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TOXICOLOGY

Gambierol Acts as a Functional Antagonist of Neurotoxin Site 5 on Voltage-Gated Sodium Channels in Cerebellar Granule Neurons

Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana (K.T.L.); Department of Chemistry, University of Utah, Salt Lake City, Utah (J.D.R., H.W.B.J.); Center for Marine Science, University of North Carolina at Wilmington, Wilmington, North Carolina (D.G.B.); and Department of Pharmacology, School of Medicine, Creighton University, Omaha, Nebraska (T.F.M.)

Received for publication April 11, 2007
Accepted June 26, 2007.

	Abstract

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The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 µM, gambierol alone had no effect on the intracellular Ca²⁺ concentration [Ca²⁺]_i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM–10 µM). However, gambierol was a potent inhibitor (IC₅₀ = 189 nM) of the elevation of [Ca²⁺]_i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC₅₀ = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca²⁺ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca²⁺]_i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca²⁺]_i in a concentration-dependent manner (IC₅₀ = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.

Cultures of G. toxicus also produce gambierol, which contains a transfused octacyclic polyether core, 18 stereogenic centers, and a skipped triene (Satake et al., 1993; Morohashi et al., 1999; Johnson et al., 2006) (Fig. 1). Investigations into the pathological and pharmacological characteristics of gambierol have been facilitated by chemical synthesis of the compound (Fuwa et al., 2002, 2004; Kadota et al., 2003; Johnson et al., 2006). Pathological effects in several tissues have been reported in mice following systemic exposure to gambierol. Gambierol has been shown to be a potent toxin with a minimal lethal dose ranging from 50 to 80 µg/kg (i.p.) in mice (Fuwa et al., 2003, 2004; Ito et al., 2003). Primary pathological effects of gambierol in mice were noted in the lung, with secondary effects in the heart (Ito et al., 2003). Gambierol also induced hypersecretion and ulceration in the stomach (Ito et al., 2003). Gambierol has also been reported to elicit aberrant behavior consistent with a neurotoxic insult (Ito et al., 2003). However, there are no reports of pathological changes in gambierol-exposed rodent brains. Recent investigations have established that gambierol inhibits voltage-gated potassium currents in mouse taste cells (Ghiaroni et al., 2005). Based on the production of gambierol by G. toxicus and the described pathological sequelae, gambierol is thought to contribute to the symptoms of ciguatera (Ito et al., 2003).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Structure of gambierol.

Neurotoxin site 5 of the VGSC has been identified as the molecular target for both PbTx and CTX (Gawley et al., 1992; Baden and Adams, 2000). Inoue et al. (2003) demonstrated that gambierol inhibits the binding of [³H]PbTx-3 to VGSCs with a K_i value of 1.4 µM in synaptosomes derived from rat brains. These authors suggested that the ability of gambierol to inhibit the binding of [³H]PbTx-3 indicates that gambierol may also interact with neurotoxin site 5 (Inoue et al., 2003).

Herein, we take advantage of the recent synthesis of gambierol (Johnson et al., 2006) to extend the characterization of the toxin using functional responses in cerebellar granule neuron (CGN) cultures. Specifically, the effect of gambierol on VGSC activator (PbTx-2)-induced elevation of [Ca²⁺]_i was examined. Gambierol interaction with the VGSC was further assessed by determining the ability of this compound to inhibit PbTx-2-induced neurotoxicity in intact neurons. Finally, we compared the effects of gambierol and brevenal as antagonists of PbTx-2-induced elevation of [Ca²⁺]_i.

	Materials and Methods

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Cerebellar Granule Cell Culture. CGN cultures were harvested from 8-day-old Sprague-Dawley rats, and they were cultured as described by Berman and Murray (1996). Cells were plated onto poly-L-lysine (mol. wt. = 393,000)-coated 9-mm clear-bottomed black 96-well culture plates (Corning Life Sciences, Acton, MA) at a density of 9.6 x 10⁴ cells/well and 12-well (Corning Life Sciences) culture dishes at 1.25 x 10⁶ cells/well, respectively. Then, cells were incubated at 37°C in a 5% CO₂ and 95% relative humidity atmosphere. CGN cultures at 8–13 days in culture (DIC) were used for lactate dehydrogenase efflux and Ca²⁺ influx studies.

Intracellular Ca²⁺ Monitoring. CGN cultures grown in 96-well plates were used for [Ca²⁺]_i measurements at 10 to 13 DIC as described by Berman and Murray (2000). In brief, the growth media were removed, and they were replaced with a dye-loading medium (100 µl/well) containing 4 µM Fluo-3 (Invitrogen, Carlsbad, CA) and 0.04% Pluronic acid (Invitrogen) in Locke's buffer, pH 7.4. After 1-h incubation in a dye-loading medium, the neurons were washed four times in fresh Locke's buffer (200 µl/well) using an automated cell washer (Labsystems, Helsinki, Finland) and transferred to the fluorescence laser plate reader (FLIPR; Molecular Devices, Sunnyvale, CA) incubation chamber. The final volume of Locke's buffer in each well was 100 µl. Neurons were excited by the 488-nm line of the argon laser, and Ca²⁺-bound fluo-3 emission in the 500- to 600-nm range was recorded with a charge-coupled device camera with shutter speed set at 0.4 s. FLIPR operates by illuminating the bottom of a 96-well microplate with an argon laser and by measuring the emissions from cell-permeant dyes in all 96 wells simultaneously using a cooled charge-coupled device camera. Gambierol ([final] = 10 nM–10 µM) or brevenal ([final] = 0.1 nM–10 µM) was added to neurons from one source plate in a volume of 30 µl at the rate of 20 µl/s. Fluorescence readings were taken every 10 s for 1 min to establish a baseline. After incubation with gambierol, PbTx-2 ([final] = 20 nM) or Locke's buffer was added from another source plate as described previously. Fluorescence readings were taken every second for the first minute and every 8 s for the duration of the experiment (25 min). Background fluorescence was automatically subtracted from all Fluo-3 fluorescence measurements.

LDH Activity Assay. All assays were carried out in 1% dimethyl sulfoxide, which has no independent effect on these measures in CGN cultures (data not shown). Growth media were removed, and the cultures were washed two times in 1 ml of Locke's incubation buffer (154 mM NaCl, 5.6 mM KCl, 1 mM MgCl₂, 2.3 mM CaCl₂, 8.6 mM HEPES, 5.6 mM glucose, and 0.1 mM glycine, pH 7.4). CGN cultures were then exposed to gambierol (1 nM–10 µM) alone or in the presence of 40 nM PbTx-2 for 2 h at 22°C. At the termination of toxin exposure, exposure buffer was collected and assayed for LDH efflux. Cytotoxicity was assessed by quantifying the activity of LDH released by injured neurons into the exposure buffer at 2 h after the termination of toxin exposure. LDH activity was determined spectrophotometrically as described by Koh and Choi (1987). Data are reported as LDH activity units per well.

Preparation of Intact Synaptosomes. Rat synaptosomes were prepared using a modification of the protocol described by Dodd et al. (1981). In brief, adult female Sprague-Dawley rats were sacrificed, and the cortices were immediately dissected out over ice. The cortices were weighed, and then they were homogenized in 20 volumes of an ice-cold (4°C) 0.32 M sucrose buffer using a glass Dounce fitted with a loose Teflon pestle. The homogenate was centrifuged at 1000g at 4°C for 10 min. The supernatant was collected and centrifuged at 20,000g for 20 min. The resultant pellet was resuspended in an isotonic buffer (132 mM choline chloride and 25 mM Tris, pH 7.4), and then it was centrifuged at 20,000g for 20 min. The wash/centrifugation step was repeated, and the final pellet was resuspended in the isotonic buffer. The concentration of protein was determined using a modification of the method described by Lowry et al. (1951). The synaptosomes used in all binding assays were prepared on the day of the experiment.

[³H]PbTx-3 Equilibrium Competition Assay. The potency and efficacy of gambierol as a competitor of high-affinity [³H]PbTx-3 (PerkinElmer Life and Analytical Sciences, Boston, MA) binding to intact synaptosomes was evaluated via a centrifugation assay. In brief, increasing concentrations of gambierol (0.3 nM–30 µM) were incubated with [³H]PbTx-3 for 90 min in microcentrifuge tubes. The reactions were carried out in an isotonic buffer (132 mM choline chloride and 25 mM Tris, pH 7.4). Reactions were allowed to proceed for 3 h at 4°C. Assay tubes were then centrifuged at 21,000g for 2 min. The incubation buffer was aspirated, and the pellet was washed gently three times with ice-cold buffer. The pellet was placed into scintillation fluid, and it was agitated overnight to solubilize the pellet. The amount of [³H]PbTx-3 bound to the synaptosomal preparation was assessed by placing the samples in a scintillation counter (Beckman Coulter, Fullerton, CA).

Analysis of Experimental Data. Concentration-response data were analyzed by nonlinear least-square regression analysis with the GraphPad software suite, version 4.0 (GraphPad Software Inc., San Diego, CA) using a three-parameter logistic equation.

Reagents. Gambierol was synthesized as described by Johnson et al. (2006). This gambierol was purified by silica gel chromatography, and it was >95% pure by proton NMR. All reagents not mentioned in the text were obtained from Sigma-Aldrich (St. Louis, MO).

	Results

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Brevetoxin exposure produces a rapid elevation of [Ca²⁺]_i that contributes to an acute necrotic cell death in CGN cultures (Fig. 2). As shown in Fig. 2, 20 nM PbTx-2 induced a robust elevation of [Ca²⁺]_i in CGN cultures. Conversely, gambierol alone did not alter [Ca²⁺]_i in CGN cultures during the 25-min exposure period (Fig. 2). Given this lack of activity of gambierol as an activator of neurotoxin site 5 on the VGSC, we evaluated the possibility that gambierol acts as an antagonist of this site. To this end, CGN cultures were exposed to 20 nM PbTx-2 in the absence and presence of various concentrations of gambierol. The results of these experiments depicted in Fig. 3 demonstrated that gambierol produced a concentration-dependent inhibition of PbTx-2-induced Ca²⁺ influx in CGN cultures. Nonlinear regression analysis of the concentration dependence of gambierol inhibition of the maximal PbTx-2-induced Ca²⁺ influx indicated that the IC₅₀ value for gambierol was 189 nM (88.4–403.8 nM, 95% CI) (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Lack of effect of various concentrations of gambierol on Fluo-3 fluorescence in 10 to 13 DIC cerebellar granule neurons. Data depict means ± S.E.M. of a representative experiment performed in triplicate and repeated three times.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Increases in Fluo-3 fluorescence produced by exposing 10 to 13 DIC cerebellar granule neurons to a submaximal concentration (20 nM) of PbTx-2 in the presence or absence of various concentrations of gambierol (GB). Data depict means ± S.E.M of a representative experiment performed in quintuplicate and repeated twice.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Nonlinear regression analysis of gambierol antagonism of the maximal increment in Fluo-3 fluorescence evoked by 20 nM PbTx-2. Data depict pooled means from two experiments that were performed in quintuplicate. The IC50 value derived from this fit was 189 nM (88–404 nM, 95% CI).

In an effort to assess the specificity of gambierol antagonism of PbTx-2-induced Ca²⁺ influx, we assessed the ability of gambierol to block Ca²⁺ influx produced by a depolarizing concentration of KCl (50 mM) (Berman and Murray, 2000). We found that gambierol had no effect on the increase of intracellular Ca²⁺ in CGN cultures exposed to 50 mM KCl, indicating that gambierol does not block voltage-dependent Ca²⁺ channels (Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Increases in Fluo-3 fluorescence produced by exposing 10 to 13 DIC cerebellar granule neurons to a depolarizing concentration (50 mM) of KCl in the presence or absence of various concentrations of gambierol. Data depict means ± S.E.M. of a representative experiment performed in quintuplicate and repeated twice.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Nonlinear regression analysis of gambierol antagonism of PbTx-2-induced neurotoxicity in cerebellar granule neurons. Neurons were exposed to PbTx-2 and/or gambierol for 2 h at room temperature. LDH efflux was then assessed as an indicator of cytotoxicity. Data depicted represent the means of two to three determinations. The IC50 value derived from this fit was 471 nM (165–1344 nM, 95% CI).

We further confirmed that gambierol inhibits the binding of [³H]PbTx-3 to VGSCs expressed on intact rat synaptosomes with a K_i value of 4.8 µM (2.5–9.4 µM, 95% CI) (Fig. 7). Our findings are consistent with the study of Inoue et al. (2003), who reported a K_i value of 1.4 µM for gambierol as a competitor of high-affinity [³H]PbTx-3 binding in rat synaptosomes. The current results support the contention that gambierol binds to neurotoxin site 5 of the VGSC.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Inhibition of specific [3H]PbTx-3 binding by gambierol. Increasing concentrations of gambierol were added to intact rat synaptosomes. The concentration of [3H]PbTx-3 was 3 nM. The figure is derived from data from a representative experiment that was repeated twice in duplicate. Error bars represent ± S.E.M.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Nonlinear regression analysis of brevenal antagonism of the maximal total Fluo-3 fluorescence evoked by 20 nM PbTx-2. Data points represent the percentage of the total Ca2+ response of CGN cultures exposed to a 20 nM concentration of PbTx-2. Data from four independent cultures are depicted as mean values ± S.E.M.

	Discussion

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Brevetoxins induce rapid neuronal depolarization by affecting both the activation- and inactivation-gating properties of VGSCs (Baden and Adams, 2000). Brevetoxin-induced autocrine excitotoxicity in cerebellar granule cells is associated with an evoked glutamate release, rapid influx of Ca²⁺, and a sustained Ca²⁺ load in exposed neurons (Berman and Murray, 2000). Excitotoxic responses to PbTx-1 are correlated with the magnitude of this Ca²⁺ load, although neuronal vulnerability is governed selectively by the N-methyl-D-aspartate receptor Ca²⁺ influx pathway that is indirectly activated indirectly by PbTx-1. In the present study, PbTx-2 elicited a rapid elevation in [Ca²⁺]_i (Fig. 2). This Ca²⁺ influx is secondary to PbTx-2 activation of VGSCs, with subsequent activation of voltage-sensitive Ca²⁺ channels, the reverse mode of operation of the Na⁺/Ca²⁺ exchanger, and Ca²⁺ influx through N-methyl-D-aspartate receptors (Berman and Murray, 2000). We demonstrate here that gambierol acts as a functional antagonist of neurotoxin site 5 in that it effectively inhibits both PbTx-2-induced neurotoxicity and elevation of [Ca²⁺]_i in CGN cultures. The mechanism by which gambierol acts as a PbTx-2 antagonist is unknown. The most parsimonious explanation is that gambierol binds to the VGSC at neurotoxin site 5 and acts as a competitive antagonist of PbTx-2 at this site. The finding that gambierol inhibits the specific binding of [³H]PbTx-3 is consistent with this suggestion. Alternatively, gambierol interaction with the VGSC may occlude the channel pore in a manner similar to that of tetrodotoxin or allosterically inhibit PbTx-2 interaction with neurotoxin site 5 (Cestèle and Catterall, 2000).

Gambierol differed from PbTx-2 in that, at concentrations ranging from 0.01 to 1.0 µM, it did not produce an influx of Ca²⁺ in CGNs. Inasmuch as we have previously shown that brevetoxin-induced Ca²⁺ influx is triggered by the activation of VGSCs, it is reasonable to infer that gambierol is not an activator of VGSC in CGNs. In contrast to these results, a recent report has found that gambierol produced a modest, relative to veratridine, increment in Ca²⁺ influx in neuroblastoma cells (Louzao et al., 2006). Based on the results of pharmacological experiments, these authors concluded that gambierol acts as a partial agonist at neurotoxin site 5 of the VGSC. However, the gambierol concentration-response curve for membrane depolarization in neuroblastoma cells was exponential with no defined maximum. Therefore, the EC₅₀ value could not be determined, but it would be >30 µM, indicating that this response may be a nonspecific effect. In the absence of a demonstration of a tetrodotoxin- or saxitoxin-induced rightward shift of the gambierol concentration-response curve, the role of VGSCs in this response is uncertain. Louzao et al. (2006) reported that neosaxitoxin produced a very modest (15%) attenuation of gambierol-induced depolarization, but they did not provide a demonstration of antagonism of the gambierol-induced Ca²⁺ response. The absence of a positive allosteric coupling between gambierol and the neurotoxin site 2 activator veratridine in that report further indicates that the observed response to gambierol in neuroblastoma cells may not involve VGSCs. In addition, neuroblastoma cell lines may not provide sufficient sensitivity to this toxin, given their relatively low expression levels of VGSCs compared with neurons (LePage et al., 2005).

All natural brevetoxins are thought to bind to neurotoxin site 5 in a "head-down" orientation into the channel, intercalating with -helices of domains III and IV of the VGSC (Poli et al., 1986; Gawley et al., 1995). Four distinct effects of brevetoxins on the VGSC have been elucidated, and they include a shift of the activation potential to more negative values, a prolongation of mean open time, an inhibition of channel inactivation, and the induction of multiple subconductance states (Jeglitsch et al., 1998). These alterations in sodium channel function promote the depolarization of neurons at resting membrane potential; thus, they account for the acute central and peripheral effects of brevetoxins observed in vivo. Gambierol is composed of eight fused polyether rings, and it is smaller than ciguatoxins or brevetoxins (11–13 rings) (Lewis, 2001; Yasumoto, 2001; Baden et al., 2005; Johnson et al., 2006). The truncated size of gambierol is similar to that of brevenal, a recently described component of K. brevis cultures that acts as an antagonist of PbTx-induced toxic effects (Bourdelais et al., 2004). Bourdelais et al. (2004) demonstrated that brevenal displaced [³H]PbTx-3 binding to brain synaptosomes, with a K_i value of 685 nM; moreover, it seemed to protect fish from PbTx-2- or PbTx-3-induced lethality. We have demonstrated a brevenal concentration-dependent antagonism of PbTx-2-induced Ca²⁺ influx in CGN cultures. Therefore, these data suggest that brevenal and gambierol are similar in that they both possess modest affinity for the [³H]PbTx-3-labeled neurotoxin site 5 and that they act as functional antagonists at this site. Interestingly, neither gambierol nor brevenal fit the "binding" motif for neurotoxin site 5 of the VGSC as proposed by Gawley et al. (1995). The functional data reported here are consistent with at least one position of the gambierol molecular target coinciding or overlapping with neurotoxin site 5 of the VGSC. Gambierol at a concentration of 0.1 µM recently has been shown to have no influence on the VGSC in taste cells (Ghiaroni et al., 2005). Given the K_i value of 1.4 µM for gambierol binding to neurotoxin site 5, a concentration of 0.1 µM may not have produced sufficient fractional occupancy of the VGSC to demonstrate antagonism. Gambierol binding to neurotoxin site 5 may prevent access of brevetoxins to this site; yet, it may have no influence on the VGSC in the absence of brevetoxins. The development of radiolabeled gambierol analogs may facilitate the identification of the precise site(s) of ligand interaction with the VGSC.

In contrast to the lack of effect on mouse taste cell VGSCs, Ghiaroni et al. (2005) have shown that gambierol potently inhibited voltage-gated K⁺ channels in the same cells. Voltage-gated K⁺ currents evoked by depolarizing pulses were inhibited by gambierol in these cells, with nanomolar potency. The influence of gambierol on I_K in taste cells was therefore characterized by an IC₅₀ value (1.8 nM) that is 2 orders of magnitude lower than that determined in the present study for antagonism of site 5 on VGSCs (189 nM). Notwithstanding the potential influence of differing membrane potentials in the two studies, these data indicate that voltage-gated K⁺ channels may represent the primary molecular target involved in the toxic actions of gambierol. A more recent study using mouse taste cells has demonstrated that ciguatoxin (CTX3C) differs from gambierol in that it did not affect voltage-gated K⁺ channels (Ghiaroni et al., 2006). However, these results indicate that the actions of polyether toxins may differ as a function of cellular context inasmuch as ciguatoxin has been shown to inhibit K⁺ currents in dorsal root ganglion neurons (Birinyi-Strachan et al., 2005).

Gambierol is acutely toxic to mice, and it produces pathological sequelae in the lung and heart (Ito et al., 2003). Hypersecretion and ulceration of the stomach also has been identified as a pathological effect of gambierol in mice (Ito et al., 2003). These toxic responses may be a consequence of gambierol inhibition of voltage-gated K⁺ channels.

Although gambierol seems to possess structural components that allow the binding of the toxin to neurotoxin site 5 of the VGSC, this interaction does not seem to result in the activation of the channel, as is the case with brevetoxins and ciguatoxins. Therefore, it is reasonable to infer that gambierol lacks efficacy at neurotoxin site 5 of the VGSC. This investigation has described two measures of functional antagonism of gambierol at neurotoxin site 5 of the VGSC. First, gambierol inhibits brevetoxin-induced elevation of [Ca²⁺]_i in CGN cultures. Second, the well established neurotoxicity of brevetoxins in CGN cultures is also reduced in a concentration-dependent manner by preincubation with gambierol. The gambierol IC₅₀ values reported herein as a functional antagonist of PbTx-2 in CGN cultures are in reasonable agreement with the K_i value for gambierol inhibition of [³H]PbTx-3 binding reported both here and by Inoue et al. (2003). Finally, we have also demonstrated that gambierol, like brevenal, although being capable of binding to neurotoxin site 5 of the VGSC, does not elicit a neurotoxic response as is typically observed for activators of this site. As a ligand of the therapeutically relevant VGSC, gambierol may provide a valuable tool in efforts to more clearly define the structure-function relationships of this voltage-sensitive ion channel, and it may provide possible leads for development of novel analgesics, neuroprotectants, and drugs used to treat cystic fibrosis.

	Acknowledgements

 
We thank Dr. Andrea Bourdelais for providing the brevenal sample.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants GM56677 (to J.D.R.) and NS053398 (to T.F.M.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.124271.

ABBREVIATIONS: CTX, ciguatoxin; PbTx, brevetoxin; VGSC, voltage-gated sodium channel; CGN, cerebellar granule neuron; DIC, days in culture; LDH, lactate dehydrogenase; CI, confidence interval.

Address correspondence to: Dr. Thomas F. Murray, Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178. E-mail: tfmurray{at}creighton.edu

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