Development of a Novel Enhanced Biosensor System for Real-Time Monitoring of Fish Stress Using a Self-Assembled Monolayer.

Wireless biosensor systems were developed in our lab for monitoring blood glucose concentrations in fish as an indicator of fish stress. However, uniform immobilization of the enzyme on the surface of the electrode is difficult, so the sensor response is typically reduced at a range of high glucose concentrations during the stress monitoring. In this study, we attempted to enhance sensor response by using a self-assembled monolayer-immobilized enzyme. Glucose oxidase was immobilized on a working electrode modified with a self-assembled monolayer. The proposed biosensor showed a good correlation between the output current and the glucose concentration range of 10⁻3500 mg dL-1 under an optimized working condition. The dynamic measurement range of this newly developed sensor is significantly improved, especially over a high concentration range, which helps the sensor to achieve better performance in dramatic changes in the stress response of fish. In addition, we used biological samples from test fish and obtained a good correlation coefficient between the sensor output current and the glucose concentration using a conventional method. The proposed wireless biosensor system was also applied to monitor fish stress responses in real time through different stressors and to obtain some precise data that reflect real fish stress responses.

Streptococcus sanguinis Noncoding cia-Dependent Small RNAs Negatively Regulate Expression of Type IV Pilus Retraction ATPase PilT and Biofilm Formation.

Small noncoding RNAs (sRNAs) have been identified as important regulators of gene expression in various cellular processes. cia-dependent small RNAs (csRNAs), a group of sRNAs that are controlled by the two-component regulatory system CiaRH, are widely conserved in streptococci, but their targets have been identified only in Streptococcus pneumoniaeStreptococcus sanguinis, a pioneer colonizer of teeth and one of the most predominant bacteria in the early oral biofilm, has been shown to have six csRNAs. Using computational target prediction and the luciferase reporter assay, we identified pilT, a constituent of the type IV pilus operon, as a negative regulatory target for one of the csRNAs, namely, csRNA1-1, in S. sanguinis RNA-RNA electrophoretic mobility shift assay using a nucleotide exchange mutant of csRNA1-1 revealed that csRNA1-1 binds directly to pilT mRNA. In addition, csRNA1-1 and csRNA1-2, a putative gene duplication product of csRNA1-1 that is tandemly located in the S. sanguinis genome, negatively regulated S. sanguinis biofilm formation. These results suggest the involvement of csRNAs in the colonization step of S. sanguinis.

Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages.

Melatonin produced by the pineal gland suppresses inflammatory responses in innate immune cells. However, the mechanism of how melatonin affects inflammatory gene regulation remains unclear. Here we performed comprehensive microarray analysis combined with transcription factor binding site (TFBS) analysis using LPS-induced mouse macrophages to investigate the effect of melatonin treatment. The results showed that melatonin preferentially downregulated interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) related signaling. The results also showed that melatonin strongly suppressed virus infection related gene expression. Furthermore, TFBS analysis implicated that melatonin downregulated the binding activity of hypoxia inducible factors (HIFs), following destabilizing actin cytoskeleton which are indispensable for induction of the TRIF-dependent signaling pathway. Indeed, it was demonstrated that melatonin treatment caused impaired phagocytosis in macrophages. Thus, melatonin regulates inflammatory responses by inhibiting specific subsets of transcription factors (TFs) by disrupting actin dynamics in the macrophage.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide.

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Human myo-inositol monophosphatase 2 rescues the nematode thermotaxis mutant ttx-7 more efficiently than IMPA1: functional and evolutionary considerations of the two mammalian myo-inositol monophosphatase genes.

Mammals express two myo-inositol monophosphatase (IMPase) genes, IMPA1/Impa1 and IMPA2/Impa2. In this study, we compared the spatial expression patterns of the two IMPase gene transcripts and proteins in mouse tissues. Results indicated discrete expression of the two IMPase genes and their protein products in various organs, including the brain. In Caenorhabditis elegans, loss of the IMPase gene, ttx-7, disrupts cellular polarity in RIA neurons, eliciting abnormal thermotaxis behavior. We performed a rescue experiment in mutant nematodes using mammalian IMPases. Human IMPA2 rescued the abnormal behavioral phenotype in the ttx-7 mutants more efficiently than IMPA1. These results raise a question about the phylogenetic origin of IMPases and the biological roles of mammalian IMPase 2 in mammals. Impa2 knockout mice generated in our laboratory, exhibited neither behavioral abnormalities nor a significant reduction in myo-inositol content in the brain and other examined tissues. Given the ability of human IMPA2 to rescue the ttx-7 mutant, and its genetic association with multiple neuropsychiatric disorders, close scrutiny of IMPA2 function and the evolutionary origin of IMPase genes is warranted.

Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium.

Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications.

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.

Surface lipoprotein PpiA of Streptococcus mutans suppresses scavenger receptor MARCO-dependent phagocytosis by macrophages.

Streptococcus mutans is associated with the initiation and progression of human dental caries and is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. For the pathogen to survive in the infected host, surface lipoproteins of S. mutans are likely to play important roles in interactions with the innate immune system. To clarify the role that a putative lipoprotein, peptidyl-prolyl cis/trans-isomerase (PpiA), of S. mutans plays in the macrophage response, we investigated the response of THP-1-derived macrophages to S. mutans challenge. The deletion of the gene encoding Lgt eliminated PpiA on the cell surface of S. mutans, which implies that PpiA is a lipoprotein that is lipid anchored in the cell membrane by Lgt. Human and murine peritoneal macrophages both showed higher phagocytic activities for the ppiA and lgt mutants than the wild type, which indicates that the presence of PpiA reduces S. mutans phagocytosis. In addition, infection with S. mutans markedly induced mRNAs of macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) in human macrophages. In particular, transcriptional and translational levels of MARCO in human macrophages infected with the ppiA mutant were higher than those in macrophages infected with the wild type. Phagocytosis of S. mutans by human macrophages markedly decreased after treatment with anti-MARCO IgG. These results demonstrate that the S. mutans lipoprotein PpiA contributes to suppression of MARCO-mediated phagocytosis of this bacterium by macrophages.

Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells.

Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells.

Fish stress become visible: a new attempt to use biosensor for real-time monitoring fish stress.

To avoid fish mortality and improve productivity, the physiological conditions including stress state of the cultured fish must be monitored. As an important indicator of stress, glucose concentrations are monitored using in vitro blood analysis. The physiological processes of fish under environmental conditions are harsher in many ways than those experienced by terrestrial animals. Moreover, the process of anaesthetizing and capturing the fish prior to analysis may produce inaccurate results. To solve these problems, we developed wireless biosensor system to monitor the physiological condition of fish. This system enables artificial stress-free and non-lethal analysis, and allows for reliable real-time monitoring of fish stress. The biosensor comprised Pt-Ir wire as the working electrode and Ag/AgCl paste as the reference electrode. Glucose oxidase was immobilized on the working electrode using glutaraldehyde. We used the eyeball interstitial sclera fluid (EISF) as the in vivo implantation site of the sensor, which component concentration correlates well with that of blood component concentration. In the present study, we investigated stress due to alterations in water chemistry, including dissolved oxygen, pH, and ammonia-nitrogen compounds. Stress perceived from behavioural interactions, including attacking behaviour and visual irritation, was also monitored. Water chemistry alterations induced increases in the glucose concentration (stress) that decreased with removal of the stimulus. For behavioural interactions, stress levels change with avoidance, sensory behaviour and activity. We believe that the proposed biosensor system could be useful for rapid, reliable, and convenient analysis of the fish physiological condition and accurately reflects the stress experienced by fish.

Effects of Hangeshashinto on Growth of Oral Microorganisms.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis.

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.

Role of protease maturation lipoprotein in osmoadaptation of Streptococcus mutans.

Osmoadaptation may be an important trait for the pathogenicity of Streptococcus mutans. However, how this organism adapts to changes in osmolality in the oral cavity remains unclear. In this study, we showed that S. mutans utilizes K(+) for osmoadaptation, in which protease maturation lipoprotein (PrtM) plays an important role. Although growth of the wild-type strain was impaired in a hyperosmotic medium [brain heart infusion (BHI) containing 0.3 M NaCl] compared with that in an unmodified BHI, the prtM mutant grew much more poorly in 0.3 M NaCl BHI. Comparison of growth behavior in the hyperosmotic medium supplemented with different osmoprotectants revealed that only the addition of K(+) allowed the bacteria to overcome the impairment of growth caused by the high osmolality. These results suggest that K(+) is an important compatible solute for S. mutans. Moreover, K(+) -associated recovery of growth was not observed for the prtM mutant, indicating that PrtM plays a critical role in the utilization of K(+) . Quantitative reverse-transcriptase polymerase chain reaction analysis showed that prtM was induced by osmotic stress, implying that prtM is an osmoresponsive gene. These findings suggest that K(+) is an important compatible solute for S. mutans, and that the osmoresponsive lipoprotein PrtM is involved in K(+) utilization, contributing to osmoadaptation of S. mutans.

Rothia dentocariosa induces TNF-alpha production in a TLR2-dependent manner.

Previous work suggested that Rothia dentocariosa is associated with periodontal inflammatory disease. However, little is known about the pathogenicity of this bacterium. To characterize host response to this bacterium, we measured (via ELISA) the amount of TNF-α in the culture supernatant following the stimulation of THP-1 cells (a human acute monocytic leukemia cell line) with R. dentocariosa cells (ATCC14189 and ATCC14190). Exposure to bacterial cells induced the production of TNF-α in a dose-dependent manner. The bacterial induction of TNF-α in THP-1 cells was mediated by the Toll-like receptor 2 (TLR2), as demonstrated by gene-specific knockdown via siRNA, which successfully suppressed TLR2 expression and significantly inhibited the production of TNF-α in the culture supernatant. To confirm the role of TLR2, we examined TLR2-dependent NF-κB activation by R. dentocariosa cells in a distinct cell line. Specifically, HEK293 cells were transiently cotransfected with the human TLR2 gene and an NF-κB-dependent luciferase-encoding reporter gene. The bacterial cells induced NF-κB activation in the transfected HEK293 cells in a dose-dependent manner. In contrast, bacterial cells failed to induce NF-κB activation in cells transfected with pEF6 control vector. Taken together, these results suggest that R. dentocariosa induces host TNF-α production by a TLR2-dependent mechanism.

Effects of surface roughness and dimorphism on the adhesion of Candida albicans to the surface of resins: scanning electron microscope analyses of mode and number of adhesions.

AIM: Candida albicans is a common oral fungus but can cause serious conditions such as Candida stomatitis. We investigated C. albicans adhesion to the surface of denture-base resins at two growth phases. METHODS: Fungal suspensions of logarithmic (9 h) and stationary phase (24 h) C. albicans (JCM2085) were used. Scanning electron microscopy (SEM) confirmed that yeast and mycelial forms were predominant in 9-h and 24-h cultures, respectively. Resin strips were polished to three surface roughness levels (Ra 3.2 µm, Ra 0.48 µm and Ra 0.06 µm) and were then immersed in C. albicans suspensions for both phases. The SEM images were taken at five sites on each strip. RESULTS: Adhesion of mycelial-form C. albicans on rough surfaces (Ra = 3.2) was 2.2 times higher than on smooth surfaces (Ra = 0.06; 7030 vs 3580 adhesions/mm(2), P < 0.01). The hyphae of these mycelial forms fully penetrated the surface cracks. Fewer adhesions occurred for yeast-form C. albicans, regardless of surface type (440-620 adhesions/mm(2), P = n.s.). CONCLUSION: Adhesion of yeast-form C. albicans was indifferent to surface roughness. In contrast, mycelial adhesion increased with surface roughness of the resin because mycelia infiltrated the minute protuberances on rough surfaces.

Differences in recognition of wild-type and lipoprotein-deficient strains of oral Streptococci in vitro and in vivo.

Whole cells of wild-type strains of Streptococcus gordonii and Streptococcus mutans induced Toll-like receptor 2 (TLR2)-mediated nuclear factor-κB (NF-κB) activation, whereas those of lipoprotein (LP)-deficient strains did not. All strains upregulated the proliferation of TLR2(+/+) splenocytes more strongly than TLR2(-/-) splenocytes. However, significant differences were not observed between the cytokine-inducing activities of wild-type and LP-deficient strains toward TLR2(+/+) and TLR2(-/-) splenocytes. Muramyl dipeptide as well as whole cells not only induced nucleotide-binding oligomerization domain 2 (NOD2)-mediated activation of NF-κB but also enhanced the proliferation of TLR2(-/-) as well as TLR2(+/+) splenocytes. Wild-type strains of these streptococci were more resistant to clearance from blood and organs (liver and spleen) in TLR2(+/+) but not TLR2(-/-) mice and induced production of larger amounts of blood TNF-α than the LP-deficient strains. Wild-type strains of both species adhered to human vascular endothelial cells more strongly than did the LP-deficient strains. Thus, this study suggested that LP plays an important role in the recognition of these streptococci by the host in vivo as well as in vitro and that these streptococci possess some components recognized by NOD2 and/or TLR2 that are involved in the mitogenic activity toward splenocytes.

Role of chloride formed on anodized titanium surfaces against an oral microorganism.

A modified titanium surface, anodized after being discharged in electrolytes, provides antibacterial activity against oral bacteria as well as osteoconductivity. However, the mechanism of this antibacterial effect against oral bacteria is still unclear. This study aims to investigate whether it is the chloride or the hydrophilicity properties of the anodized titanium, which is effective against the oral bacteria. Titanium plates are anodized in various electrolytes with or without chloride and are characterized. Then the survival of Streptococcus mutans on each specimen is evaluated. The results demonstrate that the peroxidation effects of HClO generated from the TiCl(3) formed on the titanium surface anodized in various chloride solutions efficiently killed adherent S. mutans on the surface whereas the presence of hydrophilicity alone do not demonstrate antibacterial activity. This method of anodizing titanium surface in a chloride solution may provide a novel strategy for use in orthopedic or dental implant systems.