Plasmid pcDNA3 Research Articles

An improved method for the establishment of a model of Graves' disease in BALB/c mice.

The present study aimed to develop a stable Graves' disease (GD) model in BALB/c mice by immunization and electroporation (EP). A total of 90 mice were divided into experimental (n=50), control (n=20) and blank (n=20) groups. The recombinant plasmid pcDNA3.1/thyroid-stimulating hormone (TSH) receptor 268 was constructed and injected into the bilateral gastrocnemius of experimental group mice at weeks 1, 4, 7 and 10. Equal volumes of saline were injected into the control and blank groups at the same time. The experimental and control groups were subjected to EP at the same time and location to enhance immunization. The levels of total serum thyroxine (T4) and serum TSH were examined by radioimmunoassay and immunoradiometric assay, respectively. The levels of serum thyrotropin receptor N-terminal (TRAb N) and C-terminal (TRAb C) antibodies were assessed by ELISA. Whole body pertechnetate (99mTcO4-) imaging was performed. Mouse weight and thyroid morphology and pathology were analyzed. The GD BALB/c mouse model was successfully established, with a positive rate of 79.17% (38/48). T4 levels increased from baseline levels of 12.05±4.23 to 52.51±23.58 ng/ml by week 12 (P<0.0001). TSH levels decreased from baseline levels of 5.53±2.78 to 1.43±0.89 µIU/ml by week 12 (P<0.0001). TRAb N antibody levels increased from baseline levels of 0.006±0.002 to 0.278±0.106 mIU/ml by week 12 (P<0.0001). TRAb C antibody levels increased from baseline levels of 11.111±2.808 to 46.701±26.436 arbitrary units/ml by week 12 (P<0.0001). At week 21, TSH levels remained reduced compared with pre-immunization levels (P<0.0001). Although T4, and TRAb N and C levels decreased, they remained increased compared with preimmunization levels (P<0.0001, P<0.0001, P=0.001). There were no significant alterations in antibody levels between the control and blank groups. Following four immunizations, the uptake of 99mTcO4- by the thyroid was significantly increased in the experimental group. The mean weight of the experimental mice was significantly reduced compared with the control and blank groups (all P<0.0001). Furthermore, the thyroid glands of the immunized mice were enlarged and exhibited lymphocyte infiltration, fewer colloid nodules and an increased height of epithelial cells. In conclusion, by injecting recombinant plasmid pcDNA3.1/TSHR268 and EP, a GD mouse model was successfully established.

The transfection of A20 gene prevents kidney from ischemia reperfusion injury in rats.

Ischemia/reperfusion may induce inflammation and cell death through the nuclear factor (NF)‑κB signaling pathway. As a negative regulator of NF‑κB, zinc finger A20 exhibits anti-apoptotic and anti‑inflammatory effects invitro. The present study was designed to upregulate A20 expression using an A20 transfection approach to investigate the invivo protective effects of the A20 gene on renal ischemia reperfusion injury. The A20 gene was cloned into a pcDNA3.1 vector to construct the expression plasmid pcDNA3.1‑A20. The plasmid was wrapped with a liposome and injected intravenously into rats 48h prior to establishing the models of renal ischemia reperfusion injury. Saline and the empty plasmid pcDNA3.1 were used as controls. Following 24h post‑operation, A20 expression was determined using reverse transcription‑quantitative polymerase chain reaction and western blotting. The renal function and structure were assessed by analyzing the concentrations of serum creatinine (Scr), blood urea nitrogen (BUN) and histological features. Renal tissues were additionally examined for renal tubular cell apoptosis and NF‑κB activity. The results demonstrated that invivo transfection of pcDNA3.1‑A20 induced renal A20 expression in rats. A20 overexpression invivo significantly reduced renal injury as demonstrated by the improved levels of Scr and BUN and the reduction in histological damage. These improvements were accompanied by a suppression of renal proximal tubular epithelial cell apoptosis and an inhibition of NF‑κB activity. These results indicated that transfection of the A20 gene upregulates the expression of A20 invivo and protects the kidneys from ischemia reperfusion injury via inhibition of the NF‑κB signal transduction pathway.

Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism

Oxidative DNA damage has been implicated in the pathogenesis of neurological disorders, cancer and ageing. Owing to the established link between labile copper concentrations and neurological diseases, it is critical to explore the interactions of neurotransmitters and drug supplements with copper. Herein, we investigate the pro-oxidant DNA damage induced by the interaction of L-DOPA and dopamine (DA) with copper. The DNA binding affinity order of the compounds has been determined by in silico molecular docking. Agarose gel electrophoresis reveals that L-DOPA and DA are able to induce strand scission in plasmid pcDNA3.1 (+/−) in a copper dependent reaction. These metabolites also cause cellular DNA breakage in human lymphocytes by mobilizing endogenous copper, as assessed by comet assay. Further, L-DOPA and DA-mediated DNA breaks were detected by the appearance of post-DNA damage sensitive marker γH2AX in cancer cell lines accumulating high copper. Immunofluorescence demonstrated the co-localization of downstream repair factor 53BP1 at the damaged induced γH2AX foci in cancer cells. The present study corroborates and provides a mechanism to the hypothesis that suggests metal-mediated oxidation of catecholamines contributes to the pathogenesis of neurodegenerative diseases.

HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1‑pAFP-TK and targeted killing effect.

Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non-toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1-pAFP-TK, carrying the suicide gene driven by the alpha-fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription-polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1-pAFP-TK plasmid, whereas HSVtk gene expression was not detected in normal HL-7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1-pAFP-TK decreased in a dose-dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1-pAFP-TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase-3 staining demonstrated that activated caspase-3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase-3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

Green Tea Polyphenols Ameliorate the Early Renal Damage Induced by a High-Fat Diet via Ketogenesis/SIRT3 Pathway.

Scope Several reports in the literature have suggested the renoprotective effects of ketone bodies and green tea polyphenols (GTPs). Our previous study found that GTP consumption could elevate the renal expression of the ketogenic rate-limiting enzyme, which was decreased by a high-fat diet (HFD) in rats. Here, we investigated whether ketogenesis can mediate renoprotection by GTPs against an HFD. Methods and Results Wistar rats were fed a standard or HFD with or without GTPs for 18 weeks. The renal oxidative stress level, kidney function, renal expression, and activity levels of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2) and sirtuin 3(SIRT3) were detected. The increased renal oxidative stress and the loss of renal function induced by the HFD were ameliorated by GTPs. Renal ketogenesis and SIRT3 expression and activity levels, which were reduced by the HFD, were restored by GTPs. In vitro, HEK293 cells were transfected with the eukaryotic expression plasmid pcDNA HMGCS2. GTP treatment could upregulate HMGCS2 and SIRT3 expression. Although SIRT3 expression was not affected by HMGCS2 transfection, the 4-hydroxy-2-nonenal (4-HNE) level and the acetyl-MnSOD (K122)/MnSOD ratio were reduced in HMGCS2-transfected cells in the context of H2O2. Conclusion The ketogenesis/SIRT3 pathway mediates the renoprotection of GTPs against the oxidative stress induced by an HFD.

A DNA vaccine encoding the viral hemorrhagic septicemia virus genotype IVb glycoprotein confers protection in muskellunge (Esox masquinongy), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and lake trout (Salvelinus namaycush).

BackgroundThe viral hemorrhagic septicemia virus (VHSV) is one of the most serious fish pathogens. In 2003, a novel sublineage (genotype IVb) of this deadly virus emerged in the Great Lakes basin causing serious fish kills. We have previously demonstrated that a DNA plasmid (pcDNA), containing a cytomegalovirus (CMV) promoter and the viral hemorrhagic septicemia virus (VHSV) genotype IVb glycoprotein (G) gene insert (designated pVHSivb-G) confers moderate protection in muskellunge (Esox masquinongy), a highly susceptible species upon challenge. In order to achieve optimal protection, we investigated a number of factors including the incubation time [i.e. the number of degree days (° days)] before challenge, and viral challenge dose and route. Additionally, we tested if pVHSivb-G provides protection against VHSV-IVb to less susceptible salmonids such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and lake trout (Salvelinus namaycush).ResultsAn increase in the period lapsed between vaccination and challenge to 1880° days resulted in 95% relative percent protection (RPS) in muskellunge following a single administration of the pVHSivb-G plasmid and viral challenge. An RPS of 100% for muskellunge was achieved with a longer incubation period (2400° days) and in conjunction with a booster dose of the plasmid. The pVHSivb-G vaccine also elicited significant protection in all three salmonid species, reaching 100% RPS in lake trout following an incubation period of 1001° days prior to viral challenge. Vaccination with pVHSivb-G was also associated with the development of significant levels of circulating VHSV-binding antibodies in muskellunge as measured by indirect ELISA, which reached peak levels 6–7 weeks post-vaccination. Viral shedding in vaccinated survivors was minimal and of transient nature.ConclusionsThe study shows that the pVHSivb-G plasmid can elicit a protective response against the wild virus strain in a range of species important in recreational and commercial Great Lakes fisheries.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0662-8) contains supplementary material, which is available to authorized users.

Interleukin-8 holds promise to serve as a molecular adjuvant in DNA vaccination model against Streptococcus iniae infection in fish.

DNA vaccines had been widely used in animal models against various viral infections, while it was not so convincing for many infectious diseases especially bacterial disease in aquaculture. Interleukin-8(IL-8) as one of the CXC chemokines, its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, recombination plasmid pcDNA3.1/IL-8(pcIL-8) was conducted and the capacity of IL-8 as molecular adjuvant was explored from several aspects by co-injecting with a DNA vaccine encoding a-enolase(pcENO) against Streptococcus iniae infection in channel catfish. The results suggested that co-injection of pcIL-8 with DNA vaccine increased the innate immunity and specific antibody levels, as well as increased the immune-related genes involving in pro-inflammatory response, humoral and cellular immunity. Moreover, pcIL-8 enhanced the immunoprotection of pcENO with the relative percent survival(RPS) of 60% to 80% against S.iniae infection at 4 week post vaccination(p.v.), with the significantly higher RPS of 73.33% in pcENO+pcIL-8 group compared with that of pcENO alone(53.33%) at challenge test of 8 weeks p.v. Taken together, these results indicate pcIL-8 as a molecular adjuvant co-injected with DNA vaccine not only improves the immunoprotection but also maintains long period of immunity for channel catfish against S.iniae infection. Our study signifies that IL-8 holds promise to serve as a potential adjuvant in DNA vaccines against bacterial infections for long time.

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. Biological significanceDengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.

Weak cell cycle dependency but strong distortive effects of transfection with Lipofectamine 2000 in near‐physiologically synchronized cell culture

Previously, we reported a method to generate and validate cell cycle-synchronized cultures of multiple mammalian suspension cell lines under near-physiological conditions. This method was applied to elucidate the putative interdependencies of the cell cycle and recombinant protein expression in the human producer cell line HEK293s using Lipofectamine 2000 and the reporter plasmid pcDNA3.3 enhanced green fluorescent protein, destabilized using PEST sequence. A population-resolved modeling approach was applied to quantitatively assess putative variations of cell cycle dependent expression rates based on the obtained experimental data. We could not confirm results published earlier by other groups, based on nonphysiological synchronization attempts, reporting transfection efficiency being strongly dependent on the cell cycle phase at transfection time point. On the other hand, it is demonstrated that transfection and protein expression distort the progression of the cell cycle.

Role of Phosphotyrosine Interaction Domain Containing 1 in Porcine Intramuscular Preadipocyte Proliferation and Differentiation

ABSTRACTPhosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation.

A recombinant plasmid containing CpG motifs as a novel vaccine adjuvant for immune protection against herpes simplex virus 2.

The aim of the present study was to evaluate the efficacy of a herpes simplex virus type 2 (HSV-2) DNA vaccine co‑immunized with a plasmid adjuvant containing CpG motifs. A novel eukaryotic expression plasmid vector containing kanamycin resistance gene (pcDNA3Kan) was acquired from pET‑28a(+) and pcDNA3 plasmids. A gene encoding full length HSV‑2 glycoprotein D (gD) was amplified from the pcDNA3‑gD plasmid, which was cloned into pcDNA3Kan resulting in the construction of the recombinant plasmid pcDNA3Kan‑gD (pgD). A DNA segment containing 8CpG motifs was synthesized, and cloned into pcDNA3Kan, resulting in the recombinant plasmid pcDNA3Kan‑CpG (pCpG). Mice were co‑inoculated with pgD (used as a DNA vaccine) and pCpG (used as an adjuvant) by bilateral intramuscular injection. Mice inoculated with pgD+pCpG showed higher titers of antibodies than those inoculated with the DNA vaccine alone (P<0.05). In addition, mice inoculated with pgD+pCpG showed the highest percentage of CD4+ T cells in the blood of all the groups (P﹤0.05). Thus, the present study demonstrated that pCpG could stimulate the HSV‑2 DNA vaccine to induce a stronger cell‑mediated immune response than the DNA vaccine alone. The aim of the present study was to evaluate the efficacy of a HSV‑2 DNA vaccine (pgD) co‑immunized with a plasmid adjuvant containing CpG motifs (pCpG). Whether the pCpG would be able to stimulate the pgD to induce a stronger immune response compared with pgD alone.

Effect of fibroblast growth factor receptors 1-dominant negative strategy on alkaline phosphatase activity of bone marrow stromal cells after osteogenic induction

Objective To explore the effect of fibroblast growth factor receptors 1 -dominant negativestrategy(FGFR1-DN)on alkaline phosphatase(ALP)activity of bone marrow stromal stem cells(BMSCs)after osteogenic induction. Methods BMSCs were transfected with eukaryotic expression plasmid pcDNA3. 1(+)-DN FGFR1 and pcDNA3. 1(+)-FGFR1. The experiment was conducted in 4 groups: FGFR1-DN transfection group, FGFR1 transfection group, pcDNA3. 1(+)empty vector transfection group and non-transfection group. The ALP activity of BMSCs was detected in logarithmic growth phase after osteogenic culture. The qualitative detection of ALP activity was carried out immunohistochemically while the quantitative detection by cALP kit. The ALP activity was compared between the 4 groups at 7 and 14 days after osteogenic induction. Results Compared with 7 days, the ALP activity at 14 days was significantly increased in the4 groups, and the increase in FGFR1-DN transfection group was significantly higher than in the other 3 groups(P< 0. 05). At both 7 and 14 days, the ALP activity in FGFR1-DN transfection group was the highest while that in FGFR1 transfection group was the lowest(P< 0. 05). Conclusions FGFR1-DN can promote the ALP activity of BMSCs during osteogenesis. This may provide an experimental basis for the joint application oflocal gene therapy and tissue engineering and for construction of tissue engineered bone with better biocom-patibility. Key words: Bone marrow cells; Fibroblast growth factors; Fibroblast growth factor receptor 1; Dominant negative effect

Apoptotic Effects of the B Subunit of Bacterial Cytolethal Distending Toxin on the A549 Lung Cancer Cell Line

Cytolethal distending toxin (CDT) is a secreted tripartite genotoxin produced by many pathogenic gram-negative bacteria. It is composed of three subunits, CdtA, CdtB and CdtC, and CdtB-associated deoxyribonuclease (DNase) activity is essential for the CDT toxicity. In the present study, to design a novel potentially antitumor drug against lung cancer, the possible mechanisms of cdtB anticancer properties were explored in the A549 human lung adenocarcinoma cell line. A recombinant plasmid pcDNA3.1/cdtB was constructed expressing CdtB of human periodontal bacterium Aggregatibacter actinomycetemcomitans and investigated for toxic properties in A549 cells and possible mechanisms. It was observed that plasmid pcDNA3.1/cdtB caused loss of cell viability, morphologic changes and induction of apoptosis. Furthermore, measurement of caspase activity indicated involvement of an intrinsic pathway of cell apoptosis. Consequently, the recombinant plasmid pcDNA3.1/cdtB may have potential as a new class of therapeutic agent for gene therapy of lung cancer.

Construction of a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene and its effects on apoptosis and autophagy of A375 melanoma cells

Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene, to investigate its expression in A375 melanoma cells after transfection, and to evaluate its effects on apoptosis and autophagy of A375 cells. Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR (RT-PCR)with total RNA extracted from A375 cells as the template, then inserted into the eukaryotic expression plasmid PCDNA3.1(+)to construct a recombinant plasmid, PCDNA3.1(+)-PKCI-1/HINT1. Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid (PCDNA3.1 (+)-PKCI-1/HINT1 group)or the empty plasmid PCDNA3.1(+)(control group). After additional 48-hour culture, RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively, Hoechst 33342 staining was conducted to detect apoptosis of A375 cells, Western blot analysis to detect the expressions of intracellular caspase-3 and autophagyassociated protein beclin1, and cell autophagy was observed by using the green fluorescent protein (GFP)microtubule-associated protein 1 light chain 3 (LC3)labelling method combined with confocal laser scanning microscopy. Methyl thiazolyl tetrazolium (MTT)assay was performed to evaluate the proliferative activity of A375 cells at 24, 48, 72 and 96 hours after transfection. Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1(+)-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells. MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells, and the number of live cells was decreased by 17.0%, 25.6% and 29.4% in the PCDNA3.1(+)-PKCI-1/HINT1 group at 48, 72 and 96 hours, respectively, compared with the control group (all P < 0.05). Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells. Confocal laser scanning microscopy demonstrated that the overexpression of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells. In addition, Western blot analysis indicated that PKCI-1/HINT1 upregulated the protein expressions of caspase-3 and beclin1 in A375 cells. Conclusions The eukaryotic expression plasmid PCDNA3.1 (+ )-PKCI-1/HINT1 was successfully constructed, and PKCI-1/HINT1 could be effectively expressed in A375 cells. High-level expression of PKCI-1/HINT1 could suppress cellular proliferation, promote apoptosis, and induce autophagy, of A375 cells. Key words: Melanoma; Cell line, tumor; Apoptosis; Autophagy; PKCI-1/HINT1

DNA vaccination based on pyolysin co-immunized with IL-1β enhances host antibacterial immunity against Trueperella pyogenes infection

Trueperella pyogenes is a commensal and opportunistic pathogen normally causes mastitis, liver abscesses and pneumonia of economically important livestock. To date, no specific control measure was reported to prevent T. pyogenes infections. In this study, we first constructed a recombinant plasmid pVAX1-PLO based on the main virulent factor pyolysin gene as DNA vaccine against T. pyogenes infection. Subsequently, transient expression of pVAX1-PLO and pcDNA3.1/V5-fIL-1β were identified in Human embryonic kidney cells (HEK293T) by immunofluorescence assay. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity between different immunized groups. The results showed that the successful expression of PLO or fIL-1β protein was detected by confocal microscopy for cells transfected with plasmid pVAX1-PLO and/or pcDNA3.1/V5-fIL-1β. The mice immunized with pVAX1-PLO elicited a higher titer of PLO-specific antibody than the control group. The levels of IFN-γ and IL-2 were significantly increased in the pVAX1-PLO immunized mice, while the expression level of IL-4 was slightly increased but not significant. These findings suggested that the DNA vaccine pVAX1-PLO can primarily induce Th1 immune response. The residual Colony-Forming Units (CFUs) from the liver and peritoneal fluid were decreased obviously in the pVAX1-PLO treated mice compared with the control. Importantly, co-immunization with pcDNA3.1/V5-fIL-1β and pVAX1-PLO could enhance host humoral and cellular immune responses and significantly protect mouse from T. pyogenes infection. In conclusion, our study provides a promising strategy against T. pyogenes infections and implies the potential clinical application of combined DNA vaccines in diseases control.

TAK1 promotes epithelial-mesenchymal transition of lens epithelial cells

Transforming growth factor-β-activated kinase-1 (TAK1) is thought to play a key role in the initiation of Smad-independent TGF-β signaling. This study investigated the role of TAK1 in the epithelial-mesenchymal transition (EMT) lens epithelial cells. TAK1 was overexpressed in the HLE B-3 cell line by transfecting TAK1-pcDNA3 and TAK1-binding protein 1 (TAB1)-pcDNA3 plasmids. The expression levels of TAK1, phospho-TAK1, E-cadherin, and fibronectin were detected by Western blot analysis and immunocytofluorescence to analyze the effects of overexpression. The levels of α-SMA and type I collagen were analyzed by real-time PCR. Quantitative data were analyzed by Student's t test or one-way analysis of variance (ANOVA) (multiple comparisons using LSD test). Western blot analysis showed in the TAK1-pcDNA3 plasmids group, expression of TAK1 proteins (1.00±0.03) with a maximum upregulation of approximately 80% at 24 h than it was in the control group (0.19±0.09)(t=8.02, P< 0.01); Western blot analysis showed in the TAB1-pcDNA3 plasmids group, expression of TAB1 proteins (1.00±0.02) with a maximum upregulation of approximately 78% at 24 h than it was in the control group (0.22±0.08)(t=7.63, P<0.01). The levels of E-cadherin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (1.00±0.02, 0.12±0.03, 0.98±0.09, 0.92±0.08;F=31.03, P<0.01). The levels of fibronectin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (0.11±0.03, 1.00±0.05, 0.16±0.04, 0.21±0.05;F=35.12, P<0.01). Overexpression of TAK1 with TAB1 resulted in upregulated expression of fibronectin, and downregulated expression of E-cadherin. The expression of E-cadherin was increased and the expression of fibronectin was decreased by TAK1 siRNA and TAK1 chemical inhibitors in the presence of TGF-β2. These data reveal that TAK1 can induce the EMT of HLE cells, and the inhibition of TAK1 phosphorylation may be a potential novel therapeutic target for the prevention and treatment of posterior capsule opacification. (Chin J Ophthalmol, 2016, 52: 278-284).

A recombinant plasmid of composite cysteine proteinase inhibitor/ glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host

Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-γ) and interleukin-4 (IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. Results: The pcDNA3.1(+)-BmCPI/ BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-γ and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-γ of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Characterization of local, systemic and mucosal immune responses after oral DNA vaccination against spring viremia of carp virus

DNA vaccination through i.m. injection of DNA plasmid encoding the G protein of SVCV (SVCV-G) is very successful in carp but this method of vaccine delivery is not practical. Alternatively, oral vaccination would be highly preferred due to its stress-free, labour-extensive and easy delivery. On the downside, oral vaccines are often less efficacious when compared to traditional injection vaccines and little is known about their activation of immune mechanisms. In an effort to generate an oral DNA vaccine against SVCV, carp of 2 g were orally vaccinated three times with alginate microspheres containing either 8 ug pcDNA3 plasmid, 8 ug pcDNA3-SVCVG plasmid, or PBS. Owing to the availability of several carp leukocyte-specific monoclonal antibodies, we revealed the early recruitment of specific populations of leukocytes to the intestine and spleen following oral vaccination. The combined use of cell type-specific antibodies and an antibody against the SVCV-G protein allowed us for the first time, to identify the sites and kinetics of expression of the SVCV-G protein, and to concomitantly recognize the cell types involved in antigen uptake and processing. The use of a second panel of antibodies enabled us to distinguish between various populations of both B and T lymphocytes, allowing the study of their specific roles during vaccination and infection. Using this panel we were able to characterise kinetics of recruitment of specific B and T lymphocyte populations to local sites after vaccination, as well as their distribution in systemic and other mucosal immune organs. Besides the characterization of local and systemic immune responses after oral DNA vaccination, using a wide array of techniques including RT-qPCR, histology, flow cytometry and in vitro macrophage-T cell cultures, we are currently studying the induction of immune memory after oral vaccination. This combination of techniques enables us to establish an unique overview of the kinetics of the immune response of carp after oral DNA vaccination against SVCV, at cell, protein and gene expression level and in a broad selection of immune-relevant organs. Together we will present the promising potential of an oral DNA vaccine against SVCV to not only induce an early local immune response, but also a systemic responses followed by long-term memory formation.

Bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro.

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.