Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
Submitted 11 September 2002 ; accepted in final form 3 November 2003
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ABSTRACT |
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Rana catesbeiana; sodium bicarbonate cotransport; injury and repair
Glycolytic activity during restitution poses a special problem to migrating epithelial cells after injury because lactic acid (lactate/H+) is produced by lactate dehydrogenase and must be extruded or neutralized to maintain intracellular pH (pHi). Cell acidification after injury is also a problem specific to the stomach, because gastric acid secretion results in a high luminal H+ concentration that can diffuse freely into migrating epithelial cells. Thus the activation of ion transport activity must be required to maintain a neutral pHi in migrating cells after injury. Specifically, Na+/H+ exchange may be required to reduce the H+ concentration in migrating cells. Alternatively, /
cotransport may be required to neutralize pHi or to influence volume regulation that would be involved in cell flattening and subsequent repolarization during restitution. However, little is known about the pHi requirements for cell migration in general or the role of ion transport in restitution after injury in the gastric mucosa or skin.
Thus the present study was done to determine which ions/ion transporters are required during repair after injury by using the bullfrog restitution model. We found that amiloride, bumetanide, or Cl--free conditions do not inhibit restitution, suggesting that Na+/H+ exchange, /
exchange, and Na+-K+-2Cl- cotransport are not required for restitution in the bullfrog gastric mucosa. In contrast, repair of mucosal transepithelial resistance (TER) and barrier function after injury are dependent on Na+. Our data demonstrate, however, that Na+ is required for attachment of cells that repopulate the gastric surface after injury rather than having a specific role in mechanisms that regulate restitution per se. Our data show that DIDS, which blocks Na+-driven
transport, completely inhibits recovery of TER and barrier function after injury. We show that restitution fails to occur because DIDS retards and/or blocks epithelial cell migration after injury. Because ion substitution for
, required for activity of the
/
cotransporter, did not inhibit restitution, we conclude that DIDS must block a yet undefined pathway not involved in
ion transport but essential for cell migration after injury and restitution in the gastric mucosa.
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MATERIALS AND METHODS |
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Electrophysiology experiments. For most of the studies, tissues were used in the resting state with no added stimulant or inhibitor of acid secretion. However, to determine whether is required for restitution, tissues were treated for 1 h before injury with 0.3 mM omeprazole in 0.1% DMSO, which will inhibit acid secretion completely for 24 h. Tissues were injured by exposure of the luminal surface to 1 M NaCl for 10 min as described in detail by Svanes et al. (21). After injury, the tissues were washed extensively, and both luminal and nutrient solutions were replaced. One tissue was used as a control and incubated in luminal and nutrient solutions, described in Preparation of tissues for restitution studies. The other tissue was subjected to one of the following treatments. First, Na+-free conditions were established. The luminal solution consisted of (in mM): 87.8 lithium, 4.0 K+, 0.8 Mg2+, 91.8 Cl-, 2.0 Ca2+, 2.8
, and 41.2 mannitol and was continuously gassed with 100% O2. The nutrient solution consisted of (in mM) 84.6 lithium, 17.8 choline, 4.0 K+, 0.8 Mg2+, 91.8 Cl-, 0.8
, and 10.0 glucose (pH 7.4) and was continuously gassed with 95% O2-5% CO2. Second, 1 mM amiloride in 0.1% DMSO was added to both luminal and nutrient solutions (control solutions, above) to inhibit activity of the Na+/H+ exchanger. DMSO (0.1%) was added to both luminal and nutrient solution in control tissues. Amiloride was used at a luminal pH (pHL) of 4.0 and 7.4. Third, 100 µM bumetanide in 0.1% DMSO was added to both luminal and nutrient solutions (control solutions, above) to inhibit activity of the Na+-K+-2Cl- cotransporter. DMSO (0.1%) was added to luminal and nutrient solutions in control tissues. Fourth, Cl--free conditions were established. The luminal solution consisted of (in mM) 104.1 Na+, 4.0 K+, 0.8 Mg2+, 108.1 gluconate, 2.0 Ca2+, 2.6
, and 10 mannitol and was continuously gassed with 100% O2. The nutrient solution consisted of (in mM) 104.1 Na+, 4.0 K+, 0.8 Mg2+, 89.5 gluconate, 2.0 Ca2+, 2.6
, and 10 glucose (pH 7.4) and was continuously gassed with 95% O2-5% CO2. Fifth, 500 µM DIDS was added (directly) to both luminal and nutrient solutions (control solutions, above) to inhibit activity of the
/
cotransporter and
/
exchanger. Sixth,
-free conditions were established. The luminal solution was the same as the control luminal solution, described above. The nutrient solution consisted of (in mM) 102.4 Na+, 4.0 K+, 0.8 Mg2+, 91.8 Cl-, 2.0 Ca2+, 0.8
, 17.8 HEPES, and 10 glucose (pH 7.4) and was continuously gassed with 95% O2-5% CO2. Tissues were monitored under the above conditions for 4 h (240 min) to evaluate recovery of transmucosal epithelial resistance (TER) after injury. TER was calculated from Ohm's law by using measurements of potential difference that were monitored continuously by KCl-saturated agar bridges connected via two calomel electrodes to a voltmeter. All ion transport inhibitors and buffer components were purchased from Sigma (St. Louis, MO).
Mannitol flux studies. The flux of [3H]mannitol from luminal to serosal solutions after injury was used to determine how blockade of ion transport activity affects mucosal permeability after injury. [3H]mannitol (50 µCi, 1530 Ci/mmol; New England Nuclear Life Science Products, Boston, MA) was added to the luminal solution (containing 19.3 mM of cold mannitol, except for Na+-free conditions in which there was 41.2 mM mannitol) immediately after injury. Aliquots (0.25 ml) were taken every 30 min from the nutrient solution and replaced with an equal volume of unlabeled nutrient buffer. Samples were diluted with 3 ml of scintillation fluid (Atomlight; New England Nuclear Life Science Products), and the amount of 3H in each sample was determined in a liquid scintillation counter (Packard Instruments, Meriden, CT). Mucosal-to-serosal flux was calculated by standard techniques.
Preparation and analysis of tissues after restitution studies. Tissues were fixed for 10 min at room temperature in the chamber and then overnight at 4°C in 2.0% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Tissues were postfixed with 1% OsO4 in 0.1 M cacodylate buffer, stained en bloc with aqueous 2% uranyl acetate, dehydrated through graded alcohols and propylene oxide, and then embedded in LX112 resin.
Thick sections (0.5 µm), stained with toluidine blue for light microscopy, were used to evaluate restitution in each experiment. All samples were coded, and morphological evaluation was conducted by four investigators without foreknowledge of the tissue source. Each tissue was evaluated and scored from 0 to 5, as follows. A score of 0 indicated that the apical surface of the mucosa was denuded without cell migration from the gastric pits (no restitution). A score of 1 indicated that <25% of the apical surface was covered with flattened surface epithelial cells. A score of 2 indicated that 2550% of the apical surface was covered with flattened surface epithelial cells. A score of 3 indicated that 5075% of the apical surface was covered with flattened epithelial cells, and a score of 4 indicated that 100% of the apical surface was covered with flattened epithelial cells. Finally, a score of 5 indicated that 100% of the apical surface was covered by cuboidal or columnar, rather than flattened, epithelial cells. After analysis by each investigator, the code was broken, and the results were analyzed as described below.
Statistical analysis of results. Combined data were expressed as means ± SE. Statistical analysis was done with SigmaStat software (Jandel Scientific Software, San Rafael, CA) using the unpaired t-test. Differences were regarded as significantly different at P < 0.05.
RESULTS
Injury and repair in control gastric mucosa. Exposure of the bullfrog gastric mucosa to 1 M NaCl (luminal) for 10 min induced an immediate decrease in TER (Fig. 1A). After the tissues were washed extensively and both luminal and nutrient buffers replaced with fresh buffer, TER recovered gradually to 86% of preinjury levels within 240 min (Fig. 1A). Flux of mannitol was 0.44 µM·h-1·cm-2 for the first 90 min after injury (initial flux) but was reduced eightfold (final flux) to 0.055 µM·h-1·cm-2 by 120240 min after injury (Fig. 1B). From here forward, flux for the first 90 min after injury will be called initial flux and the flux 150240 min after injury will be called final flux. Morphological analysis of control tissues 4 h after injury showed that 100% of the denuded surface was populated with surface epithelial cells that were cuboidal or columnar in shape (Figs. 1C and 2A). The mean histological score of control tissues was 4.31 (Fig. 1C).
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Effect of Na+-free solutions on repair of the gastric mucosa after injury. Tissues were incubated for 60 min with Na+-free solutions before injury. With the use of a combination of lithium and choline, it was found that TER did not increase significantly under Na+-free conditions (Fig. 1A). Exposure of tissues to 1 M NaCl for 10 min induced an immediate decrease in TER (Fig. 1A). After the tissues were washed extensively and both luminal and nutrient buffers were replaced with Na+-free solutions, there was only minimal recovery of TER within 240 min (Fig. 1A). Flux of mannitol under Na+-free conditions was linear over time (30240 min) with a rate of 0.71 µM·h-1·cm-2 (Fig. 1B). Rate of mannitol flux was higher under Na+-free conditions due to a higher concentration of mannitol in Na+-free luminal buffer (41.8 mM) compared with control luminal buffer (17.8 mM). Morphological analysis of tissues in Na+-free conditions after injury showed that most (75%) of the denuded surface was populated with surface epithelial cells that were columnar or cuboidal in shape (Figs. 1C and 2B). However, surface epithelial cells were not attached to the underlying lamina propria (Fig. 2B). The mean histological score of tissues treated with Na+-free conditions was 3.80 (Fig. 1C).
To determine whether NaCl injury after preincubation with Na+-free solutions is comparable with injury in control buffer, we evaluated the morphology of tissues in Na+-free to control buffers 60 min after injury. This concern arose because all of the known transporters required for cell shrinkage and swelling, which forms the basis of osmotic injury caused by NaCl, were blocked in Na+-free conditions. In control tissues, the surface was denuded of epithelial cells, and cell migration was minimal at this time point (not shown). Similarly, the surface was denuded of epithelial cells, and cell migration was minimal in tissues incubated with Na+-free solutions at 60 min after injury (Fig. 2C). Thus surface epithelial cells that are present 240 min after injury in Na+-free conditions (Fig. 2B) must represent new cells that populate the surface after injury.
Effects of amiloride on repair of the gastric mucosa after injury. Before injury, tissues were incubated for 60 min with 1 mM amiloride to block activity of the Na+/H+ exchanger. It was of interest to determine whether this exchanger would be required for restitution when surface cells are exposed to an acidic pHL and/or to a neutral pHL. Thus we examined the role of amiloride in restitution at pHL 4.0 or 7.4 (Fig. 3).
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Before injury, incubation with amiloride at either pHL increased TER compared with control tissues (Fig. 3, A and B). Exposure of amiloride-treated tissues to 1 M NaCl for 10 min induced an immediate decrease in TER (Fig. 3, A and B). After the tissues were washed extensively and both luminal and nutrient buffers were replaced with buffer containing amiloride, TER recovered to 55 (pHL 7.4)65% (pHL 4.0) of preinjury levels within 240 min (Fig. 3, A and B). This rate of recovery after injury was not significantly different from that of tissues treated with buffer alone. Flux of mannitol was identical at a pHL of 4.0 (not shown) or 7.4 in which the initial flux of 0.548 µM·h-1·cm-2 was reduced fourfold to 0.135 µM·h-1·cm-2 by 150240 min after injury (Fig. 3C). Morphological analysis of control tissues 240 min after injury showed that 100% of the denuded surface was populated with surface epithelial cells that were cuboidal or columnar in shape (Fig. 3D). Tissues treated with amiloride at either pHL 4.0 or 7.4 looked similar to the control tissue in Fig. 2A and had a mean histological score of 4.49 (4.0) and 4.23 (7.4), respectively (Fig. 3D).
Effects of bumetanide on repair of the gastric mucosa after injury. Before injury, tissues were incubated for 60 min with 100 µM bumetanide to block activity of the Na+-K+-2Cl- cotransporter (Fig. 4A). Exposure of tissues to 1 M NaCl for 10 min induced an immediate decrease in TER (Fig. 4A). After the tissues were washed extensively and both luminal and nutrient solutions were replaced with buffer containing bumetanide, TER recovered gradually within 240 min (Fig. 4A). There was no significant difference in the recovery of tissues treated with bumetanide compared with tissues treated with DMSO alone. The initial flux of mannitol was 0.498 µM·h-1·cm-2, and the final flux was reduced 5.6-fold to 0.097 µM·h-1·cm-2 (Fig. 4B). This rate of flux was nearly identical to that from tissues treated with DMSO alone. Morphological analysis of control tissues 4 h after injury showed that 100% of the denuded surface was populated with surface epithelial cells (Fig. 4C). Tissues treated with bumetanide looked similar to control tissues in Fig. 2A and had a mean histological score of 4.17 (Fig. 4C).
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Effects of Cl--free solutions on repair of the gastric mucosa after injury. Before injury, tissues were incubated for 60 min with Cl--free solutions in which Cl- was replaced with gluconate. Under this condition, TER increased significantly compared with control tissues (Fig. 4D). This would be an expected response in Cl--free conditions, because parietal cell acidic and nonacidic Cl- secretion is inhibited, which greatly affects (increases) TER (6). Exposure of tissues treated with Cl--free solutions to 1 M NaCl for 10 min induced an immediate decrease in TER (Fig. 4D). After the tissues were washed extensively and both luminal and nutrient buffers were replaced with Cl--free solutions, TER recovered gradually within 240 min (Fig. 4D). There was no significant difference in recovery of tissues treated with Cl--free conditions compared with tissues treated with buffer alone (Fig. 4D). Even if the data, due to the large starting resistance, were plotted as percentage of initial resistance (not shown), recovery is not significantly different after injury (P = 0.1215 at 240 min). Morphological analysis of tissues confirmed that restitution of the surface occurred with a mean histological score of 4.39 ± 0.14 (not shown).
Effects of DIDS on repair of the gastric mucosa after injury. Before injury, tissues were incubated for 60 min with 500 µM DIDS to block activity of the /
cotransporter and
/
exchanger. This concentration of DIDS did not reduce the viability of surface epithelial cells in a cell viability assay using rat gastric mucosal (RGM-1) cells. Exposure of tissues to 1 M NaCl for 10 min induced an immediate decrease in TER (Fig. 5A). After the tissues were washed extensively and both luminal and nutrient buffers were replaced with solutions containing DIDS, there was virtually no recovery of TER (Fig. 5A). Flux of mannitol in the presence of DIDS was linear over time (30240 min) with a rate of 1.1706 µM·h-1·cm-2 (Fig. 5B). This rate was significantly higher than either the initial or final rate of flux in control tissues (Fig. 5B). Morphological analysis of DIDS-treated tissues 240 min after injury had two results. First, more than half of the tissues (57%) showed no migration of surface epithelial cells in which the surface consisted only of a denuded basement membrane that was similar to Fig. 2C. Second, 38% tissues showed that some of the surface was covered with epithelial cells that were long and narrow with thin projections at the apical surface and no lamellapodia along the basement membrane (Fig. 6). In addition, many of the cells appeared to be attached only by small processes to the basement membrane or not attached at all (Fig. 6). The mean histological score of tissues treated with DIDS was 1.21 ± 0.259 (Fig. 5C).
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Effects of HEPES on repair of the gastric mucosa after injury. To determine whether blocking activity of the cotransporter and/or
/
exchanger affects restitution or whether the action of DIDS is independent of ion transport, HEPES was substituted for
in this set of experiments.
For the first study, tissues were washed exhaustively before injury until acid secretion decreased as much as possible, which was to 1.989 ± 0.546 µeq·h-1·cm-2. Under this condition, there was complete recovery of TER after injury, even in the absence of (Fig. 7A). Although initial and final fluxes of mannitol increased significantly in the absence of
(Fig. 7B), there was a distinct (3-fold) decrease in final flux that occurred in
-free conditions that did not occur with DIDS (compare Fig. 7B with Fig. 5B). In control tissues, initial and final rates of mannitol flux were 0.288 ± 0.012 and 0.084 ± 0.006 µmol·h-1·cm-2, respectively. In
-free conditions, initial and final rates of mannitol flux were 0.372 ± 0.018 and 0.126 ± 0.012 µmol·h-1·cm-2, respectively. Morphological analysis of tissues treated with
-free buffer showed no difference from control tissues, in that 100% of the surface was covered with cuboidal epithelial cells (Fig. 8). The mean histology score of tissues incubated with
-free buffer was 4.53 ± 0.102 compared with 4.47 ± 0.09 in control tissues (not shown).
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Because the small amount of that is generated during acid secretion may be adequate to facilitate restitution in our model, we inhibited acid secretion completely with omeprazole (before injury) to confirm the previous findings (Fig. 7, CD). With omeprazole, recovery of TER after injury was identical to that in control tissues (Fig. 7D). Likewise, recovery of tissues in
-free buffer with omeprazole was identical to tissues in control buffer and in
-free buffer without omeprazole (compare Fig. 7, A and C). Tissues incubated with
-free buffer with omeprazole had a mean histological score of 4.32 (Fig. 7C).
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DISCUSSION |
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Results presented here concerning the dependence of restitution on are in contrast to the seminal work by Svanes et al. (22), who showed that HEPES, substituted for
, completely inhibits restitution after injury in the bullfrog gastric mucosa. In that study, nutrient 5 mM HEPES was substituted for 18 mM
, and no restitution occurred at a pHL of 4.0. In contrast, 17.8 mM Na+-HEPES was substituted for 17.8 mM
-
in the present study, and restitution was not impaired at pHL 4.0. Our experimental design is different from that used by Svanes et al. (22), because we balanced osmolarity and Na+ concentrations in nutrient and luminal solutions. In addition, our procedure provides similar buffering capacity, which may be essential so that H+ back-diffusion and the resulting mucosal acidosis can be neutralized. In fact, our data support the notion that bicarbonate, HEPES, or any strong buffer would be effective at neutralizing acidosis and that bicarbonate, per se, is not required for restitution. This was supported experimentally in the bullfrog gastric mucosa by increasing the concentration of
from 18 to 47.8 mM, which allowed restitution to occur at a much lower pHL due to an increase in luminal alkalinization (22). In addition, Jousti et al. (10) showed no impairment of restitution in the guinea pig stomach when HEPES was exchanged equally for
, either with NaCl or Triton X-100 injury. Yanaka and colleagues (20, 26) routinely perform restitution experiments (using guinea pig stomach) in HEPES buffer rather than in
, providing further support that restitution is not dependent on
. Because restitution experiments can be performed in the absence of
, it is unlikely for restitution to rely solely on
transport to maintain pHi, including transport by the
/
exchanger or
/
cotransporter.
Studies by Jousti et al. (10) and Yanaka et al. (26), both using the guinea pig gastric mucosa, demonstrate that amiloride inhibits restitution and clearly identify a role for Na+/H+ exchange and the regulation of pHi in restitution. In contrast, our results in the bullfrog show that amiloride does not block restitution, either at neutral or acidic pHL. In the study by Jousti et al. (10), tissues that were subjected to blockade of Na+/H+ exchange with 1 mM amiloride showed only minimal recovery of TER after injury at a neutral pHL (pH 6.8). In contrast, Yanaka et al. (26) showed that amiloride blocked recovery of TER after injury by only 10% at pHL 7.0 and by 20% at pHL 3.0, suggesting that the activation of Na+/H+ exchange is particularly important for pH regulation when surface cells are subjected to an acidic pHL. Consideration must be given, however, to the experimental design in that study (26) in which HEPES buffer was used in experiments with amiloride so that
transport was also blocked. Thus it is unclear whether amiloride alone would inhibit restitution in the guinea pig stomach when
transport is active. Because amiloride did not block restitution in
-containing buffer at either pHL 4.0 or 7.4 in the present study, we suggest that at least one of the transporters must be active to facilitate restitution so that simultaneously blocking both H+ efflux via Na+/H+ exchange and
influx via
/
cotransport inhibits restitution after injury presumably by affecting pHi. Alternatively, it is possible that amiloride did not block the isoforms of Na+/H+ exchangers that are present in surface cells in our study. Because blocking both (Na+-dependent) pHi regulatory pathways in the guinea pig mucosa inhibited restitution by only 20% and not completely (26), there may be one or more redundant H+-exchange pathway(s) that is(are) Na+-independent. This was suggested in a study (18) in which blockade of Na+/H+ exchange with amiloride did not result in acidification of isolated rabbit surface cells subjected to Na+-free conditions, in which this treatment would be predicted to cause the uptake of H+ and cell acidification as it did in isolated gastric chief cells. Our finding that cell migration after injury can occur in Na+-free conditions when all of the currently known ion transporters that regulate pHi are blocked also supports the idea that a Na+-independent pHi regulatory pathway must exist in gastric surface cells.
Results presented here are also different from those recently reported by Jousti et al. (10) concerning the role of /
exchange in restitution. In that study, it was shown that incubation of the guinea pig gastric mucosa with 1 mM SITS, a stilbene compound that blocks
cotransport and
/
exchange, completely inhibits recovery of TER after injury induced by either NaCl or Triton X-100 (10). The morphology of tissues treated with SITS showed that there was localized but mainly incomplete restitution, and it was concluded that
/
exchange is required for restitution (10). However, ion substitution experiments exchanging HEPES for
did not inhibit restitution in that study (10), suggesting either that SITS significantly reduced the viability of surface epithelial cells so that restitution was impaired or that SITS inhibits a pathway that regulates restitution in the guinea pig mucosa by a mechanism other than by inhibiting ion transport. Recent studies (16, 18) with isolated cells showed that gastric surface cells have only a small amount of mRNA for AE2, the
/
exchanger, and that this exchanger is not involved in pH regulation in surface mucous cells. Thus because surface mucous cells are responsible for restitution of the gastric mucosa after injury, it is unlikely that blocking activity of the
/
exchanger would significantly influence restitution. It is more likely that both SITS and DIDS, a stilbene compound similar to SITS that was shown in this study to completely block restitution, may influence restitution by a mechanism independent of their action on ion transport. DIDS, in addition to blocking ion transport, inhibits ryanodine receptors (12, 13, 19, 24), purino(ATP) receptors (20), ATP-dependent K channels (17), and monocarboxylate transporters (MCTs) (9). The later are involved in H+-coupled lactate transport, a proton efflux pathway in white skeletal muscle, red blood cells, and many tumor cells that utilize glycolysis to generate ATP. Interestingly, MCT-1 strongly localizes to the basolateral membrane of gastric surface cells (7). Because restitution in the gastric mucosa depends on glycolysis to generate ATP (1) and is inhibited so potently by SITS and DIDS, future studies concerning the role of MCT-1 in the regulation of pHi and in restitution will be of great importance.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15681 (to S. J. Hagen) and DK-34854 (to Harvard Digestive Diseases Center).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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