Comparing Protein Expression in Erwinia amylovora Strain TS3128 Cultured under Three Sets of Environmental Conditions

Erwinia amylovora, the causal agent of fire-blight disease in apple and pear trees, was first isolated in South Korea in 2015. Although numerous studies, including omics analyses, have been conducted on other strains of E. amylovora, studies on South Korean isolates remain limited. In this study, we conducted a comparative proteomic analysis of the strain TS3128, cultured in three media representing different growth conditions. Proteins related to virulence, type III secretion system, and amylovoran production, were more abundant under minimal conditions than in rich conditions. Additionally, various proteins associated with energy production, carbohydrate metabolism, cell wall/membrane/envelope biogenesis, and ion uptake were identified under minimal conditions. The strain TS3128 expresses these proteins to survive in harsh environments. These findings contribute to understanding the cellular mechanisms driving its adaptations to different environmental conditions and provide proteome profiles as reference for future studies on the virulence and adaptation mechanisms of South Korean strains.     

Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblast co-cultures

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Quantitative Detection of Microbial Genes by Using DNA Microarrays

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.     

Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.