Identification of two novel T cell epitopes on the E2 protein of classical swine fever virus C-strain.

C-strain, also known as the HCLV strain, is a well-known live attenuated vaccine against classical swine fever (CSF), a devastating disease caused by classical swine fever virus (CSFV). Vaccination with C-strain induces a rapid onset of protection, which is associated with virus-specific gamma interferon (IFN-γ)-secreting CD8+ T cell responses. The E2 protein of CSFV is a major protective antigen. However, the T cell epitopes on the E2 protein remain largely unknown. In this study, eight overlapping nonapeptides of the E2 protein were predicted and synthesized to screen for potential T cell epitopes on the CSFV C-strain E2 protein. Molecular docking was performed on the candidate epitopes with the swine leukocyte antigen-1*0401. The analysis obtained two highly conserved T cell epitopes, 90STEEMGDDF98 and 331ATDRHSDYF339, which were further identified by enzyme-linked immunospot assay. Interestingly, the mutants deleting or substituting the epitopes are nonviable. Further analysis demonstrated that 90STEEMGDDF98 is crucial for the E2 homodimerization, while CSFV infection is significantly inhibited by the 331ATDRHSDYF339 peptide treatment. The two novel T cell epitopes can be used to design new vaccines that are able to provide rapid-onset protection.

A Multifunctional Photoacoustic/Fluorescence Dual-Mode-Imaging Gold-Based Theranostic Nanoformulation without External Laser Limitations.

Theranostics is a new type of biomedical technology that organically combines the diagnosis and therapy of diseases. Among molecular imaging techniques, the integration of photoacoustic (PA) and fluorescence (FL) imaging modes with high sensitivity and imaging depth provides precise diagnostic outcomes. Gold nanorods (Au NRs) are well-known contrast agents for PA imaging and photothermal therapy. However, their high toxicity, poor biocompatibility, rapid clearance, and the need for an external laser source limit their application. Therefore, modification of Au NRs with carbon-based nanomaterials (CBNs) is done to obtain a multifunctional dual-mode gold-based nanoformulation (mdGC), which preforms dual-mode imaging of PA and FL. The results show that mdGC promotes tumor cell apoptosis and exhibits good antitumor performance through the mitochondria-mediated apoptotic pathway by increasing the production of intracellular reactive oxygen species, reducing mitochondrial membrane potential, and regulating the expression of apoptosis-related genes. The targeting rate of mdGC to tumor tissue is up to 20.71 ± 1.94% ID g-1 ; the tumor growth inhibition rate is as high as 80.44% without external laser sources. In general, mdGC is a potential multifunctional diagnostic and therapy integrated nanoformulation.

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.

Novel ORF5 deletion of NADC30-like porcine reproductive and respiratory syndrome viruses circulating in northern China from 2016 to 2018.

North American porcine reproductive and respiratory syndrome virus (NA-PRRSV), especially NADC30-like PRRSV, has evolved and is prevalent in China. We collected 503 samples from pig breeding farms across 4 provinces in northern China from 2016 to 2018. The samples were screened by PCR testing with specific primers that could differentiate groups of NA-PRRSV; phylogenetic trees were constructed and analyzed. Overall, 175 of 503 (34.8%) samples were positive for NA-PRRSV. Dual (NADC30-like and highly pathogenic [HP]-PRRSV; NADC30-like and typical PRRSV; HP and typical PRRSV) and triple (NADC30-like, HP, and typical PRRSV) infections (92 of 175, 52.6%) were common in coinfections by NADC30-like and HP-PRRSV. Notably, 18 of 125 (14.4%) semen samples were positive for PRRSV, and 17 of the 18 positive semen samples contained NADC30-like PRRSV. Phylogenetic analysis based on GP5 amino acids revealed that the novel NADC30-like PRRSV with a unique single amino acid deletion at position 34 has become widespread and has evolved into a new subgroup.

Prevalence and risk factors for pterygium: a cross-sectional study in Han and Manchu ethnic populations in Hebei, China.

AIMS: To investigate the prevalence, ethnic differences and associated risk factors for pterygium in Han and Manchu populations aged 40-79 years in Hebei province, China. DESIGN: Cross-sectional study, as a part of the China National Health Survey. SETTING: Hebei province, China. PARTICIPANTS: A multistage cluster sampling method with urbanisation level-based stratification was used to select participants for this study. A total of 4591 individuals over 40 years were recruited for this study. Inclusive criteria: (1) residents who had been living in Hebei for more than 1 year; (2) Han individuals with both parents being Han, or Manchu individuals with both parents being Manchu; (3) underwent ophthalmic examinations and (4) information in the questionnaire was complete. MAIN OUTCOME MEASURES: Multiple logistic regression analysis was used to evaluate the association between pterygium prevalence and factors of interest. RESULTS: A total of 3790 individuals (2351 Hans and 1439 Manchus) met the study criteria, of which 248 were diagnosed with pterygium (6.5%). There was no significant difference between the prevalence rates in Hans (6.2%) and Manchus (7.2%) (p=0.232). Multivariate analysis revealed that the risk factors for grade 2 or higher pterygium were increasing age (p<0.001) and rural residence (OR 1.83; 95% CI 1.11 to 3.02; p=0.018), while the protective factors include gender (female) (OR 0.58; 95% CI 0.37 to 0.88; p=0.011), cigarette smoking (OR 0.53; 95% CI 0.34 to 0.83; p=0.005) and myopia (OR 0.50; 95% CI 0.33 to 0.77; p=0.002). Premature menopause (OR 2.66; 95% CI 1.05 to 6.72; p=0.038) increased the risk of grade 2 or higher pterygium in females, while higher high-density lipoprotein (HDL) (OR 1.94; 95% CI 1.08 to 3.47; p=0.027) was a risk factor of grade 2 or higher pterygium in males. CONCLUSION: The overall prevalence of pterygium in Han and Manchu population in Hebei, China was approximately 6.1%. There were no differences in the prevalence of pterygium between Hans and Manchus, and the race was not a risk factor. This is the first study to report on the positive association between premature menopause and pterygium in females and between higher HDL levels and pterygium in males.

Increased total iron and zinc intake and lower heme iron intake reduce the risk of esophageal cancer: A dose-response meta-analysis.

Several epidemiological studies investigated the relationship between dietary intake of essential trace elements and the risk of esophageal cancer (EC), yielding inconsistent results. We therefore conducted a systematic meta-analysis to investigate and quantify the putative association between the intake of various essential trace elements and the risk of EC. We searched Embase, PubMed, and Web of Science for eligible articles published through April 2018 reporting the odds ratio (OR) with 95% confidence interval (95% CI). Pooled results were then calculated using fixed and random effect models. A total of 20 articles containing 4855 cases from 1 387 482 participants were included in our analysis. We found a significant inverse correlation between total iron intake and the risk of EC (OR = 0.81, 95% CI: 0.70-0.94), particularly in Asian populations. A dose-response analysis revealed that each 5 mg/day increase in total iron intake was associated with a 15% reduction in EC risk (OR = 0.85, 95% CI: 0.79-0.92). In contrast, each 1 mg/day increase in heme iron intake was associated with a 21% increase in EC risk (OR = 1.21, 95% CI: 1.02-1.45). Lastly, a pooled risk estimate revealed that each 5 mg/day increase in zinc intake was associated with a 15% reduction in EC risk (OR = 0.85, 95% CI: 0.77-0.93). Taken together, our analysis indicates that increased dietary intake of total iron and zinc, as well as decreased heme iron intake, may be associated with a lower risk of developing esophageal cancer. These findings have important public health implications with respect to preventing this relatively common form of cancer.

Intake of Dietary One-Carbon Metabolism-Related B Vitamins and the Risk of Esophageal Cancer: A Dose-Response Meta-Analysis.

Several B vitamins are essential in the one-carbon metabolism pathway, which is central to DNA methylation, synthesis, and repair. Moreover, an imbalance in this pathway has been linked to certain types of cancers. Here, we performed a meta-analysis in order to investigate the relationship between the intake of four dietary one-carbon metabolism-related B vitamins (B2, B6, folate, and B12) and the risk of esophageal cancer (EC). We searched PubMed, Web of Science, and Embase for relevant studies published through 1 March 2018. The odds ratio (OR) with 95% confidence interval (CI) for the highest versus the lowest level of each dietary B vitamin was then calculated. From 21 articles reporting 26 studies including 6404 EC cases and 504,550 controls, we found an inverse correlation between the consumption of vitamin B6 and folate and the risk of EC; this association was specific to the US, Europe, and Australia, but was not found in Asia. A dose-response analysis revealed that each 100 μg/day increase in folate intake reduced the risk of EC by 12%. Moreover, each 1 mg/day increase in vitamin B6 intake decreased the risk of EC by 16%. Surprisingly, we found that each 1 μg/day increase in vitamin B12 intake increased the risk of esophageal adenocarcinoma by 2%, particularly in the US and Europe, suggesting both geographic and histological differences. Together, our results suggest that an increased intake of one-carbon metabolism-related B vitamins may protect against EC, with the exception of vitamin B12, which should be consumed in moderation.

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.

Systematic Study in Mammalian Cells Showing No Adverse Response to Tetrahedral DNA Nanostructure.

The advent of DNA technology has demonstrated great potential in a wide range of applications, especially in the field of biology and biomedicine. However, current understanding of the toxicological effects and cellular responses of DNA nanostructures remains to be improved. Here, we chose tetrahedral DNA nanostructures (TDNs), a type of nanocarriers for delivering molecular drugs, as a model for systematic live-cell analysis of the biocompatibility of TDNs to normal bronchial epithelial cells, carcinoma cells, and macrophage. We found that the interaction behaviors of TDNs in different cell lines were very different, whereas after internalization, most of the TDNs in diverse cell lines were positioned to lysosomes. By a systematic assessment of cell responses after TDN exposure to various cells, we demonstrate that internalized TDNs have good innate biocompatibility. Interestingly, we found that TDN-bearing cells would not affect the cell cycle progression and accompany cell division and that TDNs were separated equally into two daughter cells. This study improves our understanding of the interaction of DNA nanostructures with living systems and their biocompatibility, which will be helpful for further designing DNA nanostructures for biomedical applications.

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs. The envelope glycoprotein E2 of CSFV is the major antigen that induces neutralizing antibodies and confers protection against CSFV infections. Previously, we developed a murine monoclonal antibody (MAb), HQ06, against the E2 protein of CSFV. To produce the antibody conveniently and stably, the genes coding for the variable regions of the heavy and light chains of HQ06 and constant region genes from the swine antibody were fused and cloned into lentiviral expression vectors to express a recombinant porcinized MAb (rHQ06Sw) in mammalian cells. rHQ06Sw was able to react with the E2 protein or the CSFV virions specifically in different assays. Notably, rHQ06Sw could neutralize CSFV infection in a dose-dependent manner. Taken together, the functional porcinized MAb rHQ06Sw was generated, which can be used to develop novel diagnostic assays or to investigate the structure and functions of the E2 protein.

[Expression and antiviral activity of a chimeric porcinized monoclonal antibody (cHQ06) against E2 protein of classical swine fever virus].

Classical swine fever (CSF), one of OIE-listed diseases, is a highly contagious and economically important disease of pigs. Classical swine fever virus (CSFV) is the causative agent of CSF. The capsid (C) protein and the glycoproteins Erns, E1 and E2, are structural components of the virus. E2 is the most immunogenic protein of the CSFV glycoproteins, inducing neutralizing antibodies that provide protection against lethal CSFV challenge. In a previous study, we developed a murine MAb HQ06 against the E2 protein of CSFV. In this study, the variable region genes from HQ06 and constant regions gene of swine antibody are fused and cloned into the eukaryotic expression vectors to establish a cell line which can stably express a chimeric porcinized MAb (cHQ06) against E2 in CHO cell. The purified cHQ06 antibody protein was determined to be successfully generated, which exhibited high reactivity between cHQ06 and the E2 protein of CSFV by enzyme-linked immunosorbent assay (ELISA) and Western blotting. More importantly, we investigated the neutralizing activity of cHQ06 against CSFV. In conclusion, this study generated cHQ06 for efficient and stable production which can be used against to develop novel diagnostic assays, investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment.

Antioxidants Mediate Both Iron Homeostasis and Oxidative Stress.

Oxidative stress is a common denominator in the pathogenesis of many chronic diseases. Therefore, antioxidants are often used to protect cells and tissues and reverse oxidative damage. It is well known that iron metabolism underlies the dynamic interplay between oxidative stress and antioxidants in many pathophysiological processes. Both iron deficiency and iron overload can affect redox state, and these conditions can be restored to physiological conditions using iron supplementation and iron chelation, respectively. Similarly, the addition of antioxidants to these treatment regimens has been suggested as a viable therapeutic approach for attenuating tissue damage induced by oxidative stress. Notably, many bioactive plant-derived compounds have been shown to regulate both iron metabolism and redox state, possibly through interactive mechanisms. This review summarizes our current understanding of these mechanisms and discusses compelling preclinical evidence that bioactive plant-derived compounds can be both safe and effective for managing both iron deficiency and iron overload conditions.

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.

A mitochondria-mediated apoptotic pathway induced by deoxynivalenol in human colon cancer cells.

Deoxynivalenol (DON), known colloquially as "vomitoxin", is a pathogenic mycotoxin produced by Fusarium fungi. Human food poisoning outbreaks, with nausea, diarrhea, and vomiting as primary symptoms, have been associated with Fusarium-infected cereals. Therefore, this study was designed to determine the molecular aspects of DON in human colon cancer cells (HT-29). To this aim, we have monitored the effects of DON on (i) cellular morphological changes via optical and transmission electron microscopy, especially in regards to cell viability and mitochondria changes, and (ii) its effects on key regulators of cell apoptosis, including cytochrome c, caspase-9, caspase-3, Bcl-2, Bax, and Bid. Our results showed that DON treatment inhibited cell proliferation, induced significant morphological changes, and promoted the activation of cytochrome c and caspases. Furthermore, changes in Bcl-2, Bax, and Bid expression were detected. The relative expression profile of Bcl-2 was contrary to that of Bax and Bid, as Bcl-2 expression decreased as the concentrations DON increased, reaching a minimum at the highest concentration of DON. We concluded that DON-induced apoptosis was caused by mitochondrial dysfunction and subsequent release of cytochrome c into the cytoplasm and successive activation of caspases, and this was likely regulated by Bcl-2 family proteins.