A novel recombinant adenovirus expressing apoptin and melittin genes kills hepatocellular carcinoma cells and inhibits the growth of ectopic tumor.

HCC is the most common fatal malignancy. Although surgical resection is the primary treatment strategy, most patients are not eligible for resection due to tumor heterogeneity, underlying liver disease, or comorbidities. Therefore, this study explores the possibility of multi-molecular targeted drug delivery in treating HCC. In this study, we constructed the recombinant adenovirus co-expressing apoptin and melittin (MEL) genes. The inhibitory effect of the recombinant adenovirus on hepatocellular carcinoma cells was detected through experiments on cell apoptosis, migration, invasion, and other factors. The tumor inhibitory effect in vivo was assessed using subcutaneous HCC mice. Results showed that recombinant adenovirus co-expressing anti-tumor genes TAT and apoptin, RGD and MEL can significantly inhibit the proliferation, migration, and invasion of HCC cells by inducing an increase in reactive oxygen species (ROS) levels, upregulation of apoptotic proteins such as Bax, cleaved caspase-3, and cleaved caspase-9, and downregulation of the anti-apoptotic protein Bcl-2. In subcutaneous HCC mice, recombinant adenovirus induced significant apoptosis in tumor, and inhibited tumor growth. In conclusion, recombinant adenovirus co-expressing apoptin and MEL can inhibit the growth and proliferation of tumor cells both in vivo and in vitro.

Chicken Heat Shock Protein 70 Is an Essential Host Protein for Infectious Bursal Disease Virus Infection In Vitro.

Infectious bursal disease virus (IBDV) infection causes pathogenicity and mortality in chickens, leading to huge economic losses in the poultry industry worldwide. Studies of host-virus interaction can help us to better understand the viral pathogenicity. As a highly conservative host factor, heat shock protein 70 (Hsp70) is observed to be involved in numerous viral infections. However, there is little information about the role of chicken Hsp70 (cHsp70) in IBDV infection. In the present study, the increased expression of cHsp70 was observed during IBDV-infected DF-1 cells. Further studies revealed that Hsp70 had similar locations with the viral double-stranded RNA (dsRNA), and the result of pull-down assay showed the direct interaction between cHsp70 with dsRNA, viral proteins (vp)2 and 3, indicating that maybe cHsp70 participates in the formation of the replication and transcription complex. Furthermore, overexpression of cHsp70 promoted IBDV production and knockdown of cHsp70 using small interfering RNAs (siRNA) and reducedviral production, implying the necessity of cHsp70 in IBDV infection. These results reveal that cHsp70 is essential for IBDV infection in DF-1 cells, suggesting that targeting cHsp70 may be applied as an antiviral strategy.

Antineoplastic Effects and Mechanisms of a New RGD Chimeric Peptide from Bullfrog Skin on the Proliferation and Apoptosis of B16F10 Cells.

Malignant melanoma, an increasingly common form of skin cancer, poses a significant threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. In this work, a new anti-tumor peptide, temporin La (T-La), was selected from a cDNA library generated from bullfrog skin. Two new derivative antitumor peptides, T-La (S) and T-La (FS), were designed by bioinformatics analysis and coupled with the RGD small molecule peptide to create chimeric RGD peptides, (RGD-T-La [S] and RGD-T-La [FS]). Preliminary experiments showed that the new antitumor peptides had significant antitumor effects. After coupling to the chimeric RGD peptide, the targeted treatment of mouse melanoma was significantly improved. Our data demonstrate that the 4 peptides tested herein significantly inhibited the proliferation, migration, and invasion of B16F10 cells; with an increase in polypeptide concentration, the proportion of melanoma cells in the G0/G1 phase decreased or increased significantly, respectively, the reactive oxygen species (ROS) content increased significantly, the mitochondrial membrane potential decreased significantly, and the expression of pro-apoptotic Bax, Caspase-3, and Caspase-9 increased, and anti-apoptotic Bcl-2 decreased significantly. Tyr and MITF genes were significantly downregulated. In conclusion, the use of these new anti-tumor peptides, when combined with a chimeric RGD peptide, may increase ROS levels and decrease mitochondrial membrane potential by inhibiting the activity of mitochondria, thus releasing apoptosis-promoting factors in B16F10 cells. The present study describes a new potential strategy for the application of promising peptides in the treatment of various cancers.

Molecular design and anti-melanoma activity of a novel bullfrog antibacterial peptide RGD-chimera.

Melanoma is a common malignant skin tumor, which is the only fatal skin tumor at present. Melanoma has a high degree of malignancy and metastasis. The activity of modified Temporin-La (T-La) peptides from bullfrog skin were evaluated for antitumor activity and improved targeting in melanoma cells. The amino acid sequence of T-La was modified, resulting in the antitumor peptide, T-La (FS). T-La and T-La (FS) were coupled to the RGD small molecule polypeptide to form the chimeric peptides RGD-T-La and RGD-T-La (FS), respectively. The secondary structures for the peptides, evaluated using circular dichroism, were found to be α-helical. The structure of T-La was evaluated using bioinformatics. In addition, the antitumor effects of the modified peptide and the targeting of RGD chimeric peptide to the tumor in vivo and in vitro were analyzed. Antitumor activity was measured in vitro using the MTT assay. Tumor cells with high integrin αvß3 expression were detected using flow cytometry, and tumor cells were screened for sensitivity to RGD-T-La (FS) to establish a tumor model in nude mice. The effects of the peptides on tumor cells were measured using laser confocal microscopy in real-time. The mechanism of the peptide antitumor activity in tumor cells was evaluated with scanning electron microscopy. B16 melanoma cells were the most sensitive to the peptides, for which the cell survival rate was 24.65% for 10 µg/ml RGD-T-La (FS). RGD-La (FS) had a rapid effect on tumor cells. RGD chimeric polypeptides exhibited site-targeting cytotoxic effects in tumor cells. In the B16 melanoma mouse model, the peptides exhibited antitumor effects against early melanoma development and induced tumor apoptosis, possibly by inhibiting VEGF and promoting caspase-3 expression. Overall, the present study provides a scientific basis for the application of small molecule antimicrobial peptides as targeted antitumor agents and lays the foundation for the clinical application of these peptides as antitumor drugs.

Effects of Selenium-Enriched Yeast Improved Aflatoxin B1-Induced Changes in Growth Performance, Antioxidation Capacity, IL-2 and IFN-γ Contents, and Gene Expression in Mice.

Sixty Kunming mice were randomly assigned into three groups. Mice in a control group were fed a basal diet, while mice in AFB1 group and AFB1-Se group were fed the basal diet supplemented with 250 µg/kg AFB1 or the basal diet supplemented with 250 µg/kg AFB1 and 0.2 mg/kg selenium as selenium-enriched yeast, respectively. On day 30 of the experiment, growth performance, glutathione peroxidase (GSH-Px) activities, total antioxidant capacity (T-AOC) levels, and malondialdehyde (MDA) contents in liver, interleukin-2 (IL-2), and interferon-γ (IFN-γ) contents in serum, and cytochrome P3a11 (Cyp3a11), IL-2, IFN-γ, and GSH-Px1 mRNA levels in liver were determined. The results showed that final weights, weight gains, T-AOC levels, GSH-Px1, and IFN-γ mRNA levels in AFB1-Se group and control group were higher or significantly higher than those in AFB1 group (P < 0.05 or P < 0.01), respectively. Body length gains in AFB1 group were lower than those in the control group (P < 0.05), while there was no significant difference between the AFB1-Se and control groups (P > 0.05). IL-2 contents and liver IL-2 mRNA levels in AFB1-Se group were significantly higher than those in the AFB1 group and control group (P < 0.01), and IL-2 contents in the control group were also significantly higher than those in the AFB1 group (P < 0.01). IFN-γ contents in AFB1-Se group and AFB1 group were significantly higher than those in control group (P < 0.01), while IFN-γ contents in AFB1-Se group were significantly lower than those in AFB1 group (P < 0.01). Cyp3a11 mRNA levels in AFB1-Se group and AFB1 group were significantly higher than those in the control group (P < 0.01). The results indicated that selenium-enriched yeast could partly reduce the toxicity induced by AFB1 in mice, including improving growth performance, antioxidation capacity, IL-2 and IFN-γ contents, and enhancing IL-2, IFN-γ, and GSH-Px1 mRNA levels.

Study of biological activity and targeted distribution of catesbeianalectin in mouse tissue.

Lectin has attracted attention because of its ability to serve as a carrier for targeted drug delivery. Large lectins isolated from marine invertebrates and crustaceans have strong immunogenicity and adverse effects, which limit their usefulness. This study reports the identification of catesbeianalectin via screening a bullfrog skin cDNA library. The catesbeianalectin polypeptide has a molecular weight of 1.47 kD, making it the smallest known lectin in terms of molecular weight. Circular dichroism analysis showed a PPII helix secondary structure. Catesbeianalectin strongly induces agglutination of rabbit erythrocytes and a variety of pathogens include Staphylococcus aureus, Streptococcus suis type 2, Actinobacillus pleuropneumoniae, and piglet paratyphoid Salmonella. The mean serum titer in catesbeianalectin-immunized Balb/c mice was 1:25, which was significantly lower than that of positive controls immunized with wheat germ agglutinin. Surface plasmon resonance indicated an S-type lectin. 125I-labeled catesbeianalectin did not pass the blood-brain barrier. This study provides a basis for further research on the potential of catesbeianalectin as a carrier in targeted drug delivery.

Modification and Characterization of Fe3O4 Nanoparticles for Use in Adsorption of Alkaloids.

Magnetite (Fe3O4) is a ferromagnetic iron oxide of both Fe(II) and Fe(III), prepared by FeCl2 and FeCl3. XRD was used for the confirmation of Fe3O4. Via the modification of Tetraethyl orthosilicate (TEOS), (3-Aminopropyl)trimethoxysilane (APTMS), and Alginate (AA), Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Fe3O4@SiO2-NH2-AA nanoparticles could be obtained, and IR and SEM were used for the characterizations. Alkaloid adsorption experiments exhibited that, as for Palmatine and Berberine, the most adsorption could be obtained at pH 8 when the adsorption time was 6 min. The adsorption percentage of Palmatine was 22.2%, and the adsorption percentage of Berberine was 23.6% at pH 8. Considering the effect of adsorption time on liquid phase system, the adsorption conditions of 8 min has been chosen when pH 7 was used. The adsorption percentage of Palmatine was 8.67%, and the adsorption percentage of Berberine was 7.25%. Considering the above conditions, pH 8 and the adsorption time of 8min could be chosen for further uses.

Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions.

This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (ß)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.

Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens.

Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P < 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

Effects of astragalus polysaccharide on the adhesion-related immune response of endothelial cells stimulated with CSFV in vitro.

BACKGROUND: Astragalus polysaccharide (APS) has immunomodulatory activities on porcine peripheral blood mononuclear cells. The immunomodulatory effects of APS on porcine endothelial cells (ECs) expose to classical swine fever virus (CSFV) remain unknown. METHODS: The virus was titrated using an indirect immune biotin enzyme standard method to confirm that porcine ECs were susceptible to CSFV infection and to determine the TCID50 of CSFV (C-strain). Porcine ECs were cultured with CSFV in the presence of APS. Relative quantitative PCR was used to assess the mRNA expression of factors that influence EC adhesion and immunity. RESULTS: The expression of adhesion factors mRNA increased following stimulation with CSFV; this effect was inhibited by pre-exposing the cells to APS. In addition, the expression of growth factors and some immune factors increased after infection with CSFV; this increase in tissue factor (TF), transforming growth factor (TGF-ß), and interleukin-8 (IL-8) could be inhibited by the addition of APS. The immune response mediated by Toll-like receptor 4 (TLR4) in ECs may be unregulated by CSFV as it was also inhibited by pre-treatment with APS. DISCUSSION: The addition of APS to the culture can obviously regulate the expression of molecules related to the adhesion, growth, and immune response of ECs, as well as the production of cytokines. Therefore, it may have the potential to be an effective component in vaccines against CSFV.

Determination of 11 quinolones in bovine milk using immunoaffinity stir bar sorptive microextraction and liquid chromatography with fluorescence detection.

A sensitive, selective and reproducible immunoaffinity stir bar sorptive microextraction (SBSME) coupled with liquid chromatography-fluorescence method for determination of 11 quinolones (QNs) in bovine milk was developed and validated. It is first report of a broad-specificity monoclonal antibody to QNs that has been immobilized to glass bar for preparation of a re-usable immunoaffinity stir bar. Analytes were extracted by placing stir bar in milk and shaking on a rotary shaker for 30min at 30rpm, followed by liquid chromatography and fluorescence detection. The newly developed method has limits of detection for each QN from 0.05 to 0.1ng/g with intra-day and inter-day precision ranging from 3.2 to 11.9% and from 5.2 to 12.5%, respectively. This allowed us to quantitatively analyze drugs in bovine milk with the advantage of significantly simplified sample preparation. The proposed method was successfully applied to the bovine milk samples analyses with QNs, demonstrating its rare application in animal food safety analysis.

Adenovirus-mediated co-expression of the TRAIL and HN genes inhibits growth and induces apoptosis in Marek's disease tumor cell line MSB-1.

BACKGROUND: The objective of this study was to determine the in vitro tumor-inhibitory effect of a recombinant adenovirus expressing a fusion protein of tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) and hemagglutinin-neuraminidase (HN) genes on the MSB-1 Marek's disease tumor cell line. METHODS: TRAIL and HN genes were amplified from lymphocytes in the peripheral blood of chickens and the LaSota strain of Newcastle disease virus (NDV), respectively, using RT-PCR. The two genes were connected with a 2A connecting peptide by site-directed mutagenesis and gene splicing by overlap extension (SOE). The target gene TRAIL-2A-HN was cloned into the shuttle vector pShuttle-CMV. Homologous recombination was carried out with the vector pAdeasy-1 in the bacterium BJ5183 to construct the recombinant adenovirus plasmid pAd-TRAIL-2A-HN. After linearization, the plasmid was transfected into AD293 cells and packaged. Real-time quantitative PCR (RT-PCR) and fluorescence microscopy confirmed the introduction of the recombinant adenovirus into AD293 cells. The TCID50 method (50% tissue culture infectious dose) was employed to determine viral titers for the exprimental and control viruses, which met criteria for use. The Marek's disease tumor cell line MSB-1 was transfected with the constructed recombinant adenovirus. The infectivity of the recombinant adenovirus and the expression levels of exogenous genes were detected with RT-PCR and western blotting. The effects of the recombinant adenovirus on the growth of MSB-1 cells and cellular apoptosis were determined using flow cytometry. RESULTS: The recombinant adenovirus infected the cultured cells in vitro, and replicated and expressed exogenous genes in the cells. The recombinant adenovirus Ad-TRAIL-2A-HN inhibited the growth of MSB-1 cells and induced apoptosis by expressing exogenous genes. The rate of induced MSB-1 cell apoptosis reached 11.61%, which indicated that TRAIL and HN produced synergistic tumor-inhibiting effects. CONCLUSION: The constructed TRAIL-2A-HN fusion gene combined the apoptosis-inducing function of TRAIL and the adsorptive capacity of HN from NDV for tumor cells, and the capacity of the recombinant adenovirus expressing this fusion gene to induce tumor cell apoptosis was reported. These results provide a basis for future in vivo tumor suppression studies using recombinant adenoviruses.

[Lateral superior genicular flap combined with Tuihuang xiaozhong decoction for the treatment of soft tissue defects around the knee joint].

OBJECTIVE: To evaluate the early combination of Chinese and Western medicine for anti-inflammation and lateral superior genicular flap for the treatment of soft tissue defects around the knee joint. METHODS: From June 2004 to September 2008, 8 patients with soft tissue defects around the knee joint were treated with lateral superior genicular flap. Among the patients, 5 patients were male and 3 patients were female, ranging in age from 32 to 56 years, with an average of 35.2 years. The defected area ranged from 7.6 cm x 4.5 cm to 15.2 cm x 7.5 cm. The disease course ranged from 3 months to 3 years. Three patients had the defects at the posterior of the knee, 2 patients had the defects at the popliteal fossa, and 3 patients had the defects at the lateral side of the knee. At the early stage, all the patients were treated with Tuihuang Xiaozhong decoction and antibiotics for 3 to 5 days. RESULTS: All the flaps survived, and the knee function recovered. One patient had epidermis necrosis at the distal end of the flap of lateral side of the knee. CONCLUSION: The early combination of Chinese and Western medicine for anti-inflammation is a simple, easy to promote, and no special microsurgical instruments are needed.