The oxidized phospholipid PGPC impairs endothelial function by promoting endothelial cell ferroptosis via FABP3.

Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O2•-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O2•-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.

Infrared Singularities of Multileg QCD Amplitudes with a Massive Parton at Three Loops.

We derive the structure of three-loop anomalous dimensions governing infrared singularities of QCD amplitudes with one massive and an arbitrary number of massless external partons. The contributions of tripole and quadrupole correlations involving a massive parton are studied in detail. The analytical expression of tripole correlations between one massive and two massless partons is obtained at three loops for the first time. We regularize the infrared divergences in the soft matrix element in a novel approach, where no extra scale dependence is involved, and the calculation can be performed in momentum space. Our results are essential to improve the theoretical predictions of single top and top quark pair productions at hadron colliders.

[Transurethral decompression and drainage with holmium laser for prostatic abscess: Analysis of clinical therapeutic effect].

OBJECTIVE: To investigate the clinical effect and safety of transurethral decompression and drainage with holmium laser in the treatment of prostatic abscess. METHODS: We retrospectively analyzed the clinical data on 13 cases of prostatic abscess treated in our hospital from January 2015 to May 2019. One of the patients was cured by drug therapy while the other 12 underwent transurethral decompression and drainage with holmium laser after failure in medication. We recorded such postoperative symptoms as fever, frequent urination, urgent urination, painful urination, tenteria, dysuria and abdominal distension, obtained the dynamical indices of blood and urine routine and culture after surgery, and performed MRI during the follow-up for possible recurrence and complications. Those with disappearance of the clinical symptoms, negative results of urine leukocyte and pathogen examinations, and no recurrence revealed by MRI were considered to be cured. RESULTS: After operation, the clinical symptoms were improved significantly and the urinary catheters removed within 5－10 days in all the cases. At 3－5 days after removal of the catheters, all the patients experienced smooth urination, with no urinary retention or urethral stricture. The patients were followed up for 3－16 months, during which no recurrence was observed. CONCLUSIONS: Transurethral decompression and drainage with holmium laser can achieve a definite clinical effect in the treatment of prostatic abscess and therefore deserves to be promoted in clinical practice.

Three-Loop Quark Jet Function.

We calculate the massless quark jet function to three-loop order. The quark jet function is a universal ingredient in SCET factorization for many collider and decay processes with quark initiated final state jets. Our three-loop result contributes to the resummation for observables probing the invariant mass of final state quark jets at primed next-to-next-to-next-to-leading-logarithmic accuracy. It represents the first complete three-loop result for a factorization ingredient describing collinear radiation. Furthermore it constitutes a major component of the N-jettiness subtraction method at next-to-next-to-next-to-leading order accuracy, which eventually may enable the calculation of fully differential cross sections with a colorful final state at this order.

Activation of STAT3-mediated CXCL12 up-regulation in the dorsal root ganglion contributes to oxaliplatin-induced chronic pain.

Oxaliplatin-induced chronic painful neuropathy is the most common dose-limiting adverse event that negatively affects cancer patients' quality of life. However, the underlying molecular mechanisms are still unclear. In the present study, we found that the intraperitoneal administration of oxaliplatin at 4 mg/kg for five consecutive days noticeably upregulated the expression of CXC motif ligand 12 (CXCL12) in the dorsal root ganglion, and the intrathecal injection of an anti-CXCL12 neutralizing antibody or CXCL12 siRNA attenuated the mechanical allodynia and thermal hyperalgesia induced by oxaliplatin. We also found that the signal transducers and transcription activator 3 (STAT3) was activated in the dorsal root ganglion, and inhibition of STAT3 with S3I-201 or the injection of AAV-Cre-GFP into STAT3flox/flox mice prevented the upregulation of CXCL12 expression in the dorsal root ganglion and chronic pain following oxaliplatin administration. Double-label fluorescent immunohistochemistry findings also showed that p-STAT3 was mainly localized in CXCL12-positive cells in the dorsal root ganglion. Furthermore, the results of a chromatin immunoprecipitation assay revealed that p-STAT3 might be essential for oxaliplatin-induced CXCL12 upregulation via binding directly to the specific position of the CXCL12 gene promoter. Finally, we found that cytokine TNF-α and IL-1ß increases mediated the STAT3 activation following oxaliplatin treatment. Taken together, these findings suggested that the upregulation of CXCL12 via TNF-α/IL-1ß-dependent STAT3 activation contributes to oxaliplatin-induced chronic pain.

Charm-Quark Production in Deep-Inelastic Neutrino Scattering at Next-to-Next-to-Leading Order in QCD.

We present a fully differential next-to-next-to-leading order calculation of charm-quark production in charged-current deep-inelastic scattering, with full charm-quark mass dependence. The next-to-next-to-leading order corrections in perturbative quantum chromodynamics are found to be comparable in size to the next-to-leading order corrections in certain kinematic regions. We compare our predictions with data on dimuon production in (anti)neutrino scattering from a heavy nucleus. Our results can be used to improve the extraction of the parton distribution function of a strange quark in the nucleon.

Effects of Geographical Origin and Tree Age on the Stable Isotopes and Multi-Elements of Pu-erh Tea.

Pu-erh tea is a famous tea worldwide, and identification of the geographical origin of Pu-erh tea can not only protect manufacture's interests, but also boost consumers' confidence. However, tree age may also influence the fingerprints of Pu-erh tea. In order to study the effects of the geographical origin and tree age on the interactions of stable isotopes and multi-elements of Pu-erh tea, 53 Pu-erh tea leaves with three different age stages from three different areas in Yunnan were collected in 2023. The δ13C, δ15N values and 25 elements were determined and analyzed. The results showed that δ13C, δ15N, Mg, Mn, Fe, Cu, Zn, Rb, Sr, Y, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu had significant differences among different geographical origins (p < 0.05). Mn content was significantly influenced by region and tree age interaction. Based on multi-way analysis of variance, principal component analysis and step-wised discriminant analysis, 24 parameters were found to be closely related to the geographical origin rather than tree age, and the geographical origin of Pu-erh tea can be 100.0% discriminated in cross-validation with six parameters (δ13C, δ15N, Mn, Mg, La, and Tb). The study could provide references for the establishment of a database for the traceability of Pu-erh tea, and even the identification of tea sample regions with different tree ages.

Endothelial extracellular vesicles induce acute lung injury via follistatin-like protein 1.

Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles (eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of eEVs after cardiopulmonary bypass, remains unclear. Plasma plasminogen-activated inhibitor-1 (PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice (C57BL/6, Toll-like receptor 4 knockout (TLR4-/-) and inducible nitric oxide synthase knockout (iNOS-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3 (JAK2/3)-signal transducer and activator of transcription 3 (STAT3)-interferon regulatory factor 1 (IRF-1) pathway, along with iNOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors (AG490 or S3I-201, respectively), and was relieved in TLR4-/- and iNOS-/- mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1 (FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.

Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice.

The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 participants were recruited for the screening of differentially expressed plasma microRNAs (miRNAs). We found that miR-1204 is significantly increased in both the plasma and aorta of elder patients with AAD and is positively correlated with age. Cell senescence induces the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induces vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. Furthermore, miR-1204 aggravates angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuates ß-aminopropionitrile monofumarate-induced AAD development in mice. Mechanistically, miR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by VSMCs and loss of their contractile phenotype. MYLK overexpression reverses miR-1204-induced VSMC senescence, SASP and contractile phenotypic changes, and the decrease of transforming growth factor-ß signaling pathway. Our findings suggest that aging aggravates AAD via the miR-1204-MYLK signaling axis.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA.

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

[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.

Combination of molecularly targeted therapies and immune checkpoint inhibitors in the new era of unresectable hepatocellular carcinoma treatment.

Multikinase inhibitors (MKIs) have been the only first-line treatment for advanced hepatocellular carcinoma (HCC) for more than a decade, until the approval of immune checkpoint inhibitors (ICIs). Moreover, the combination regimen of atezolizumab (anti-programmed cell death protein ligand 1 antibody) plus bevacizumab (anti-vascular endothelial growth factor monoclonal antibody) has recently been demonstrated to have superior efficacy when compared with sorafenib monotherapy. The remarkable efficacy has made this combination therapy the new standard treatment for advanced HCC. In addition to MKIs, many other molecularly targeted therapies are under investigation, some of which have shown promising results. Therefore, in the era of immuno-oncology, there is a significant rationale for testing the combinations of molecularly targeted therapies and ICIs. Indeed, numerous preclinical and clinical studies have shown the synergic antitumor efficacy of such combinations. In this review, we aim to summarize the current knowledge on the combination of molecularly targeted therapies and immune checkpoint therapies for HCC from both preclinical and clinical perspectives.

[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.

[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.