Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30.

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.

Water extract of Cnidii Rhizoma suppresses RANKL-induced osteoclastogenesis in RAW 264.7 cell by inhibiting NFATc1/c-Fos signaling and prevents ovariectomized bone loss in SD-rat.

BACKGROUND: Cnidii Rhizoma is the dried root stem of Cnidium officinale Makino. Cnidii Rhizoma (CR) has been used to treat menstrual irregularity, menstrual pain, and menopause in Korea. However, the effects and mechanisms of CR on RANKL-induced osteoclastogenesis pathway remain to be elucidated. In this study, we investigated the effects of CR on the inhibition of bone resorption of osteoclast and its mechanism RANK signaling pathway. METHODS: The anti-osteoclastogenesis of water extract of CR was measured using RAW 264.7 cell. Tartrate-resistant acid phosphatase (TRAP) assay, pit assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot were performed. Moreover, the effects of CR were determined with an in vivo model using ovariectomized (OVX) rats. RESULTS: CR extract suppressed osteoclastogenesis, its activity and bone resorption activity through decreasing gene of osteoclast-related such as nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, etc. Moreover, CR extract prevented the bone loss in OVX rats. CONCLUSION: These results show that CR has a positive effect on menopausal osteoporosis by suppressing osteoclastogenesis.

Preparation and Characterization of an Olive Flounder (Paralichthys olivaceus) Skin Gelatin and Polylactic Acid Bilayer Film.

Olive flounder skin gelatin (OSG) was used as a film base material. A bilayer film of OSG and polylactic acid (PLA) was prepared using solvent casting method to enhance the film properties. Physical properties of the OSG-PLA film were increased compared with the nonaugmented OSG film. In particular, the PLA lamination decreased water vapor permeability from 2.17 to 0.92 × 10-9 g·m/m2 ·s·Pa, as well as of the water solubility from 16.62% to 9.27%, in the bilayer film relative to the OSG film. The oxygen permeability of the OSG-PLA bilayer film was held low by the OSG film, compensating for the high oxygen permeability of the PLA layer. Therefore, the OSG-PLA bilayer film with its enhanced physical properties and high water and oxygen barrier properties can be applied as a food packaging material.

Characterization of red ginseng residue protein films incorporated with hibiscus extract.

An edible film was prepared from red ginseng residue protein (RGRP) and incorporated with hibiscus extract (HE). RGRP was extracted from red ginseng residue, which is an inexpensive by-product of the red ginseng processing industry. Different concentrations of HE were added to an RGRP film-forming solution as a natural antioxidant. The prepared RGRP films without HE had a tensile strength of 16.9 MPa and an elongation at break of 25.1%. The antioxidant activity of the RGRP film increased with increasing concentration of HE. In addition, the RGRP film with 1% HE exhibited the lowest value of water vapor permeability (1.88×10-9 g·m/m2·s·Pa), which indicates that the film has high water barrier property. The results present the production of edible films from discarded red ginseng residue, and the antioxidant activity of RGRP films as a packaging material can prevent lipid oxidation and quality loss of food products.

Antimicrobial activity of gelatin films based on duck feet containing cinnamon leaf oil and their applications in packaging of cherry tomatoes.

Duck feet gelatin (DFG) films were prepared and applied to the packaging of cherry tomatoes. Cinnamon leaf oil (CLO) was incorporated into the DFG films at concentrations of 0.5, 1.0, and 1.5% to provide antimicrobial activity. The DFG films with 1.0% CLO incorporation exhibited the most desirable tensile strength (41.6 MPa) and elongation at break (18.5%). Regarding the antimicrobial activity, the DFG films containing CLO significantly inhibited the growth of foodborne pathogens. In addition, the DFG film with 1.0% CLO was employed in the coating and wrapping of cherry tomatoes inoculated with Salmonella typhimurium. The DFG film with 1.0% CLO incorporation reduced the population of the bacteria to below the detection limit. Moreover, the DFG film with CLO delayed the color change on cherry tomatoes. Overall, the DFG film with CLO enhanced the shelf life of cherry tomatoes and can be used as an antimicrobial packaging.

Application of a puffer fish skin gelatin film containing Moringa oleifera Lam. leaf extract to the packaging of Gouda cheese.

This study aims to develop a puffer fish skin gelatin (PSG) film that contains Moringa oleifera Lam. leaf extract (ME) as a new biodegradable film. With the increase in ME concentration, the tensile strength and elongation at break of the PSG film increased, whereas the oxygen permeability and water vapor permeability decreased. In addition, the PSG film with ME exhibited antimicrobial activity against Listeria monocytogenes and antioxidant activity. To apply the ME-containing PSG film to food packaging, Gouda cheese was wrapped with the ME-containing PSG film. During storage, the cheese packaging with the ME-containing PSG film effectively inhibited the microbial growth and retarded the lipid oxidation of cheese compared with the control sample. Thus, the ME-containing PSG film can be used as an antimicrobial and antioxidative packaging material to improve the quality of food products.

Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity.

Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 µg ml(-1)) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections.

Characterization of a starfish gelatin film containing vanillin and its application in the packaging of crab stick.

To explore the use of starfish gelatin (SFG) films as a biodegradable material, SFG from starfish was extracted and used as a film material. In addition, to provide antimicrobial activity and enhanced flavor of SFG films, vanillin was incorporated. As the concentration of vanillin increased, the tensile strength of the films increased and water vapor permeability decreased. With regard to the structural characteristics of SFG films containing vanillin, the microstructure of the SFG films was not affected by the addition of vanillin. In addition, the SFG films containing vanillin exhibited antimicrobial activity against Listeria monocytogenes. As the application of the SFG films, crab sticks were packed with SFG films containing 0.05% vanillin. During storage, the populations of L. monocytogenes inoculated on crab sticks wrapped with SFG films containing vanillin were lower than those on the control sample, suggesting that SFG films containing vanillin can be useful in active food packaging.

Xanthii Fructus inhibits allergic response in the ovalbumin-sensitized mouse allergic rhinitis model.

BACKGROUND: Xanthii Fructus (XF) is widely used in traditional anti-bacterial and anti-inflammatory Asian medicine. Allergic rhinitis is a common inflammatory disease characterized by markedly increased levels of anti-inflammatory factors and the recruitment of inflammatory cells into the nasal mucosa. We investigated the effects of XF in the allergen-induced rhinitis model. MATERIALS AND METHODS: Following ovalbumin (OVA)/alum intraperitoneal injection on days 0, 7 and 14, the BALB/c mice (albino, laboratory-bred strain of the house mice) were challenged intranasally with OVA for 10 days a week after the last sensitization. The number of sneezes was recorded for 10 days; additionally, the levels of cytokines, histamine, immunoglobulin E (IgE) and OVA-specific serum IgE were estimated. Eosinophil infiltration, thickness of nasal mucosa and expression of caspase-1 were determined by immunohistochemistry. We also evaluated the effect of XF on the phosphorylation of nuclear factor kappa-B (NF-κB) and inhibitor of nuclear factor kappa B-alpha (IκB-α) in human mast cell-1 (HMC-1), by Western blotting. RESULTS: The administration of XF significantly decreased sneezing and the serum levels of histamine, IgE, OVA-specific IgE, and cytokines such as tumor necrosis factor-alpha (TNF-α), interleukine-1 beta (IL-1ß), IL-5, IL-6, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2). XF inhibited the changes in thickness of the nasal septum, influx of eosinophils and expression of capase-1. In addition, XF inhibited the phosphorylation of IκB-α and NF-κB in phorbol-myristate-acetate plus calcium ionophore A23187 (A23187) stimulated HMC-1. CONCLUSION: This study suggests that XF acts a potent anti-allergic drug which alleviates the allergic responses in ovalbumin-sensitized mouse allergic rhinitis model.

Temperature-dependent control of Staphylococcus aureus biofilms and virulence by thermoresponsive oligo(N-vinylcaprolactam).

Bacterial biofilms are associated with persistent infections because they are highly tolerant of antimicrobial agents, and in the case of Staphylococcus aureus, which is a leading cause of nosocomial infections because of its resistance to diverse antibiotics, biofilm formation is a known mechanism of drug resistance. In the present study, we investigated the ability of thermoresponsive oligo (N-vinylcaprolactam) (OVCL) to control biofilm formation by and the virulence of S. aureus. One synthetic and four commercial OVCLs (MW ≤ 240,000) at 50 µg/mL were found to increase S. aureus biofilm formation 7-fold at 25 °C, but to markedly inhibit S. aureus biofilm formation at 37 °C. Confocal and scanning electron microscopy confirmed the temperature-dependent effect of OVCL on S. aureus biofilms. It was found that the addition of OVCL to S. aureus culture caused cells to become dramatically more hydrophilic at 37 °C, which partially supports the biofilm reduction. Also, transcriptional analysis showed that OVCL temperature-dependently regulated biofilm-related genes (aur, agrA, and icaA) in S. aureus. In addition, it was found surface coatings containing OVCL effectively controlled S. aureus biofilm formation on solid glass surfaces. Furthermore, OVCL inhibited the hemolysis of human red blood cells by S. aureus at 37 °C and attenuated S. aureus virulence in the nematode Caenorhabditis elegans. These results suggest that OVCL has potential use for controlling bacterial biofilm formation and virulence.

Anti-biofilm, anti-hemolysis, and anti-virulence activities of black pepper, cananga, myrrh oils, and nerolidol against Staphylococcus aureus.

The long-term usage of antibiotics has resulted in the evolution of multidrug-resistant bacteria. Unlike antibiotics, anti-virulence approaches target bacterial virulence without affecting cell viability, which may be less prone to develop drug resistance. Staphylococcus aureus is a major human pathogen that produces diverse virulence factors, such as α-toxin, which is hemolytic. Also, biofilm formation of S. aureus is one of the mechanisms of its drug resistance. In this study, anti-biofilm screening of 83 essential oils showed that black pepper, cananga, and myrrh oils and their common constituent cis-nerolidol at 0.01 % markedly inhibited S. aureus biofilm formation. Furthermore, the three essential oils and cis-nerolidol at below 0.005 % almost abolished the hemolytic activity of S. aureus. Transcriptional analyses showed that black pepper oil down-regulated the expressions of the α-toxin gene (hla), the nuclease genes, and the regulatory genes. In addition, black pepper, cananga, and myrrh oils and cis-nerolidol attenuated S. aureus virulence in the nematode Caenorhabditis elegans. This study is one of the most extensive on anti-virulence screening using diverse essential oils and provides comprehensive data on the subject. This finding implies other beneficial effects of essential oils and suggests that black pepper, cananga, and myrrh oils have potential use as anti-virulence strategies against persistent S. aureus infections.

Stilbenes reduce Staphylococcus aureus hemolysis, biofilm formation, and virulence.

Stilbenoids have a broad range of beneficial health effects. On the other hand, the emergence of antibiotic-resistant Staphylococcus aureus presents a worldwide problem that requires new antibiotics or nonantibiotic strategies. S. aureus produces α-hemolysin (a pore-forming cytotoxin) that has been implicated in the pathogenesis of sepsis and pneumonia. Furthermore, the biofilms formed by S. aureus constitute a mechanism of antimicrobial resistance. In this study, we investigated the hemolytic and antibiofilm activities of 10 stilbene-related compounds against S. aureus. trans-Stilbene and resveratrol at 10 µg/mL were found to markedly inhibit human blood hemolysis by S. aureus, and trans-stilbene also inhibited S. aureus biofilm formation without affecting its bacterial growth. Furthermore, trans-stilbene and resveratrol attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is normally killed by S. aureus. Transcriptional analysis showed that trans-stilbene repressed the α-hemolysin hla gene and the intercellular adhesion locus (icaA and icaD) in S. aureus, and this finding was in line with observed reductions in virulence and biofilm formation. In addition, vitisin B, a stilbenoid tetramer, at 1 µg/mL was observed to significantly inhibit human blood hemolysis by S. aureus.

Antiallergic effects of Scutellaria baicalensis on inflammation in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis (SB) is one of the most widely used medicinal herbs for the treatment of inflammation. In this study, we investigated the antiallergic effect of SB in vivo and in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats received intradermal injections of anti-DNP IgE at each of three dorsal skin sites. Forty-eight hours later, each rat received an injection of DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. One hour before injection, SB extract was administered orally. The dorsal skin of the rats was removed and the pigment area measured. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, human mast cells (HMC-1) were pretreated with SB extract for 30min before stimulation with phorbol 12-myristate 13-acetate (PMA) plus A23187. The effects on pro-inflammatory cytokine expression and mitogen activated protein (MAP) kinase expression were investigated using TNF-α and IL-8 assays, and Western blotting analysis of HMC-1 cells. RESULTS AND CONCLUSIONS: SB treatment inhibited the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following treatment of RPMCs with SB. In HMC-1 cells, SB restored IL-8 and TNF-α expression and inhibited MAP kinase expression in compound 48/80-induced HMC-1 cells. These data suggest that SB may prove to be a useful anti-inflammatory agent through its downregulation of the expression of various inflammatory mediators.