis: Meta-analysis.

BACKGROUND: Advanced rectal cancer with submesenteric lymph node metastasis is a common complication of advanced rectal cancer, which has an important impact on the treatment and prognosis of patients. AIM: To investigate the clinical and pathological characteristics of inferior mesenteric artery (IMA) root lymph node metastases in patients with rectal cancer. The findings of this study provided us with fresh medical information that assisted us in determining the appropriate treatment for these patients. METHODS: Our study searched PubMed, Google Scholar, and other databases and searched the relevant studies and reports on the risk factors of IMA root lymph node metastasis of rectal cancer published in the self-built database until December 31, 2023. After data extraction, the Newcastle-Ottawa scale was used to evaluate the quality of the included literature, and RevMan5.3 software was used for meta-analysis and heterogeneity testing. The fixed effect modules without heterogeneity were selected to combine the effect size, and the random effect modules with heterogeneity were selected to combine the effect size. The cause of heterogeneity was found through sensitivity analysis, and the data of various risk factors were combined to obtain the final effect size, odds ratio (OR) value, and 95% confidence interval (CI). Publication bias was tested by drawing funnel plots. RESULTS: A total of seven literature were included in this study. By combining the OR value of logistic multivariate regression and the 95%CI of various risk factors, we concluded that the risk factors for lymph node metastasis in the IMA region of rectal cancer were as follows: Preoperative carcinoembryonic antigen (CEA) > 5 ng/mL (OR = 0.32, 95%CI: 0.18-0.55, P < 0.05), tumor located above peritoneal reflexive (OR = 3.10, 95%CI: 1.78-5.42, P < 0.05), tumor size ≥ 5 cm (OR = 0.36, 95%CI: 0.22-0.57, P < 0.05), pathological type (mucinous adenocarcinoma/sig-ring cell carcinoma) (OR = 0.23, 95%CI: 0.13-0.41, P < 0.05), degree of tumor differentiation (low differentiation) (OR = 0.17, 95%CI: 0.10-0.31, P < 0.05), tumor stage (T3-4 stage) (OR = 0.11, 95%CI: 0.04-0.26, P < 0.05), gender and age were not risk factors for IMA root lymph node metastasis in rectal cancer (P > 0.05). CONCLUSION: Preoperative CEA level, tumor location, tumor size, tumor pathologic type, tumor differentiation, and T stage were correlated with IMA root lymph node metastasis.

Stabilizing Zinc Hexacyanoferrate Cathode by Low Contents of Cs Cations for Aqueous Zn-Ion Batteries.

Exploring cathode materials with excellent electrochemical performance is crucial for developing rechargeable aqueous zinc ion batteries (RAZIBs). Zinc hexacyanoferrate (ZnHCF), a promising candidate of cathode materials for RAZIBs, suffers from severe electrochemical instability issues. This work reports using low contents of alkaline metal cations as electrolyte additives to improve the cycle performance of ZnHCF. The cations with large sizes, particularly Cs+, changes the intercalation chemistry of ZnHCF in RAZIBs. During cycling, Cs+ cations co-inserted into ZnHCF stabilize the host structure. Meanwhile, a stable phase of CsZn[Fe(CN)6] forms on the ZnHCF cathode, suppressing the loss of active materials through dissolution. ZnHCF gradually converts to an electrochemically inert Zn-rich phase during long-term cycling in aqueous electrolyte, leading to irreversible capacity loss. Introducing Cs+ in the electrolyte inhibits this conversion reaction, resulting in the extended lifespan. Owing to these advantages, the capacity retention rate of ZnHCF/Zn full batteries increases from the original 7.0 % to a high value of 54.6 % in the electrolyte containing 0.03 M of Cs2SO4 after 300 cycles at 0.25 A ⋅ g-1. This research provides an in-depth understanding of the electrochemical behavior of ZnHCF in aqueous zinc electrolyte, beneficial for further optimizing ZnHCF and other metal hexacyanoferrates.

[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.

[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.

[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.

[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.

[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.

[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.