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It has prior to now been suggested that there is a web page distinctive from CB1R and CB2R, which impacts CB2R-mediated migration in human blood neutrophils and that this site could be GPR55 31. furthermore, an additional contemporary examine confirmed that the extremely metastatic MDA-MB231 breast melanoma mobilephone line expresses GPR55 and migrates efficiently against LPI forty. therefore, we got down to examine the results of LPI on the migration of neutrophils. As determine 1Ai shows, neutrophils readily migrated towards a gradient of LPI (figure 1Ai, ▪). Likewise, the extra stable artificial cannabinoid GPR55 agonist and CB1R antagonist AM251 21 brought on a awareness-dependent migration of neutrophils (figure 1Ai, •). The chemotactic properties of these ligands had been corresponding to that of the CB2R agonist 2-AG (figure 1Ai, ♦). subsequent, we verified no matter if the migration of neutrophils towards either LPI or 2-AG may be blocked by way of pretreating the cells with selective receptor antagonists. Pre-incubation of neutrophils with either the GPR55 antagonist cannabidiol (5 μM) 41 (determine 1Aii, CBD) or the selective CB2R antagonist AM630 (figure 1Aii, 5 μM) for 10 min enormously diminished the migratory houses of neutrophils in opposition t LPI (3 μM) or 2-AG (1 μM), respectively. In contrast, neutrophil migration was no longer impaired when cells were pretreated with the 'wrong' antagonists, i.e., 5 μM of CBD adopted with the aid of 1 μM 2-AG stimulation or 5 μM AM630 adopted by using 3 μM LPI stimulation, respectively (statistics now not proven).figure 1
GPR55 and CB2R agonists result in the directional migration and polarization of neutrophils. (A) Human blood neutrophils were placed to the higher wells of a microBoyden chamber and (i) were allowed emigrate towards expanding concentrations of LPI (▪), AM251 (•) or 2-AG (♦) within the backside wells for 1 h. Migrated cells within the bottom wells were counted via a movement cytometer. (ii) Neutrophils have been pre-incubated with DMSO (0.05%), CBD (5 μM) or AM630 (5 μM) for 10 min at 37 °C and their migration towards LPI (3 μM) or 2-AG (1 μM) turned into assessed as in panel Ai. (iii) Chemotaxis of neutrophils towards DMSO (0.01%, white bar), LPI (three μM, easy gray bar), 2-AG (1 μM, darkish gray bar) or LPI and a pair of-AG combined (3 μM and 1 μM, respectively, black bar) become assessed as in panel Ai. (iv) Chemotaxis of neutrophils became assessed as in panel iii, apart from that AM251 (three μM) was used as an alternative of LPI. (v) Neutrophils have been pre-incubated with DMSO (0.05%), CBD (5 μM) and/or AM630 (5 μM) for 10 min at 37 °C and their migration in opposition t DMSO (0.01%) and LPI (3 μM) + 2-AG (1 μM) changed into assessed as in panel Ai. Representatives of 3-6 independent experiments, performed in quadruplicates, are shown for all subpanels. data are mean±SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) Neutrophils were seeded on fibronectin-coated glass coverslips and handled with a gradient of 0.01% DMSO (handle; i) or ligands for 5 min at 37 °C and stained with methanolic Texas-pink Phalloidin (purple) and DAPI (blue). (ii) LPI treatment (3 μM) caused fuzzy protrusions (arrows), whereas (iii) 2-AG (1 μM) caused an elongation of the neutrophils (arrows). (iv) Extending head (arrow) and tail formation (dashed arrow) point out a polarization of neutrophils in accordance with a mixture of LPI (3 μM) and 2-AG (1 μM). Cells have been analyzed the use of a Zeiss LSM510 META Axioplan confocal microscope (common magnification: one hundred×). Scale bars: 10 μm. consultant photos of three impartial experiments are proven. (C) Neutrophils have been seeded on fibronectin-covered glass coverslips and handled with LPI (three μM) and a couple of-AG (1 μM) for 5 min at 37 °C and stained with Texas-crimson Phalloidin (purple) and DAPI (blue). (i) Cells displayed a transparent directional and polarized structure when migrating against a native source of LPI/2-AG (white dot). (ii) In distinction, no cytoskeleton remodeling passed off after a uniform addition of agonists to the medium. Cells had been analyzed as in panel B (customary magnification: sixty three×). representative photographs of three unbiased experiments are proven. Scale bars: 10 μm.
In lots of the outdated stories, 2-AG has been used by myself 12, 35 or in combination with chemokines to induce neutrophil migration 14, 42. because each 2-AG and LPI are endogenous lipid mediators launched by using stimulated macrophages 7, 29, 30, 43, we investigated the concomitant effect of 2-AG and LPI on the migration of neutrophils. The aggregate of LPI (three μM) and 2-AG (1 μM) confirmed a major impact on neutrophil migration, when in comparison to LPI or 2-AG alone (figure 1Aiii). more prominently, the migration of neutrophils towards AM251 (three μM), when combined with 2-AG (1 μM), changed into synergistically superior in comparison to neutrophils migrating in opposition t AM251 or 2-AG on my own (figure 1Aiv). These outcomes could be significantly diminished by pretreating the cells with the GPR55 antagonist CBD (5 μM), the CB2R antagonist AM630 (5 μM) or a combination of each for 10 min (figure 1Av).
in line with chemokines, neutrophils bear cytoskeletal rearrangement and form change, which culminates in cellular polarization and thereby makes it possible for the cells to migrate successfully 44. We therefore subsequent assessed the cytoskeletal rearrangement of neutrophils in response to a 5-min publicity to a gradient of LPI or 2-AG alone, or a gradient of their mixture thereof. Cytoskeletal rearrangement was assessed with the aid of F-actin phalloidin staining. In neutrophils treated with automobile, actin became found at the periphery of the cells, which had a circular spherical shape (determine 1Bi). LPI (three μM) caused random non-directional protrusions within the neutrophils (figure 1Bii, arrows). per old reports 14, medication of neutrophils with 2-AG (1 μM) resulted in an elongation of cells, besides the fact that children, devoid of displaying the attribute polarity of migrating leukocytes (determine 1Biii, arrows). handiest when neutrophils had been concomitantly incubated with a gradient of LPI (three μM) and 2-AG (1 μM), the ordinary polarity of migrating neutrophils – i.e. extending head (arrow) and retracting tail (dashed arrow) – could be detected (determine 1Biv). This effect become now not because of the larger awareness of the mixed ligands per se, due to the fact that neither LPI nor 2-AG on my own, up to five μM, could evoke the identical cytoskeletal rearrangement (records no longer proven), however become stylish on the gradient of compounds (see figure 1C).
When LPI (3 μM) and 2-AG (1 μM) have been simultaneously utilized to the media the use of the right of a narrow tip (figure 1Ci, white dot), a directional move in opposition t the source of ligands turned into accompanied. The uniform addition of ligands into the subculture medium, youngsters, didn't evoke any shape exchange in the neutrophils (determine 1Cii). Likewise, when proven in a Boyden-migration assay, no migration of neutrophils may be accompanied in the absence of a ligand gradient (Supplementary assistance, figure S1).
Taken together, these statistics demonstrate that LPI, AM251 or 2-AG alone may every evoke neutrophil migration – albeit to a lesser extent than when utilized in combination – and with out inducing the commonplace morphology of migrating neutrophils. really, most effective the mixture of LPI and a couple of-AG evoked a strong migratory response in human blood neutrophils by way of the institution of a rear-front asymmetric morphology.GPR55 is expressed in human blood neutrophils
next, we investigated the expression of the cannabinoid receptors CB1 and CB2 in addition to that of GPR55 in human blood neutrophils at both mRNA and protein levels. It has up to now been stated that the sample of expression stages of CB1 and CB2 receptors in neutrophils is dependent upon the isolation method forty five, forty six, forty seven. here we used an untouched, non-column based equipment at room temperature in the absence of Ca2+ and Mg2+ ions to avoid a stimulation of the cells. GPR55 mRNA replica numbers (figure 2Ai, black bar) had been tremendously better than those of the CB2R (figure 2Ai, white bar). certainly, both CB2R and GPR55 mRNA might even be detected with the aid of RT-PCR (determine 2Aii). The expression of CB1R changed into not detectable in either RT-PCR or actual time PCR (records not proven). additionally, we demonstrated the expression of GPR55 in differentiated neutrophil-like HL60 cells (dHL60), which expressed the differentiation marker CD11b at tiers similar to these of neutrophils (Supplementary counsel, figure S2). true time PCR analysis showed a 5.5-fold raise in GPR55 mRNA levels in dHL60 cells (figure 2Aiii, black bar) compared to undifferentiated HL60 cells (uHL60) (determine 2Aiii, white bar).figure 2
GPR55 is incredibly expressed in human blood neutrophils and neutrophil-like HL60 cells. (A) GPR55 and CB2R mRNA expression in freshly isolated human blood neutrophils became assessed by (i) quantitative real-time PCR and (ii) RT-PCR. PCR products had been analyzed on a three% agarose gel. (iii) Relative expression of GPR55 mRNA in undifferentiated (uHL60) and HL60 cells differentiated with 1.seventy five% DMSO for four days (dHL60) changed into measured by means of precise-time PCR. Representatives of three independent experiments are shown for all subpanels. information are imply±SEM (**P< 0.01). (B) (i) GPR55 and CB2R protein expression in neutrophils, uHL60 and dHL60 cells become assessed by using western blotting the use of rat anti-GPR55 and rabbit anti-CB2R antibodies. Lysates have been probed for β-actin as a loading handle. A representative blot of three impartial experiments is shown. (ii) Western blotting of GPR55 and CB2R in lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells was carried out as in panel Bi. A representative blot of three impartial experiments is shown. (C) GPR55 expression in human blood neutrophils, uHL60 and dHL60 cells changed into demonstrated with the rat anti-GPR55 antibody (1:250) and an Alexa Fluor-594 goat anti-rat secondary antibody (1:250). The dHL60 cells exhibit multilobular nuclei (arrows). Cells have been analyzed the use of a Zeiss LSM510 META Axioplan confocal microscope (long-established magnification: 100×). Scale bars: 10 μm. representative photos of 2-3 unbiased experiments are proven.
We next assessed the protein degrees and cellular expression of both GPR55 and CB2 receptors in neutrophils, in addition to in uHL60 and dHL60 cells. HEK293 mobilephone traces stably expressing the CB2 receptor (HEK-CB2R), the GPR55 receptor (HEK-GPR55) 18 on my own or in aggregate with the CB2 receptor (HEK-CB2R/GPR55) served as controls. the use of antibodies peculiarly focused on the respective receptors, we found that GPR55 and CB2R proteins were expressed in neutrophils, uHL60 and dHL60 cells on the acceptable protein sizes (determine 2Bi, ∼37 kDa for GPR55 and ∼45 kDa for CB2R). The specificity of the antibodies became tested through western blot using the lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells. As determine 2Bii shows, the antibodies reacted with their respective objectives handiest. moreover, we verified the expression of GPR55 in freshly isolated neutrophils and HL60 cells with the aid of immunofluorescence using the rat anti-GPR55 antibody (determine 2C). As accompanied in different simple cells forty eight, GPR55 was predominantly found intracellulary in each neutrophils and HL60 cells. This staining became specific, since handiest HEK-GPR55 cells, but now not untransfected HEK293 cells showed a good immunoreactivity with the rat anti-GPR55 antibody (Supplementary tips, figure S3).Gα13/RhoA and Gαi mediate GPR55 and CB2R cytoskeletal remodeling/chemotactic outcomes, respectively
It has been confirmed that GPR55 mediates its downstream signaling routine by means of Gα13 and the RhoA small GTPase in HEK293 cells 15, 18, 19. furthermore, we lately showed that – amongst all Gα subunits – GPR55 couples entirely to Gα13 in HEK293 cells 49. in order to test which Gα protein subunits are involved within the GPR55 and CB2R mediated signaling effects in neutrophils, we used the toxins C3 and pertussis toxin to inhibit the exercise of Gα13/RhoA and Gαi, respectively. Pre-incubation of neutrophils with C3 toxin (three μg/ml, 2 h) significantly inhibited the LPI-brought on migration of neutrophils, however confirmed no effect on the two-AG-stimulated migration (determine 3Ai and 3Aii). even so, pertussis toxin (three μg/ml, 2 h) avoided the migration of neutrophils against 2-AG, but had no significant effect on LPI-caused migration (figure 3Ai and 3Aii). furthermore, migration of neutrophils against a mixture of LPI and 2-AG turned into enormously inhibited when the cells have been pre-incubated with either C3 or pertussis toxin (figure 3Aiii).figure three
GPR55 and CB2R mediate chemotaxis and cytoskeletal home improvement via coupling to Gα13/RhoA and Gαi proteins. Neutrophils have been pre-incubated with cell-permeable C3 toxin (C3, 3 μg/ml) or pertussis toxin (PTX, three μg/ml) for 2 h at 37 °C in PBG buffer. (A) Cells were allowed to migrate towards (i) LPI (3 μM), (ii) 2-AG (1 μM) or (iii) a combination of LPI (three μM) and a couple of-AG (1 μM) for 1 h. Migrated cells within the bottom wells were counted with the aid of a circulate cytometer. The chemotactic index become calculated as variety of cells migrated in opposition t agonists divided by the variety of cells migrated towards automobile (DMSO 0.01%). Representatives of two unbiased experiments, performed in quadruplicates, are proven. information are imply±SEM (*P< 0.05; **P< 0.01). n.s.: no longer huge. (B) Cells had been seeded on fibronectin-covered glass coverslips and handled with 0.01% DMSO (handle), LPI (3 μM), 2-AG (1 μM) or a mix of LPI (3 μM) and 2-AG (1 μM) for 5 min at 37 °C and stained with Texas-crimson Phalloidin (purple) and DAPI (blue). Cells have been analyzed the usage of an OLYMPUS fluorescence microscope geared up with a Hamamatsu ORCA CCD camera (common magnification: 60×). Scale bars: 10 μm. representative images from 2 independent experiments are proven.
subsequent, we demonstrated no matter if the influence of these inhibitors on neutrophil migration was because of an impairment of cytoskeletal home improvement. C3 toxin (three μg/ml, 2 h) inhibited the formation of protrusions triggered by means of LPI (evaluate figure 3Bvi and 3Bii), but had no impact on the elongated morphology of two-AG-handled neutrophils (examine figure 3Bvii and 3Biii). in addition, inhibition of RhoA avoided the polarization of neutrophils in accordance with a combination of LPI and a couple of-AG (compare determine 3Bviii and 3Biv). Pertussis toxin (3 μg/ml, 2 h) inhibited the elongation of 2-AG-encouraged cells (evaluate figure 3Bxi and 3Biii), but didn't affect the non-directional protrusions stimulated by using LPI (evaluate determine 3Bx and 3Bii). Inhibition of Gαi signaling abrogated the polarized head/tail morphology of neutrophils upon remedy with a mix of LPI and 2-AG (examine determine 3Bxii and 3Biv).
In abstract, the exclusive inhibitory results both (i) of C3 toxin on GPR55-mediated responses or (ii) of pertussis toxin on CB2R-mediated responses, provide proof for the involvement of Gα13/RhoA in GPR55-mediated and Gαi in CB2R-mediated signaling in neutrophils.Rac1 and Cdc42 are worried in the cytoskeletal rearrangement of neutrophils after concomitant activation of GPR55 and CB2R
The migration of leukocytes in opposition t chemotactic agents occurs through a coordinated collection of pursuits, usually together with a cytoskeletal rearrangement that depends on the function of the Rho family unit of small GTPases 50. for example, neutrophil-like dHL60 cells have been reported to elongate according to CB2R agonists (i.e. JWH015 and a pair of-AG), thereby activating Rac1 and Cdc42, whereas repressing RhoA 14. although, under these circumstances, dHL60 cells did not exhibit the commonplace rear/entrance polarity of chemotaxing neutrophils.
We demonstrated whether Rac1 and Cdc42 were differentially activated via either LPI, 2-AG or the mixed utility of both ligands in neutrophils. per old stories of GPR55-mediated Rac1 activation in HEK293 cells 15, LPI (1 μM) caused a speedy – albeit modest – activation of Rac1 in neutrophils (determine 4Ai, lane 2). the two-AG (1 μM) triggered a swift and important raise in activated Rac1 (figure 4Aii, lane 3), whereby a highest of Rac1 activity turned into reached within 60 s (Supplementary information, figure S4). This time body of activation is in step with CB2R-mediated Rac1 activation in dHL60 cells 14. youngsters, the incubation of neutrophils with both LPI and a couple of-AG did not effect in a synergistic activation of Rac1 (figure 4Ai, lane 4).figure four
Cytoskeletal rearrangement of (A) neutrophils and (B) HEK293 cells requires the concomitant activation of GPR55 and CB2R. (A) Neutrophils were inspired with LPI (1 μM), 2-AG (1 μM) and LPI (1 μM) + 2-AG (1 μM) for 1 min at 37 °C. lively GTP-bound Rac1 and Cdc42 GTPases were extracted from the lysates with PAK area-gluthatione agarose beads. GTP-certain and total GTPase levels have been visualized by using western blotting using mouse anti-Rac1 (i) and rabbit anti-Cdc42 (ii) antibodies. The β-actin served as a loading manage. The ratio of GTP-certain vs complete GTPase tiers turned into assessed with ImageJ software (graphs). consultant blots from 3-four impartial experiments are shown. information are suggest±SEM. (*P< 0.05; ***P< 0.001). (B) HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells had been seeded on 1% PDL-coated glass coverslips. Serum-starved cells had been incubated with agonists (1 μM) for 10 min in a serum-free medium. The fixed cells were stained for F-actin through methanolic Texas-crimson Phalloidin (crimson) and with DAPI (blue). Cells were analyzed the usage of an OLYMPUS fluorescence microscope outfitted with a Hamamatsu ORCA CCD digital camera (common magnification: 60×). Scale bars: 20 μm. representative photographs from 3-4 experiments are shown. (C) (i) HEK-CB2R/GPR55 cells were transfected with 200 ng of NFAT-luciferase reporter plasmid and 24 h later cells have been prompted with agonists (1 μM) for three h in a serum-free medium. The luciferase endeavor was visualized the use of a steadylite plus kit (PerkinElmer). Luminescence (relative easy units (RLU)) become measured in a TopCounter (appropriate count number NXT; Packard) for 5 s. data are imply±SEM from three independent experiments carried out in quadruplicate (**P< 0.01) (ii) HEK-CB2R/GPR55 cells had been challenged with ligands (1 μM) and the ensuing picometer shifts of mirrored easy wavelength in opposition t the time (s) have been monitored. Transformation of optical signatures have been made by using the enviornment below the curve (AUC) values between the 1 200 and three 600 s time features. data had been normalized and expressed as p.c of maximum activation brought on via LPI + 2-AG. facts are suggest ±SEM from three impartial experiments carried out in quadruplicate (***P< 0.001).
Activated Cdc42 became reported to be involved within the polarization and directional migration of neutrophils 51. once again, 1 μM LPI caused a modest activation of Cdc42 (determine 4Aii, lane 2), whereas 1 μM 2-AG changed into able to promote GTP-binding to Cdc42 in neutrophils inside 1 min (determine 4Aii, lane 3). apparently, incubation of neutrophils with both agonists led to an extra enhance in Cdc42 pastime in comparison to 2-AG alone (determine 4Aii, lane 4).
In abstract, the mixed application of each the GPR55 agonist LPI and the CB2R agonist 2-AG extra potentiated Cdc42 recreation. This technique may accordingly underlie the polarized morphology (see determine 1Biv and 4A, right of lane 4) and the trafficking of neutrophils in opposition t a gradient of each ligands (see figure 1Aiii).RhoA-dependent F-actin formation and activation of the downstream transcription element NFAT are mediated by means of GPR55 and stronger within the presence of activated CB2R
next, we desired to verify the extent to which every of the respective receptors – i.e. GPR55 or CB2R – is concerned in the formation of RhoA-dependent F-actin. on the grounds that the brief lifestyles-time of purified neutrophils does not allow for manipulations corresponding to siRNA knockdown, we took knowledge of HEK293 cells stably expressing these receptors by myself, or a mixture thereof. We and others have prior to now proven that in HEK293 cells, GPR55 stimulation results in the activation of RhoA, Rac1 and Cdc42 15, 18.
The GPR55 agonist LPI (1 μM, 10 min) precipitated prominent F-actin fibers in HEK-GPR55 (determine 4Biv) however not in HEK-CB2R cells (determine 4Bv). This impact was dependent on the activity of the Gα13/RhoA axis, on the grounds that the transient transfection of HEK-GPR55 cells with dominant negative mutants of Gα13 (Supplementary assistance, figure S5i) and RhoA (Supplementary assistance, figure S5ii) or a ten-min pretreatment with 10 μM ROCK inhibitor Y27632 (Supplementary guidance, determine S5iii) averted actin polymerization in line with 1 μM LPI. enormously, GPR55-mediated F-actin formation became modestly attenuated in the presence of non-activated CB2R in HEK-CB2R/GPR55 cells (determine 4Bvi).
The CB2R agonist 2-AG (1 μM) couldn't induce actin rearrangement in the HEK-GPR55 cells (figure 4Bvii), however resulted in some accumulation of polymerized actin in the periphery of HEK-CB2R cells (determine 4Bviii, arrows). This impact might also be followed in HEK-CB2R/GPR55 cells (figure 4Bix, arrow). medicine of HEK-CB2R/GPR55 cells with each LPI (1 μM) and 2-AG (1 μM) vastly multiplied the formation of filamentous actins (determine 4Bxii) when in comparison with LPI (determine 4Bvi) or 2-AG (figure 4Bix) by myself. Co-administration of LPI and 2-AG did not display a change in actin formation in HEK-GPR55 (determine 4Bx) or HEK-CB2R (determine 4Bxi) cells in comparison to remedy of these cells with any of the agonists on my own (compare determine 4Bx and 4Biv and 4Bxi and 4Bviii, respectively).
we've currently proven that in HEK293 cells, the stimulation of GPR55 triggers multiple signaling pathways, eventually leading to the activation of transcription elements such because the nuclear aspect of activated T cells (NFAT), the nuclear element-κB and the cAMP responsive point binding (CREB) protein 21. in addition, we've suggested that GPR55-mediated NFAT activation is crucially based on the feature of RhoA 18. hence, we subsequent demonstrated whether NFAT-transcription element pastime became differentially regulated in the presence of activated GPR55 and/or CB2 receptors in our HEK-CB2R/GPR55 cell mannequin. Concomitant activation of GPR55 and CB2R with LPI (1 μM) and 2-AG (1 μM) ended in a significant enhancement of NFAT-activation when in comparison to cells encouraged with 1 μM LPI only (figure 4Ci, compare light gray and black bars). the 2-AG (1 μM) didn't set off NFAT exercise in HEK-CB2R/GPR55 cells (determine 4Ci, darkish gray bar). This was expected, when you consider that CB2 receptors typically mediate their signaling activities predominantly via Gαi-pathways, which haven't been suggested to result in NFAT-exercise. in an effort to verify the signaling pursuits in the HEK-CB2R/GPR55 mannequin on a greater 'international' scale, we subjected our cells to a Dynamic Mass Redistribution Assay (DMR, Epic®). we have previously reported the suitability of this equipment for label-free measurement of signaling hobbies of each GPR55 21 and Gαi-coupled 7TM/GPCRs forty nine. actually, corresponding to our findings within the NFAT-assay, concomitant activation of GPR55 and CB2R with LPI (1 μM) and a couple of-AG (1 μM) ended in a enormously higher DMR response in these cells when in comparison to cells encouraged with 1 μM LPI simplest (determine 4Cii, examine light grey and black bars). once again, 2-AG (1 μM) by myself didn't induce any DMR in HEK-CB2R/GPR55 cells (determine 4Cii, darkish gray bar).
In summary, these facts indicate that the LPI-brought about activation of GPR55 is essential for the RhoA-elegant rearrangement of the actin cytoskeleton and downstream signaling events akin to activation of the transcription component NFAT. despite the fact, these consequences are stronger within the presence of a 2-AG-activated CB2 receptor.Activated GPR55 inhibits CB2R- and C5aR-mediated respiratory burst in neutrophils
A dramatic increase in ROS tiers – well-known as the 'respiratory burst' – is a mechanism used by neutrophils to get to the bottom of infection. This system is catalyzed by the NADPH oxidase advanced fifty two and is regulated by using the small GTPase Rac2 38. In neutrophils and HL60 cells, 2-AG and the complement part 5a (C5a) had been suggested to activate Rac2 by the use of their cognate Gαi-coupled receptors, i.e., the CB2R and the C5aR 14, fifty three, 54. right here, we tested no matter if the activation of GPR55 and CB2 receptors had an effect on ROS creation in neutrophils.
GPR55 agonists LPI (300 nM) or AM251 (300 nM) did not induce ROS creation in neutrophils per se (determine 5Ai and 5Aii, easy gray bars). In contrast, 2-AG (10 μM) caused ROS production in neutrophils (figure 5Ai and 5Aii, darkish grey bars), an effect that can be inhibited with 10 μM selective CB2R antagonist AM630 (Supplementary assistance, determine S6). apparently, 2-AG-triggered ROS production became drastically diminished when neutrophils had been concomitantly stimulated with LPI (300 nM) or AM251 (300 nM) (determine 5Ai and 5Aii, black bars). This impact become dose stylish (figure 5Aiii, a hundred nM-300 nM LPI) and will also be followed in neutrophils activated with C5a (determine 5Aiii). similar to neutrophils, we observed that ROS formation inspired with the aid of 2-AG (10 μM) become enormously inhibited by means of co-administration of LPI (figure 5Aiv) in dHL60 cells, despite the fact greater concentrations of LPI (1 μM-10 μM) have been necessary to look an effect. youngsters, like in neutrophils, LPI by myself could not induce ROS production in dHL60 cells (determine 5Aiv).figure 5
GPR55 activation inhibits (A) CB2R-mediated respiratory burst and (B) C5a-precipitated degranulation in neutrophils. (A) ROS construction in neutrophils changed into measured by move cytometry. (i) Cells have been loaded with 1 μM 2′,7′-DCF-DA and then incubated with DMSO (0.1%), LPI (300 nM), 2-AG (10 μM) or a mix of LPI and 2-AG for 20 min at 37 °C. ROS construction turned into measured as a transformation in fluorescence within the FL1 channel. (ii) ROS production in neutrophils was measured as in panel Ai apart from that AM251 (300 nM) changed into used as an alternative of LPI. (iii) Neutrophils were incubated with C5a (5 nM) or 2-AG (10 μM) and treated with buffer (manage) or LPI (100 nM or 300 nM) for 20 min. ROS production turned into assessed as in panel Ai. (iv) Serum-starved dHL60 cells were loaded with 5 μM 2′,7′-DCF-DA for 10 min at 37 °C after which incubated with 2-AG (10 μM) in mixture with assay buffer (manage) or LPI (1 or 10 μM). LPI (10 μM) used in combination with DMSO (0.1%) did not result in adjustments in ROS levels. ROS construction become recorded in a Flex-Station II device (Ex. 485nm, Em. 535 nm) 20 min after ligand addition. Representatives of three-four unbiased experiments, carried out in quadruplicates are proven for all subpanels. records are imply± SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) (i) Neutrophils have been incubated with LPI (300 nM) or assay buffer (manage) for 1 h at 37 °C. MPO liberate changed into caused by increasing concentrations of C5a for 30 min and measured as the exchange in absorbance at 630 nm in a colorimetric assay. (ii) Neutrophils have been incubated with expanding concentrations of LPI for 1 h at 37 °C. MPO free up changed into caused with C5a (300 nM) for 30 min and assessed as in panel Bi. statistics are mean±SEM of three unbiased experiments performed in triplicates (*P< 0.05; **P< 0.01; ***P< 0.001). The MPO free up induced by way of 300 nM C5a become set to 100%.
In summary, these facts exhibit that – as soon as activated – GPR55 inhibited the CB2R-mediated ROS construction in both neutrophils and dHL60 cells.Activated GPR55 inhibits the C5a-triggered degranulation of neutrophils
in an effort to be capable of destroy infectious agents, neutrophils keep a big variety of enzymes in azurophilic granules 3. Upon activation via C5a and/or different inflammatory mediators, these granules release their enzymes – e.g., myeloperoxidase (MPO) – to the milieu three. considering the fact that activated GPR55 may block C5a-mediated ROS creation in neutrophils (determine 5Aiii), we next validated no matter if LPI may likewise regulate C5a-precipitated MPO unlock. in reality, pretreatment of neutrophils with 300 nM LPI for 1 h (determine 5Bi, black bars) greatly inhibited the MPO release triggered via diverse concentrations of C5a (determine 5Bi, white bars). expanding doses of LPI reduced MPO release mediated via 300 nM C5a as much as a highest of seventy five% (determine 5Bii). No MPO liberate changed into accompanied when neutrophils have been incubated with LPI alone (determine 5Bi, black bar at 0 awareness of C5a). accordingly, activated GPR55 can prevent C5a-mediated degranulation of neutrophils.Activated GPR55 inhibits ROS creation and degranulation in neutrophils by way of repression of Rac2 exercise
It has often been said that the small GTPase Rac2 regulates degranulation and the NADPH oxidase endeavor in neutrophils by the use of its translocation to the plasma membrane and incorporation in the NADPH oxidase complicated fifty three, fifty five. moreover, JWH015, an artificial CB2R-specific agonist, became proven to set off Rac2 in dHL60 cells and first neutrophils 14. to peer no matter if the GPR55-mediated inhibition of ROS creation and degranulation is based on Rac2, we investigated the activation and translocation of Rac2 in keeping with each GPR55 and CB2R agonists in neutrophils and dHL60 cells.
remedy of neutrophils with LPI (1 μM) for 1 min reduced the pastime of Rac2 when in comparison to automobile (determine 6A, lanes 1 and 2). In distinction, 2-AG (1 μM) resulted in a major activation of Rac2 (figure 6A, lane 3), which reached the optimum undertaking at 1 min and back to its basal endeavor after 2 min (Supplementary guidance, determine S7). youngsters, concomitant stimulation of neutrophils with 2-AG (1 μM) and LPI (1 μM) resulted in a major inhibition of Rac2 endeavor when compared to cells handled with 2-AG on my own (determine 6A, compare lanes 3 and four). Likewise, in dHL60 cells Rac2 pastime turned into decreased within the presence of 1 μM LPI (determine 6B, lane 2) and superior within the presence of 1 μM 2-AG (figure 6B, lane 3). once more, within the presence of both ligands, the Rac2 pastime become decreased when compared to dHL60 cells handled with 2-AG alone (determine 6B, examine lanes 3 and four).figure 6
GPR55 activation suppresses the CB2R-mediated activation and translocation of Rac2. (A) Neutrophils had been motivated with agonists (1 μM) for 1 min at 37 °C. The energetic GTP-sure Rac2 was extracted from the lysates with PAK domain-gluthatione agarose beads. GTP-certain and total GTPase tiers have been visualized with the aid of western blotting the use of a rabbit anti-Rac2 antibody, β-actin served as a loading control. The ratio of GTP-sure vs total GTPase stages turned into assessed with ImageJ application (graph). records are mean±SEM of three unbiased experiments. (*P< 0.05; ***P< 0.001). (B) Serum-starved dHL60 cells have been influenced with agonists (1 μM) for 1 min at 37 °C. The extraction of active GTP-sure Rac2 become carried out as in panel (A). statistics are suggest±SEM of three independent experiments (**P< 0.01; ***P< 0.001). (C) Neutrophils were seeded on fibronectin-lined glass coverslips and treated with 0.01% DMSO (handle; i) or ligands for five min at 37 °C. fastened cells had been incubated with rabbit anti-Rac2 antibody and stained with Alexa Fluor-488 goat anti-rabbit antibody (green), Texas-purple Phalloidin (purple), and DAPI (blue). manage (i) and LPI (three μM, ii) handled cells show a nuclear/perinuclear localization of Rac2 (arrows). Upon 2-AG stimulation (1 μM, iii), Rac2 distributed evenly within the cytosol (arrow) and in part colocalized with actin at the plasma membrane (yellow, arrowhead). (iv) medication with a mixture of LPI (three μM) and a couple of-AG (1 μM) confirmed a nuclear localization of Rac2 in polarized neutrophils. Cells have been analyzed using an OLYMPUS fluorescence microscope fitted with a Hamamatsu ORCA CCD digital camera (long-established magnification: 60×). Scale bars: 10 μm. consultant photos from 2-three experiments are shown.
The feature of Rac2 in ROS construction and degranulation is based on its translocation from the nuclear and/or perinuclear zones to the plasma membrane. In neutrophils, Rac2 was particularly discovered in or closely around the nucleus (figure 6Ci, arrow, Rac2 in eco-friendly, DAPI/nuclear staining in blue). medication of neutrophils with three μM LPI didn't alter the perinuclear distribution of Rac2 (determine 6Cii, arrow). In distinction, stimulation of neutrophils with 2-AG (1 μM) prompted a redistribution of Rac2 to the cytosol (determine 6Ciii, eco-friendly, arrow) and partly resulted in a colocolization with the peripheral actin (figure 6Ciii, yellow, arrowhead). This effect, however, could be averted by way of concomitant utility of 3 μM LPI with 1 μM 2-AG, leading to a perinuclear distribution of Rac2 in these cells (figure 6Civ, arrow).
In abstract, these statistics imply that GPR55 regulates both ROS and MPO creation in neutrophils via suppressing the activity of the small GTPase Rac2.
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When the Kansas metropolis Chiefs kick off the 2017 NFL typical season at Arrowhead Stadium on Sept. 17 against the Philadelphia Eagles, the stadium will have lots of new foods and drinks offerings to fuel lovers.
each concessionaires, Aramark and Levy eating places, will characteristic new ideas this season. The Chiefs also have rolled out some new additions, together with the new Locker Room club determined backyard the participant’s locker room on the container level.
reduce and upper Concourse Concessions (Aramark)
▪ BBQ Alley at the Chiefs hall of Honor. presented through Spectrum in sections 118-122, it is going to characteristic three Kansas city metro barbecue restaurants: Smoke Brewing Co. featuring its deep fried burnt ends; Plowboys barbeque that includes its American Royal Championship pulled pork sandwich; and The Little Pig BBQ that includes its BBQ Sundae with barbecue beans topped with coleslaw, pulled pork and a pickle wedge.
Plowboys also will greatest its new pork rind nachos (pork rinds with apartment-made cheese sauce, bitter cream, jalapenos and a choice of pork or pork). It also will promote its packaged pork rinds pro with its Yardbird rub.
▪ BRGR Kitchen + Bar. At section 102, it's going to function four burgers: The huge Mock, the quantity three, The BRGR and The S&P. other signature menu gadgets will consist of Pig Wings, Chile Fondue with delicate pretzel sticks; and the no longer-So-commonplace tots with BRGR’s version of Totchos.
▪ Zoom food. Aramark’s newest prototype featuring favorite fan foods akin to burgers, fowl tenders and fries is delivered in new approaches. The concession stand in part 111 will present fans extra manage through self-ordering kiosks and beverage stations, and delivered in color-coded select-up stations.
▪ Chickie’s & Pete’s. headquartered in Philadelphia in 1977, it will offer a constrained menu in part a hundred thirty of Arrowhead, serving its Crabfries (crinkle-cut French fries sprinkled with a secret blend of spices and served with its white cream cheese sauce for dipping), Cheese Steak Sandwiches, and boneless Buffalo wings.
▪ Dickey’s Barbecue Pit. In area 301, it will characteristic such objects as chopped brisket, waffle fries, half-pound of rack ribs and Frito pie.
▪ Craft Corners. An additional 13 bar areas were introduced serving blended drinks, select wines, draft and packaged beers, refillable souvenir soda, and bottled water.
among these 13 areas are six Bar categorical stands (sections one zero one, 136, 309, 332, 345 and 321), 5 Kansas metropolis Cocktail Co. bar portables (sections 108, a hundred and fifteen, 126, 133 & 318) and two bars at the CommunityAmerica credit Union Huddle (area 123).
fanatics also will see new menu objects at accepted concession stands. They consist of The Arrowhead Biscuit, a biscuit sandwich with hash brown patty, Canadian 1st baron beaverbrook, fried egg, 1st baron beaverbrook jam and cheese topped with mini pancakes and 1st Baron Beaverbrook (sections 103, one hundred thirty five); the Loaded Potato epidermis Burger, fried potato skins lined with cheese, Baron Verulam bits and sour cream and topped with a burger, lettuce and tomato slice (sections 109, 127, 303); the Coca-Cola Braised brief Rib Mac and Cheese (Mac Shack, sections 104, 117, 301, 326); the publisher 1st baron verulam Alfredo hen Sandwich (The Coop, sections 112, 126, 303, 326); the Carved Roast pork Sandwich (The Carvery, area 109); and the Carved Turkey Sandwich (The Carvery, part 109).
Scout Investments club degree Concessions (Levy)
Levy restaurants took two stands and repurposed them, changing a regular Grill Stand into the Stadium Stockyard with half-pound burgers from the Kansas city Steak Co. with top rate toppings on a salt and pepper brioche bun, branded with the Arrowhead logo. It additionally serves hen wings in three patterns: a dry ranch rub wing, Thai BBQ or Buffalo, in section 220.
the brand new Kingdom Kantina will characteristic a “south of the border” menu, including a Nacho Grande featuring home made chips and road tacos topped with queso fresco are only the starting. aspects will consist of a spicy shrimp cocktail, and Elote Mexican highway corn with chili butter topped with cotija cheese and chili lime salt. Spicy queso dip and fresh made guacamole can be available at section 238.
Levy has additionally introduced cashless lanes at two places, stand 210 and 215.
Arrowhead Stadium facilities
container box visitors and lovers, who use points in the course of the Chiefs Kingdom Rewards application, can accumulate in the Locker Room club, on the stadium’s lowest stage at the 50-yard line at the back of the Chiefs bench. it's steps away from the players’ locker room entrance with a view of the postgame press convention room, and geared up with a full-provider bar and televisions throughout the room.
different new elements consist of an more suitable sound system, a brand new viewing platform and a one-approach glass wall permitting lovers to monitor postgame press conferences.
The country wide football League and Intel sport group additionally partnered to install “FreeD” video expertise in opt for stadiums, including Arrowhead Stadium. it will create new the right way to watch video and replays via calibrating a series of 38 in-stadium cameras so that pixels captured by every digital camera are lined up in house with every different.
The Tower at Charles Bridge in Prague, Czech Republic.
Prague, Cradle of way of life
everybody who visits the Czech Republic begins with Prague, the cradle of Czech subculture and considered one of Europe's most charming cities. Prague presents a close-intact medieval core of Gothic structure that may transport you returned 500 years – the 14th-century Charles Bridge, connecting two ancient neighbourhoods across the Vltava River, with the citadel ramparts and the spires of St Vitus Cathedral rising above, is one of the basic sights of world commute. however the metropolis isn't virtually heritage; it be a a must have city centre with a prosperous array of cultural offerings, and a newly emerging foodie scene.
Castles & Chateaux
attractive view to the tower and chateau of Cesky Krumlov, within the Czech Republic.
The Czech Republic's vicinity on the coronary heart of the previous Austro-Hungarian Empire has viewed a long history of raiding tribes, conquering armies and effective dynasties. This turbulent past has left a legacy of a whole lot of castles – in all places you seem to be there appears to be a turreted fortress perched above a city, or a summer time palace lazing peacefully amid manicured parkland. The number and variety of castles is effortlessly awe-inspiring – everything from grim Gothic ruins clinging to a dizzy pinnacle of rock, to majestic, baroque mansions full of the superior furniture that Europe's artisans may give.
Folklore & subculture
The Czech Republic may well be a contemporary, ahead-considering nation riding into the future on the again of the ecu and NATO, however it is also a country wealthy in way of life. this is most obvious in South Bohemia and Moravia, the place a nevertheless-thriving folk culture sparks into lifestyles throughout the summer festival season. all the way through this time, communities from Cesky Krumlov to Telc to Mikulov don common garb, pick up their musical contraptions – and wine glasses – and sing and dance themselves foolish, animating historical traditions in one of the top of the line examples of 'residing heritage' in the Czech Republic.
Buskers on Prague's Charles Bridge.
the place Beer is God
The most excellent beer on this planet just obtained stronger. for the reason that the invention of Pilsner Urquell in 1842, the Czechs have been famous for producing some of the world's most efficient brews. however the internationally noted company names – Urquell, Staropramen and Budvar – have been equalled, and even surpassed, via a bunch of regional Czech beers and microbreweries that are catering to a renewed interest in common brewing. by no means before have Czech pubs provided such a wide array of ales – names you'll now should get your head round encompass Kout na Sumave, Svijansky Rytír and Velkopopovicky Kozel.
PRAGUE & THE CZECH REPUBLIC'S good 10
a cafe in Prague's ancient town.
1. Counting Statues on Charles Bridgeadvert remarks
even if you talk over with alone within the early morning mist or shoulder your means throughout the afternoon crowds, crossing Charles Bridge is the quintessential Prague event. inbuilt 1357, its 16 stylish arches withstood wheeled site visitors for 500-ordinary years – thanks, legend claims, to eggs combined into the mortar – unless it was made pedestrian-simplest after World struggle II. via day, the noted baroque statues stare down with stony indifference on a desirable parade of buskers, jazz bands and postcard marketers; at crack of dawn, they regain whatever of the secret and magic their creators sought to seize.
2. Gawking at Prague fort
traditional houses on the leading rectangular of Telc, Czech Republic.
A thousand years of background is cradled within the walls of Prague's hilltop castle, a posh of churches, towers, halls and palaces it is virtually a village in its personal right. here's the cultural and historic heart of the Czech Republic, comprising no longer simplest collections of actual treasures such because the golden reliquaries of St Vitus Treasury and the Bohemian crown jewels, but additionally the websites of exquisite historical pursuits such because the murder of St Wenceslas and the 2d Defenestration of Prague.
3. Prague's historic city square
despite the swarms of tourists, crowded pavement cafes and over-the-correct commercialism, it's inconceivable no longer to enjoy the spectacle of Prague's premier public house: tour leaders thrusting through the crowds gathered to watch the Astronomical Clock; college students handing out flyers for a drama construction; core-aged couples in rain jackets and good shoes, frowning at red-haired, leather-clad punks; and a bored-searching man with a placard promoting a museum of torture instruments. Verily, all of human existence is right here.
A young musician performs on the John Lennon wall in Prague. The wall is filled with John Lennon-impressed graffiti and items of lyrics from Beatles songs.
4. Renaissance Splendour of Cesky Krumlov
This sleepy, southern Bohemian town is arguably the Czech Republic's most effective other world-type, should-see sight apart from Prague. None apart from countrywide Geographic has dubbed this former medieval stronghold one of the "world's most suitable locations", and when you trap a glimpse of the rocky, rambling Renaissance fort with its mesmerising multicoloured tower, you're going to believe the attraction. yes, this definitely is that fairy-story city the tourist brochures promised.
5. Kutna Hora's Bone Church
in the 14th-century Kutna Hora rivalled Prague because the most critical city in Bohemia, growing to be wealthy on the veins of silver ore that laced the rocks under it. today it's a stunning town with a few fascinating and weird historical points of interest. Get an perception into the life of a medieval miner on a tour of a former silver mine, or marvel at the ingenuity of the man who created art out of human is still on the grimly captivating "bone church" of Sedlec.
6. Fairy-story Karlstejn citadel
Karlstejn castle was born of a grand pedigree, beginning existence in 1348 as a hideaway for the crown jewels and treasury of the Holy Roman Emperor, Charles IV. Perched excessive on a crag overlooking the Berounka River, this cluster of turrets, sheer walls and looming towers is as immaculately maintained because it is powerfully evocative. The brightest megastar among the constellation of castles that lie scattered throughout Bohemia, Karlstejn will fulfil even your wildest expectations as to what a principal European fairy-story fortress should appear to be.
7. up to date art at Veletrzní Palac
In 1996 the large, grimly functionalist Veletrzní Palac inbuilt 1928 to condo international alternate gala's, grew to be the new domestic of the countrywide Gallery's museum of twentieth and 21st century artwork. This significant, ocean-liner-like constructing can now lay declare to being certainly one of Prague's surest (and largest) galleries, including works with the aid of Van Gogh, Picasso, Klimt, Mucha and the impressionists, in addition to masterpieces by using Czech expressionist, cubist and surrealist artists, chiefly the remarkable Nineteen Thirties art work of Prague via Austrian artist Oskar Kokoschka.
Telc is a quiet and fairly city, a great place to relax by means of the waterside with a e-book and a tumbler of wine. The ancient city, ringed via medieval fish ponds and unspoilt by way of up to date constructions, is a Unesco World Heritage web page with a sprawling, cobblestoned town square where which you can stroll alongside Gothic arcades and admire based Renaissance facades. within the delicate light of a summer night, when the tour agencies have gone, it be a peaceful, magical vicinity where photographers and artists find countless proposal.
9. Czech Beer
'where beer is brewed, life is decent', in response to an ancient Czech proverb. Which means that life within the Czech Republic have to be very decent indeed, as the nation is awash in breweries each massive and small. Czech beer has been noted for its first-class and flavour since the invention of Pilsner Urquell in 1842, but in fresh years there has been a renaissance of microbreweries and craft beers, and you may now savour every thing from classic lezák (faded lager) to kvasnicove (yeast beer) and kavove pivo (coffee-flavoured beer).
10. Underrated Olomouc
Olomouc, set in a extensive, fertile stretch of the Morava River basin, is one of the Czech Republic's most underrated cities. Legend says it changed into situated by means of Julius Caesar. nowadays it's a youthful, laid-returned college city, friendly and cheap, with cobbled streets and the greatest trove of historical structure outdoor Prague – and infrequently a tourist in sight. regardless of a somewhat bedraggled, sooty outskirts, its historic centre is certainly northern Moravia's most attractive city. remember to try the cheese, Olomoucky syr, reputedly the smelliest in the country.
When to move
- timber start to bud the first week of April and by way of may also, cities and cities are in full bloom
- The Prague Spring competition begins in mid-can also, so purchase tickets and booklet resort rooms well in improve
- Easter weekend can carry glorious sunshine however also troves of travellers.
- Prague comes lower back to lifestyles after the summer spoil and the concert season resumes in earnest.
- still numerous mild and sun to get in a full day of sightseeing.
- The climate is mainly amazing for running in West Bohemia's spa towns.
- The break season kicks off on the fifth of the month, the eve of St Nicholas Day (Sv Mikulas), when kids costume up as angel, devil or St Nick himself.
- enjoy a cup of svarak (mulled wine) at Christmas markets on the town squares throughout the country.
every day finances
forex: Czech crown (CZK kč)
Midrange: €70-180 (NZ$117-&301)
- Double room: €one hundred-140
- Three-path dinner in informal restaurant: €25
- live performance ticket: €10-30
more counsel Czech Tourism: (www.czechtourism.com) authentic vacationer guidance for the Czech Republic
Reproduced with permission from the 12th edition of Lonely Planet's Prague & the Czech Republic guidebook, researched and written by using Mark Baker and Neil Wilson, 2017. published this month, www.lonelyplanet.com, RRP: NZD$34.ninety nine
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AICPA [1 Certification Exam(s) ]
AIIM [1 Certification Exam(s) ]
Alcatel-Lucent [13 Certification Exam(s) ]
Alfresco [1 Certification Exam(s) ]
Altiris [3 Certification Exam(s) ]
American-College [2 Certification Exam(s) ]
Android [4 Certification Exam(s) ]
APC [2 Certification Exam(s) ]
APICS [1 Certification Exam(s) ]
Apple [69 Certification Exam(s) ]
Arizona-Education [1 Certification Exam(s) ]
ARM [1 Certification Exam(s) ]
Aruba [6 Certification Exam(s) ]
ASIS [2 Certification Exam(s) ]
ASQ [3 Certification Exam(s) ]
ASTQB [6 Certification Exam(s) ]
Autodesk [2 Certification Exam(s) ]
Avaya [85 Certification Exam(s) ]
Axis [1 Certification Exam(s) ]
Banking [1 Certification Exam(s) ]
BEA [5 Certification Exam(s) ]
BICSI [2 Certification Exam(s) ]
BlackBerry [17 Certification Exam(s) ]
BlueCoat [2 Certification Exam(s) ]
Business-Objects [11 Certification Exam(s) ]
Business-Tests [4 Certification Exam(s) ]
CA-Technologies [20 Certification Exam(s) ]
Certification-Board [9 Certification Exam(s) ]
Certiport [3 Certification Exam(s) ]
CheckPoint [31 Certification Exam(s) ]
CIPS [4 Certification Exam(s) ]
Cisco [270 Certification Exam(s) ]
Citrix [35 Certification Exam(s) ]
CIW [17 Certification Exam(s) ]
Cloudera [10 Certification Exam(s) ]
Cognos [19 Certification Exam(s) ]
College-Board [2 Certification Exam(s) ]
CompTIA [33 Certification Exam(s) ]
ComputerAssociates [6 Certification Exam(s) ]
CPP-Institute [1 Certification Exam(s) ]
CWNP [12 Certification Exam(s) ]
Dassault [2 Certification Exam(s) ]
DELL [7 Certification Exam(s) ]
DMI [1 Certification Exam(s) ]
ECCouncil [18 Certification Exam(s) ]
ECDL [1 Certification Exam(s) ]
EMC [122 Certification Exam(s) ]
Enterasys [13 Certification Exam(s) ]
Ericsson [5 Certification Exam(s) ]
Esri [2 Certification Exam(s) ]
ExamExpress [15 Certification Exam(s) ]
Exin [39 Certification Exam(s) ]
ExtremeNetworks [3 Certification Exam(s) ]
F5-Networks [19 Certification Exam(s) ]
Filemaker [9 Certification Exam(s) ]
Financial [35 Certification Exam(s) ]
Fortinet [10 Certification Exam(s) ]
Foundry [6 Certification Exam(s) ]
Fujitsu [2 Certification Exam(s) ]
GAQM [7 Certification Exam(s) ]
Genesys [4 Certification Exam(s) ]
Google [4 Certification Exam(s) ]
GuidanceSoftware [2 Certification Exam(s) ]
H3C [1 Certification Exam(s) ]
HDI [9 Certification Exam(s) ]
Healthcare [3 Certification Exam(s) ]
HIPAA [2 Certification Exam(s) ]
Hitachi [27 Certification Exam(s) ]
Hortonworks [1 Certification Exam(s) ]
Hospitality [2 Certification Exam(s) ]
HP [712 Certification Exam(s) ]
HR [1 Certification Exam(s) ]
HRCI [1 Certification Exam(s) ]
Huawei [20 Certification Exam(s) ]
Hyperion [10 Certification Exam(s) ]
IBM [1491 Certification Exam(s) ]
IBQH [1 Certification Exam(s) ]
ICDL [6 Certification Exam(s) ]
IEEE [1 Certification Exam(s) ]
IELTS [1 Certification Exam(s) ]
IFPUG [1 Certification Exam(s) ]
IIBA [2 Certification Exam(s) ]
IISFA [1 Certification Exam(s) ]
Informatica [2 Certification Exam(s) ]
Intel [2 Certification Exam(s) ]
IQN [1 Certification Exam(s) ]
IRS [1 Certification Exam(s) ]
ISACA [4 Certification Exam(s) ]
ISC2 [6 Certification Exam(s) ]
ISEB [24 Certification Exam(s) ]
Isilon [4 Certification Exam(s) ]
ISM [6 Certification Exam(s) ]
iSQI [7 Certification Exam(s) ]
Juniper [54 Certification Exam(s) ]
Legato [5 Certification Exam(s) ]
Liferay [1 Certification Exam(s) ]
Lotus [66 Certification Exam(s) ]
LPI [21 Certification Exam(s) ]
LSI [3 Certification Exam(s) ]
Magento [3 Certification Exam(s) ]
Maintenance [2 Certification Exam(s) ]
McAfee [8 Certification Exam(s) ]
McData [3 Certification Exam(s) ]
Medical [25 Certification Exam(s) ]
Microsoft [228 Certification Exam(s) ]
Mile2 [2 Certification Exam(s) ]
Military [1 Certification Exam(s) ]
Motorola [7 Certification Exam(s) ]
mySQL [4 Certification Exam(s) ]
Network-General [12 Certification Exam(s) ]
NetworkAppliance [35 Certification Exam(s) ]
NI [1 Certification Exam(s) ]
Nokia [2 Certification Exam(s) ]
Nortel [130 Certification Exam(s) ]
Novell [37 Certification Exam(s) ]
OMG [9 Certification Exam(s) ]
Oracle [232 Certification Exam(s) ]
P&C [1 Certification Exam(s) ]
Palo-Alto [3 Certification Exam(s) ]
PARCC [1 Certification Exam(s) ]
PayPal [1 Certification Exam(s) ]
Pegasystems [10 Certification Exam(s) ]
PEOPLECERT [4 Certification Exam(s) ]
PMI [15 Certification Exam(s) ]
Polycom [2 Certification Exam(s) ]
PostgreSQL-CE [1 Certification Exam(s) ]
Prince2 [6 Certification Exam(s) ]
PRMIA [1 Certification Exam(s) ]
PTCB [2 Certification Exam(s) ]
QAI [1 Certification Exam(s) ]
QlikView [1 Certification Exam(s) ]
Quality-Assurance [7 Certification Exam(s) ]
RACC [1 Certification Exam(s) ]
Real-Estate [1 Certification Exam(s) ]
RedHat [8 Certification Exam(s) ]
RES [5 Certification Exam(s) ]
Riverbed [8 Certification Exam(s) ]
RSA [13 Certification Exam(s) ]
Sair [8 Certification Exam(s) ]
Salesforce [3 Certification Exam(s) ]
SANS [1 Certification Exam(s) ]
SAP [78 Certification Exam(s) ]
SASInstitute [15 Certification Exam(s) ]
SAT [1 Certification Exam(s) ]
SCO [9 Certification Exam(s) ]
SCP [6 Certification Exam(s) ]
SDI [3 Certification Exam(s) ]
See-Beyond [1 Certification Exam(s) ]
Siemens [1 Certification Exam(s) ]
Snia [6 Certification Exam(s) ]
SOA [15 Certification Exam(s) ]
Social-Work-Board [1 Certification Exam(s) ]
SUN [63 Certification Exam(s) ]
SUSE [1 Certification Exam(s) ]
Sybase [17 Certification Exam(s) ]
Symantec [132 Certification Exam(s) ]
Teacher-Certification [3 Certification Exam(s) ]
The-Open-Group [8 Certification Exam(s) ]
TIA [3 Certification Exam(s) ]
Tibco [18 Certification Exam(s) ]
Trend [1 Certification Exam(s) ]
TruSecure [1 Certification Exam(s) ]
USMLE [1 Certification Exam(s) ]
VCE [5 Certification Exam(s) ]
Veeam [2 Certification Exam(s) ]
Veritas [25 Certification Exam(s) ]
Vmware [51 Certification Exam(s) ]
Wonderlic [1 Certification Exam(s) ]
XML-Master [3 Certification Exam(s) ]
Zend [5 Certification Exam(s) ]
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