COMMUNICATION
Autophosphorylation-dependent Targeting of Calcium/ Calmodulin-dependent Protein Kinase II by the NR2B Subunit of the N-Methyl- D-aspartate Receptor*

Stefan Strack and Roger J. ColbranDagger

From the Department of Molecular Physiology and Biophysics and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0615

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Activation and Thr286 autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx via N-methyl-D-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+ influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+ channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

CaMKII is a multifunctional, calcium-activated kinase (1, 2), whose alpha  and beta  isoforms are particularly abundant in brain cytosol and in postsynaptic densities (PSDs),1 submembranous scaffolds for receptors, ion channels, and signal transducers (3, 4). Postsynaptic calcium influx triggers autophosphorylation of CaMKII at a threonine residue in the autoinhibitory domain (Thr286 in CaMKIIalpha ) (5), which renders the kinase persistently active and causes a translocation of soluble CaMKII to the PSD (6). Multiple lines of evidence indicate Thr286 autophosphorylation of postsynaptic CaMKII is necessary for NMDA receptor-dependent LTP (7-11), a cellular model of learning and memory. PSD-associated CaMKII phosphorylates ionotropic glutamate receptors (6, 12-14), providing a mechanism for increased synaptic strength during LTP (15).

Mechanisms by which CaMKII is targeted to its postsynaptic substrates are poorly understood. Previous gel overlay analyses revealed a candidate PSD-associated CaMKII-anchoring protein, p190, that binds selectively to the Thr286-autophosphorylated kinase ([P-T286]CaMKIIalpha ) (16). The NR2A and NR2B subunits of the NMDA receptor share several properties with this CaMKII-binding activity, including apparent size, enrichment in PSDs, and regional and developmental expression profiles2 (17). Here, we demonstrate a direct and specific interaction between [P-T286]CaMKIIalpha and NR2B and show that NR2B targets CaMKII in intact cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Immunoprecipitations-- PSD isolation and immunoprecipitation of sodium dodecyl sulfate (SDS)-solubilized PSD proteins were carried out as described (6) using 2 µg/ml NR2A/B antibodies (Chemicon) and protein phosphatase 1 antibodies (18). For CaMKII·NMDA receptor coimmunoprecipitation, PSDs (1 mg/ml) were cross-linked (45 min, 4 °C) with 0.25 mM dithiobis(succinimidyl suberate), dissolved by sonication in 2% SDS, and diluted 15-fold in 1% Nonidet P-40, 200 mM NaCl, 50 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µM microcystin-LR. The supernatant after ultracentrifugation (30 min, 100,000 × g) was immunoprecipitated with 3 µg/ml goat anti-CaMKII (16) or preimmune IgG (19). The cross-linker was cleaved and proteins eluted from the beads by boiling in reducing SDS sample buffer.

CaMKII Gel Overlays-- Purified recombinant CaMKIIalpha was autophosphorylated with [gamma -32P]ATP (8,000-40,000 cpm/pmol) in the presence of calcium/calmodulin or EGTA at Thr286 or Thr305/306, respectively, and desalted (16). Stoichiometries ranged between 0.17 and 0.39 (Thr286) and 0.24 and 0.47 (Thr305/306). Protein blots to be analyzed for CaMKII binding were blocked and incubated with 100-200 nM [32P]CaMKII in 5% milk for 3 h, washed extensively, and autoradiographed.

Immunofluorescence-- 18-Day-old cultures of dissociated neonatal rat cortex were fixed in acetone:methanol (1:1), blocked, and incubated 10-14 h in 1:500 dilutions of goat anti-CaMKII (16), rabbit anti-NR1 (20), and mouse anti-synaptophysin (Boehringer Mannheim) in 1% normal donkey serum, 10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Triton X-100. Cultures were treated with species-specific donkey antibodies conjugated to Cy3, Cy2, and Cy5 (Jackson Laboratories) and imaged on a Zeiss laser scanning confocal microscope.

Generation and Analysis of NMDA Receptor Fusion Proteins-- The entire cytoplasmic domains (C terminus starting immediately after transmembrane region IV) of NR1 (splice variant A containing both C1 and C2 exon cassettes), NR2A, and NR2B subunits, as well as shorter NR2B constructs, were subcloned from full-length cDNAs by polymerase chain reaction using Pfu polymerase and primers containing restrictions sites or by restriction digests. Fragments were sequenced and ligated into pRSET-A His6 tag (Qiagen) or pGEX-2T glutathione S-transferase (GST) (Amersham Pharmacia Biotech) fusion vectors. His6 tag fusions were expressed, and GST fusions were expressed and purified according to the manufacturers' instructions. His6 tag fusion protein lysates were subjected to CaMKII overlay (see above) or immunoblotted with anti His6 tag antibodies (CLONTECH) and 125I-labeled secondary antibodies for expression levels, followed by PhosphorImager quantification.

Microtiter Plate Solution Binding-- Ni2+-coated 96-well plates (HisSorb strips, Qiagen) were adsorbed for 2 h with soluble His6 tag NR2B fusion protein expressing or nonexpressing bacterial extracts (0.25 mg/ml) in blocking buffer (5 mg/ml bovine serum albumin, 200 mM NaCl, 50 mM Tris, pH 7.5, 0.1% Tween 20, 5 mM beta -mercaptoethanol). After extensive washes, [32P-T286]CaMKIIalpha diluted in blocking buffer (200 µl) was allowed to bind to the tethered fusion protein for 2 h, followed by 10-12 more washes. Bound CaMKII was solubilized in 1% SDS, 0.2 N NaOH, 50 mM EDTA, and quantified by liquid scintillation counting. Nonspecific binding to control bacterial extracts was subtracted from total binding to obtain specific binding. No specific binding was observed using [32P-T306]CaMKIIalpha .

GST Pull-down Analysis-- GST fusion proteins were incubated (1 h, 4 °C) with either purified CaMKIIalpha (Fig. 2D, see caption) or with a freshly prepared rat brain cytosolic extract (~3 mg/ml extract protein, 10 µg/ml GST fusion protein) containing 2 µM microcystin-LR and 0.5% Triton X-100, precipitated with glutathione-agarose, washed extensively, and eluted with SDS sample buffer. CaMKIV antibodies were from Transduction Laboratories.

HEK293 Cell Colocalization-- HEK293 cells were seeded on coverslips in 35-mm dishes, transfected with a total of 3 µg/dish DNA (1 µg of SRalpha promotor-CaMKIIalpha expression plasmid, 2 µg of cytomegalovirus promotor plasmids with NMDA receptor subunits at a mass ratio of 1:3 NR1a and NR2A/B subunits), and grown for 48 h as described (21). Robust expression of NMDA currents was verified by patch-clamp recording of parallel cultures.3 Cells were washed and incubated in Mg2+-free Hanks' balanced saline containing 2 mM CaCl2 and either the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 50 µM) or NMDA/glycine (100/10 µM) for 15 min. Cultures were fixed and processed for immunofluorescence (see above) using 1:500 antibody dilutions of goat anti-CaMKII (16), mouse anti-NR1 (PharMingen), and rabbit anti-NR2A/B (Chemicon). Between 2 and 5% of cells were strongly positive for at least one label; only those cells expressing high levels of each antigen (>50% of transfected cells) were included in the analyses. Under basal conditions, CaMKIIalpha expression was diffusely cytoplasmic. Irrespective of agonist treatment, NR1 and NR2A/B strictly colocalized (mean scores >3.4, see below) in a patchy or reticular, often perinuclear pattern as seen previously in heterologous cells (22). Cultures were randomized prior to sampling digital images on a confocal microscope to prevent operator bias. Coded images (as in Fig. 3) were assigned a colocalization score by a second, naive observer: 0, mutual exclusion; 1, coincidental overlap; 2 or 3, increasing degrees of colocalization, 4, complete overlap of labels. For reference, the cells in Fig. 3 scored a 0, 1, 2, 2, and a 3 (from left to right, top to bottom).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

To determine whether NR2 subunits contribute to the previously characterized "p190" overlay binding activity (16), we analyzed immunoprecipitated NR2A/B by gel overlay with [32P-T286]CaMKIIalpha (Fig. 1A). A CaMKII-binding activity comigrating with NR2A and NR2B was immunoprecipitated with NR2A/B antibodies, but not control antibodies, indicating that NR2A and/or NR2B are CaMKII-binding proteins.


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Fig. 1.   Identification of a CaMKII·NMDA receptor complex in neurons. A, SDS-solubilized PSD proteins (input) were immunoprecipitated with the indicated antibodies and analyzed by [32P-T286]CaMKIIalpha overlay and autoradiography (left) or NR2B immunoblot (right). Arrows point to the position of NR2A/B (NR2A comigrates with NR2B, not shown). B, cultured cortical neurons were triple-labeled with antibodies to CaMKII (red), NR1 (green), and the synaptic vesicle marker synaptophysin (blue) and imaged by confocal fluorescence microscopy. Arrowheads point to representative synapses where the three labels overlap (white in the merged image). C, double-immunofluorescence staining of a dendritic branch of a cultured cortical neuron demonstrates localization of CaMKII (red) in dendritic spines adjacent to synaptophysin-positive presynaptic terminals (blue). D, reversibly cross-linked PSDs (input) were solubilized in SDS and immunoprecipitated with either CaMKII antibodies or preimmune IgG, followed by immunoblotting with the indicated antibodies. Data are representative of at least three experiments.

This interaction may be physiologically relevant, because triple immunofluorescent labeling of cultured cortical neurons demonstrated that CaMKII colocalizes with NMDA receptors in many punctae along dendritic shafts, identified as synapses by the adjacent or overlapping presence of synaptophysin (Fig. 1B). Higher magnification revealed a mostly postsynaptic localization of CaMKII in dendritic spines (Fig. 1C). Moreover, a complex of CaMKII with NMDA receptor subunits can be immunoprecipitated from PSDs using CaMKII antibodies, but not preimmune IgG (Fig. 1D). NR2B was more efficiently coprecipitated than NR1, likely because association of CaMKII with NR1 is indirect (i.e. via NR2B, see below). Recovery of the receptor-kinase complex required pretreatment of PSDs with a reversible cross-linker prior to essentially complete PSD solubilization in 2% SDS, indicating that the interaction of CaMKII with NMDA receptors is not stable in harsh detergents. The specificity of the cross-linking procedure was demonstrated by the absence of other abundant PSD proteins in the immunoprecipitate, including the catalytic subunit of protein phosphatase 1 (Fig. 1D).

NMDA receptor subunits have a common transmembrane topology with three membrane-spanning regions and a C-terminal tail of variable length, which forms the intracellular portion of the receptor (Fig. 2A, diagram). Bacterial lysates expressing the cytoplasmic domains of the predominant forebrain NMDA receptor subunits, NR1, NR2A, and NR2B, as His6 tag fusion proteins were screened for [32P]CaMKIIalpha binding by overlay (Fig. 2A). The NR2B cytoplasmic domain bound about six times more [32P-T286]CaMKIIalpha than the corresponding region of NR2A; neither NR1 nor any endogenous bacterial proteins showed detectable binding. Interactions with NR2A and NR2B were specific for autonomously active CaMKII, as CaMKIIalpha phosphorylated in the absence of calcium/calmodulin at Thr305/306 ([P-T306]CaMKIIalpha ) bound only weakly (<5%). Because NR2B displayed the most robust interaction with CaMKII, we mapped its CaMKII-binding domain by creating a series of truncation and internal deletion constructs. Only constructs containing NR2B residues 1260-1309 showed CaMKII binding similar to the full-length cytoplasmic tail. Fusion of NR2B-(1260-1309) to GST demonstrated that this domain is also sufficient for interaction with autonomous CaMKII (Fig. 2B).


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Fig. 2.   Identification of a CaMKII-binding domain in NR2B. A, full-length cytosolic domains of NR1 (splice variant A, 834-T), NR2A (838-T), and NR2B (839-T) (where T indicates terminus) and the diagrammed NR2B constructs were screened for overlay binding of [32P]CaMKIIalpha phosphorylated at either Thr286 (T286) or Thr305/306 (T306). Data were corrected for expression levels and autophosphorylation stoichiometries, normalized to NR2B-(839-T) and expressed as means ± S.E. of four to eight experiments. B, a blot of 0.5 µg of the indicated NR2A or NR2B residues fused to GST or 1.5 µg of GST alone was first stained for protein with Ponceau S (top) and then analyzed for [32P-T286]CaMKIIalpha binding (bottom). C, indicated soluble His6 tag NR2B fusion proteins were affinity-tethered to a microtiter plate and incubated with the indicated concentrations of [32P-T286]CaMKIIalpha . Shown are means ± S.D. of duplicate determinations from one experiment representative of three. Inset, linear fit of Scatchard plot of same data. D, CaMKIIalpha and GST-NR2B-(1260-1309) fusion protein (0.5 µM each) with or without calcium/calmodulin (0.5 mM/3 µM) and Thr286 autophosphorylation were sedimented with glutathione-agarose and analyzed by Ponceau S staining of protein blots. E, a rat brain cytosolic extract was incubated with GST-NR2B or GST alone, purified with glutathione-agarose, and immunoblotted with the indicated antibodies. Data (D, E) are representative of three experiments.

A solution interaction assay was employed to examine binding of CaMKII to NR2B that had not undergone denaturation/renaturation for gel overlay analysis. [32P-T286]CaMKIIalpha bound saturably to a His6 tag NR2B fusion protein containing residues 1260-1309, but not to a construct that starts at residue 1310, C-terminal of this domain (Fig. 2C). Scatchard analysis indicated that binding involves a simple bimolecular interaction with a Kd of 138 ± 60 nM (n = 3) (Fig. 2C, inset). This Kd is ~100 times lower than the average concentration of CaMKIIalpha in forebrain (16, 23), suggesting that the interaction can readily occur in neurons.

The CaMKII-binding domain in NR2B contains a high-affinity phosphorylation site, Ser1303, which is phosphorylated by CaMKII in vitro and is also phosphorylated in vivo (13). However, three lines of evidence indicate that the binding of CaMKII to NR2B-(1260-1309) is not dependent on a substrate interaction. First, the model peptide substrate syntide-2 only weakly inhibits CaMKII binding (~30%) at concentrations of ~100-fold the Km for phosphorylation (not shown). Second, even though NR2A residues 1255-1298 are 36% identical to NR2B-(1260-1309), and sequences surrounding the phosphorylation site are almost perfectly conserved (NR2B, LRRQHSYD; NR2A, INRQHSYD) (13), CaMKII binding to NR2A-(1255-1298) is ~10-fold weaker under our overlay conditions (10.7 ± 1.8%, n = 3, Fig. 2B), suggesting that nonconserved residues in NR2B-(1260-1309) are important for high-affinity CaMKII binding. Third, "pull-down" experiments, in which GST-NR2B fusion protein was purified with glutathione-agarose, showed that calcium/calmodulin alone did not promote CaMKII interaction with NR2B, but that stoichiometric interaction was instead strictly dependent on CaMKIIalpha autophosphorylation at Thr286 (Fig. 2D). On the other hand, calcium/calmodulin binding is sufficient for full CaMKII activation, and Thr286 autophosphorylation stabilizes the active conformation of the kinase in the absence of calcium/calmodulin (1, 2). Thus, CaMKII residues outside the substrate binding site are involved in the interaction with NR2B.

Further evidence for specific association of CaMKII with NR2B was obtained by performing GST-NR2B pull-downs from brain cytosolic extracts. alpha  and beta  isoforms of CaMKII were isolated following incubation with GST-NR2B-(1260-1309), but not GST alone. Affinity-purified CaMKIIalpha displayed an upward electrophoretic mobility shift characteristic of autophosphorylation (Fig. 2E). CaM kinase IV, a related kinase with a similar phosphorylation site preference (24), as well as other kinases and phosphatases tested, were not detected in the precipitated material, strongly indicating that NR2B-(1260-1309) binds selectively to CaMKII.

The NR2B subunit of the NMDA receptor was shown to target Thr286 autophosphorylated CaMKII in HEK293 cells. CaMKIIalpha was coexpressed with various NMDA receptor subunit combinations, and their distributions were compared by immunofluorescence (Fig. 3). Whereas NR1 alone does not form functional NMDA receptors in HEK293 cells, activation of both NR1/NR2A and NR1/NR2B receptors leads to massive calcium influx (25). Coexpression of CaMKIIalpha and NR1 alone resulted in low colocalization scores that were unaffected by acute treatment with the receptor coagonists NMDA/glycine (Fig. 3A). Perhaps reflecting the low but detectable CaMKII binding activity of NR2A (Fig. 2, A and B), additional expression of the NR2A subunit led to a small increase in CaMKIIalpha and NR1/NR2A colocalization, which was not significantly increased by NMDA/glycine treatment (Fig. 3B). In cells expressing NR2B with CaMKIIalpha and NR1, we observed a similarly modest increase in colocalization in the absence of agonist treatment compared with CaMKIIalpha and NR1 alone (Fig. 3, C and D). In contrast to NR2A-containing NMDA receptors, activation of NR1/NR2B receptors with NMDA/glycine caused a highly significant redistribution of CaMKIIalpha into receptor-positive patches (Fig. 3, C and D), strongly suggesting that receptor activation induced the formation of a CaMKII·NR2B complex. Replacing extracellular calcium with barium, which is receptor-permeable but binds only poorly to calmodulin, completely blocked the effect of NMDA (Fig. 3D). Thus, opening of NMDA receptors is not sufficient for complex formation, but calcium influx is essential, presumably to stimulate calcium/calmodulin-dependent autophosphorylation of CaMKII. Consistent with this interpretation, an autophosphorylation-incompetent form of the kinase, T286A-CaMKIIalpha (26, 27), expressed at similar levels of wild-type CaMKIIalpha failed to show activity-induced colocalization with NR1/NR2B containing NMDA receptors (Fig. 3D). Thus, NR2B mediates targeting of CaMKII to NMDA receptors in a calcium- and Thr286 autophosphorylation-dependent manner in intact cells.


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Fig. 3.   NR2B targets CaMKII in intact cells. HEK293 cells were transiently transfected with CaMKIIalpha together with either NR1 alone (A), NR1/NR2A (B), or NR1/NR2B (C, D). After 48 h, cells were treated for 15 min with either the NMDA receptor antagonist APV, or the coagonists NMDA and glycine as indicated, and processed for double immunofluorescence confocal microscopy. Only merged images of two antigens are shown in A and B; individual fluorescence channels are additionally shown in C. Colocalization (yellow in the merged images) of red (CaMKII) and green labels (NR1, NR2A, or NR2B) in individual cells was ranked from 0 to 4 (see "Experimental Procedures," two to three experiments for each condition). The means ± S.E. (numbers of cells in parentheses) is shown at the bottom of the images in A (pooled data from APV and NMDA treatment) and B and in D. D also shows the score distribution of cells transfected with CaMKIIalpha (w.t., wild-type or T286A mutant) and NR1/NR2B under the indicated conditions. Significant increase (dagger p < 0.01; *, p < 0.0001) by Student's t test compared with NR1 alone (A); **, p < 0.0001, compared with all other conditions.

Our data support a model in which dendritic calcium influx induced by synaptic activity triggers CaMKII autophosphorylation at Thr286 and subsequent binding to residues 1260-1309 in the NR2B subunit of the NMDA receptor. What are the functional consequences of this interaction? Autonomous CaMKII in the PSD is inactivated by PSD-associated serine/threonine phosphatases (18, 28, 29). Once dephosphorylated at Thr286, CaMKII positioned near the mouth of the NMDA receptor calcium channel is likely to undergo rapid re-autophosphorylation even during periods of low level NMDA receptor activation. Thus, an interaction of CaMKII with NMDA receptors is predicted to boosts autonomous kinase activity, leading to enhanced phosphorylation of nearby downstream effectors of synaptic plasticity (15). Furthermore, recruitment of CaMKII into the PSD structure (6), possibly via association with NR2B, may play a role in the rapid ultrastructural changes of synapses that undergo LTP (30, 31). The developmental appearance of NR2A and down-regulation of NR2B in the mammalian visual system correlate with the end of the "critical period" of synapse maturation (32, 33). Preferential association of CaMKII with NR2B over NR2A may therefore provide a mechanism by which NMDA receptor subunit composition can impact developmental plasticity.

    ACKNOWLEDGEMENTS

We thank L. MacMillan for scoring cells; M. Bass for invaluable technical assistance; V. Rema, F. Ebner, and M. Maguire (Vanderbilt) for NR1 antibodies and cortical cultures; D. Lynch (Penn State) for NR expression plasmids; T. Soderling (Vollum Institute) for CaMKII expression plasmids; DNAX, Inc. (Palo Alto, CA) for use of the pME18S expression vector; L. Popp, S. Sessoms, and D. Lovinger (Vanderbilt) for help with HEK cell transfections; and R. Blakely, F. Ebner, J. Exton, L. Limbird, J. Lisman, D. Lovinger, and B. Wadzinski for helpful suggestions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM47973 (to R. J. C.) and American Heart Association Grant-in-aid 96010040 (to R. J. C., an Established Investigator of the American Heart Association). Confocal microscopy was performed using the Vanderbilt University Medical Center Cell Imaging Resource (supported by National Institutes of Health Grants CA68485 and DK20593).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.

Dagger To whom correspondence should be addressed: Dept. Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232-0615. Tel.: 615-936-1630; Fax: 615-322-7236; E-mail: roger.colbran{at}mcmail.vanderbilt.edu.

The abbreviations used are: PSD, postsynaptic density; CaMKII, calcium/calmodulin-dependent protein kinase II; CaMKIIalpha /beta , alpha /beta isoform of CaMKII; [P-T286]CaMKIIalpha , CaMKIIalpha autophosphorylated at threonine 286; [P-T306]CaMKIIalpha , CaMKIIalpha autophosphorylated at threonine 305 and/or threonine 306; NMDA, N-methyl-D-aspartateAPV, 2-amino-5-phosphonovaleric acidGST, glutathione S-transferaseLTP, long term potentiation.

2 S. Strack, R. B. McNeill, and R. J. Colbran, unpublished data.

3 R. L. Popp and D. M. Lovinger, personal communication.

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Abstract
Introduction
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Results & Discussion
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