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Kazuyuki Ishihara - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the Treponema denticola Virulence Factor Dentilisin.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Yuichiro Kikuchi, Kazuyuki IshiharaAbstract:Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas gingivalis and Tannerella forsythia. It has many virulence factors, yet there are only a few reports detailing these factors. Among them, dentilisin is a well-documented surface protease. Dentilisin is reported to be involved in nutrient uptake, bacterial coaggregation, complement activation, evasion of the host immune system, inhibition of the hemostasis system, and cell invasion as a result of its action, in addition to its original proteolysis function. Therefore, characterization of dentilisin, and clarifying the relationship between T. denticola and the onset of periodontal disease will be important to better understanding this disease. In this chapter, we explain the methods for analysis of dentilisin activity and pathogenicity.
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Treponema denticola transcriptional profiles in serum-restricted conditions.
FEMS microbiology letters, 2018Co-Authors: Mariko Tanno-nakanishi, Yuichiro Kikuchi, Eitoyo Kokubu, Satoru Yamada, Kazuyuki IshiharaAbstract:Treponema denticola is a major pathogen in periodontal disease and is frequently isolated from the lesions of patients with chronic periodontitis. Treponema denticola utilizes serum components as nutrient sources so as to colonize and proliferate in the gingival crevice. However, the mechanisms of serum utilization remain unclear. Therefore, the aim of the present study was to identify T. denticola serum utilization genes. Precultured T. denticola cells were suspended in a tryptone-yeast extract-gelatin-volatile fatty acids medium containing 0, 1% and 10% serum, respectively, and incubated anaerobically for 17 h. Total RNA was isolated, and T. denticola gene expression was compared by microarray and reverse transcription-polymerase chain reaction. In serum-depleted conditions, the expression levels of a potential hydroxylamine reductase, several ABC transporters, and phosphoenolpyruvate synthase were increased, while those of genes encoding methyl-accepting chemotaxis proteins and a transcriptional regulator were decreased. These results suggest that T. denticola may uptake serum components mainly through the action of ABC transporters. In particular, the decrease in the dmcA expression level with decreasing serum concentration suggests its involvement in chemotaxis toward serum-rich environments.
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Response of epithelial cells infected by Treponema denticola
Oral diseases, 2018Co-Authors: Eitoyo Kokubu, Takashi Inoue, Kazuyuki IshiharaAbstract:Objective In the gingival crevice, the interaction between epithelial cells and periodontopathic bacteria is important for the development of periodontitis. Treponema denticola is a major pathogen of chronic periodontitis and possesses several virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine-specific protease (dentilisin). Here, we investigated the behaviours of epithelial cells infected with T. denticola by measuring the expression of interleukin (IL)-1β, IL-6, β defensin 2 (BD-2) and heat-shock protein 70 (HSP70). Methods Epithelial cells were infected with T. denticola wild-type strain, Msp-deficient mutant or dentilisin-deficient mutant, and the expression levels of the above targets were analysed by polymerase chain reaction. Results Infection with T. denticola wild-type strain and mutants induced the production of IL-6 and HSP70. The level of BD-2 induced by T. denticola wild-type strain at 24 hr was significantly higher than that of the dentilisin-deficient mutant. The level of IL-1β mRNA in the wild-type strain and dentilisin-deficient mutant was slightly lower than that in the uninfected control. Conclusion These results suggest that the levels of BD-2 were affected by Msp and dentilisin. This effect may contribute to the disruption of the response of epithelial cells to eradicate T. denticola.
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Treponema denticola invasion into human gingival epithelial cells
Microbial Pathogenesis, 2016Co-Authors: Satoru Inagaki, Eitoyo Kokubu, Ryuta Kimizuka, Atsushi Saito, Kazuyuki IshiharaAbstract:Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-β-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.
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hemagglutinin adhesin domains of porphyromonas gingivalis play key roles in coaggregation with Treponema denticola
Fems Immunology and Medical Microbiology, 2010Co-Authors: Rieko Ito, Kazuyuki Ishihara, Mikio Shoji, Koji Nakayama, Katsuji OkudaAbstract:Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.
Mena Soory - One of the best experts on this subject based on the ideXlab platform.
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The influence of cholesterol, progesterone, 4-androstenedione and testosterone on the growth of Treponema denticola ATCC 33520 in batch cultures.
Anaerobe, 2006Co-Authors: Douglas T Clark, Mena SooryAbstract:Previously, we have shown that reference and freshly isolated Treponema denticola cultures are capable of metabolising cholesterol, progesterone, 4-androstenedione and testosterone by means of 5alpha-reductase, 3beta-and 17beta-hydroxysteroid dehydrogenase activity [Clark DT, Soory M. The metabolism of cholesterol and certain hormonal steroids by Treponema denticola. Steroids. 2006;71:352-63. ]. The aim of the work presented in this paper was to investigate the modulation of T. denticola growth in batch cultures by these steroids, using T. denticola ATCC 33520 as a model system. Growth curves were summarised using statistics based on optical density and protein yield. Cholesterol was found to stimulate growth at concentrations of 10 and 25microg/mL. Certain hormonal steroids inhibited the maximum achievable optical density at concentrations of 1 and 10microg/mL while the minimum concentration shown to inhibit protein yield was 0.001microg/mL of progesterone. The potential of the hormonal steroids to inhibit growth was in the order of progesterone, 4-androstenedione and testosterone.
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The influence of cholesterol, progesterone, 4-androstenedione and testosterone on the growth of Treponema denticola ATCC 33520 in batch cultures.
Anaerobe, 2006Co-Authors: Douglas T Clark, Mena SooryAbstract:Abstract Previously, we have shown that reference and freshly isolated Treponema denticola cultures are capable of metabolising cholesterol, progesterone, 4-androstenedione and testosterone by means of 5α-reductase, 3β-and 17β-hydroxysteroid dehydrogenase activity [Clark DT, Soory M. The metabolism of cholesterol and certain hormonal steroids by Treponema denticola. Steroids. 2006;71:352–63. [10] ]. The aim of the work presented in this paper was to investigate the modulation of T. denticola growth in batch cultures by these steroids, using T. denticola ATCC 33520 as a model system. Growth curves were summarised using statistics based on optical density and protein yield. Cholesterol was found to stimulate growth at concentrations of 10 and 25 μg/mL. Certain hormonal steroids inhibited the maximum achievable optical density at concentrations of 1 and 10 μg/mL while the minimum concentration shown to inhibit protein yield was 0.001 μg/mL of progesterone. The potential of the hormonal steroids to inhibit growth was in the order of progesterone, 4-androstenedione and testosterone.
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The metabolism of cholesterol and certain hormonal steroids by Treponema denticola.
Steroids, 2006Co-Authors: D T Clark, Mena SooryAbstract:Abstract The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5α-reduce and further metabolise testosterone, 4-androstenedione, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography–mass spectroscopy. All the substrates were 5α-reduced. Both reference cultures and fresh isolates of T. denticola presented 3β- and 17β-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.
Eric C. Reynolds - One of the best experts on this subject based on the ideXlab platform.
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Metabolic cooperativity between Porphyromonas gingivalis and Treponema denticola.
Journal of oral microbiology, 2020Co-Authors: Lin Xin Kin, Catherine A. Butler, Nada Slakeski, Brigitte Hoffmann, Stuart G. Dashper, Eric C. ReynoldsAbstract:Porphyromonas gingivalis and Treponema denticola are proteolytic periodontopathogens that co-localize in polymicrobial subgingival plaque biofilms, display in vitro growth symbiosis and synergisti...
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Virulence Factors of the Oral Spirochete Treponema denticola
Journal of dental research, 2010Co-Authors: Stuart G. Dashper, Christine A. Seers, Kheng H. Tan, Eric C. ReynoldsAbstract:There is compelling evidence that treponemes are involved in the etiology of several chronic diseases, including chronic periodontitis as well as other forms of periodontal disease. There are interesting parallels with other chronic diseases caused by treponemes that may indicate similar virulence characteristics. Chronic periodontitis is a polymicrobial disease, and recent animal studies indicate that co-infection of Treponema denticola with other periodontal pathogens can enhance alveolar bone resorption. The bacterium has a suite of molecular determinants that could enable it to cause tissue damage and subvert the host immune response. In addition to this, it has several non-classic virulence determinants that enable it to interact with other pathogenic bacteria and the host in ways that are likely to promote disease progression. Recent advances, especially in molecular-based methodologies, have greatly improved our knowledge of this bacterium and its role in disease.
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Major proteins and antigens of Treponema denticola.
Biochimica et biophysica acta, 2009Co-Authors: Paul D. Veith, Stuart G. Dashper, Neil M. O'brien-simpson, Rita A. Paolini, Rebecca Orth, Katrina A. Walsh, Eric C. ReynoldsAbstract:Treponema denticola is a Gram-negative, motile, asaccharolytic, anaerobic spirochaete which along with Porphyromonas gingivalis and Tannerella forsythia has been shown to form a bacterial consortium called the Red Complex that is strongly associated with the clinical progression of chronic periodontitis. T. denticola was grown in continuous culture in a complex medium with a mean generation time of 15.75 h. Samples from two different membrane-enriched preparations and a cytoplasm-enriched preparation were separated by two-dimensional gel electrophoresis and the proteins identified by MALDI-TOF/TOF mass spectrometry. In total, 219 non-redundant proteins were identified including numerous virulence factors, lipoproteins, ABC transporter proteins and enzymes involved in the metabolism of nine different amino acids of which glycine seems to be of particular importance. Novel findings include the identification of several abundant peptide uptake systems, and the identification of three flagellar filament outer layer proteins. Two-dimensional Western blot analysis using sera from mice immunized with formalin-killed T. denticola cells suggested that Msp, PrcA, OppA, OppA10, MglB, TmpC and several flagellar filament proteins are antigenic.
Katsuji Okuda - One of the best experts on this subject based on the ideXlab platform.
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hemagglutinin adhesin domains of porphyromonas gingivalis play key roles in coaggregation with Treponema denticola
Fems Immunology and Medical Microbiology, 2010Co-Authors: Rieko Ito, Kazuyuki Ishihara, Mikio Shoji, Koji Nakayama, Katsuji OkudaAbstract:Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.
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Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola
FEMS immunology and medical microbiology, 2010Co-Authors: Rieko Ito, Kazuyuki Ishihara, Mikio Shoji, Koji Nakayama, Katsuji OkudaAbstract:Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.
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The Treponema denticola Surface Protease Dentilisin Degrades Interleukin-1β (IL-1β), IL-6, and Tumor Necrosis Factor Alpha
Infection and immunity, 2006Co-Authors: Meguru Miyamoto, Kazuyuki Ishihara, Katsuji OkudaAbstract:Dentilisin is a major surface protease and virulence factor of the bacterium Treponema denticola. In this study, we found that T. denticola reduced inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha, in peripheral blood mononuclear cells through degradation by dentilisin.
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A 43-kDa protein of Treponema denticola is essential for dentilisin activity.
FEMS microbiology letters, 2004Co-Authors: Kazuyuki Ishihara, Howard K Kuramitsu, Katsuji OkudaAbstract:A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3′ region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.
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Detection of Treponema denticola in Atherosclerotic Lesions
Journal of clinical microbiology, 2001Co-Authors: Katsuji Okuda, Kazuyuki Ishihara, Taneaki Nakagawa, Akihiko Hirayama, Yoshiyuki Inayama, Kenji OkudaAbstract:We examined 26 atherosclerotic lesions and 14 nondiseased aorta specimens to detect the periodontopathogenic part of the bacterial 16S rRNA locus by PCR. Treponema denticola sequence of the 16S rRNA locus was found in 6 out of 26 DNA samples (23.1%) from the formalin-fixed, paraffin-embeded atherosclerotic lesions obtained during surgery but not in any of the 14 nondiseased aorta samples from deceased persons. Utilizing immunofluorescence microscopy, we observed aggregated antigenic particles reacting with rabbit antiserum against T. denticola in thin sections of the PCR-positive samples, but we could not detect any reacting particles in the PCR-negative thin sections.
Daniel Grenier - One of the best experts on this subject based on the ideXlab platform.
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Binding properties of Treponema denticola lipooligosaccharide
Journal of oral microbiology, 2013Co-Authors: Daniel GrenierAbstract:Background and objective : The cell-surface lipooligosaccharide (LOS) of Treponema denticola possesses several biological properties. The aim of this study was to investigate the binding properties of T. denticola LOS to extracellular matrix (ECM) proteins, mucosal cells, and oral bacteria. Design : LOS was isolated from T. denticola and labeled with tritium. Tritium-labeled LOS was placed in ECM protein-, epithelial cell-, fibroblast-, or bacterium-coated wells of a 96-well microplate. Following incubation, unattached LOS was removed by extensive washing, and the amount of bound LOS was determined by measuring the radioactivity in the wells. Peptostreptococcus micros coated with LOS was used to stimulate fibroblasts, and the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) by the fibroblasts was determined by ELISA. Results : T. denticola LOS had a high affinity for laminin. It also bound to gingival epithelial cells and fibroblasts. Soluble CD14 significantly increased the binding of LOS to fibroblasts. More LOS bound to P. micros than the other oral bacterial species tested. Stimulating fibroblasts with LOS-coated P. micros induced the secretion of IL-6 and IL-8. Conclusions : Our study provided evidence that T. denticola LOS possesses the capacity to bind to ECM proteins, mucosal cells, and oral bacteria. In addition, LOS binding to bacteria may increase their pro-inflammatory potential. Keywords : adherence; cytokine; epithelial cells; fibroblasts; laminin; lipooligosaccharide; periodontal disease; Peptostreptococcus micros; Treponema denticola (Published: 16 September 2013) Citation: Journal of Oral Microbiology 2013, 5 : 21517 - http://dx.doi.org/10.3402/jom.v5i0.21517
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Cleavage of Human Immunoglobulin G by Treponema denticola
Anaerobe, 2001Co-Authors: Daniel Grenier, Denis MayrandAbstract:Abstract Mechanisms by which microbial proteases may counteract the local host immune system include the degradation of immunoglobulins. In this study, we report the capacity of the periodontopathogen Treponema denticola to degrade immunoglobulin G (IgG). Intact IgG was not hydrolysed by whole cells, as revealed by SDS-PAGE analysis. When IgG molecules were treated with endoglycosidase F to remove the carbohydrate moiety, significant degradation was observed. However, pre-treatment with glycosidases possessing specificities different from endoglycosidase F (lysozyme or neuraminidase) did not render the molecule susceptible to cleavage by T. denticola. SDS-PAGE analysis of the IgG degradation products suggests that T. denticola cleaves inside the heavy chain polypeptide. Serine-specific protease inhibitors were highly effective in inhibiting the degradation of glycosidase-treated IgG molecules by T. denticola. The synergistic effect of glycolytic enzymes andT. denticola proteases on IgG may occur during periodontitis since both glycolytic activities and spirochete numbers significantly increase in diseased periodontal sites.
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Antagonistic effect of oral bacteria towards Treponema denticola.
Journal of clinical microbiology, 1996Co-Authors: Daniel GrenierAbstract:This study was designed to isolate oral bacteria exhibiting antagonism towards Treponema denticola and to characterize the inhibitory activity. Eleven bacterial isolates obtained from subgingival sites and identified as either Staphylococcus aureus or Streptococcus mutans were found to inhibit the growth of T. denticola. When the activity spectra of these isolates were analyzed, two additional periodontopathogens (Porphyromonas gingivalis and Prevotella intermedia) were found to be affected, whereas most gram-positive bacteria were not. Strains of S. aureus produce a bacteriocin-like inhibitory substance (heat stable and protease sensitive), whereas the inhibitory effect of S. mutans appears to be related to the production of lactic acid. The negative interactions reported in this paper may govern population shifts observed in subgingival sites.
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Cytotoxic effect of peptidoglycan from Treponema denticola
Microbial pathogenesis, 1993Co-Authors: Daniel Grenier, Veli-jukka UittoAbstract:Abstract Peptidoglycan isolated from pathogenic bacteria has been previously found to exhibit various biological activities. The aim of this investigation was to evaluate the toxicity of Treponema denticola peptidoglycan towards epithelial cells. The cytotoxicity of a lipopolysaccharide-like material was also determined. Epithelial cells were incubated with the test substances and cell viability was assayed by measuring lactate dehydrogenase activity and the conversion of tetrazolium salt into blue formazan. Morphological changes in the epithelial cells were monitored by scanning and transmission electron microscopy. While lipopolysaccharide-like material exerted negligible toxic effects on the epithelial cells, peptidoglycan was highly toxic. This cytotoxicity was both time-and concentration-dependent and was higher in the presence of serum. The epithelial cells appeared to be unable to recover following a short period of incubation with peptidoglycan. The study demonstrated that peptidoglycan from T. denticola is a potent toxic factor for epithelial cells. This represents a potential new virulence factor for this suspected periodontopathogen.
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Characterization of the wheat germ agglutinin‐binding property of Treponema denticola
Journal of periodontal research, 1993Co-Authors: Daniel Grenier, D. Groleau, Antonio NanciAbstract:Lectins were used to characterize glycoconjugates on the cell surface of Treponema denticola, a suspected periodontopathogen. Bacteria were first screened by light microscopy using fluorescein isothiocyanate-coupled lectins. Wheat germ agglutinin (WGA) showed a high reactivity to T. denticola. While the WGA-binding activity was accentuated following heating or detergent treatments of bacterial cells, the reaction was inhibited by incorporation of competing carbohydrates. Scanning and transmission electron microscope studies were conducted in order to characterize the distribution of the WGA-binding sites on the cell surface of T. denticola. Data from these studies confirmed that heat treatment increases the percentage of labeled profiles and suggest that the WGA-binding sites are concentrated on specific regions on the spirochete surface. Initial biochemical analysis indicated that the high reactivity to WGA resides in a peptidoglycan fraction.
