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Gamma interferon (IFN-γ) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-γ-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-γ and protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-γ receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-γ signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.
Enterohemorrhagic Escherichia coli (EHEC) refers to a family of bacterial enteropathogens that can contaminate food and water to cause outbreaks of diarrhea and hemorrhagic colitis (10, 25). In addition, the Shiga-like toxins expressed by EHEC have been associated with causing hemolytic uremic syndrome, a leading cause of acute renal failure among the pediatric population (10, 56). Multiple serotypes of EHEC, including O157:H7 and non-O157 serotypes, elicit these diseases. Indeed, non-O157 serotypes are increasingly recognized as important human enteropathogens (23), although they often lack the well-characterized virulence factors present in O157:H7 strains (12). Non-O157:H7 EHEC, such as serotype O113:H21, are generally negative for the locus for enterocyte effacement (LEE) pathogenicity island and therefore do not express the outer membrane protein intimin or a functional type III secretion system. However, both O157:H7 and O113:H21 express Shiga-like toxins (26, 13), indicating these pathogens can utilize both similar and divergent strategies to infect host cells. These data highlight a need to better understand the different EHEC serotypes and how they interact with the enterocyte.
Cholesterol-enriched microdomains in the lipid bilayer are biochemically distinct regions of the eukaryotic plasma membrane and also contain sphingolipids and proteins important in signal transduction (48). Such microdomains provide a platform for host cell signal transduction cascades that initiate from an extracellular stimulus (33). For instance, functional gamma interferon (IFN-γ) receptor (IFNGR) domains 1 and 2 reside in membrane microdomains of epithelial cells, and pharmacological disruption of microdomains inhibits IFN-γ-induced signal transducer and activator of transcription 1 (Stat1) tyrosine phosphorylation in the cytosol and DNA binding in the nucleus (40, 51, 52). In addition to serving as platforms for host-driven signal transduction, bacteria and their products manipulate membrane microdomains as part of their pathogenic strategy (31, 38). For example, when intimately attaching to host cells, enteropathogenic E. coli causes the clustering of microdomain-associated host cell proteins (59). Epithelial cell vacuolization by the vacuolating cytotoxin A of H. pylori occurs in a microdomain-dependent manner (39). However, whether bacterial infection can also target microdomains to disrupt components of host cell signal transduction cascades, such as cytokine receptors, is largely unknown.
IFN-γ plays a central role in the Th1 cellular immune response against a wide variety of microbes (44). Citrobacter rodentium is a murine attaching and effacing pathogen model for EHEC O157:H7 infection that elicits a Th1-response typified by IFN-γ production (19). Indeed, IFN-γ knockout mice infected with C. rodentium demonstrate worse symptoms of disease than their wild-type littermates (47). Previously, we showed that infection with EHEC O157:H7 and O113:H21 disrupts IFN-γ-induced Stat1 signal transduction in epithelial cells in vitro (4). These inhibitory effects were independent of the locus of enterocyte effacement pathogenicity island (PAI), the type III secretion system, Shiga-toxins type 1 and type 2, and the pO157 plasmid (4). Moreover, live bacteria, but not heat-killed organisms, are required to elicit the inhibition of IFN-γ-stimulated Stat1-tyrosine phosphorylation. Taken together, these observations suggest that IFN-γ-Stat1 signal transduction likely is important to host defense in response to attaching-effacing bacterial enteropathogens.
The objectives of this study were to further delineate both bacterial and host cell factors that mediate EHEC suppression for IFN-γ-induced Stat1 signal transduction. The data presented herein suggest a role for EHEC adhesion, bacterial protein synthesis, and a contact-dependent soluble mediator in suppression of IFN-γ signal transduction. In addition, EHEC infection disrupts the subcellular localization of IFN-γ receptor 1 in human epithelial cells.
Eukaryotic tissue culture.T84 colonic epithelial cells were cultured as previously described (4). Briefly, cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2% penicillin-streptomycin, 2% sodium bicarbonate, and 0.6% l-glutamine (all obtained from Life Technologies, Grand Island, N. Y.). Approximately 3 × 106 T84 cells were seeded onto 6-cm petri dishes (Falcon) or six-well plates (Costar) and grown to confluence for whole-cell protein extraction. Cells were grown at 37°C in a 5% CO2 atmosphere. Prior to bacterial infection or protein extraction, the cells were incubated in medium containing no antibiotics for ∼20 h.
Bacteria and bacterial growth conditions.EHEC of serotypes O157:H7 (strain CL-56) and O113:H21 (strain CL-15) were kept on 5% sheep blood agar plates at 4°C and cultured in static, nonaerated Penassay broth (Difco Laboratories, Detroit, MI) overnight at 37°C (4, 22). EHEC serotype O157:NM (NM, nonmotile), including strain E32511, confirmed not to express flagellin (13), and strain MOH67 were grown under the same conditions (kind gifts from M. A. Karmali, Health Canada, Guelph, Ontario, Canada). To infect epithelial cells, bacteria were pelleted from broth cultures by centrifugation at 3,000 rpm for 5 min and washed in cell culture medium. The bacteria were resuspended in a final volume of 25 μl of tissue culture medium without antibiotics and then added to host cells, where the multiplicity of infection (MOI) was 100 bacteria per epithelial cell (4). The same volume of medium alone served as a vehicle control treatment. In some experiments, bacteria in tissue culture medium were physically separated from T84 epithelial cells grown on the bottom of a six-well tissue culture plate by placing the bacteria in a compartment above the cells, separated by a 0.4-μm Transwell filter (Costar). Medium from the T84 compartment beneath the filter was cultured on 5% sheep blood agar plates to ensure lack of bacterial growth.
Bacterial culture supernatants and conditioned medium.To collect bacterial culture supernatants, 1 ml of EHEC O157:H7 grown overnight in Penassay broth (10 ml, static) was centrifuged (3,000 rpm, 5 min) and resuspended in 15 ml of T84 tissue culture medium with FBS and without antibiotics. After 6 h growth at 37°C in a 5% CO2 atmosphere, the medium was centrifuged (3,000 rpm, 15 min), filtered (0.45 μm) and stored at −20°C. To collect conditioned medium, EHEC O157:H7 strain CL-56 grown overnight in 10 ml Penassay broth (37°C, static) was used to infect confluent monolayers of T84 cells (MOI 100:1, 6 h), while T84 cells receiving medium only served as a control. After 6 h, medium was cleared by centrifugation (3,000 rpm, 15 min), filtered (0.45 μm), and stored at −20°C until use. This conditioned medium contained factors secreted either by T84 cells alone or by bacterium-T84 cell contact. In both cases, the effectiveness of filtration was confirmed by lack of bacterial growth from 100 μl of this conditioned medium plated onto 5% sheep blood agar plates and then incubated overnight at 37°C.
Whole-cell protein extraction.T84 cells were either infected with Escherichia coli or incubated with conditioned medium. Subsequently, some cells were stimulated with IFN-γ (50 ng/ml) at 37°C for 30 min. Next, T84 cells were washed three times with ice-cold phosphate-buffered saline (PBS) (pH 7.4) and the whole-cell protein extracts were collected and stored at −80°C as previously described (4).
Isolation of EHEC O157:H7 flagellin.EHEC flagellin was isolated as previously described (2, 43). Briefly, overnight cultures of EHEC O157:H7 strain CL-56 were resuspended in a hydrochloric acid (pH 2.0) solution containing 1.0 mM phenylmethylsulfonyl fluoride for 30 min at room temperature. Bacteria were then pelleted at 5,000 × g for 30 min. Protein from the flagellin-rich supernatant was concentrated using a Millipore centrifugal filter device (5-kDa cutoff), protein concentration was determined by the Bio-Rad assay, and samples were stored at −80°C until use. An aliquot (5 or 10 μg) of the sample was electrophoresed through a 10% Tris-HCl gel (Bio-Rad), and the gel subsequently Coomassie brilliant blue-stained to confirm that the flagellin subunit (approximately 66 kDa) was successfully isolated (43).
Pharmacological inhibitor studies.T84 cells were preincubated with the eukaryotic cell protein synthesis inhibitor cycloheximide (10 μg/ml, 45 min) (57), with inhibitor remaining in the medium throughout the infection period. In addition, epithelial cells and bacteria were incubated with the bacterial protein synthesis inhibitor chloramphenicol (100 μg/ml) at 0, 30, 90, and 180 min after infection with EHEC O157:H7 (MOI 100:1, 6 h) (4). MG-132 (50 μM; preincubation for 45 min, left in the medium for the infection period) was employed to determine the role of the host proteasome (42). Sodium orthovanadate (1,000 μM for the entire 6 h infection or for 30 min following infection) treatment of infected and uninfected T84 cells was used to determine the effects of protein tyrosine phosphatases (41). In all cases, the effects of vehicle alone were also evaluated and found to have no effect on the tyrosine phosphorylation status of Stat1 or expression levels of native Stat1 (data not shown).
Infected and uninfected T84 cells were also coincubated with either the quorum sensing mimetic norepinephrine (10 to 500 μM) (49) or the quorum sensing inhibitor propranolol (10 to 250 μM) (49) at 37°C for 3.5 h to determine the role of quorum sensing in the ability of EHEC O157:H7 to suppress IFN-γ-induced Stat1 tyrosine phosphorylation.
Proteinase and heat inactivation treatment of conditioned medium.Conditioned medium from T84 cells infected with EHEC O157:H7 strain CL-56 was incubated with proteinase K (200 μg/ml, 1 h, 37°C). Subsequently, proteinase K activity was neutralized by adding the serine protease inhibitor Pefabloc (Roche/Boehringer) (5 mM, 2 h, 37°C). In addition, trypsin digestion of conditioned medium was similarly performed, except that FBS-free T84 culture medium was studied. Conditioned medium from T84 cells infected with EHEC O157:H7 strain CL-56 was employed. Following incubation with trypsin (200 μg/ml, 1 h, 37°C) in the medium, soybean trypsin inhibitor (400 μg/ml, 30 min, 37°C) was added to neutralize proteinase activity (15). Such medium was also prepared with the addition of 10% FBS.
Conditioned medium from T84 cells alone and from T84 cells infected with EHEC O157:H7 strain CL-56 was heat inactivated by boiling (100°C, 2 h). Subsequently, conditioned medium was cooled to 37°C and employed in protein extraction experiments.
Immunoblotting.Equal volumes of whole-cell protein extracts were analyzed for tyrosine phosphorylated Stat1 and native Stat1, as previously described (4) or with the following modifications: sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer was added to whole-cell protein extracts in a 1:2 (vol/vol) ratio, samples were boiled for 3 min and then loaded into wells of precast 10% polyacrylamide gels (Ready Gel; Bio-Rad Laboratories, Hercules, CA). Gels were electrophoresed at 111 V for 1 to 1.5 h, and migrated proteins were then transferred onto nitrocellulose membranes (BioTrace NT; Pall Corporation, Ann Arbor, MI) at 100 V for 1.5 to 2 h at 4°C. The nitrocellulose membranes were subsequently incubated in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20°C. The blocking buffer was decanted and the membranes were incubated with either anti-native Stat1 primary antibody (Santa Cruz Biotechnologies, Santa Cruz, CA; 1:5,000 dilution in Odyssey buffer) or anti-phospho-Stat1 primary antibody (Cell Signaling, Beverly, MA; 1:1,000 dilution) and anti-β-actin primary antibody (1:5,000 dilution; Sigma) at 4°C overnight on a shaker. Primary antibody solutions were subsequently decanted and the membranes were washed four times with PBS plus 0.1% Tween (5 min per wash).
The membranes were then incubated with secondary antibodies: IRDye 800 goat anti-rabbit immunoglobulin G (IgG) (1:20,000 dilution; Rockland Immunochemicals, Gilbertsville, PA) and Alexa Fluor 680 goat anti-mouse IgG (1:20,000 dilution; Molecular Probes, Eugene, OR) at 20°C for 1 h on a shaker. Nitrocellulose membranes were again washed four times with PBS-Tween with a final wash in PBS without Tween. Immunoblots were also probed with anti-Jak1 (AnaSpec, San Jose, CA; 1:1,000 dilution) and anti-Jak2-phosphospecific (pTyr10097/1008) (AnaSpec; 1:1,000 dilution) antibodies to determine if the inhibitory activity occurred upstream of Stat1-phosphorylation (45). Bands were detected by scanning the nitrocellulose membranes into the Odyssey system (LI-COR Biosciences) with both 700-nm and 800-nm channels, at a resolution of 169 μm (58).
Densitometry.The integrative intensity of all bands was calculated using automated software provided with the Odyssey Infrared Imaging System (LI-COR Biosciences). For immunoblots with dual antibody staining, integrative intensity values were normalized to the integrative intensities obtained for either anti-native Stat1 or β-actin (58). Normalized values obtained from bands of uninfected cells stimulated with IFN-γ were then set to 100%, with samples derived from EHEC-infected cells then calculated as a percentage relative to uninfected stimulated cells.
Localization of IFN-γ receptor 1 to microdomains using buoyant density ultracentrifugation.T84 cells were grown to confluence in 3-cm tissue culture dishes and detergent resistant microdomains were isolated, as described previously (51). After a 6-h infection period, T84 cells were washed three times in ice-cold PBS, scraped with a rubber policemen, pipetted into 15-ml conical tubes, and centrifuged at 1,000 rpm for 2 min. Pelleted cells (∼106) were resuspended in 0.8 ml of extraction buffer (25 mM Tris-HCl, 150 mM NaCl, 10% sucrose, 1% Triton X-100, 1 mM dithiothreitol, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 10 μg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate; all from Sigma Aldrich, Oakville, Ontario, Canada). The lysate was then passed through a 25-gauge needle 10 times and incubated for 30 min on ice.
Optiprep stock solution (Invitrogen, Burlington, Ontario, Canada) (60%) was then added to cell lysates (2:1, vol/vol); 2.5 ml of the 40% solution, loaded into the bottom of a 12-ml ultracentrifuge tube (catalogue 344059; Beckmann, Palo Alto, CA), and then overlaid with 6.5 ml of 35% and 3.0 ml of 5% Optiprep solutions, diluted in extraction buffer. Samples were centrifuged for 20 h (at 36,000 rpm) at 160,000 × g at 4°C in an SW41 Ti rotor (Beckman). After ultracentrifugation, eight 1.5-ml fractions were collected and snap-frozen in liquid nitrogen for storage at −80°C, prior to analysis by immunoblotting (4, 51). For Western blotting, membranes were incubated with polyclonal rabbit anti-human antibody against IFNGR1 (1:200; C-20) or caveolin-1 (1:500; N-20) (all from Santa Cruz). Immunoblots were then washed and incubated with secondary antibody, IRDye 800 goat anti-rabbit IgG (1:10,000 dilution; Rockland Immunochemicals).
Confocal microscopy.T84 cells grown on glass coverslips were washed three times with ice-cold PBS (pH 7.4), fixed in 4% paraformaldehyde for 30 min, permeabilized for 4 min with 0.1% Triton X-100, and blocked for 30 min in 2% bovine serum albumin/0.1% Triton X-100 (4, 28). Subsequently, cells were probed for IFNGR1 with 1:100 anti-IFNGR1 (Santa Cruz) in 2% bovine serum albumin overnight at 4°C, washed in PBS, incubated with 1:500 fluorescein isothiocyanate-red goat anti-Armenian hamster antibody (Jackson Labs) in bovine serum albumin/Triton X-100 for 1 h at room temperature, and washed in PBS (pH 7.4). Vectashield (Vector Labs, Burlington, Ontario, Canada) mounting medium for fluorescence was added, and slides were sealed with coverslips and examined using a confocal microscope (Leica DMIRE2 spinning disk confocal microscope).
Live EHEC must be in contact with epithelial cells to suppress Stat1 activation.Adhesion of microbes to host cells can be integral to causing disease (14). Our previous studies showed that live, but not heat-killed, EHEC O157:H7 suppressed Stat1 activation (4). Here we determined whether direct contact between live EHEC serotype O157:H7 or O113:H21 and epithelial cells was required. Analysis of whole-cell protein extracts showed that IFN-γ induced tyrosine phosphorylation of Stat1 in uninfected T84 cells, whereas infection with EHEC O157:H7 or O113:H21 in the lower chamber resulted in complete inhibition of Stat1 tyrosine phosphorylation (Fig. 1). In contrast, when either strain was placed in the upper chamber, separated from the T84 monolayer by insertion of a Transwell filter, Stat1 activation was not inhibited. These data suggest that direct contact between the bacterium and epithelial cell is required to inhibit Stat1 activation.
Conditioned medium, but not bacterial culture supernatant, suppresses IFN-γ-induced Stat1 tyrosine phosphorylation.To confirm the necessity of direct contact between bacteria and epithelial cells in suppressing IFN-γ-induced Stat1 activation, T84 cells were incubated with EHEC O157:H7 bacterial culture supernatant, or with conditioned medium taken either from T84 cells alone or from T84 cells infected with EHEC O157:H7. Both T84 cells treated with bacterial culture supernatant alone or with T84 media alone still supported IFN-γ activation of Stat1 tyrosine phosphorylation (Fig. 2A). In contrast, incubation of T84 cells with conditioned medium from tissue culture cells infected with EHEC O157:H7 resulted in reduced (16% of control) activation of Stat1 tyrosine phosphorylation by IFN-γ relative to uninfected epithelial cells (Fig. 2B). These results indicate that a soluble factor(s) generated from contact between bacteria and epithelial cells, but not from bacteria or epithelial cells alone, suppresses Stat1 activation. Also, these data complement the results shown in Fig. 1 that direct contact between EHEC and the epithelial cell is necessary to interrupt Stat1 activation.
Soluble factor(s) is insensitive to proteinase and heat inactivation treatment.To determine if the soluble factor(s) responsible for suppression of IFN-γ-induced Stat1 activation was a peptide or nonpeptide, we incubated conditioned medium, taken from T84 cells only or T84 cells infected with EHEC, with either proteinase K or trypsin. Subsequently, proteinase K activity was neutralized by adding the serine protease inhibitor Pefabloc to prevent cytotoxicity from the protease, while trypsin was neutralized using soybean trypsin inhibitor. Results indicate that proteinase K treatment did not reverse the suppressive effect of conditioned medium from T84 cells infected with EHEC O157:H7 on IFN-γ-induced Stat1 tyrosine phosphorylation (Fig. 3A). Similarly, treatment with trypsin and the soybean trypsin inhibitor did not reverse the inhibition of IFN-γ-induced Stat1 tyrosine phosphorylation by conditioned medium (Fig. 3B). These results suggest that the factor responsible for suppression of Stat1 activation is proteinase insensitive.
In addition, conditioned medium taken from T84 cells with EHEC O157:H7 strain CL-56 was heat inactivated and then incubated with T84 cells. Heat-inactivated conditioned medium from T84 cells infected with EHEC still suppressed IFN-γ-induced Stat1 activation (Fig. 3C). Together with the proteinase data, this finding suggests that the factor(s) responsible for suppressing IFN-γ-induced Stat1 tyrosine phosphorylation is nonpeptide.
Bacterial but not epithelial cell protein synthesis mediates suppression of Stat1 activation.Bacterial infection can induce expression of suppressors of cytokine signaling (SOCS) in host cells, which can then suppress subsequent cytokine signal transduction (8, 50). Thus, we tested the role of bacterial and host cell protein synthesis during Stat1 inhibition. First, inhibition of bacterial protein synthesis by coincubation with chloramphenicol completely blocked the ability of EHEC O157:H7 to suppress IFN-γ-induced Stat1 tyrosine phosphorylation (Fig. 4A). This effect was time dependent, as addition of chloramphenicol more than 30 min after infection was unable to reverse the inhibition of Stat1 tyrosine phosphorylation by EHEC. In contrast, inhibition of host cell protein synthesis using cycloheximide did not block the ability of EHEC O157:H7 infection to suppress Stat1 activation (Fig. 4B). Therefore, time-dependent bacterial but not host cell protein synthesis is required to suppress Stat1 activation by IFN-γ.
Mechanisms of EHEC-mediated suppression of Stat1 activation.Stat activity is down-regulated by the host cell proteasome (37). Also, proteasome inhibition can prevent NF-κB activation, and NF-κB activity is implicated in suppression of Stat1 activation (27) and SOCS expression (32). Preincubation with the proteasome inhibitor MG-132 did not reverse the ability of EHEC to suppress IFN-γ-induced Stat1 tyrosine phosphorylation, indicating that the effect is proteasome-independent (Table 1).
Protein tyrosine phosphatases are known to deactivate phosphorylated Stat1 (18, 53). To determine if the inhibition of Stat1-tyrosine phosphorylation by EHEC O157:H7 was due to either a bacterial protein tyrosine phosphatase or the upregulation of a host cell protein tyrosine phosphatase, infected and uninfected T84 cells were treated with sodium orthovanadate. Since a phosphorylated Stat1 band was not recovered in cell extracts of EHEC-infected cells, the inhibitory effects of EHEC O157:H7 on Stat1-tyrosine phosphorylation likely is not due to a bacterial protein tyrosine phosphatase or the up-regulation of a host cell protein tyrosine phosphatase (Table 1).
Bacterium-mediated mechanisms.Activation of Toll-like receptors can suppress cytokine signal transduction in macrophages (8), and T84 cells express the functional receptor for flagellin, TLR5 (2). However, treatment of T84 cells with flagellin isolated from EHEC O157:H7 did not inhibit subsequent IFN-γ-induced Stat1 activation (Table 1). Furthermore, infection of T84 cells with two nonmotile EHEC O157:H- strains, E32511 and MOH67, also resulted in suppression of IFN-γ-induced Stat1 tyrosine phosphorylation (Table 1). These data indicate that EHEC suppression of cytokine signaling does not require expression of flagella and reinforce the NF-κB-independent nature of Stat1 inhibition.
Quorum sensing by EHEC O157:H7 regulates expression of multiple EHEC virulence factors, including type III secretion and attaching-effacing lesion formation on host cells (49). To determine if quorum sensing was involved in the EHEC-mediated suppression of Stat1 activation, we coincubated T84 cells with either propanolol, an inhibitor of quorum sensing, or norepinephrine, a quorum-sensing mimetic (49). Norepinephrine did not enhance the ability of EHEC O157:H7 to suppress IFN-γ-induced Stat1 tyrosine phosphorylation. Moreover, propanolol did not reverse the EHEC-mediated inhibition of Stat1 activation. Taken together, these data suggest that expression of the bacterial factor(s) responsible for suppressing Stat1 activation is not regulated by EHEC quorum sensing (Table 1).
EHEC O157:H7 infection disrupts Jak phosphorylation.IFN-γ signals through the IFN-γ receptor, a cell surface receptor comprised of two functional domains: IFN-γ receptor domain 1 provides the binding site for IFN-γ, while IFN-γ receptor domain 2 initiates downstream signaling events (1). The cytoplasmic tails of both domains are linked to Jak proteins, which upon phosphorylation provide docking sites for latent Stat1 proteins. Jak1 is linked to IFN-γ receptor domain 1 and Jak2 is linked to IFN-γ receptor domain 2 (1). To determine if EHEC O157:H7 inhibition of Stat1 phosphorylation occurs upstream of Stat1, cell extracts of infected and uninfected T84 cells were probed with an anti-Jak1 and an anti-Jak2 phosphospecific (pTyr10097/1008) antibody (Fig. 5). Relative to uninfected cells, the constitutive amounts of Jak1 and phospho-Jak2 in EHEC-infected cells were 67 and 96%, respectively, as determined by densitometry. In response to IFN-γ stimulation, levels of both Jak1 and phospho-Jak2 were reduced in EHEC-infected cells to 21 and 49%, respectively, of those in uninfected controls.
EHEC infection disrupts subcellular localization of IFN-γ receptor domain 1. Mycobacterium avium infection decreases IFN-γ receptor domain 1 and 2 expression in macrophages (20). Thus, we determined the expression and subcellular localization of the IFN-γ receptor domain 1 using confocal microscopy. In an uninfected T84 monolayer, prominent IFN-γ receptor domain 1 immunoreactivity occurred in the basal and mid-regions, but not the apical region of the cell (Fig. 6A, C, and E). However, T84 cells infected with EHEC O157:H7 strain CL-56 exhibited decreased staining in the basal and mid-regions, but increased staining in the apical region, compared with uninfected cells (Fig. 6B, D, and F). In the lateral view of a three-dimensional reconstruction from the T84 monolayer, IFN-γ receptor domain 1 expression was seen throughout the uninfected cell (Fig. 6G), whereas the expression predominately localized to the apical aspect of EHEC-infected cell (Fig. 6H).
Functional IFN-γ receptor domain 1 localizes to caveolin-enriched plasma membrane microdomains.IFN-γ receptor domain 1 localized to membrane microdomains has been suggested to contain the functional signal-transducing receptor for IFN-γ (51). Fractionation of the Triton X-100 protein extract on a density gradient followed by immunoblot analysis demonstrated that a portion of the cellular pool of IFN-γ receptor domain 1 was found in the caveolin-1-enriched membrane microdomain fraction of unstimulated T84 cells (fraction 3) (Fig. 7). Subsequent analysis of this membrane microdomain fraction showed that infection of T84 cells with both EHEC serotypes O157:H7 and O113:H21 caused decreased microdomain expression of IFN-γ receptor domain 1 compared with uninfected cells (Fig. 7).
EHEC serotypes O157:H7 and O113:H21 both disrupt cytokine signal transduction in epithelial cells (4), but the mechanisms underlying this observation remain to be delineated. Herein, we show that bacteria physically separated from a T84 cell monolayer were unable to suppress Stat1 activation by IFN-γ. In contrast, we provide mechanistic evidence that EHEC O157:H7 and O113:H21 both suppressed IFN-γ-induced Stat1 activation when allowed to contact host cells. Reinforcing the notion that contact is necessary, conditioned medium from infected T84 cells, but not medium from T84 cells or bacterial culture supernatant alone, inhibited IFN-γ-induced Stat1 activation. Furthermore, bacterial, but not host cell, protein synthesis was required to inhibit Stat1 activation, and inhibition was independent of protein tyrosine phosphatases, flagella, quorum sensing, or host cell proteasome activity. Lastly, with respect to host factors, EHEC O157:H7 infection disrupted the subcellular distribution of IFN-γ receptor domain 1.
Adhesion of microbes to epithelial cells can be a critical step in the infection process (14). For example, Listeria monocytogenes binds specific host cell surface receptors before internalizing into epithelial cells (42). Adhesion is also critical in a different manner for bacteria that actively avoid internalization, such as EHEC O157:H7 (25). This attaching-and-effacing bacterium adheres to the cell surface and injects its own receptor known as translocated intimin receptor (Tir), or EspE, into the host cell via a type III secretion system (9, 21). Host membrane-based EspE then intimately binds to the bacterial outer membrane protein intimin, encoded by the eae gene on the LEE pathogenicity island. However, it is likely that the mechanism behind EHEC inhibition of IFN-γ-induced Stat1 activation is distinct from the products of the LEE pathogenicity island and the attaching-effacing lesion, since a type III secretion mutant also suppressed IFN-γ-induced Stat1 activation (4) and since O113:H21 is negative for the LEE marker eaeA (11). As contact between the bacteria and host cell is required, candidate bacterial factors include outer membrane proteins common to the two serotypes involved in adhesion, such as the chromosomally encoded long polar fimbriae (35, 55), a hypothesis that awaits testing.
Bacterial contact with eukaryotic cells induces the secretion of multiple proteins of both bacterial (6) and host cell (5) origin. We now describe, for the first time, that EHEC O157:H7 infection of T84 cells led to secretion of a factor(s) capable of suppressing Stat1 activation. This soluble factor is not dependent on quorum sensing for expression nor is it present in bacterial culture supernatants, providing further support for the contention that interaction between EHEC and T84 cells is necessary to suppress Stat1 activation. Furthermore, this factor may not be a peptide, since its ability to suppress Stat1 activation was resistant to treatment with proteinase K, trypsin, heat inactivation, and sodium orthovanadate. Indeed, bacteria can secrete nonpeptide factors that interact with host cells, such as the monocyte chemoattractant diethyl phthalate by Helicobacter pylori (27). However, since peptides can be heat inactivation resistant (16), and since protease treatment may leave behind an active, smaller peptide, the exact molecular identity of the factor(s) requires further determination.
We also found that the bacterial protein synthesis inhibitor chloramphenicol completely blocked the ability of EHEC O157:H7 to suppress IFN-γ-induced Stat1 tyrosine phosphorylation in a time-dependent manner, whereas the inhibition of host cell protein synthesis using cycloheximide did not block the ability of EHEC O157:H7. Taken together, the present data suggest that the soluble mediator(s) inhibiting Stat1 signal transduction is shed or secreted from the bacteria in a protein synthesis-dependent manner.
Cytokine-induced signal transduction is negatively regulated by various complementary pathways, including induction of SOCS protein expression and phosphatase activity (45). For example, infection with L. monocytogenes (50) or treatment with Toll-like receptor ligands (8) inhibits IFN-γ activation of Stat1 tyrosine phosphorylation in macrophages by inducing SOCS expression. Indeed, SOCS proteins may degrade Jak proteins through a proteasome-dependent mechanism to suppress cytokine signaling (45). Recently, respiratory syncytial virus-mediated suppression of Stat2 activity was shown to be dependent on the proteasome (37), and NF-κB activation has also been implicated in SOCS induction (32).
In this study, we provide multiple lines of evidence that induction of SOCS protein expression or proteasome activity by EHEC is likely not involved. First, two different EHEC O157:NM strains suppressed IFN-γ-induced Stat1 activation, while flagellin did not, indicating a TLR5-independent mechanism. Next, inhibition of host cell protein synthesis by cycloheximide did not reverse the effects of EHEC-mediated Stat1 suppression, suggesting a SOCS-independent mechanism. Also, pharmacological inhibition of proteasome activity by MG-132 did not reverse EHEC-mediated Stat1 suppression. Additionally, because MG-132 can function as an NF-κB inhibitor, NF-κB activation by EHEC O157:H7 (2, 7) is likely not involved.
Since host cell phosphatases negatively regulate Stat1 tyrosine phosphorylation (18, 53), the possibility that EHEC O157:H7 and O113:H21 directly induced host cell phosphatase activity was also tested. Protein tyrosine phosphatases regulate IFN-γ-Stat1 signaling by dephosphorylating activated Stat1 and Jak proteins (3, 41). Stat1 dephosphorylation was originally described at the host cell nucleus (18); however, the precise mechanism and host cell factor were not described.
Subsequently, a ubiquitous Stat1 protein tyrosine phosphatase was identified: T-cell protein tyrosine phosphatase (TcPTP) activity is present in cytoplasmic and nuclear extracts of hepatoma (Hep3B) cells, HeLa cells, and mouse embryonic fibroblasts (41, 53). Inhibition of TcPTP activity by orthovanadate results in Stat1 phosphorylation and Stat1 DNA binding in both cytoplasmic and nuclear extracts of IFN-γ- and interleukin-6-stimulated cells (41).
In the current study, sodium orthovanadate treatment of infected T84 cells did not reverse the inhibitory effects of EHEC O157:H7 infection on Stat1 activation. Therefore, in accordance with the cycloheximide data, upregulation of a host cell protein tyrosine phosphatase likely is not responsible for the inhibitory effects of EHEC O157:H7 on activation of Stat1. Tyrosine phosphorylation of Stat1 did not occur when T84 cells were treated with sodium orthovanadate for 30 min or when the tissue culture cells were simultaneously treated with sodium orthovanadate and EHEC for 6 h. Taken together, these results argue against the involvement of a bacterial protein tyrosine phosphatase. In accordance with the proteinase K, trypsin, and heat inactivation data, the findings provide further evidence for the involvement of a nonpeptide bacterially derived factor in the inhibition of Stat1 tyrosine phosphorylation.
IFN-γ-induced Stat1 signaling also can be regulated upstream of Stat1 proteins; for instance, at the level of Jak proteins, or further upstream by degradation, or down-regulation, of the IFN-γ receptor. Jak1 and Jak2 are cytoplasmically linked to IFN-γ receptor domains 1 and 2, respectively (1). Sodium orthovanadate specifically abolishes the interaction of Jak2 and TcPTP (46) and thereby allows for continued Jak phosphorylation and Stat protein activation (17). In the current study, Jak2 phosphorylation was reduced following EHEC infection relative to uninfected T84 cells. However, Jak2 phosphorylation was unchanged in EHEC-infected cells treated with sodium orthovanadate, suggesting that a host cell-derived protein tyrosine phosphatase may not be responsible for the observed inhibitory effects of EHEC infection on Stat1 activation. Instead, the results provide support for the concept that EHEC disruption of IFN-γ-stimulated Stat1 activation occurs further upstream, at the level of the IFN-γ receptor.
Recent evidence shows that multiple bacteria prevent activation of IFN-γ-induced Stat1 activity, including Mycobacterium avium (20), Mycobacterium tuberculosis (54), L. monocytogenes (50), and EHEC O157:H7 and EHEC O113:H21 (4). Mechanistically, M. avium infection of macrophages leads to reduced mRNA and whole-cell protein expression of IFN-γ receptor domains 1 and 2 and to decreased IFN-γ-induced Stat1 tyrosine phosphorylation (20). Adenovirus also disrupts IFN-γ signal transduction by decreasing IFN-γ receptor domain 1 mRNA and cell surface protein expression (24). On the other hand, M. tuberculosis inhibits IFN-γ-induced Stat1 activation in macrophages without affecting Stat1 tyrosine phosphorylation or DNA-binding ability (29, 54). Rather, M. tuberculosis decreases the association of activated Stat1 with transcriptional coactivator proteins (54) in a process dependent on bacterially induced interleukin-6 release from the macrophage (34). The present study provides evidence that EHEC O157:H7- and EHEC O113:H21-mediated suppression of Stat1 activation by IFN-γ involves the production of a secreted mediator(s) and disrupted IFN-γ receptor domain 1 expression in caveolin-enriched membrane microdomains.
Cholesterol and sphingolipid-enriched plasma membrane microdomains are thought to serve as platforms for signal transduction originating at the host cell surface (40). Protein receptors such as CC chemokine receptor 5 for macrophage inflammatory protein 1β (36), gp130 for interleukin-6 signaling, and the IFN-γ receptor domain 1 for IFN-γ (40) all require localization to microdomains to retain functionality. It has recently become appreciated that microbes exploit membrane microdomains to their advantage (31, 38). For example, the cystic fibrosis transmembrane conductance regulator (CFTR) serves as a cell surface receptor for Pseudomonas aeruginosa on the apical aspect of epithelial cells, and CFTR must be localized to microdomains to allow bacterial internalization and activation of the proinflammatory NF-κB signal transduction cascade (30).
Here, we confirm that IFN-γ receptor domain 1, which is important for binding of IFN-γ (1), localizes to membrane microdomains of intestinal epithelial cells. Furthermore, EHEC O157:H7 and EHEC O113:H21 infection was shown to disrupt this localization to caveolin-enriched fractions. Whether conditioned medium mimics this effect or inhibition of bacterial protein synthesis using chloramphenicol reverses this effect remains to be determined. Nevertheless, these results indicate that bacteria can alter the distribution and function of signaling receptors in addition to exploiting the proteins present in membrane microdomains.
In summary, this study provides novel observations describing the mechanisms underlying EHEC suppression of cytokine signaling that are important for human defense against infection. Developing a better understanding of the pathogenesis of infection will help to guide new approaches to future therapeutic intervention strategies.
We thank Danny Aguilar at the Hospital for Sick Children Graphics Center for assistance preparing the figures.
N.J. was the recipient of a Cell Signals Trainee Award and an Open Fellowship award from the University of Toronto. P.J.M.C. was the recipient of a Canadian Institutes of Health Research (CIHR) Doctoral Student Award and a Cell Signals Trainee Award. D.M.M. is a CIHR scholar. P.M.S. is the recipient of a Canada Research Chair in Gastrointestinal Disease. This work was supported by operating grants from the CIHR.
- Received 14 September 2005.
- Returned for modification 24 October 2005.
- Accepted 12 December 2005.
- Copyright © 2006 American Society for Microbiology


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Instructions in English .
Instruction Manual in GREEK
Written Warranty for 2 Years .
Completely New Model of an Attitude Design Basics but Build Internal Circuit and Capabilities . Easy handling without complex settings is a Perfect Machine for Detecting Coins and Treasures . Equipped with Graphical Display Goal, Excellent Depth Detection , Technology Operation with the push of a button , without complicated setting the ACE-150 is the best detector in its class . Continuous Measurement Depth , Sound Separation Metal , Three Pre - set programs and Separation Of course the Most Advanced Liquid Crystal Display for quick visual identification of targets are few more of the new features of this stunning model .
Handwriting Analyst Identity goal with the method the cursor. The analysis is done in five ( 5) Departments Conductivity .
Depth Currency .
Audio Split Identity Objective . Different Sound Response Depending on the Conductivity of Metal (Low , Medium, High) . Three (3 ) Different audio performance .
Sensitivity Setting - Depth . Four (4 ) Settings.
Indication Low battery level .
External Speaker with very clean performance across the spectrum of tonal Odds ( Bass, Treble )
Excellent New Releases "Pro-Formance" scanning disks replacement .
Headphone .
Large Liquid Crystal Display showing all settings .
Testing of Electronic Circuits Microprocessor .
Handy Keyboard with Soft Push .
Operation with the touch of a button . Without Unnecessary or complex configuration mode starts immediately .
Easy Disassembly into three pieces .
Detection Method for All Metals .
Detection Method for Jewelry.
Detection Method for Currencies .
Sensitivity Setting - Depth .
Select Method Detection through Three ( 5) Pre - set programs Separation .
Changing length of 1.06 m to 1.29 m
Adjustable Arm rest .
Weight 1.2 kg .
Operating Frequency 6,5 kHz.
Function with Four AA-size batteries (Minion).
Manual Centering Target .
Even with the ACE-150 is not fitted with a centering target (pinpoint) is very easy each operator to locate the exact point of a metal target .
As you pass over a metal target the audible signal will sound at the finish of the disc in the movement over the target. In other words, if you pass the disc onto the target from left to right you will hear a beep when the left edge of the tray is above the target. Similarly, when you move your disk from right to left bumped from the target then the alarm will sound when the right edge of the tray is above the metal target .
Once you have finished tracing from right to left and from left to right move by 90 ° relative to the target and repeat the same procedure as mentioned above. You will find that your goal at the intersection of the two axes.
Watch the Video: http://www.youtube.com/watch?v=d5zbojUYGxs

subset: commoncrawl


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All is not good.
MLM companies may sound good but there is usually trouble hounding them at the door. When you look to answer what is MelaLeuca you will find that it was a troubled company from the get go.
Do to certain law violations the company almost folded before it started. But fortunately for the founders, one of their top officers was able to rectify the situation and the world is now able to get MelaLeuca products if they want.
After that very shaky start, the company has grown to be a corporation that has revenues reaching about $2 billion. Not bad for a company participating in the very competitive beauty industry.
Thanks To Affiliate Marketing This Blog Is Paying My Rent, Food, Cars And Vacations
I've learned everything about making money online & affiliate marketing from this online community.
A Brief Glimpse Into Melaleuca The Company
The original company company was formed by Allen and Roger Ball in 1984 and called it Oil of Melaleuca. They had heard about a potent Australian plant called Melaleuca which had the potential to help people feel better and be healthier.
Unfortunately for them, they were not great business people. They had a lot of trouble with the law initially and it took a friend of theirs, current CEO Frank Vandersloot, to turn the company around and make it the success it is today.
Based in Idaho Falls the company has grown under the leadership of Mr. Vandersloot but it does not document a lot of milestones along its way. In its brief history, it has developed over 400 organic and healthy products to make your feel better, Plus, it has grown to be in about 16 markets around the world.
Two keys about the company that should be mentioned is that there are rumors that the top brass do not push its sales people to recruit others. And the other is that the top brass does not encourage their sales associates to buy more products than they can sell.
It is a soft sell spin on a hard sell industry. This approach has helped them to continue to grow as an MLM company while their MLM counterparts are decreasing in revenue.
Melaleuca Products
The main ingredient of Melaleuca products is Melaleuca Alternifolia oil or commonly known as tea tree oil. In some ways the company has been compared to Amway due to the Melaleuca products they sell.
These products range from nutrition, home care, cosmetics, and personal care. There are about 400 in all and that number may be growing. One of the more popular Melaeuca products is their Renew dry skin therapy.
They also have a product that is a morning and evening vitamin pill regime you take regularly. With their environmentally friendly cleaning products it is not hard to see why they are compared to Amway.
One of their products seems to be very effective against eczema. So the overall news about their products seems to be good. That may be because of their strict attitude concerning the ingredients they put in their products.
They have built a 4 point philosophy that has guided their product development over the years. It goes like this:
- It must be safe and not harm your health
- It must be effective and do the job well
- It must be eco-friendly
- It must be affordable
They have about 6 main natural ingredients that they rely on to make their products.
- Citric acid
- Melaleuca oil
- Sodium citrate
- Essential oils
- Glycolic acid
- Thyme oil
They do have a list of 9 ingredients that they say they never use:
- Abrasives
- Parabens
- Phosphates
- Formaldehyde
- Ammonia and more
Signing Up With Melaleuca
It can be a bit difficult to sign up with the company to market their products. The sign up can be confusing as there seems to be no direct sales rep sign up form. There are a lot of regular positions available or at least advertised on their career opportunities web page.
You need to contact the company to find out how you can sign up as a sales rep. If you go through one of their reps you may experience some hard sales tactics to sign you up. One of the issues involved is that the company name may not be mentioned at all.
The focus of the sign up discussion may be on how great the products are and how great the business opportunity is. Also how life changing the new opportunity is. To buy the products, if you do not want to start a business can be difficult if you do not have a membership.
The key to signing up is that you are on the hook to buy up to 35 points of product every month. Another red flag is the back up order that the company sends you whether you need it or order it or not. It arrives and yo have to pay for it.
One former sales rep was losing $80 a month because of this issue. You can't get away from it either as it is a requirement to being a sales rep. Plus, if you want to get out, that may be more difficult than getting in.
It seems that the company will drag its feet on quitting announcements sent to them. As they take their time processing the request, they continue to send you back up product and charge your credit card or bank account.
This information is not found on their website either so you need to do your research before sending the company any of your information.
Is It Possible To Make Money At Melaleuca
This is the same question that is asked for every MLM company. Don't be fooled because Melaleuca is an MLM company and it also uses a confusing and complicated format to determine your compensation.
Selling Melaleuca products is not going to be easy. While it only has about 150,000 representatives the compensation package can be difficult to navigate. Especially when they are charging you for monthly product you can't sell right away.
Like every other MLM company you can make a commission by doing your own direct selling. Also, you have to recruit more people if you want to make any real money.
Of course, they have to sell and continue to order products on a monthly basis if you are going to see any of that money. The company also uses a point system and you and your recruits need to hit 35 points every month.
Another way to make money is to sign up people to be customers. Once you do that you get about 7% of product points for all the customers you signed up or they singed up.
The biggest commission is in the first month. If your customer makes a lot of purchases, then you get about 50% commission. After that, it drops fast. For 1-7 customers you enrolled, you get 7% commission from the second month on. Of course, they have to continue buying the product for you to make some money.
The commission rate goes up if you enroll 8 to19 customers and higher yet if you enroll over 20. Here is the link to the whole compensation scheme. There are other ways to make money, but to do so requires a heavy time commitment on your part.
As usual, there is along list of bonuses you may get if you are lucky. It is all laid out for you at that compensation link. What also works against you is the usual MLM pricing plan. The products are higher priced than you can get locally at your favorite store.
Then Melaleuca is like every other MLM business - it has about a 95%+ failure rate even though their are not as many sales reps as other MLM companies. Just remember that for you to make money, your customers have to buy and buy every month.
The Pros And Cons Of Melaleuca
Like every MLM company there are pros and cons that are a part of the business. Here are some of those pros and cons that come when you sing up with this company:
- Eco friendly products
- Good product selection to sell
- You can shop without being a a sales rep
- Been around for a long time
- Has healthy ingredients in its products
- Set your own hours
- Recruit customers not new sales reps
- Be your own boss
- Products may not be the best
- Products may not work
- Higher price on products
- Backup products sent to you
- Monthly payment on products you never ordered
- Hard to get out of
- Lose a lot of money
- 95% failure rate
- Need serious salesmanship skills
To Be Fair It May Not Be A Scam
The company is an MLM corporation. There is no doubt about it. Its sign up, recruitment and levels of compensation including bonuses can be confusing, complicated and not lucrative for you.
Yet despite those faults, there are many people who like this style of employment. It gives them freedom to be their own boss, set their own hours and it does provide them with some extra income.
To those that fail at selling their products and who do not get fairly compensated this company may seem like a scam to them. And the way it is structured it certainly looks like one. But to be fair, there are sales reps who sign up customers and make money.
The products are healthy and full of natural ingredients. Plus, you do get some support from the person who signed you up. Yet is it all worth it when you have to pay $80 a month to remain active in the business? And pay $80 a month until they cut you loose?
Those are two questions you need to ask yourself. A lot of reviewers are not calling this company a scam but to us it definitely looks like one. Because of the difficulty in getting paid the process to make money is more of a scam than not.
All the good reviews aside, if your reps are not making a decent wage, then you are cheating your reps while earning money hand over foot.
There Is A Better Way To Make Money
There is always a better way to make money, even a regular traditional job is better because you know how much you are making, when that salary comes in and so on. MLM style of employment is risky and a crap shoot when it comes to earning a decent living.
But there is a better way to go about earning some extra cash and you do not have to quit your day job to do it. You can earn money on the side while still working 9 to 5 jobs. That is legitimate money that does not have a confusing or complicated compensation plan.
It is called affiliate marketing. Everything is on the up and up in this growing industry plus, you know exactly when and how much you will be paid. There is no mystery, no secret compensation rules to follow and no hidden costs. Make a sale and you get paid.
The good news is that you know everything about affiliate marketing before you sign up. You can even scan the different affiliate platforms to see the commission rates before you take the plunge. You get to choose your payment heights, not the company.
Ask Your Questions
That is the way to learn all about affiliate marketing. All you need to do is contact us and ask your questions. You will have questions that we can guarantee. We will gladly answer them to the best of our ability. Then the rest is up to you.
Affiliate marketing is one of the fastest growing industries out there because more people are connecting to the Internet every month. What that means is your potential audience is growing even before you sign up.
So don't hesitate. Contact us now and get the answers to your questions. Then see how affiliate marketing is far better than trying to sign up with an MLM company. Nothing ventured means nothing gained.
Thank you for reading.


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Start your Saturday mornings with some free fitness in Hilliard! Mat Happy Yoga and SHED Fitness will be at Hilliard Station Park on Saturday mornings to teach fun yoga and fitness classes for the Motivation at the Station series.
Motivation at the Station Schedule
Motivation at the Station runs each Saturday from July 11 – August 29. The classes are open to all fitness levels and are 50 minutes long. Bring a mat, towel, or a blanket, plus water.
Motivation at the Station with Shed Fitness
Saturdays at 7 a.m. SHED Fitness will offer full-body circuit classes designed for people of all fitness levels at 7 a.m.
Motivation at the Station with Mat Happy Yoga
Saturdays at 8 a.m. Classes will begin at 8 a.m. with a vinyasa flow that includes standing and seated postures, back bends, twists, and inversions.
Participants must register and sign a waiver before attending. Classes are limited to 50 people spaced six feet apart. Participants are encouraged to wear a face mask.
Looking for more free fitness? Check out this Free Fitness in Columbus roundup with options throughout the summer.
Other articles of interest
- Free or cheap events this weekend in Columbus
- Drive in Theatres in Ohio
- Fun and Tasty Adventure Trails in Ohio
- Farmers Markets in Columbus (and the changes for 2020)
- The most beautiful public gardens in Columbus
- Planetariums and Astronomy: Best places to see the stars in Ohio
- Walking labyrinths in Columbus and Central Ohio
- Caverns, animals, and more at Olentangy Indian Caverns
- Easton Drive-in Movies Sunset Series
- Explore the Magical Fairy Trail in Granville
- Details about Splash Pads in Columbus for 2020
- Best places to pick berries in Columbus
- The best Dog Parks in Columbus
- Best photography locations in Columbus
- Nature walks and scenic hiking trails in Columbus
- Free outdoor movies around Columbus this Summer
Hilliard, OH 43026
Hilliard, OH 43026


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At a time when the top Indian NBFCs are shying away from engaging with the credit-starved rural consumers, Indo-Korean FinTech player True Balance is aggressively focussing and expanding its lending business in that market.
True Balance is operated by SoftBank-backed Balancehero India, which is a wholly-owned subsidiary of Balancehero Co. Ltd. Korea. The Gurugram-based FinTech firm, which received an NBFC license from the Indian central bank last year, plans to influx much need liquidity in the rural market even as the COVID-19 triggered pandemic caused a massive global socio-economic crisis. India, Asia's third-largest economy, has been worst hit as the country went for a total lockdown in a bid to contain the spread of the COVID19 virus.
COVID19 was a double whammy for the country that was already facing a liquidity crisis since December 2018 and with the International Monetary Fund (IMF) slashing the economic growth to 1.9% from the previously estimated 5.8%, the situation has become grimmer. In a bid to revive the economy, the Indian government has announced some fiscal stimulus but lenders are being cautious, especially in the rural markets.
However, True Balance thinks this is the right opportunity to engage with the unbanked and uneducated rural consumers through a digital channel. There are over 4000 NBFCs in the country but only a mere 30 of them are lending digitally.
Victor Choi, Chief Executive Officer at Balance Hero India, told IBS Intelligence, " These are unprecedented times. Even as the COVID19 has made the economy suffer, the first line of banks has become very picky in lending and most of the NBFCs have either tightened their credit lines or have stopped disbursements. We think it is an opportunity for us to be nearer to the needy during the time of crisis."
Choi further added that True Balance is focused and committed to serving the rural market as he feels that the market is largely agriculture-driven and has not been impacted much.
The full-stack fintech platform, which claims to have 20 million direct and indirect users, had recently announced that despite COVID19 impact, the digital lender has already disbursed loans worth of INR 230 million in March 2020 with an average ticket size of INR 5,000-7,000 credit amount.
"We are committed to solving the last mile issue of credit in the rural market and all our disbursements are 100% digital. We hope to solve the issue of liquidity in this market, which accounts for 45% of the country's GDP. We aim to strengthen our data and alternative credit scoring strategy to target the next billion Indian users coming out of the rural and small towns of India," Choi said adding that the company is in the process of two lending products –Level-up Loan and Refer a Loan, in the July-September quarter. The 'level-up loan' is being run as a pilot at present.
The company had last year raised $23 million in its Series C financing round from seven Korean investors — NH Investment & Securities, IBK Capital, D3 Jubilee Partners, SB Partners, Shinhan Capital, and existing partners IMM Investment and HB Investment.
True Balance began as a digital payments company offering mobile balance, or top up pre-pay mobile credit in 2014. However, over the years it has evolved into a digital wallet app that helps users pay their mobile and electricity bills, and offer credit to customers so that they can pay later for their digital purchases. The company clocks over 300,000 digital transactions on its app each day. The start-up, which recently introduced e-commerce shopping services on its app to sell products like smartphones, has clocked $100 million in GMV sales so far.


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On December 14th in the City Sogo Gym in Kishiwada fans will see Japanese Youth Featherweight champion Kyohei Tonomoto (9-2, 4) make his first defense, as he takes on Ryotaro Motohashi (9-1, 2) in a very interesting looking match up. The bout isn't one that will get headline treatment, but is an evenly matched contest between two men who are both still in the formative years of their career and both promise a lot going forward.
The champion won the belt back in May, when he defeated previous champion Hikaru Matsuoka in Hyogo, with a majority decision. The win was, by far, the biggest win of Tonomoto's career and saw him show the early potential we had seen in 2014, when he lost in the All Japan Rookie of the Year final to Reiya Abe. Sadly for Tonomoto his career lost a lot of steam in 2015, and despite his talent he didn't fight for over 3 years, before resurfacing in 2018 with a win over a Thai novice. Given his inactivity the win over Matsuoka was a huge surprise, though a very legit win at this level.
Although Tonomoto doesn't do anything amazingly eye catching he does the basics really well. He's a solid technical boxer who moves well, controls distance well and really does love doubling up his accurate jab, controlling the tempo well with it. He's busy, accurate and very relaxed in the ring, though does look like he perhaps lacks in the physical side of things and if someone tries to bully him it does appear that he might struggle to force them to back off.
The 23 year old Motohashi is a really obscure Japanese fighters, even for us, and the Kyoto based Osakan born youngster has managed to remain under the radar after his debut in September 2015. That was despite getting relatively far in the 2017 Rookie of the Year. He was sadly unable to compete in the West Japan final, which ended his tournament just a fight from the All Japan final. Sadly this is only his third bout since his Rookie tournament came to an end, and it's hard to be too confident in him given how little we've managed to see.
Despite all the footage of Japanese boxing being available Motohashi is a bit of an enigma with the only fan cam footage being available of him, and strangely Boxing Raise have seemingly mislabeled a fight that is supposed to feature him but is actually a Koki Tyson fight. From what little footage there is of him, which is admittedly from 2017, he looks rather slow and although more physical than Tonomoto he looks clumsier, and the sort of fighter who, at time, was happier to engage in close combat. The footage of him is so old that it's hard to read much into, but it was certainly fun to see and he did look like he could be in some very fun bouts if matched right.
Making a prediction on such little footage of one of the fighters can be tricky, but from what we have seen this does look like a stylistically straight forward bout for Tonomoto, who should be too sharp, too accurate and too busy. Motohashi will likely have moments when he does get up close, but overall we think those moments might be often enough for the challenger to bully the champion or rack up the rounds.
If Motohashi had scored a stoppage or two in his last 5 bouts we may have been convinced that his gruelling style would break down Tonomoto, but his lack of power suggests that won't be the case.
Prediction - UD8 Tonomoto
The Japanese Youth title scene is a genuinely intriguing one, even if it doesn't feature the huge names that compete in Japan. This coming Sunday we get the chance to see some Youth title bouts, and again the bouts are really interesting, without being huge news.
One of those bouts this weekend will see Japanese Youth Featherweight champion Hikaru Matsuoka (15-4-3, 2) make his first defense, as he takes on Kyohei Tonomoto (8-2, 4). Neither of these two are big names, but both will certainly see this bout as a chance to help make a name for themselves.
The 24 year old champion, a member of the Taisei Gym, win the title last year with a technical decision win over Noboru Osato. That was his third straight win and his 7th win in his last 8. Whilst that sounds impressive, his competition hasn't been the best, and more worryingly he has shown a real lack of durability, with 3 stoppage losses, including 2 stoppage losses in his last 9. Whilst he has shown a shaky chin he also has a lack of power, with only 1 stoppage in his last 7 wins and only 1 stoppage in th elast 6 years. Like some other fighters however his focus isn't on punching through the target or inflicting damage. He knows he 's not a puncher. Instead he looks to box behind a jab, fight at range and control the fight with his jab and movement.
Although no world beater it's clear that Matsuoka is a talented fighter. He's well schooled, a good mover and he fights to his strengths. Looking through his record he has fought plenty of notable opponents, scoring wins over Richard Pumicpic, Yu Konomura and of course Osato. On the other hand he has lost to the likes of Seizo Kono and Yuki Strong Kobayashi. From those losses it's clear if he needs to avoid punchers, and if he can do that he could have a pretty successful career. Luckily for him, Tonomoto is no huge puncher.
Aged 23 Tonomoto is someone who has been really over-looked and hasn't really had much attention at all. That's despite reaching the 2014 Rookie of the Year final, where he lost to the then unheralded Reiya Abe. Part of the reason why Tonomoto hasn't had much attention following his Rookie of the Year run is due to inactivity, and he took more than 3 years out, following a blow out win against Namchoke Meesri. Thankfully for the youngster he was young enough to have that 3 year break and still being a young kid when he returned to the ring last December, when he stopped Nanthipat Kesa inside a round.
Given his long break there isn't a lot of recent footage available of Tonomoto, though his last fight is available on Boxing Raise. That fight lasted just 164 seconds but it was clear Tonomoto was a pretty well schooled fighter, firing off hard and crisp jabs, flowing combinations, nice movement and although there was flaws he looked fun and exciting. He looked defensively questionable, but exciting, aggressive and like someone with the potential to go a very long way. That was however a bout against some one not fit to be in the ring with him, whilst his upcoming bout is a contest against a national youth champion.
Despite the inactivity we're actually backing Tonomoto here. We suspect that both will match each other well in terms of speed, though Matsuoka may have the slight edge, however Tonomoto appears to have the variation in his work, and the more aggressive mentality. Those, we suspect, will be his keys in a very close and competitive bout.
Prediction SD8 Tonomoto, in an bit of an over-looked and hotly contested fight
Having canned the old "Full Schedule" of Asianboxing we have instead decided to concentrate more on the major bouts. This section, the "Preview" section will look at major bouts involving OPBF and national titles. Hopefully leading to a more informative style for, you the reader.
August 2020


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The Adventures of Winston Freer CD by Miracle Factory - Trick
This is a CD containing scanned booklets of Winston Freer materials. As some of the booklets are so old, the prints might not be very clear in certain books. Plenty of materials inside.
Of all the routines/ ideas in this CD I particularly like Freer tile deal. This is a very interesting topological puzzle. A set of geometrical shapes forms a rectangle. When a small square is removed from this set and re-arrange again, yet they still form a rectangle seemingly without losing any area. This is repeated 2 more times, at the end a total of 3 squares are removed and yet the rectangle still seems to be the same shape and size. You'd have to construct this yourself which would be quite easy since the template is already provided.
At the end of the CD, footage of Winston Freer performing a levitation is also shown. The video is in black and white and is not very clear, but you can still see what's going on. Plenty of materials for you to choose from. I'm sure you'll be able to find some stuff that is usable.
Rating 3.5/5
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text: Source: Pool / Getty UPDATED: 2:55 p.m. EST — Bill Cosby has been taken away in handcuffs and was headed straight to prison after he was sentenced on Tuesday. Live footage was played on TV showing what amounted to a perp walk following the sentencing. Bill Cosby was just perp-walked in handcuffs out of the… via … Continue reading Bill Cosby Sentenced To 3 to 10 Years in Prison for Sexual Assault Conviction — Cassius | born unapologetic | News, Style, Culture


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Sony Pictures & Disney have finally struck a new short term deal regarding Spider-Man. This is the crazy, & messy story of how Sony ended up with the rights to Spider-Man in the first place. A story about Bankruptcy, lawsuits,… Read More ›
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Man yells 'I'm Spider-Man' before jumping through second story window.
After being dumped by his girlfriend. The world now knows that a Michigan man is no Peter Parker. Oh, it gets much worse! Before I read the article any further, I thought maybe this kid or guy was trying to look… Read More ›


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For new releases go to
As we transition to our new site, you'll still find some Stats NZ information here on this archive site.
9 May 2018: We revised the Māori farm tables, see Agricultural production statistics: June 2016 (final) on our new website.
See the Excel tables in the 'Downloads' box on this page. If you have problems viewing the files, see opening files and PDFs.
Tables for Federation of Māori Authorities
Access more data on Infoshare and NZ.Stat
Infoshare and NZ.Stat allow you to organise data in the way that best meets your needs. You can view the resulting tables onscreen or download them.
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For this release, select the following categories from the Infoshare homepage:
Subject category: Industry sectors
Group: Agriculture
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For this release, select Agriculture from the themes on the NZ.Stat homepage.
Agricultural Production Statistics: June 2017 (provisional) will be released on 14 December 2017.


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Lisakovsk is a provincial town and has no airport. That is why to get to Moscow, you need to reach a larger city close to Lisakovsk first, from there you'll be able to fly.
Lisakovsk: larger cities nearby
• Kostanay — 100.19 km.
Lisakovsk: airports nearby
Flight schedule Lisakovsk — Moscow
It is impossible to buy direct flights Lisakovsk — Moscow, because the former does not have an airport. However, pretty close the following airports are to be found:
• Kostanay — 101.18 km• UKHTA — 149.64 km.
Airports in Moscow
Find the best accommodation in Moscow on hotels.anywayanyday.com: search for hotels by price, popularity, trip ranking, accommodation type and rendered services.
IATA code — MOW.
City Latitude: 55.752.
City longtitude: 37.616.
Area: 17075400.
Population: 143300000
Population density: 8.39.
Phone code: 7.
Latitude — 52.54707.
Longitude — 62.49987.0
Population — 40000.
Latitude — .
Longitude — .
Population — .
Area: 2717300.
Population: 16197000
Population density: 5.96.


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3 November 2019
3 November 2019
Finally, the day of the Grand Finals are upon us. Before that however, we still have to go through the Grand Finals between ALPHA RED, BREN Esports, Greyhound Gaming and Team Absolute. The local Indonesian casters were Wooswaa and Wawa Mania. The English casters were BlackenBlue and Jaeger! You can catch the stream here.
Match 1: ALPHA Red vs BREN Esports (7:8; 14:16)
Winner: BREN Esports
Map: Inferno
A positive start for ALPHA Red as they get the pistol round, giving them a good head start. BREN Esports seem to be a little overconfident with their movements, and gave up a few careless kills throughout the game. However the first half ended closely, at 7:8. BREN Esports are the favorites for this game, so the first half ending that closely surprised the crowd. BREN Borkem's performance was insane, getting 28/5/20. Even till the end, it was a very close game all the way to 13:13 before BREN lurches forward to match point. Unfortunately, ALPHA Red drop the ball right towards the end at 14:15, failing to push the game to overtime and letting the victory slip out of their hands into BREN Esports' hands.
Match 2: BREN Esports vs ALPHA Red(4:11; 9:16)
Winner: ALPHA Red
Map: Dust
Although BREN ESports (CT) got the pistol round, ALPHA Red came back more fearsome than ever, pushing back hard and aggressive. Economy-wise, ALPHA Red was rolling in dough, being able to keep their arsenals stocked, locked and ready to go. Meanwhile, BREN dropped behind and had to do eco rounds time and time again early game. The first half went downhill as ALPHA Red push their advantage forward, ending at 4:11. However, Dust is a T-sided map so the outcome wasn't sealed yet as the team swap happened.
BREN Esports pick up the (much needed) pistol round in the second half. This gave them the cash injection they needed to start a snowball rolling and they catch up incredibly fast. However, ALPHA Red started to inch towards the match point and managed to steal the win with the 7 round advantage they got from the first half. Dust2 does not seem to be BREN's forte it seems
Match 3: BREN Esports vs ALPHA Red(5:11; 8:16)
Winner: ALPHA Red
Map: Overpass
Again, ALPHA Red took the pistol round of the first half, which gives them a strong position to start in. BREN seems to be tripping up over themselves, losing rounds on their full buys and putting themselves into bad eco and ALPHA Red were chugging forward steadily. The first half ends at 4:11, a full 7 rounds lead. And to make things worse, ALPHA Red take the pistol round in the second half as well. One of the casters mentioned that this isn't quite the dagger in BREN's back, only the needle. But I say Arya Stark (from Game of Thrones) only needed one Needle for all her fights. Ultimately, ALPHA Red really did get the needle that they needed to put in BREN's back, and they'll be the ones proceeding to the Grand Finals.
Meanwhile, while the game between ALPHA Red and BREN Esports were running, Grayhound Gaming had taken out Absolute 2:0. Which means the Grand Finals will be ALPHA Red versus Grayhound Gaming.
Grand Finals
Match 1: ALPHA Red vs Grayhound Gaming (9:6; 16:13)
Winner: ALPHA Red
Map: Inferno
Grayhound Gaming are the favourites for this matchup. Interestingly, ALPHA Red were able to get a very strong start on the first half of Inferno as the CT, going strong into an 8:3 scoreline. But Grayhound Gaming were able to pull the game back in by ending the first half at 9:6. On top of that, they also get the pistol round, giving them the opportunity to bounce back. Grayhound weren't quite able to reach the finish line before ALPHA Red, however, as their economy started to falter through attrition. ALPHA Red took the victory at 16:13, the first game out of this BO5.
Match 2: Grayhound Gaming vs ALPHA Red (12:3; 16:3)
Winner: Grayhound Gaming
Map: Dust
Absolutely dominant performance from Grayhound Gaming here, getting a huge lead at 12:3 by the first half. Just blazed through the first half, only giving 3 rounds away to ALPHA Red. Not much that could be said, as Grayhound's prestige shows through on the most classic map in CS:GO. After the first half, the 3 rounds that ALPHA Red got from the first half were the only ones they got by the end of the match. Grayhound Gaming shows who's the top dog by tying the match score 1:1.
Match 3: Grayhound Gaming vs ALPHA Red (11:4; 16:5)
Winner: Grayhound Gaming
Map: Overpass
The stakes are high, and this is the final match of the whole series! I wondered if I'd see another stomp like in Match 2, or if the underdogs will upset the audience with an unexpected win again. Grayhound Gaming gets the pistol round and the blitzkrieg begins. ALPHA Red get blown out of the water and the first half ends 11:4. There isn't much of a resistance that could be mounted in the second half either as Grayhound Gaming pick up the second pistol round as well. Shattering blows were delivered by Grayhound Gaming and ALPHA Red are wavering slowly. ALPHA Red tried to utilize their utility better and the smokes and mirrors were working. ALPHA Red put up a valiant effort, getting so close to victory but ultimately were struck down by Grayhound Gaming as they ran out of gas at the last few moments. Grayhound Gaming were able to hit match point JUST as ALPHA Red ran out of cash, and that's why they weren't able to push the game to overtime.
OMEN Challenger Series: 12 teams, 11 countries, 3 days and our one champion has finally been revealed. Our OCS Grand Champions are Grayhound Gaming! Congratulations to them, they will be taking home a total of $20,000 USD. But not all is lost for ALPHA Red, as their consolation prize is still a solid $10,000 USD!
At the end of the day, there was a performance my MTV, featuring RAMENGVRL on the stage with her popping energy and her soda-sweet flow as she delivers some hot bars and blew up the exhibition hall. It was a powerful performance and great note to end the day on. Thanks to everyone who followed the events of the OMEN Challenger Series! My name is Trilz and I hope to see you next year, for the next OCS.
Written by: Dexter Lim aka Trilz


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Governor Ayodele Fayose of Ekiti State has always prided himself as being the people's governor but that was not the case in Ado Ekiti the state capital on Thursday.
A number of Ekiti residents protested on the streets to demand that Fayose answer corruption allegations against him.
The protesters, comprising members of the All Progressives Congress in Ekiti, the Conference of Nigerian Political Parties, youths, artisans and traders among others, demanded the prosecution of the governor and they marched from Fajuyi park through Okeyinmi and stopped at Ijigbo area, where they were addressed by leaders of each group.
The CNPP demanded specifically that 17 members of the Ekiti State House of Assembly alleged to have falsified their certificates to submit themselves for prosecution.
Recall that the Economic and Financial Crimes Commission has frozen the governor's accounts with Zenith Bank and some of his associates after allegedly tracing N4.7bn from the Office of the National Security Adviser under Col. Sambo Dasuki (retd.) to them and those of two sons of a former Minister of State for Defence, Musiliu Obanikoro.


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