Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia

Kevin T. Bush1, Tatsuo Tsukamoto2, and Sanjay K. Nigam1

1 Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0693; 2 3rd Division, Department of Medicine, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Ischemic epithelial cells are characterized by disruption of intercellular junctions and loss of apical-basolateral protein polarity, which are normally dependent on the integrity of the adherens junction (AJ). Biochemical analysis of both whole ischemic kidneys and ATP-depleted Madin-Darby canine kidney (MDCK) cells demonstrated a striking loss of E-cadherin (the transmembrane protein of the AJ) with the appearance and accumulation of an ~80-kDa fragment reactive with anti-E-cadherin antibodies on Western blots of ATP-depleted MDCK cells. This apparent ischemia-induced degradation of E-cadherin was not blocked by either inhibitors of the major proteolytic pathways (i.e., proteasome, lysosome, or calpain), or by chelation of intracellular calcium, suggesting the involvement of a protease capable of functioning at low ATP and low calcium levels. Immunocytochemistry revealed the movement of several proteins normally comprising the AJ, including E-cadherin and beta -catenin, away from lateral portions of the plasma membrane to intracellular sites. Moreover, rate-zonal centrifugation and immunoprecipitation with anti-E-cadherin and anti-beta -catenin antibodies indicated that ATP depletion disrupted normal E-cadherin-catenin interactions, resulting in the dissociation of alpha - and gamma -catenin from E-cadherin and beta -catenin-containing complexes. Because the generation and maintenance of polarized epithelial cells are dependent upon E-cadherin-mediated cell-cell adhesion and normal AJ function, we propose that the rapid degradation of E-cadherin and dissolution of the AJ is a key step in the development of the ischemic epithelial cell phenotype. Furthermore, we hypothesize that the reassembly of the AJ after ischemia/ATP depletion may require a novel bioassembly mechanism involving recombination of newly synthesized and sorted E-cadherin with preexisting pools of catenins that have (temporally) redistributed intracellularly.

Madin-Darby canine kidney; adenosine 5'-triphosphate; adherens junction; polyacrylamide gel electrophoresis; adenosine 5'-triphosphate depletion; antimycin A; 2-deoxyglucose; plakoglobin; cell adhesion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

TO FUNCTION PROPERLY, EPITHELIAL cells depend on the integrity of intercellular junctions [e.g., adherens (AJ), tight, gap, desmosomes] and arrangement of plasma membrane lipids and proteins into strictly maintained apical and basolateral domains. Ischemia and subsequent reperfusion/reoxygenation perturb epithelial function by disrupting intercellular junctions (11, 19, 21, 30, 31), protein polarization (11, 19, 21), the actin-based cytoskeleton (1, 11), and folding mechanisms in the endoplasmic reticulum (17, 18). Although much of this dysfunction can ultimately be traced back to rapid and severe alterations in ATP, free radicals, intracellular pH, and calcium, the precise mechanisms involved in the generation of the ischemic epithelial cell phenotype remain poorly understood.

In models of polarized epithelial biogenesis, the generation and maintenance of apical/basolateral polarity has been shown to be linked to AJ assembly. In particular, calcium-dependent cell-cell adhesion via homophilic interactions of the extracellular domain of E-cadherin (the transmembrane protein of the AJ) between adjacent cells has been found to be critically important in the formation of tight polarized epithelia (12, 13, 29). For example, the addition of either calcium chelators or anti-E-cadherin antibodies to the culture medium has been shown to block the formation of intercellular junctions and the development of apical-basolateral polarity in kidney-derived Madin-Darby canine kidney (MDCK) epithelial cells (12). Furthermore, transfection of nonpolarized fibroblasts with E-cadherin has been found to induce the movement of Na-K-ATPase from a uniform distribution on the plasma membrane to sites of cell-cell contact, in a manner reminiscent of polarizing epithelial cells (20). Along with several similar studies, these findings indicate that E-cadherin-mediated cell-cell contact initiates a cascade of events leading to the formation of polarized epithelial cells (10, 13, 23, 29). E-cadherin also interacts with several cytoplasmic proteins (alpha -,beta -, and gamma -catenin) and the cytoskeleton to form the AJ. Together, these intra- and extracellular interactions provide the basis for many properties characteristic of the AJ (6, 9, 14, 22, 25, 32, 33). However, despite its importance in the generation and maintenance of the tight polarized epithelial cell, much remains to be understood about the effects of ischemia on the AJ and the proteins that comprise it.

Here we show that ischemia and/or ATP depletion induces distinct biochemical lesions in the AJ. Analysis of ischemic whole kidney and ATP-depleted cultured MDCK cells demonstrates that an ischemic insult results in the selective degradation of E-cadherin and the disruption of the protein-protein interactions between E-cadherin and the catenins that comprise the AJ. These findings suggest that alterations in E-cadherin, as well as its interactions with the cytoplasmic components of the adherens junction, constitute a key lesion in epithelial ischemia. Moreover, because the maintenance of polarized epithelial cells is critically dependent on E-cadherin-mediated cell-cell contact, the results also provide a potential unifying mechanism for generation of the ischemic epithelial cell phenotype, including the loss of polarity and the disruption of other intercellular junctions.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Reagents and chemicals. Antibodies against E-cadherin were either isolated from hybridoma cells (rr-1) kindly provided by Barry Gumbiner (Univ. of California-San Francisco) or purchased from Sigma Chemical (St. Louis, MO) and Transduction Laboratories (Lexington, KY). Antibodies against alpha -, beta -, and gamma -catenin were from either Transduction Laboratories or Sigma. Antimycin A and 2-deoxyglucose were also from Sigma.

Renal ischemia and ATP depletion. For the in vivo studies of epithelial ischemic injury, adult Sprague-Dawley rats were subjected to 0-3 h of renal ischemia without reperfusion as previously described (17). For the in vitro analysis of epithelial ischemia, confluent monolayers of MDCK cells (~106 cells/100-mm2 dish) growing in Dulbecco's modified minimal essential medium (supplemented with 5% FCS and antibiotics) were depleted of ATP for 0-6 h by incubation in PBS (supplemented with 1.5 mM CaCl2 and 2 mM MgCl2) containing 2 mM deoxy-D-glucose and 10 µM antimycin A (30, 31).

Confocal immunofluorescent microscopy. Confluent MDCK cell monolayers growing on coverslips subjected to ATP depletion were fixed in methanol, permeabilized with saponin, reacted with antibodies against components of the AJ, and examined with a Bio-Rad confocal microscope as described (30, 31).

Immunoprecipitation. Control and ATP-depleted monolayers of MDCK cells were rinsed gently with PBS, lysed for 30 min at 4°C in 1 ml of immunoprecipitation buffer (27), and subjected to immunoprecipitation with anti-E-cadherin antibodies (rr-1) and anti-beta -catenin antibodies by using methods previously described (5, 17, 30, 31).

Rate-zonal centrifugation. Monolayers of MDCK cells were grown in 6-well cluster plates and subjected to 0-4 h of ATP depletion as described above. The cells were rinsed twice in PBS, solubilized for 30 min at 4°C in 1 ml immunoprecipitation buffer (27), and cleared of insoluble matter. The supernatant was then layered on top of 5-20% linear sucrose gradients created in the immunoprecipitation buffer without detergents and subjected to rate-zonal centrifugation (17, 30, 31).


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To investigate the effects of ischemia on E-cadherin and the AJ, we employed an ischemic whole kidney preparation as well as ATP depletion of MDCK cells, a well-characterized cell culture model of epithelial cell ischemia that reproduces many of the lesions seen in epithelial ischemic injury but has the advantage of being amenable to detailed biochemical analysis (4, 8, 11, 17, 19, 30, 31, 34).

ATP depletion and whole tissue ischemia induces selective degradation of E-cadherin. Western blot analysis of ATP-depleted MDCK cell lysates revealed the highly reproducible loss of the 120-kDa band of E-cadherin and the appearance of an ~80-kDa fragment that was also recognized by the anti-E-cadherin antibody (rr-1) (Fig. 1A). This smaller band was either not present in control cells or apparent only as a faint band; however, as ATP depletion proceeded, this band progressively increased, whereas the 120-kDa band diminished (Fig. 1, A and B). More than one-half of the 120-kDa form of E-cadherin was reproducibly degraded by 2 h and, by 4 h of ATP depletion, the 80-kDa fragment represented the predominant band detectable on the blots with the anti-E-cadherin antibody (Fig. 1B). In contrast, there was no similar striking loss of the cytosolic catenins detectable even after 6 h of ATP depletion, a point at which little or no 120-kDa form of E-cadherin is detectable (Fig. 1C). Thus ATP depletion of cultured MDCK cells induced a rapid and selective loss of E-cadherin under conditions when ATP levels are extremely low (<10% of control) (4, 17, 31).


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Fig. 1.   Effect of ATP depletion (ATPd) and whole kidney ischemia on proteins of adherens junction (AJ). A: whole cell lysates of Madin-Darby canine kidney (MDCK) cells subjected to 0-6 h of ATP depletion were analyzed by SDS-PAGE and Western blot analysis for the presence of E-cadherin. B: graph indicating relative amounts of 120-kDa form of E-cadherin (open bars) and ~80-kDa fragment (grey bars) on Western blots of MDCK cells subjected to 0-6 h of ATP depletion. Summation of bars is equivalent to total amount of 120-kDa and 80-kDa bands present on immunoblots. Data are presented as a percentage of control [means ± SE (n = 3)]. C: Western blot analysis of levels of alpha -, beta -, and gamma -catenin present in cells lysates from MDCK cells depleted of ATP for 0-6 h. D: aliquots of whole kidneys subjected to 0-3 h of ischemia were analyzed by Western blot for presence of either E-cadherin or beta -catenin.

Analysis of ischemic whole rat kidney demonstrated a similar phenomenon in vivo. Western blots of ischemic or nonischemic kidneys revealed a clear and consistent reduction in the total amount of E-cadherin after 3 h of ischemia (Fig. 1D), suggesting that the loss of E-cadherin is not only a phenomenon of cultured cells. Because of interspecific differences in reactivity, the antibody used to detect E-cadherin in the whole kidney preparation was different from that used in MDCK cells; this antibody does not appear to react with the 80-kDa band on the blots. Nevertheless, as with the cell culture model, there was a clear loss in the total amount of E-cadherin detectable on Western blots after 3 h of ischemia in whole kidney. Similar to ATP depletion of MDCK cells, the amount of beta -catenin detectable on Western blots was only slightly altered by whole tissue ischemia (Fig. 1D). Thus the observed loss of E-cadherin (under conditions of ATP depletion or ischemia) appears to be a highly selective event occurring in both cultured cells and in vivo.

It is important to note that the 80-kDa band that we have identified is likely different from the soluble 80-kDa fragment of E-cadherin that is either released into the media of cultured human carcinoma cells, generated by trypsinization of epithelial cells in the presence of calcium (7), or found circulating in the serum of cancer patients (15). Because this fragment is cleaved from the cell surface and is lost into the extracellular media or serum, one would not expect to detect it on Western blots of cell lysates, as is the case with the 80-kDa fragment described here. In addition, this extracellular cleavage fragment of E-cadherin would not be able to interact with the cytosolic catenins, whereas the 80-kDa fragment described in this study can be immunoprecipitated with beta -catenin (see below) and is also coimmunoprecipitated with anti-beta -catenin antibodies (see below). Moreover, taken together with the fact that ~95% of surface-accessible E-cadherin is internalized within the first 60 min of ATP depletion of MDCK cells (19), the 80-kDa fragment described here is probably the result of an intracellular degradation event, rather than an extracellular cleavage that gives rise to the 80-kDa fragment found in the media or serum.

The single and highly reproducible 80-kDa fragment observed after the ATP depletion of MDCK cells suggests that a highly specific cleavage of E-cadherin is occurring. However, relatively high concentrations of a number of proteolytic inhibitors that block degradation via different proteolytic pathways, including proteasomal, lysosomal, and calpain-mediated mechanisms, had no apparent effect on the loss or cleavage of E-cadherin, or the generation of the 80-kDa fragment (Fig. 2, lanes 1-8). These data raise the possibility that an intracellular, potentially novel, protease capable of operating at low ATP levels (or independent of ATP) is involved in the degradation of E-cadherin. Moreover, because treatment with cell-permeant calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM; 200 µM) and dimethyl-BAPTA-AM (100 µM-data not shown), concentrations more than twice as high as those found to reduce intracellular calcium concentration [Ca2+]i in ATP-depleted MDCK cells by >60%) (34) had little or no effect on the loss of E-cadherin (Fig. 2, lanes 9-10), the candidate protease is also unlikely to be dependent on increased levels of intracellular calcium. (This is also consistent with the lack of inhibition with blockers of calpain activity.)


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Fig. 2.   Neither inhibition of proteolysis or chelation of intracellular calcium can block ATP-depletion-induced degradation of E-cadherin. Western blot analysis of E-cadherin in lysates of MDCK cells subjected to 0-4 h of ATP depletion in absence or presence of either inhibitors of cellular proteolysis (lanes 3-8) or a cell-permeant chelator of intracellular calcium (lane 10). lanes 1 and 9: controls, no ATP depletion; lane 2: control, 4 h of ATP depletion without agents; lane 3: 250 µM aLLN; lane 4: 250 µM aLLM; lane 5: 250 µM E64; lane 6: 250 nM calpeptin; lane 7: 500 µM chloroquine; lane 8: 500 µM primaquine; lane 10: 200 µM BAPTA-AM. Inhibitors block different proteolytic pathways, including proteasomal (aLLN and aLLM), calpain-mediated (aLLN, aLLM, calpeptin, and E64), and lysosomal (chloroquine and primaquine) pathways [MG132 and lactacystin, additional proteasome inhibitors, also had no effect on E-cadherin degradation or cleavage (data not shown)] .

ATP depletion perturbs the cellular localization of components of the adherens junction. To further analyze the fate of the proteins comprising the AJ, immunocytochemical analysis was performed. In control cells, E-cadherin was localized to lateral aspects of the plasma membrane in a continuous linear staining pattern with only a modest amount of apparent intracellular staining (Fig. 3A). Although there was some intracellular staining for each of the catenins in control cells, these three proteins were also primarily found in a linear pattern at the lateral portions of the plasma membrane (Fig. 3, C, E, and G). After 4 h of ATP depletion, although there was little change in the localization of gamma -catenin, (Fig. 3H), the staining pattern for E-cadherin, and beta -catenin, and to a lesser extent alpha -catenin, was altered. For example, the linear staining at the level of the plasma membrane for E-cadherin appeared to be thinner and more discontinuous (Fig. 3B) and was accompanied by the appearance of punctate intracellular staining for this protein (Fig. 3B). Although a portion of both alpha - and beta -catenin remained localized at the lateral portions of the plasma membrane, 4 h of ATP depletion resulted in an increase in the intracellular staining of both of these proteins as well (Fig. 3, D and F). Because E-cadherin is known to be inaccessible to externally added biotin after 1 h of ATP depletion in MDCK cells (19), these immunocytochemical data support the notion that ATP depletion leads to internalization of E-cadherin as well as other components of the AJ. If, as the data suggest, E-cadherin is internalized and degraded in response to ATP depletion, this raises an interesting question: how are the homophilic interactions of E-cadherin disrupted in the presence of normal levels of extracellular calcium (see EXPERIMENTAL PROCEDURES)? It is possible that alteration of the phosphorylation state of the E-cadherin-catenin complex during ATP depletion affects E-cadherin homophilic binding, allowing for the internalization of the protein and its subsequent degradation. This notion is not without precedent because the phosphorylation states of beta -catenin, gamma -catenin, p120, as well as E-cadherin have been shown to be critical to maintenance of cell-cell adhesion in v-SRC-transformed MDCK cells and in mitotic MDCK cells (2, 3, 28).


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Fig. 3.   ATP depletion alters the distribution of the proteins of the AJ. Confocal microscopic immunocytochemical localization of E-cadherin (A and B), alpha -catenin (C and D), beta -catenin (E and F) and gamma -catenin (G and H) at the level of the AJ in MDCK cells subjected to 0-4 h of ATP depletion.

ATP depletion alters E-cadherin-catenin interactions of the AJ. Under normal physiological conditions, the cytoplasmic domain of E-cadherin associates directly with either beta -catenin or gamma -catenin, which in turn associate with alpha -catenin. alpha -catenin, which can interact with alpha -actinin and actin filaments, mediates the interaction between the cadherin-catenin complex and the actin cytoskeleton (16, 24, 26). To determine whether, as a result of E-cadherin degradation, this complex of E-cadherin with the other components of the AJ was altered, sucrose density gradient analysis was performed. Western blot analysis of the gradient fractions revealed that, in control cells, E-cadherin was found in the medium density fractions of a 5-20% sucrose gradient as a 120-kDa protein (Fig. 4A). However, after 4 h of ATP depletion, the amount of the 120-kDa form of E-cadherin was markedly reduced and was now found in the lower density fractions of the gradient (Fig. 4A). The ~80-kDa form of E-cadherin was now apparent and was also found in this same low-density region of the gradient (Fig. 4A). This behavior of E-cadherin is very different from certain tight junction proteins, which fractionate as a high-density complex after ATP depletion (31), and suggests that the normal protein-protein interactions involving E-cadherin are disrupted in the face of ATP depletion.


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Fig. 4.   ATP depletion disrupts the AJ. A: aliquots of lysates of MDCK cells subjected to 0-4 h of ATP depletion were layered on top of 5-20% sucrose density gradients and centrifuged to equilibrium. Gradients were fractionated and analyzed by SDS-PAGE followed by Western blot analysis with antibodies against E-cadherin, alpha -, beta -, and gamma -catenin. Immunoblots were quantified by using a HP Scanjet II scanner and NIH-IMAGE software, and results for E-cadherin are represented graphically. B: lysates of MDCK cells subjected to 0-6 h of ATP depletion were subjected to immunoprecipitation with anti-E-cadherin antibody. Western blots of the immunoprecipitates were probed with antibodies against E-cadherin, alpha -, beta -, and gamma -catenin. C: lysates of MDCK cells subjected to 0-4 h of ATP depletion were subjected to immunoprecipitation with anti-beta -catenin antibody. Western blots of immunoprecipitates were probed with antibodies against E-cadherin.

To test this hypothesis, Western blots of immunoprecipitates of E-cadherin were analyzed. In control cells, E-cadherin antibodies were able to coimmunoprecipitate many known components of the AJ (Fig. 4B). Thus E-cadherin, as well as alpha -, beta -, and gamma -catenin were all detectable on Western blots of E-cadherin immunoprecipitates. However, after 1-2 h of ATP depletion, a time at which the 120-kDa E-cadherin is still the major form present in MDCK cells, gamma -catenin was barely detectable on Western blots (Fig. 4B). Continued ATP depletion resulted in the dissociation of alpha -catenin, whereas beta -catenin was still detectable on Western blots of E-cadherin immunoprecipitates even after 6 h of ATP depletion (Fig. 4B). Thus ATP depletion caused a rapid disruption of the protein-protein interactions between E-cadherin and alpha - and gamma -catenin. ATP depletion would thus appear to affect intracellular transduction mechanisms between E-cadherin and the actin-based cytoskeleton by dissolving links mediated by the catenins, which are thought to be crucial to apical-basolateral protein polarization. Interestingly however, the linkages between E-cadherin and beta -catenin appear to remain intact. In fact, immunoprecipitation with anti-beta -catenin antibodies was able to coimmunoprecipitate both full-length E-cadherin as well as the 80-kDa fragment of E-cadherin (Fig. 4C). This finding not only shows that the E-cadherin and beta -catenin remain associated but also provides strong support for the notion that 80-kDa cleavage fragment is different from the extracellular fragment of E-cadherin that is found in the media of trypsinized epithelial cells (7), or that that is found circulating in the serum of cancer patients (15).

We argue here that the rapid disruption of the protein-protein interactions of the components of the AJ, as well as the loss of E-cadherin itself, constitute a key lesion in epithelial ischemia. Because the integrity of intercellular junctions and protein polarization in epithelial cells is critically dependent on E-cadherin-mediated cell-cell contact, as well as E-cadherin's links to the actin-based cytoskeleton mediated through the catenins, these findings also suggest a unifying mechanistic explanation for much that has been observed in the ischemic epithelial cell phenotype, including the disruption of tight junctions and the loss of apical-basolateral polarity. The combination of degradation of full-length E-cadherin and the loss of E-cadherin-catenin interactions, together with the internalization of E-cadherin and alpha - and beta -catenin, could explain the disruption of productive cell contacts which occurs in ischemia. In a very broad sense, this model is something of a reversal of that proposed for the role of E-cadherin and the AJ in the development of polarized epithelial cells, in which the homophilic binding of E-cadherin between adjacent cells initiates a cascade of events that lead to the formation of other intercellular junctions and the development of apical-basolateral polarity.

The centrality of E-cadherin to epithelial polarization and biogenesis of the permeability barrier [two key features of the normal polarized epithelia that are disrupted in ischemia (11, 19, 31)] suggests that our results also bear in important ways on the recovery of epithelial function after ischemic injury. E-cadherin is selectively and rapidly degraded after ATP depletion. Taken together with the finding that ATP depletion disrupts the normal protein-protein interactions that comprise the AJ, these data indicate that the recovery of the tight polarized epithelial cell phenotype is dependent on the reassembly of the AJ. However, it remains unclear how the AJ is reassembled after ischemic injury. Under normal physiological conditions, E-cadherin interacts with either beta -, or gamma -catenin at the level of the ER. Soon after the arrival of this complex at the plasma membrane, alpha -catenin is loaded onto this complex, allowing for interaction with the actin cytoskeleton (13). In the cell recovering from ATP depletion (and presumably nonlethal ischemic injury), it seems reasonable to hypothesize that a preexisting cytosolic pool of catenins (that had redistributed intracellularly after ATP depletion) is recruited by newly synthesized and folded E-cadherin during recovery of the AJ. However, it remains possible that the cell initially utilizes preformed AJ proteins (catenins and any remaining 120-kDa E-cadherin) to rapidly reestablish cell-cell adhesion, with subsequent synthesis and assembly of new AJs. Nevertheless, new synthesis of E-cadherin (and, after severe injury, possibly the catenins as well) is likely to be necessary for the AJ and the polarized epithelial cell to fully recover from ischemic injury.


    ACKNOWLEDGEMENTS

This work was supported in part by a RO1 grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (to S. K. Nigam) and by a Scientist Development Award from the American Heart Association (to K. T. Bush). This work was done during the tenure of an American Heart Association Established Investigatorship (to S. K. Nigam).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. K. Nigam, Univ. of California, San Diego, Dept. of Medicine (0693), 9500 Gilman Dr., La Jolla, CA 92093-0693. E-mail: snigam{at}ucsd.edu

Received 13 August 1999; accepted in final form 30 November 1999.


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ABSTRACT
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RESULTS AND DISCUSSION
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