Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

BACKGROUND: Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously. METHODS: Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. RESULTS: We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively. CONCLUSION: Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection

Universal Stress Proteins Are Important for Oxidative and Acid Stress Resistance and Growth of Listeria monocytogenes EGD-e In Vitro and In Vivo

Background: Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species. Methods and Findings: We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (deltalmo0515, deltalmo1580 and deltalmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models. Tolerance to acidic stress was clearly reduced in Δlmo1580 and deltalmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in deltalmo1580 and deltalmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with deltalmo1580 or deltalmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection. Conclusion: This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions

M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures

Listeria monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine

Ex situ diet influences the bacterial community associated with the skin of red-eyed tree frogs (Agalychnis callidryas)

Amphibians support symbiotic bacterial communities on their skin that protect against a range of infectious pathogens, including the amphibian chytrid fungus. The conditions under which amphibians are maintained in captivity (e.g. diet, substrate, enrichment) in ex situ conservation programmes may affect the composition of the bacterial community. In addition, ex situ amphibian populations may support different bacterial communities in comparison to in situ populations of the same species. This could have implications for the suitability of populations intended for reintroduction, as well as the success of probiotic bacterial inoculations intended to provide amphibians with a bacterial community that resists invasion by the chytrid fungus. We aimed to investigate the effect of a carotenoid-enriched diet on the culturable bacterial community associated with captive red-eyed tree frogs (Agalychnis callidryas) and make comparisons to bacteria isolated from a wild population from the Chiquibul Rainforest in Belize. We successfully showed carotenoid availability influences the overall community composition, species richness and abundance of the bacterial community associated with the skin of captive frogs, with A. callidryas fed a carotenoid-enriched diet supporting a greater species richness and abundance of bacteria than those fed a carotenoid-free diet. Our results suggest that availability of carotenoids in the diet of captive frogs is likely to be beneficial for the bacterial community associated with the skin. We also found wild A. callidryas hosted more than double the number of different bacterial species than captive frogs with very little commonality between species. This suggests frogs in captivity may support a reduced and diverged bacterial community in comparison to wild populations of the same species, which could have particular relevance for ex situ conservation projects

The determination of Benzoic Acid in Lemon Flavoured Beverages by Stir Bar Sorptive Extraction-CGC-MS.

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