exos
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viability when exos activity is absent. thus, we tested the hypothesis that exos - mediated intracellular survival involves evasion of acidified intracellular compartments, and that without exos, internalized bacteria are trafficked to acidified vacuolar compartments wherein they lose viability .
on december 18, 2014 exos acquired medifit corporate services inc. [ 9 ]
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]. mutants in the t3ss that cannot express exos, e. g .
exos in eukaryotic cells. mol microbiol 37: 287 - 299. doi :
exos, the leader in proactive health and performance solutions, today announced a partnership with odell beckham jr. — the nfl’s 2014 rookie of the year — as a brand ambassador for exos’ line of high - quality nutritional supplements, exos performance nutrition .
exos and exot. rev physiol biochem pharmacol 152: 79 - 92. pubmed :
exos with endosome vesicles. j biol chem 282: 13022 - 13032. doi :
exos, formerly named athletes' performance, is an american limited liability company founded in 1999. in 2014 exos acquired medifit corporate services, which staffs corporate wellness centers nationwide. the brand names athletes' performance and the subsidiary core performance were integrated into the brand name exos as well .
requires the adp - ribosylation domain of exos. microbes infect, 14: 1366–73. pubmed :
exos earns a place on the list for its 113 - percent growth and strong job creation .
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when not expressing the adpr activity of exos, one of its t3ss effectors. evasion of this fate by exos adpr activity correlates with suppression of acidification, a feat that it can accomplish even when bacteria are trapped inside vacuoles, and are without the translocon normally used to transport t3ss effectors across host membranes. how exos accomplishes this when known targets of exos are located within the cell cytoplasm is to be determined .
exos is honored to be named to fast company' s list of the world' s top 10 most innovative companies of 2015 in fitness. exos made the list “for improving the entire health and fitness industry. ” this is the third time in five years that exos has been named to fast company’s list .
exou, exos, and exot to virulence in the lung. infect immun 72: 6969 - 6977 .
exos rhogap and gfp - rhogdi cooperatively stimulated cell rounding in cho cells— to investigate the role of rhogdi in exos rhogap - stimulated reorganization of the actin cytoskeleton, the combined effect of gfp - rhogdi and exos rhogap on actin cytoskeleton was tested. both exos rhogap and gfp - rhogdi stimulated a dose - dependent cell rounding (fig. 2 a). at high concentrations of dna transfection (> 50 ng / ml), exos rhogap elicited ∼80% cell rounding, whereas gfp - rhogdi elicited ∼50% cell rounding. at lower concentrations of dna transfection (5–20 ng / ml), neither gfp - rhogdi nor exos rhogap effectively elicited cell rounding, but upon co - transfection of gfp - rhogdi and exos rhogap, an additive effect on cell rounding was observed (fig. 2 a). controls showed that exos (1–234) r146k, a mutated form of the exos rhogap domain that does not possess rhogap activity, failed to stimulate cell rounding alone and did not enhance the rounding potential of gfp - rhogdi (fig. 2 b). this indicated that the rhogap activity of exos is required for the cooperative stimulation of actin reorganization by exos rhogap and rhogdi. exos (e381d), the full - length exos with a defective adp - ribosyltransferase activity, elicited a similar additive effect on reorganization of the actin cytoskeleton with gfp - rhogdi (fig. 2 c) .
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if you’re new to exos, this is the place to start. this series of articles and sample workouts will bring you up to speed on our innovative approach. begin experiencing the health and performance benefits of the exos system in less than five minutes .
in 2017, two more lawsuits were filed against exos citing civil rights and employment discrimination, which are still pending. [ 11 ]
out in july, the label' s 20th release also includes music by ptu, exos and pilldriver (aka marc acardipane) .
mutants (exos secretion capable) suggests they represent different compartments, consistent with the fact that only the latter can replicate inside vacuoles .
exos is required for membrane bleb niche formation and bacterial survival within epithelial cells. infect immun 78: 4500 - 4510. doi :
thousands of elite athletes, fortune 500 companies, and everyday folks use the exos system to improve health, fitness, and performance .
87 of the 335 players invited to this year’s nfl combine prepared with exos at one of its 6 facilities spread across the united states .
the cooperativity between p50rhogap and gfp - rhogdi in reorganization of the actin cytoskeleton suggests the presence of a similar mechanism of rho gtpase inactivation by eukaryotic rhogap and rhogdi. apparently, exos rhogap functions as a molecular mimic of eukaryotic rhogaps to modulate host actin cytoskeleton, and this modulation is through coordinating with eukaryotic rhogdi. although exos rhogap mimics eukaryotic rhogap, direct comparison between exos rhogap and p50rhogap indicated that exos rhogap is ∼3 - fold more potent than p50rhogap in eliciting actin reorganization (data not shown). exos rhogap also appears more resistant to the actions of dominant active rho gtpases (data not shown). this could be due to the different subcellular localization between exos rhogap and p50rhogap. exos localized to membranes through an n - terminal membrane localization domain (mld) (41), whereas p50rhogap is primarily localized in cytosol. therefore, exos rhogap can constitutively inactivate rho gtpases in membrane, whereas p50rhogap may only act on rho gtpases on membrane through temporal membrane localization (42). the differences between exos and p50rhogap in reorganization of actin cytoskeleton and in resistance to the actions of dominant active rho gtpases could also be due to their different specificity toward rho gtpases. both in vitro and in vivo studies showed that exos rhogap targeted rhoa, rac, and cdc42 equally well (5, 6), whereas p50rhogap prefers to target cdc42 relative to rho and rac (43 – 45). the crystal structures of p50rhogap (46, 47) and exos rhogap (48, 49) showed that although the two proteins shared the common catalytic arginine finger, there is little similarity between the two structures, indicating a convergent evolution between the eukaryotic rhogaps to exos rhogap .
exos adpr activity acts upon multiple cellular targets [ 40 ]. for example, inhibition of endocytic vesicle trafficking and lysosomal degradation of the epidermal growth factor receptor results from exos adp - ribosylation of rab5 and rab9 [ 39 ]. thus, exos inhibition of phagosome maturation through adp - ribosylation of rab5, and perhaps exos adpr effects on other rab gtpases (e. g. rab6 or rab9) with which it is known to interact [ 41 ], could explain our results. those effects of exos could help internalized bacteria remain in immature phagosomes rather than trafficking to inhibitory lamp3 +, acidified (mature) phagolysosomes. arguing against rabs being the critical target for this activity of exos adpr activity, however, is that popb mutants inhibit vacuolar acidification and replicate intracellularly. these mutants remain in vacuoles and should be unable to translocate exos across host (vacuolar or plasma) membranes to access rabs that are located in the cytoplasm outside of vacuoles. none of the other known targets of exos adpr activity including ras [ 42 ] and the erm proteins (ezrin, radixin, moesin) [ 43 ], however, are known to be located inside vacuoles. however, it remains possible that exos can escape vacuoles in a translocon - mutant background through an alternative mechanism to exert its effects on vacuole acidification .
some individual bacterial cells among populations of mutants lacking exos adpr activity were found in lt - (non - acidified) vacuoles. while it is possible that
“the core pillars are mindset, nutrition, movement, and recovery, ” said mark verstegen, founder of athletes' performance. “exos defines our brand in a way that is about the xs and os of the winning gameplans that we build for people everyday. that’s really the foundation of what exos is to us. ”
over the last few years, exos has trained roughly half of the nfl first round draft picks, according to verstegen. during the same time period, the training and performance company has placed anywhere between 75 to 100 players into the league each season. additionally, exos has worked with the last 3 mls cup winners .
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pseudomonas aeruginosa uses a type iii secretion system to promote development of severe disease, particularly in patients with impaired immune defenses. while the biochemical and enzymatic functions of exou, exos, and exot, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood. since exou and exos are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection. in the work described here, several isogenic mutants of a bacterial strain that naturally secretes exou, exos, and exot were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia. measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of exou had the greatest impact on virulence while secretion of exos had an intermediate effect and exot had a minor effect. it is of note that these results conclusively show for the first time that exos is a virulence factor. infection with isogenic mutants secreting wild - type exos, exos defective in gtpase - activating protein (gap) activity, or exos defective in adp - ribosyltransferase activity demonstrated that the virulence of exos was largely dependent on its adp - ribosyltransferase activity. the gap activity of this protein had only a minor effect in vivo. the relative virulence associated with each of these type iii effector proteins may have important prognostic implications for patients infected with p. aeruginosa .
in a new piece released this week, forbes discussed how several new products from exos’ new supplement line, exos performance nutrition, are now nsf certified for sport. the nsf certified for sport products include aminos (lemon and berry), whey protein isolate (chocolate and vanilla), multi - vitamin elite, and catalyte isotonic .
exos, the leader in proactive health and performance solutions, today announced, together with thorne research, the launch of exos performance nutrition, a new line of high - quality nutritional supplements to support the needs of consumers ranging from everyday adults to elite athletes. exos and thorne research, a leader in developing and manufacturing pure, high - quality nutritional supplements, have been working together to ensure the efficacy and safety of their unprecedented line of nutritional supplements to help people achieve their health and performance goals .
it has been shown that exos deficiency leads to the secretion of cleaved il - 1β in both alveolar macrophages and in the lungs of mice infected with p. aeruginosa (galle et al. , 2008). exos is a bifunctional protein containing an amino - terminal rho gtpase activating protein (gap) domain, which modifies host cell targets that control the cytoskeleton, such as cdc42, rho and rac1; and a carboxy - terminal adp - ribosyltransferase domain (adprt) with ribosylation activity causing cytoskeleton rearrangements. of note, the adprt domain, but not the gap domain of exos, is essential to inhibition of il - 1β mediated by the exoenzyme. the importance of ribosylation activity of exos in this process allows speculating that modulation of host cell cytoskeleton dynamics is a possible mechanism through which exos inhibits the inflammasome. for instance, it has been recently reported that organelle transportation activity of microtubules is critical for activation of the inflammasome mediated by nlrp3 (misawa et al. , 2013). finally, exos induces caspase - 3 dependent apoptotic cell death in response to infection but pro - inflammatory death of macrophages is also increased in the infection of macrophages with mutants lacking exos. whether this effect is due to a putative exos - mediated inhibition of caspase - 1 - dependent pyroptosis or caused by cytotoxic effects independent of inflammasome activation has yet to be addressed .
this dominant - negative mutant of rhogdi was used to test the role of rhogdi in the actin reorganization by exos rhogap. gfp - rhogdi (i177d) inhibited the membrane - cytosol translocation of gfp - cdc42 stimulated by exos rhogap alone and also inhibited the cooperative effect of gfp - rhogdi and exos rhogap on translocation of gfp - cdc42 from membrane to cytosol (fig. 4 c). this indicates that gfp - rhogdi (i177d) serves as a dominant - negative mutant for both endogenous rhogdi and gfp - rhogdi to inhibit the membrane - cytosol translocation of rho gtpases. together, the cooperativity between exos rhogap and rhogdi on the actin reorganization suggests that exos rhogap and rhogdi function in series to interact and inactivate rho gtpases, and extraction of gdp bound rho gtpases on the cell membrane by rhogdi was the rate - limiting step of rho gtpase inactivation .
exos is a bifunctional type iii cytotoxin of pseudomonas aeruginosa with n - terminal rho gtpase - activating protein (rhogap) and c - terminal adp - ribosyltransferase domains. although the exos rhogap inactivates cdc42, rac, and rhoa in vivo, the relationship between exos rhogap and the eukaryotic regulators of rho gtpases is not clear. the present study investigated the roles of rho gtpase guanine nucleotide disassociation inhibitor (rhogdi) in the reorganization of actin cytoskeleton mediated by exos rhogap. a green fluorescent protein - rhogdi fusion protein was engineered and found to elicit actin reorganization through the inactivation of rho gtpases. green fluorescent protein - rhogdi and exos rhogap cooperatively stimulated actin reorganization and translocation of cdc42 from membrane to cytosol, and a rhogdi mutant, rhogdi (i177d), that is defective in extracting rho gtpases off the membrane inhibited the actions of rhogdi and exos rhogap on the translocation of cdc42 from membrane to cytosol. a human rhogdi small interfering rna was transfected into hela cells to knock down 90% of the endogenous rhogdi expression. hela cells with knockdown rhogdi were resistant to the reorganization of the actin cytoskeleton elicited by type iii - delivered exos rhogap. this indicates that exos rhogap and rhogdi function in series to inactivate rho gtpases, in which rhogdi extracting gdp - bound rho gtpases off the membrane and sequestering them in cytosol is the rate - limiting step in rho gtpase inactivation. a eukaryotic gtpase - activating protein, p50rhogap, showed a similar cooperativity with rhogdi on actin reorganization, suggesting that exos rhogap functions as a molecular mimic of eukaryotic rhogaps to inactivate rho gtpases through rhogdi .
( effector translocation) t3ss mutants were only found in vacuoles. the acidification state of occupied vacuoles suggested a relationship with exos expression, i. e. vacuoles occupied by the
mutants, showing its viability is suppressed by vacuolar acidification. taken together, the data show that the mechanism by which exos adpr activity allows intracellular replication by pa involves suppression of vacuolar acidification. they also show that variability in exos expression by wild - type pa inside cells can differentially influence the fate of individual intracellular bacteria, even within the same cell .
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small interfering rna provides a powerful and convenient tool to investigate the roles of rhogdi in reorganization of the actin cytoskeleton mediated by exos rhogap. hela cells with knockdown expression of rhogdi were resistant to the actin reorganization elicited by type iii - delivered exos rhogap. this indicates that rhogdi is required for exos rhogap - mediated actin reorganization. therefore, rhogdi appears to be a major regulator of rho gtpases activity and actin cytoskeleton. rhogdi extracting rho gtpases off the membrane and sequestering them in cytosol may be the rate - limiting step in rho gtpases regulation .
april 20, 2017 exos extended their partnership with adidas, despite their worker rights violations in the garment industry across indonesia, cambodia, and el salvador. [ 12 ] [ 13 ]
with their speed and strength specialists, position coaches, dieticians, therapists, and array of other service offerings, it is no surprise that so many sports agents direct their clients to exos .
exos was founded as athletes' performance, inc. in tempe, arizona in 1999 by mark verstegen. [ 5 ] the same year, the company formed a partnership with adidas. [ 6 ]
rhogdi (i177d) is a dominant - negative mutant that inhibits rhogdi and exos rhogap - stimulated membrane - cytosol translocation of gfp - cdc42— interestingly, studies performed by other groups showed that rhogdi inhibited both intrinsic and rhogap - stimulated gtp hydrolysis of rho gtpases in vitro (16, 32). these in vitro data were further supported by structural studies showing that the n - terminal regulatory arm of rhogdi binds to switch i and switch ii regions of rho gtpases and interferes with rhogap binding to the same regions (18). in the present study, exos rhogap and rhogdi cooperatively inactivated rho gtpases in vivo, which indicates that rhogdi does not inhibit binding of exos rhogap to rho gtpases in vivo; instead, rhogdi and exos rhogap function in series to interact and inactivate rho gtpases. that is, exos rhogap first converted gtp - rho to gdp - rho on the cell membrane and rhogdi subsequently extracts gdp - rho from the cell membrane to the cytosol, inhibiting the reactivation of rho gtpases by gefs on membrane .
away from acidified compartments within epithelial cells that have internalized them, and that this enables intracellular replication. thus, exos mutants lacking adpr activity traffic more often to acidified compartments, where they fail to thrive .
to further investigate the roles of rhogdi in the exos rhogap - mediated actin cytoskeleton reorganization, sirna technology was applied to knock down the expression of endogenous rhogdi in hela cells. using a control sirna, which has a fluorescent conjugate, the sirna transfection efficiency reached ∼ 90% . transfection of rhogdi - specific sirna into hela cells, at 68–72 h post - transfection, reduced expression of rhogdi to 10–20% of endogenous levels (fig. 5 a). the knockdown in expression of rhogdi was confirmed by immunofluorescent staining with α - gdi antibody (fig. 5 b). compared with wild type hela cells, the cells with knockdown rhogdi appeared flatter and produced extended actin cytoskeleton structures (fig. 5 b). although type iii - delivered exos rhogap, exos (1–234) - ha, efficiently elicited cell rounding and actin reorganization in wild type hela cells, the hela cells with knockdown expression of rhogdi were resistant to the cell rounding elicited by exos rhogap (fig. 5 c), and cells with knockdown expression of rhogdi and infected with exos rhogap retained extended actin cytoskeleton structures, such as stress fibers, lamellipodia, and filopodia, indicating the presences of active rho, rac, and cdc42 (fig. 5 d). quantitative analysis showed that type iii - delivered exos rhogap elicited ∼90% actin reorganization in wild type hela cells, whereas it elicited only ∼30% actin reorganization in rhogdi knockdown cells (fig. 5 e). this indicated that rhogdi is a major regulator of rho gtpases, and rhogdi is required for exos rhogap to elicit reorganization of the actin cytoskeleton and showed that depletion of rhogdi is critical for cellular resistance to exos rhogap effect .
in 2009, the core performance brand opened the first corporate performance center with google, which had been previously contracted and staffed by plus one heath management and then by medifit corporate service prior. the company was named fast company
most innovative company in sports
in 2011, while the exos training center in az was ranked 4 among the top 10 most ludicrously expensive gyms. [ 7 ] in 2012, exos supported training for 60 london olympic medalists, including 24 gold medal winners. the same year, exos trained 14 nfl 1st round draft picks and supported the la galaxy soccer team back to back mls cup wins. [ 8 ]
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whether exos inhibits vacuolar acidification directly, or by redirecting bacteria to other compartments within the cell is yet to be directly determined. supporting the latter possibility, wild - type pao1 traffics to membrane blebs, where they are free to replicate without suppression by intravacuolar factors. however, popb (translocon) mutants, which also replicate within cells in a exos adpr activity - dependent fashion, do not traffic to blebs and instead replicate within vacuoles [ 30 ]. supporting the likelihood that exos acts locally upon the vacuole to inhibit acidification / promote intracellular replication is our data showing that popb mutant infected cells, like cells infected with wild - type bacteria, harbor a larger percentage of bacterial - occupied vacuoles that are not acidified compared to cells infected with exsa mutants. also supporting the probability of direct manipulation, rather than an escape mechanism, is that exos adpr activity, when introduced into cells without bacteria, blocks endocytic vesicle trafficking [ 39 ] .
citation: heimer sr, evans dj, stern me, barbieri jt, yahr t, fleiszig smj (2013) pseudomonas aeruginosa utilizes the type iii secreted toxin exos to avoid acidified compartments within epithelial cells. plos one 8 (9): e73111. urltoken
to further test whether the rounding phenotype elicited by gfp - rhogdi is due to disruption of actin cytoskeleton through inactivation of rho gtpases, gfp - rhogdi was co - transfected into cho cells with gfp fusion rho gtpases. the actin cytoskeleton structure was visualized by phalloidin staining after transfection. as previously described (6), expression of gfp - cdc42, gfp - rac1, or gfp - rhoa stimulated unique structures of actin cytoskeleton, whereas expression of gfp - rhogdi disrupted the unique phenotypes elicited by the respective rho gtpases (data not shown). the reorganization of actin cytoskeleton elicited by gfp - rhogdi was similar to that elicited by exos (1–234), the rhogap domain of exos, indicating that exos rhogap and rhogdi may function in the same signal pathway to modulate rho gtpase activity .
exos human capital llc is a private personalized performance training institute, with an estimated 1001 to 5000 employees and $ 100 - $ 500 million annual revenue. their primary competitors include plus one health management, health fitness, and aquila ltd. [ 3 ] [ 4 ]
environment, and express virulence traits that help the bacteria evade host defenses. in the latter regard, the type iii secretion system (t3ss) plays a major role through the expression of one or more of four known effector proteins exos, exou, exot and exoy which promote
pseudomonas aeruginosa is a gram - negative opportunistic pathogen that causes life - threatening infections in cystic fibrosis patients, the immune compromised, and burn patients (1). p. aeruginosa pathogenesis involves cell - associated and secreted virulence factors as well as intrinsic antibiotic resistance. secreted virulence factors include the type iii cytotoxins exos, exot, exoy, and exou, which are delivered by the bacterium into mammalian cells (2). exos is a bi - functional cytotoxin that contains a rho gtpase - activating protein (rhogap) 1 domain within the n terminus and a 14 - 3 - 3 - dependent adp - ribosyltransferase domain within the c terminus. transfection of the c - terminal adp - ribosyltransferase domain into cultured cells resulted in cell death (3, 4), whereas transfection of the n - terminal rhogap domain stimulated reorganization of the actin cytoskeleton (5). the n - terminal rhogap domain of exos reorganizes the actin cytoskeleton through inactivating cdc42, rac1, and rhoa in vitro and in vivo. arg 146 is a residue that is required for the exos rhogap activity (5, 6) .
the observation that acidified vacuoles were sometimes within membrane blebs suggests that vacuolar trafficking might also be impacted. if so, that may or may not involve exos. a potential mechanism could be lysosomal exocytosis, a process triggered by plasma membrane damage including that induced by a t3ss as shown for
“the biggest difference is how comprehensive the program is, ” said laboy of what differentiates exos from its competition. “there’s a lot of training options that we have at different price points. their ability to put a total program together and keep it together for as long as they have is impressive. a lot of training facilities only have strength training. others may have a little bit better physical therapy. with [ exos ], they do all of the things you would want to have during the pre - draft process for your players. they do all of them very well. ”
lacking exos adpr activity; inhibiting acidification reduces the activity of acid - dependent antimicrobial factors, e. g. acid - hydrolases, within epithelial vacuoles by drug - induced elevation of vacuolar ph and / or the prevention of phagosome maturation by inhibition of lysosome fusion as shown previously for autophagosome maturation [
was exos' s second full - length, and came at a time when dub and minimal techno from iceland—produced by artists like thor, ozy and aðalsteinn guðmundsson—was flourishing. originally released on ae recordings, the album has become a cult favourite since, and arrives at a time of increased interest in icelandic dub techno (thule
on july 24, 2015 a class action lawsuit was filed by francisco flores, individually, and on behalf of all others similarly situated against medifit. exos, also referred to as medift corporate services inc. , agreed to pay $ 1. 3 million to settle the numerous labor code and wage and hour violations. [ 10 ]
transcriptional reporter, and as expected these tended to localize to acidic vacuoles. low levels of exos expression by those bacteria might follow loss of viability because they had been trapped in acidic vacuoles. alternatively, these could be low - level expressers among the viable population and that is why they were trafficked to acidic vacuoles. the t3ss of
although the enzymatic actions of the bacterial gaps or gefs toward their specific rho gtpases are well characterized, little is known about how these bacterial gaps and gefs interact with the host regulators of rho gtpases. therefore, the present study investigated the roles of rhogdi (rhogdi - 1) in reorganization of the actin cytoskeleton stimulated by exos rhogap .
like exos, yope of yersinia and sptp of salmonella are type iii cytotoxins that possess rhogap activity. yope is a gap for cdc42, rac1, and rhoa (50, 51), whereas sptp is a gap for cdc42 and rac1 (52). it is likely that yope and sptp also modulate the actin cytoskeleton through rhogdi .
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the present study is the first report addressing the roles of rhogdi in reorganization of the actin cytoskeleton stimulated by a bacterial rhogap that noncovalently modulates the nucleotide state of rho gtpases, which linked the action of this bacterial rhogap to the host regulators of rho gtpases. although previous biochemical and structural studies indicated that rhogdi inhibited both intrinsic and rhogap - stimulated gtp hydrolysis (16, 32), the observed cooperative effect between exos rhogap and gfp - rhogdi in reorganization of actin cytoskeleton suggests that, in vivo, exos rhogap and rhogdi function in series to inactivate rho gtpases. that is, exos rhogap converts gtp - rho to gdp - rho, and rhogdi subsequently extracts gdp - rho from the cell membrane and forms a tight complex with gdp - rho in cytosol. this effectively inhibits the reactivation of rho gtpases by gefs on the cell membrane. this model is supported by the previous studies showing that rhogdi prefers to bind to gdp - rho than gtp - rho (40), and gdp - rho complexed with rhogdi is resistant to the actions of gefs (25) .
e381d) or a vector control (pucp18). data are shown as the mean (+ / - sem) values of bacteria - occupied vacuoles per cell. grey columns denote lt (-) vacuoles, black columns denote lt (+) vacuoles. calculations excluded cells showing bleb - niche formation. without exos adpr activity (complementation with pucp18 or pucp
the gram - negative bacterium p. aeruginosa is an opportunistic extracellular pathogen ubiquitously found in the environment. antibiotic - resistance and vast distribution make p. aeruginosa a major source of nosocomial acute infection of immunocompromised individuals and infection associated to the use of contaminated medical devices. p. aeruginosa is also often associated to the infection of chronic cystic fibrosis patients (garau and gomez, 2003). the bacteria express a functional t3ss through which four known effectors, exoenzyme s (exos), exot, exou, and exoy are secreted into host cell (engel and balachandran, 2009). of note, expression of the exoenzymes varies among the different strains of p. aeruginosa (engel and balachandran, 2009). activity of these effectors trigger signaling cascades, such as synthesis of camp (exoy), cleavage of phospholipids (exou), and modulation of cytoskeleton dynamics (exos, exot, exou) that potentially can lead to activation of cell death pathways, although cytotoxic effects have been described to exos and exou only (pederson and barbieri, 1998; sato and frank, 2004) .
importantly, the t3ss - dependent effector proteins of p. aeruginosa have been shown to inhibit the inflammasome activation in macrophages both in vitro and in vivo. from the four described effectors secreted by p. aeruginosa t3ss, exos, and exou inhibit inflammasome - dependent responses, arguing that if not inhibited this pathway can play a pivotal role in immune responses and bacterial clearance .
t3ss effectors (e. g. the gap activity of exos and exot) can inhibit host cell invasion and that the desirability of being internalized by a cell can depend on the host cell type and the prevailing extracellular conditions. whatever the case, the fact that not all bacteria within a population express the t3ss at a given time point could explain the paradox of how invasive
exos stands among those who represent the forefront of the electronic music nowadays. his musical masterpieces seem to be made for the intense listener. his abilites to build aesthetic minimal deep techno with dub and experimental influences impresses even the most experienced specialists. you can measure his artist potential by the density of the elements on his tracks. the musical conception of this musician suggests that he absorbed the philosophy of the basic channel crew of moritz von oswald and mark ernestus of the beginning of the nineties, but expressed it in his own way. exos can go from the most experimental compositions to the more elegant underground dancefloor tunes, alway with a very particular sense of harmony, like' maya', one of the highest examples of total quality soundwaves ...
( pa) can enter epithelial cells wherein they mediate formation of plasma membrane bleb - niches for intracellular compartmentalization. this phenotype, and capacity for intracellular replication, requires the adp - ribosyltransferase (adpr) activity of exos, a pa type iii secretion system (t3ss) effector protein. thus, pa t3ss mutants lack these capacities and instead traffic to perinuclear vacuoles. here, we tested the hypothesis that the t3ss, via the adpr activity of exos, allows pa to evade acidic vacuoles that otherwise suppress its intracellular viability. the acidification state of bacteria - occupied vacuoles within infected corneal epithelial cells was studied using lysotracker to visualize acidic, lysosomal vacuoles. steady state analysis showed that within cells wild - type pao1 localized to both membrane bleb - niches and vacuoles, while both
immunofluorescent staining of the actin cytoskeleton, rhogdi, and exos (1–234) ha— hela cells were fixed with 4% paraformaldehyde and permeabilized with 0. 1% triton - x - 100 for 10 min. the permeabilized cells were incubated with a polyclonal rabbit anti - human rhogdi antibody (santa cruz biotechnology) and / or a monoclonal mouse anti - ha antibody. subsequently, the cells were incubated with the secondary antibodies with different fluorescein conjugates. the endogenous rhogdi was labeled with a goat anti - rabbit antibody conjugated with alexa fluor 488 (green), and exos (1–234) ha was labeled with a goat anti - mouse antibody conjugated with alexa fluor 546 (red). the actin cytoskeleton was stained either by phalloidin alexa fluor 546 (red) or 448 (green) .
sports agent c. j. laboy, who just today left octagon football for relativity sports estimated that octagon has had 40 to 50 players train with exos over the past 10 years, including seattle seahawks running back marshawn lynch, and the cincinnati bengals’ leon hall. this year, laboy is representing a handful of clients, including one of the nfl draft’s top rated cornerbacks, justin verrett of tcu .
plasmid construction— glutathione s - transferase - rhogdi expressing vector, pgex - rhogdi, was a gift from dr. klaus aktories. the cdna encoding rhogdi - 1 in pgex - rhogdi was subcloned into the bamhi sites of pegfpc1 to produce a gfp - rhogdi - 1 fusion protein. the mutations i177d and l55s, l56s on pgfp - rhogdi were obtained through standard site - directed mutagenesis procedures using a quikchange mutagenesis kit (stratagene). the pcr products were confirmed by dna sequencing. pexv - p50rhogap was a gift from dr. alan hall. the dna encoding p50rhogap was amplified by pcr using primers containing hindiii / ecori sites and c - terminal ha tag (positive primer: 5′ - gat ctc gag aag ctt gcc atg gat ccg ctc tca - 3′; negative primer: 5′ - gtc gac gaa ttc tca caa gct ggc gta gtc ggg cac gtc gta ggg gta gag ccc gct ggg gtc c - 3′). the pcr product was confirmed by sequencing and cloned into the hindiii / ecori sites of pegfpn1 vector to produce p50rhogap - ha. mammalian expression vectors encoding exos (1–234), exos (1–234) r146k, exos (e381d), and gfp - cdc42 have been described previously (6) .
transcriptional output using a threshold value of 1000 units of gfp fluorescent intensity (green) based on expression levels observed under t3ss - inducing conditions (see results). prior to imaging, epithelial cells were infused with lysotracker dnd - 22 (blue). exos - expressing bacteria (high output, green) [ solid arrows ] were located primarily outside of acidified (blue) intracellular compartments, which often contained bacteria with low
rhogdi is required for exos rhogap - mediated actin reorganization. a, hela cells at 40–50% confluency were either mock transfected or transfected with control sirna or human rhogdi sirna. at 68–72 h post - transfection, whole cell lysates were collected and subjected to sds - page followed by western blots using α - rhogdi igg or α - actin antibody. b, at 68–72 h post - transfection, the cells were fixed and permeabilized, and endogenous rhogdi was probed with rabbit α - rhogdi igg followed by goat α - rabbit igg conjugated with alexa fluro 488 (green). f - actin was stained with phalloidin alexa fluro 546 (red). c, at 68–72 h post - transfection, the cells were infected with p. aeruginosa pa103 (δ exou, exot: : tc) expressing exos (1–234) ha at an multiplicity of infection of 8: 1. after a 3–4 - h infection, hela cells were fixed, permeabilized, and stained with rabbit α - rhogdi igg followed by goat α - rabbit igg conjugated with alexa fluro 488 (green) or mouse α - ha igg followed by goat α - mouse igg conjugated with alexa fluro 546 (red). d, a representative cell transfected with rhogdi sirna and then infected with p. aeruginosa expressing exos (1–234) - ha was stained by mouse α - ha igg followed by goat α - mouse igg conjugated with alexa fluro 546 (red) and by phalloidin alexa fluro 488 (green, shown in gray scale). the arrow points to bacteria. e, the percentage of hela cells alone (-) or transfected with rhogdi sirna (+) that exhibited actin reorganization upon infection by type iii - delivered exos rhogap (+) or without infection (-) was quantified from three independent experiments by determining the number of hela cells containing actin structures (f - actin staining) divided by the total number of transfected cells × 100% .
rhogdi and p50rhogap cooperatively stimulated actin reorganization in cho cells— because the relationship between mammalian rhogaps and rhogdi - 1 on actin reorganization has not been addressed, p50rhogap was co - expressed with gfp - rhogdi in cho cells. as observed for exos rhogap with gfp - rhogdi, co - expression of p50rhogap - ha and gfp - rhogdi cooperatively stimulated reorganization of actin cytoskeleton in cho cells (fig. 7 a) and translocated gfp - cdc42 from membrane to cytosol (fig. 7 b) .
those who had the pleasure of walking around the nfl combine this weekend in indianapolis saw the likes of defensive end jadeveon clowney, quarterback blake bortles, offensive lineman greg robinson, and tight end eric ebron, all among the top draft eligible prospects at their respective positions. the aforementioned quartet all trained at athletes' performance, or what is now referred to as exos. athletes' performance, arguably the leader in human and health performance, along with core performance recently combined forces last month under one unified brand .
exos and rhogdi cooperatively stimulated actin reorganization in cho cells. indicated concentrations of pegfp, pegfp - rhogdi, pexos (1–234) - ha, pflexos (e381d) - ha, or pexos (1–234) r146k - ha were transfected or co - transfected into cho cells. the total transfected dna was normalized with pcmv - luciferase. at 24 h post - transfection, the cells were examined by fluorescent microscopy, and the percentage of cell rounding was calculated. the data from at least three independent experiments are shown. a, results from cho cells transfected or co - transfected with indicated concentrations of pegfp, pegfp - rhogdi, or pexos (1–234) - ha. b, results from cho cells transfected or co - transfected with indicated concentrations of pegfp, pegfp - rhogdi, or pexos (1–234) r146k - ha. c, results from cho cells transfected or co - transfected with indicated concentrations of pegfp, pegfp - rhogdi, or pflexos (e381d) - ha (full - length exos defective in adp - ribosyltransferase) .
in 2004, exos founder, mark verstegen, released his first book
core performance. the next year, the company started a relationship with the german national soccer team in preparation for the 2006 world cup. in 2006, the company began providing solutions to the united states military. the company launched cpro and cpesd products in 2008 and entered an innovation and development partnership with adidas micoach. from inception until 2008, more than 25 first round picks were api athletes, including 8 each in the 2005 and 2006 nfl drafts .
the mechanism of cooperativity between exos rhogap and rhogdi is studied by using a dominant - negative mutant of rhogdi, rhogdi (i177d), which is capable of binding to switch i and switch ii regions of rho gtpases with the n - terminal flexible regulatory arm but is defective in extracting the isoprenyl moiety of rho gtpases off the cell membrane through the hydrophobic binding pocket. rhogdi (i177d) inhibited the membrane - cytosol translocation of rho gtpases by gfp - rhogdi and exos rhogap, suggesting that rhogdi plays essential roles in exos rhogap - stimulated rho gtpase inactivation, and extraction of the isoprenyl moiety of gdp - rho off the membrane by the hydrophobic binding pocket of rhogdi is the rate - limiting step. the present study is consistent with the previously proposed two - step model for the interaction of rhogdi with the membrane - bound cdc42 based on the kinetics and structural studies, in which the n - terminal flexible regulatory arm of rhogdi binds to the switch i and switch ii regions of cdc42 (fast step), and the c - terminal hydrophobic binding pocket of rhogdi extracts the isoprenyl moiety of cdc42 off the membrane (slow step) (18, 35). although rhogdi (i177d) inhibited the ability of rhogdi and rhogap to translocate rho gtpases from membrane to cytosol, it did not inhibit cell rounding stimulated by rhogap and gfp - rhogdi. similar to gfp - rhogdi, expression of gfp - rhogdi (i177d) in cho cells stimulated cell rounding in a dose - dependent manner, whereas gfp - rhogdi (l55s, l56s) did not stimulate cell rounding (data not shown). this indicates that the binding of the n - terminal regulatory arm of rhogdi to gdp - bound rho gtpases on the membrane also inhibits gef - stimulated activation of rho gtpases .
one demonstration of the selectivity of the export signal rules is that only the chicken adp - ribosyltransferase nrt2 chk shows major violations of the rules even though this protein is more similar to hopptos1 and s2 than either of the type iii - secreted adp - ribosyltransferases from p. aeruginosa, exos and exot (see petnicki - ocwieja et al. , proc. natl. acad. sci. usa 99: 7652–7657 (2002); u. s. patent application ser. no. 10 / 341, 180 to collmer et al. , filed jan. 13, 2003, each of which is hereby incorporated by reference in its entirety) .
gfp - rhogdi (i177d) inhibited the membrane - cytosol translocation of gfp - cdc42 by gfp - rhogdi (wt) and exos rhogap. a, upper panels, cho cells were transfected with pgfp, pgfp - rhogdi - (i177d), or pgfp - rhogdi (l55s, l56s) at a concentration of 20 ng / ml. at 24 h post - transfection, the cells were examined by fluorescent microscopy. lower panels, at 24 h post - transfection, the cells were fractionated, and the membrane (m) and cytosolic fractions (c) were subjected to sds - page followed by western blot using anti - gfp monoclonal antibody as primary antibody. b and c, cho cells were transfected with pgfp, pgfp - cdc42 (cdc42), pgfp - rhogdi (gdi - wt), pgfp - rhogdi (i177d) (gid - dn), pgfp - rhogdi - (l55s, l56s) (gdi (ll / ss) ), and pexos - (1–234) ha (exos - gap) at the indicated concentrations (ng / ml). the total transfected dna was normalized with pcmv - luciferase. at 24 h post - transfection, the cells were harvested, lysed, and fractionated into post - nuclear supernatant (p), membrane (m), and cytosol (c) fractions. the equivalent fractions were subjected to sds - page followed by western blot with anti - gfp antibody for detection of gfp and gfp fusion proteins or α - ha antibody for detection of exos (1–234) - ha. gfp was used as a cytosolic marker. the images were developed, and the density of the gfp antibody immune reactive bands was measured in fluorchem8900. the membrane - cytosol (mem / cyto) ratio of gfp - cdc42 was calculated as (0. 65 / 0. 35) i177d; (0. 15 / 0. 85) l55, 56s; (0. 08 / 0. 92) gfp. the data from three independent experiments are shown .
rhogdi is required for the exos rhogap - mediated reorganization of the actin cytoskeleton— although rhogdi (i177d) inhibited the ability of rhogdi and rhogap to translocate rho gtpases from membrane to cytosol, it did not inhibit cell rounding stimulated by rhogap and gfp - rhogdi. similar to gfp - rhogdi, expression of gfp - rhogdi (i177d) in cho cells stimulated cell rounding in a dose - dependent manner, whereas gfp - rhogdi (l55s, l56s) did not stimulate cell rounding (data not shown). this indicates that the binding of the n - terminal regulatory arm of rhogdi to gdp - bound rho gtpases on the membrane inhibits gef - stimulated activation of rho gtpases .
gfp - rhogdi and p50rhogap cooperatively stimulated actin reorganization in cho cells. a, cho cells were transfected with pegfp, pegfp - rhogdi, or p50rhogap - ha as indicated. total transfected dna was normalized with pcmv - luciferase. at 24 h post - transfection, the cells were examined by fluorescent microscopy, and the percentage of cellular rounding was calculated as previously described. the data from three independent experiments are shown. b, cho cells were transfected with pegfp - cdc42 (200 ng / ml), pegfp - rhogdi (10 ng / ml), and p50rhogap - ha (10 ng / ml) as indicated. total transfected dna was normalized with pcmv - luciferase. at 24 h post - transfection, the cells were lysed and fractionated. the equivalent membrane and cytosol fractions were subjected to sds - page followed by ecl western blotting with anti - gfp antibody (for detection of gfp or gfp fusion proteins) or anti - ha antibody (for detection of exos (1–234) - ha and exos (1–234) r146k - ha). transiently expressed gfp was used as a cytosolic fractionation marker. the density of the gfp antibody immune reactive bands were measured in fluorchem8900, and the membrane - cytosol (mem / cyto) ratios of gfp - cdc42 were calculated. the error bars represent the standard deviations from at least three independent experiments. an asterisk indicates a statistically significant difference (p < 0. 05) relative to the membrane - cytosol ratio of gfp - cdc42 elicited by gfp - rhogdi or p50rhogap individually .
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exos (1–234) and gfp - rhogdi cooperatively translocated gfp - cdc42 from membrane to cytosol. cho cells were cotransfected with pgfp - cdc42, pexos (1–234) - ha, pexos (1–234) r146k - ha, and pegfp - rhogdi at the indicated concentrations (ng / ml). the total transfected dna for each plate was normalized with pcmv - luciferase. at 24 h post - transfection, the ells were harvested, lysed, and fractionated. the equivalent membrane and cytosol fractions were subjected to sds - page followed by western blot with anti - gfp antibody as primary antibody. the images were developed, and the density of the gfp antibody immune reactive bands was measured in fluorchem8900. the membrane - cytosol (mem / cyto) ratio of gfp - cdc42 was calculated. the data from three independent experiments are shown .
exos rhogap and gfp - rhogdi cooperatively translocated cdc42 from the membrane to cytosol— gtp - bound rho gtpases are active and localize to the membrane, whereas gdp bound rho gtpases are inactive and complexed to rhogdi in the cytosol. changes in the activation states of rho gtpases can be measured using the preferential binding of rho - gtp to the rho - binding domains of rhotekin and p21 - activated kinase (23, 24), which is useful for measuring the activation of rho by toxins, such as cytotoxic necrotizing factor 1 (25, 26), but has limited resolution when measuring the inactivation of rho, because p21 - activated kinase binds only a fraction of rho - gtp within a cell extract (6). an alternative method to measure the activation state of rho gtpases is to measure the relative distribution of rho gtpases within membrane and cytosol fractions (6). transfection of pexos (1–234) or pegfp - rhogdi in cho cells at 100 ng / ml stimulated the translocation of gfp - cdc42 from membrane to cytosol, whereas lower amounts of transfected pexos (1–234) and pegfp - rhogdi (10 ng / ml or 20 ng / ml) did not affect the localization of gfp - cdc42 (fig. 3). co - transfection of pexos (1–234) and pegfp - rhogdi at 10 or 20 ng / ml stimulated the translocation of gfp - cdc42 from membrane to cytosol. as control, transfection with pexos (1–234) r146k at 100 ng / ml did not stimulate the translocation of gfp - cdc42 from membranes to cytosol, which indicated that the exos rhogap activity was required to stimulate membrane - cytosol translocation of gfp - cdc42 (fig. 3). in addition, co - transfection of pexos (1–234) r146k and pegfp - rhogdi at 10 and 20 ng / ml did not stimulate the translocation of gfp - cdc42 from membranes to cytosol (data not shown).
exos , formerly named athletes ' performance , is an american company founded in 1999 to maximize the potential of athletes . in 2014 the brand names athletes ' performance and the subsidiary core performance were integrated into one brand name , exos . the company focuses on proactive health and performance for elite athletes , the military and businesses . founded by mark verstegen in 1999 and based in phoenix , arizona , it has private training facilities in phoenix , arizona , fieldhouse usa in frisco , texas ; at stubhub center in carson , california ; the sklz headquarters in carlsbad , california and at the andrews institute in gulf breeze , florida . the company offers training and nutrition services in raleigh , north carolina and cary , north carolina through a partnership with raleigh orthopedic clinic . exos has trained 6 nfl overall # 1 picks , 94 nfl first round draft picks and 459 total nfl draftees , 9 nba first-round picks , world cup heroes ( us women 's soccer and german men 's soccer ) , and hosts of all-stars across every major sport . members of the boston red sox have spent part of their off season time with exos . it is a private personalized performance training institute , described by urltoken as " a high-tech fitness boot camp for professionals , " and by espn as " a sprawling weight room/running track/football field/swimming pool/café/spa . " training programs can include aspects such as a nutrition program , massages , metabolic tests , cardiovascular work , drills , and aptitude and media training . 
exos, formerly named athletes' performance, is an american company founded in 1999 to maximize the potential of athletes. in 2014 the brand names athletes' performance and the subsidiary core performance were integrated into one brand name, exos. the company focuses on proactive health and performance for elite athletes, the military and businesses. founded by mark verstegen in 1999 and based in phoenix, arizona, it has private training facilities in phoenix, arizona, fieldhouse usa in frisco, texas; at stubhub center in carson, california; the sklz headquarters in carlsbad, california and at the andrews institute in gulf breeze, florida. the company offers training and nutrition services in raleigh, north carolina and cary, north carolina through a partnership with raleigh orthopedic clinic. exos has trained 6 nfl overall # 1 picks, 94 nfl first round draft picks and 459 total nfl draftees, 9 nba first-round picks, world cup heroes (us women's soccer and german men's soccer), and hosts of all-stars across every major sport. members of the boston red sox have spent part of their off season time with exos. it is a private personalized performance training institute, described by urltoken as " a high-tech fitness boot camp for professionals, " and by espn as " a sprawling weight room/running track/football field/swimming pool/café/spa. " training programs can include aspects such as a nutrition program, massages, metabolic tests, cardiovascular work, drills, and aptitude and media training.
exos, formerly named athletes' performance, is an american company founded in 1999 to maximize the potential of athletes. in 2014 the brand names athletes' performance and the subsidiary core performance were integrated into one brand name, exos. the company focuses on proactive health and performance for elite athletes, the military and businesses. founded by mark verstegen in 1999 and based in phoenix, arizona, it has private training facilities in phoenix, arizona, fieldhouse usa in frisco, texas; at stubhub center in carson, california; the sklz headquarters in carlsbad, california and at the andrews institute in gulf breeze, florida. the company offers training and nutrition services in raleigh, north carolina and cary, north carolina through a partnership with raleigh orthopedic clinic. exos has trained 6 nfl overall # 1 picks, 94 nfl first round draft picks and 459 total nfl draftees, 9 nba first-round picks, world cup heroes (us women's soccer and german men's soccer), and hosts of all-stars across every major sport. members of the boston red sox have spent part of their off season time with exos. it is a private personalized performance training institute, described by urltoken as " a high-tech fitness boot camp for professionals, " and by espn as " a sprawling weight room/running track/football field/swimming pool/café/spa. " training programs can include aspects such as a nutrition program, massages, metabolic tests, cardiovascular work, drills, and aptitude and media training.