Effect of cell swelling on membrane and cytoplasmic distribution of pICln

Francesco Emma1, Sylvie Breton2, Rebecca Morrison3, Stephen Wright4, and Kevin Strange3

1 Renal Division, Children's Hospital, Boston 02115; 2 Renal Unit, Massachusetts General Hospital, Boston, Massachusetts 02129; 3 Laboratory of Cellular and Molecular Physiology, Departments of Anesthesiology and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232; and 4 Department of Physiology, University of Arizona, Tucson, Arizona 85724

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

pICln is found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies led to the proposal that pICln is a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothelial cells have shown that the protein is localized primarily to the cytoplasm. It has therefore been postulated that activation involves reversible translocation of pICln from the cytoplasm and insertion into the plasma membrane. We tested this hypothesis using several different approaches. Fractionation of C6 glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that ~90% of the recovered pICln was confined to the cytosol. Swelling had no effect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pICln is localized primarily, if not exclusively, to the cytoplasm of swollen and nonswollen cells. Similarly, transfection of cells with a green fluorescent protein-labeled pICln construct failed to reveal any membrane localization of the protein. These findings do not support the hypothesis that pICln is a volume regulatory anion channel activated by swelling-induced membrane insertion.

anion channels; organic osmolytes; cell volume regulation; osmoregulation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

AN APPARENTLY UBIQUITOUS response to swelling in vertebrate cells is activation of an outwardly rectifying anion current termed ICl,swell. The general characteristics of the ICl,swell channel include an Eisenman type I anion permeability sequence (I- > Br- > Cl- > F-), modest outward rectification, voltage-dependent inactivation at potentials above the reversal potential for Cl-, inhibition by a wide variety of compounds including conventional anion transport inhibitors, and block by extracellular nucleotides (reviewed in Refs. 18, 23). The degree of rectification, voltage sensitivity, and pharmacology can vary somewhat from cell to cell. It is not clear whether the differences observed reflect the existence of distinct channels or experimental and physiological variables. In addition to its role in Cl- transport, the channel responsible for ICl,swell has been postulated to be the major pathway for volume regulatory efflux of organic anions and electroneutral organic osmolytes (reviewed in Refs. 13, 23). Because of this role, the ICl,swell channel has been termed VSOAC (volume-sensitive organic osmolyte/anion channel).

The molecular identity of VSOAC is not known. However, the protein pICln has been proposed to be either the channel or a channel regulator (8, 14, 19). The cDNA encoding pICln was cloned and described by Paulmichl et al. (19). When expressed in Xenopus oocytes, pICln induces an outwardly rectifying anion current that is blocked by extracellular nucleotides and inactivated by depolarizing voltages, characteristics that at least superficially resemble those of VSOAC.

Paulmichl et al. (19) initially concluded that pICln is a novel anion channel-forming protein. Compelling evidence was presented to support this conclusion. Expression of a pICln cDNA in which a possible nucleotide binding site located in the putative channel pore was mutated resulted in the induction of an anion conductance that had altered voltage and Ca2+ sensitivities and was no longer inhibited by extracellular nucleotides.

A subsequent series of studies carried out by Krapivinsky and co-workers (14) led them to conclude that pICln was not the VSOAC channel but was instead a regulator of it. These investigators demonstrated that oocytes express pICln endogenously and that oocyte swelling activates an endogenous VSOAC current (see also Refs. 1, 9) that superficially resembles the current induced by heterologous expression of pICln. The oocyte ICl,swell is inhibited by microinjection of an anti-pICln monoclonal antibody into the cytoplasm. Biochemical measurements and immunofluorescence studies demonstrated that pICln is localized primarily to the cell cytoplasm. The apparent absence of the protein from the plasma membrane is unexpected if it functions as an ion channel. Although intriguing, the regulator hypothesis was troubling to many workers in the field because it did not take into account the results of mutagenesis studies described by the authors in their first report on pICln (19). Thus we proposed the alternate hypothesis that pICln is a channel activated by reversible, swelling-induced insertion into the plasma membrane (23). Studies described in this paper were carried out to test this hypothesis directly.

    MATERIAL AND METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. Rat C6 glioma cells were cultured in MEM (GIBCO, Gaithersburg, MD) with 10% fetal bovine serum (HyClone, Logan, UT) and penicillin-streptomycin as described previously (10). The osmolality of the growth medium was elevated to 440 mosmol/kgH2O by adding NaCl. Hypotonic growth medium (200 mosmol/kgH2O) was made by diluting MEM 35% with a solution containing 5.5 mM glucose, 10 mM HEPES, 0.5 mM MgSO4, 5 mM KCl, and 1.4 mM CaCl2.

Antibody production. A fusion protein consisting of full-length pICln cloned from rat C6 glioma cells ligated to glutathione-S-transferase (GST) was generated in BL21 Escherichia coli using a commercially available kit (Pharmacia Biotech, Piscataway, NJ). The GST-pICln fusion protein was purified using glutathione Sepharose 4B, pICln was cleaved from GST with thrombin, and the protein was injected intradermally and intramuscularly into rabbits to generate polyclonal antibodies. Preimmune and immune sera were harvested and stored at -20°C.

Membrane fractionation. Cells were plated on 100-mm-diameter tissue culture dishes, grown for 48 h in 440 or 200 mosmol/kgH2O MEM, and then exposed to 440 mosmol/kgH2O MEM for 2 h. After this acclimation period, cells were exposed at room temperature for 5 min to 200 or 440 mosmol/kgH2O flux medium containing 95 or 223 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES at pH 7.4. Cells were then rinsed in ice-cold flux medium of the same composition and scraped off the plate in 1 ml of ice-cold flux medium containing 0.5 mM dithiothreitol and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). This material and 200 µl of flux medium that was used to rinse the plate were transferred to a glass homogenizer tube with a Teflon pestle and homogenized on ice with 100 strokes. Nuclei and unbroken cells were removed from 1 ml of the homogenate by centrifugation for 20 min at 600 g and 4°C in an Eppendorf centrifuge. The resultant supernatant was removed, and 800 µl were centrifuged for 30 min at 16,000 g and 4°C. This centrifugation step generated a pellet that contained plasma membrane fragments and a supernatant that contained microsomes and cytoplasmic proteins. After removal of the supernatant, the pellet was rinsed briefly with 100 µl of flux medium and then resuspended in 160 µl of flux medium containing protease inhibitors. Centrifugation of 700 µl of the supernatant at 100,000 g and 4°C in a Beckman ultracentrifuge for 1 h produced a pellet and supernatant that contained microsomal and cytoplasmic proteins, respectively.

Aliquots of the various fractions were mixed with 4× Laemmli buffer (3 parts sample and 1 part buffer) and boiled for 4 min. Proteins were resolved by SDS-PAGE and then transferred to nitrocellulose membranes. pICln was visualized by anti-pICln Western analysis using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). Western blots were quantified using image analysis software (Optimas, Bioscan, Edmonds, WA). In initial studies, we observed that little or no pICln could be detected in the microsomal fraction. Therefore, further analyses of this fraction were not performed.

Green fluorescent protein-pICln transfection and localization studies. C6 glioma cells were transfected with cDNA coding for green fluorescent protein (GFP)-pICln fusion protein. The open reading frames for pICln and GFP (Green-Lantern, GIBCO BRL, Grand Island, NY) were ligated into pCDNA III (Invitrogen, Carlsbad, CA). GFP was ligated to the amino or carboxy terminus of pICln.

For transfection studies, C6 glioma cells were grown to 80% confluency in 60-mm-diameter culture dishes and incubated in 5 ml of serum-free MEM containing 5 µg of cDNA and 50 µl of Lipofectamine (GIBCO BRL). After 16 h, cells were detached by trypsinization, plated onto acid-washed glass coverslips, and grown in 440 mosmol/kgH2O MEM for 48 h. The cells were then exposed to 200 mosmol/kgH2O MEM or fresh 440 mosmol/kgH2O MEM for 5 min and fixed for 20 min in the same medium containing 4% paraformaldehyde. Fixed cells were washed in PBS, treated with 1% SDS for 4 min (2), permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated for 20 min in PBS-1% BSA to block nonspecific background staining. Primary anti-pICln antibody was applied at a dilution of 1:100 for 1.5 h at room temperature, followed by incubation with a indocarbocyanine (Cy3)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h. Coverslips were mounted in Vectashield antifading solution (Vector Labs, Burlingame, CA) diluted 1:1 in 0.1 M Tris · HCl, pH 8.0. Images were captured directly from a Nikon FXA photomicroscope using an Optronics color charge-coupled device (CCD) camera and IP Lab Spectrum (Scanalytics, Vienna, VA) acquisition and analysis software running on a Power PC 8500. Pictures were printed on a Tektronix Phaser 440 dye sublimation color printer.

GFP-pICln localization was also carried out on living C6 glioma cells. GFP fluorescence was imaged using a Zeiss Axiovert 35 inverted microscope equipped with an Achroplan ×100 oil immersion objective lens and KS-1381 microchannel plate image intensifier (Video Scope International, Washington, DC) interfaced to a CCD camera (model 200, Video Scope International). Video images were digitized by a Matrox MVP/AT image processing board (Matrox Electronics Systems, Dorvall, PQ, Canada) and Image 1 software (Universal Imaging, West Chester, PA).

Localization of endogenous pICln. Immunofluorescence visualization of pICln in nontransfected cells required tyramide signal amplification (NEN Life Science, Boston, MA). This technique uses horseradish peroxidase activity to catalyze the deposition of tyramide-conjugated FITC around antigenic sites. Cells were grown on glass coverslips in 440 mosmol/kgH2O MEM for 48 h and then fixed in the same medium containing 4% paraformaldehyde or exposed to 200 mosmol/kgH2O medium for 5 min before fixation. After treatment with 1% SDS and permeabilization in 0.1% Triton X-100, cells were incubated in TNB blocking buffer (NEN Life Science) for 30 min at room temperature. Primary antibody was applied for 1.5 h, as described above. Antibody-treated cells were washed three times for 5 min in 0.05% Tween 20-PBS, and then goat anti-rabbit antibody coupled to biotin (Vector Labs) was applied at a dilution of 1:100 for 30 min. After three washes in 0.05% Tween 20-PBS, streptavidin-horseradish peroxidase conjugate diluted 1:100 in TNB buffer was applied for 30 min. The cells were then washed in 0.05% Tween 20-PBS, and tyramide-conjugated FITC diluted 1:50 in an amplification buffer (NEN Life Science) was applied for 10 min, followed by washing in 0.05% Tween 20-PBS. Control incubations were performed by replacing the primary antibody with preimmune serum at a dilution of 1:100 for 1.5 h.

Confocal microscopy. Confocal images were captured using a Bio-Rad MRC600 microscope (Bio-Rad, Hercules, CA) controlled by a Pentium computer (Optiplex Pentium 5133, Dell Computer, Austin, TX). Z series images were taken at 1-µm intervals using fixed gain and aperture settings. Images were imported into Adobe Photoshop 3.04 for size reduction and printed on a Tektronix Phaser 440 dye sublimation color printer.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Membrane fractionation. The central goal of our studies was to relate putative changes in pICln membrane insertion to VSOAC activation. However, it is possible that cell swelling induces alterations in pICln localization that have nothing to do with channel activity and volume regulation per se. To control for this, we used a protocol that allowed us to dramatically vary VSOAC activation in two groups of cells swollen to the same extent. Briefly, C6 cells were grown in either 440 or 200 mosmol/kgH2O MEM. After 48 h, both groups of cells were transferred to 440 mosmol/kgH2O MEM for an additional 2 h. When cells acutely and chronically acclimated to hypertonicity are exposed to 200 mosmol/kgH2O medium, they swell approximately twofold (6). Because acutely acclimated cells have elevated cytoplasmic ionic strength, swelling-induced VSOAC activation, measured by [3H]taurine efflux, is extremely low. In contrast, cells acclimated chronically to hypertonic medium lose ~80% of their [3H]taurine content within 10 min after swelling (6).

Membrane fractionation studies were carried out on cells acclimated acutely or chronically to 440 mosmol/kgH2O culture medium and then exposed for 5 min to either 200 or 440 mosmol/kgH2O flux medium. Figure 1A shows a Western blot of pICln present in cytoplasm- and plasma membrane-containing fractions isolated from swollen and nonswollen C6 glioma cells. A total of three such experiments was performed. The amount of pICln in the membrane-containing fraction relative to that in the cytoplasm-containing fraction is shown in Fig. 1B. Approximately 90% of the pICln was present in the cytoplasm-containing fraction. Cell swelling had no significant effect on the distribution of pICln between the cytoplasm and plasma membrane (Fig. 1).


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Fig. 1.   Quantitation of pICln in cytoplasm- and plasma membrane-containing cell fractions. A: Western immunoblot analysis of pICln in cytoplasm (C) and plasma membrane (M) fractions isolated from swollen and nonswollen C6 glioma cells acclimated to various conditions; times and osmolalities (in mosmol/kgH2O) for acclimation conditions in experiments A, B, C, and D (see text for further details) are shown in B. All 4 experiments shown were done in parallel. Total protein contents of cytoplasm and plasma membrane fractions were similar (data not shown). Because concentration of pICln in plasma membrane fraction is low, total protein loaded on gel was 5 times that of cytoplasm fraction. B: amount of pICln in plasma membrane-containing fractions (relative to cytoplasm-containing fractions) isolated from swollen and nonswollen C6 glioma cells acclimated to various conditions. Values shown are means ± SE (n = 3 separate experiments).

Immunolocalization. Conventional immunofluorescence microscopy was utilized to assess pICln localization in intact C6 glioma cells. Cells were grown in 440 mosmol/kgH2O MEM for 48 h and then fixed in paraformaldehyde or exposed to 200 mosmol/kgH2O medium for 5 min before fixation. pICln was present in the cytoplasm and in intracellular reticulate structures that probably correspond to rough endoplasmic reticulum (Fig. 2A). No obvious plasma membrane localization was detected in either swollen or nonswollen cells (Fig. 2C). Cells incubated with preimmune serum were negative (Fig. 2, B and D).


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Fig. 2.   Localization of pICln in C6 glioma cells by conventional immunofluorescence microscopy. Cells were grown in 440 mosmol/kgH2O MEM for 48 h and then fixed in paraformaldehyde (A) or exposed to 200 mosmol/kgH2O medium for 5 min before fixation (C). pICln immunoreactivity is localized predominantly to cytoplasm and intracellular reticulate structures. No redistribution of pICln to plasma membrane was detected in swollen cells (C). Nonswollen (B) and swollen (D) cells show no staining when incubated with preimmune serum rather than anti-pICln antibody. Scale bars, 5 µm.

To further assess the possibility of a plasma membrane localization of pICln, confocal images were captured at 1-µm focal intervals. As with conventional immunofluorescence microscopy, confocal images revealed extensive pICln immunoreactivity in the cytoplasm and in intracellular reticulate structures (Fig. 3). Again, no obvious membrane localization of the protein was detected in nonswollen or swollen cells (compare Fig. 3, A-D, and Fig. 3, E-H).


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Fig. 3.   Localization of pICln in C6 glioma cells by confocal immunofluorescence microscopy. Cells were grown in 440 mosmol/kgH2O MEM for 48 h and then fixed in paraformaldehyde (A-D) or exposed to 200 mosmol/kgH2O medium for 5 min before fixation (E-H). Images were captured at 1-µm focal intervals on z-axis: A and E show planes closest to coverslip, and D and H show planes closest to cell apex. pICln immunoreactivity is localized predominantly to cytoplasm and intracellular reticulate structures in both nonswollen (A-D) and swollen (E-H) cells. Scale bars, 5 µm.

GFP-pICln transfection studies. As a final test of the hypothesis that pICln is a channel activated by reversible, swelling-induced insertion into the plasma membrane, we transfected cells transiently with a cDNA coding for GFP-pICln fusion proteins. Cells were grown in 440 mosmol/kgH2O medium for 48 h and then fixed in paraformaldehyde or exposed to 200 mosmol/kgH2O medium for 5 min before fixation. Images of cells transfected with an amino-terminal GFP-pICln fusion protein are shown in Fig. 4. GFP is detected largely in the cytoplasm and colocalizes with pICln (compare Fig. 4, A and B, and Fig. 4, C and D). Cell swelling had no effect on the distribution of the fusion protein.


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Fig. 4.   Localization of green fluorescent protein (GFP)-pICln fusion protein in C6 glioma cells by conventional fluorescence microscopy. After cDNA transfection, cells were grown in 440 mosmol/kgH2O medium for 48 h and then fixed in paraformaldehyde (A and B) or exposed to 200 mosmol/kgH2O medium for 5 min before fixation (C and D). pICln was immunolocalized with anti-pICln antibody. Both GFP (A and C) and fluorescence associated with anti-pICln immunoreactivity (B and D) colocalize (compare A with B and C with D) and are observed predominantly in cytoplasm and intracellular reticulate structures. No redistribution of GFP-pICln to plasma membrane was detected in swollen cells (C and D). Scale bars, 5 µm.

Results identical to those shown in Fig. 4 were obtained with a carboxy-terminal GFP-pICln fusion protein (data not shown). We also used video- and computer-enhanced fluorescence microscopy to examine the distribution of the carboxy-terminal fusion protein in living cells. Again, we were unable to detect membrane localization of GFP-pICln or changes in its distribution brought about by swelling. Images obtained using this approach were similar to those shown in Fig. 4 (data not shown).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Paulmichl and co-workers (8, 19) have postulated that pICln is a swelling-activated anion channel. However, Krapivinsky et al. (14) subsequently argued that pICln is not a channel but is instead a channel regulator. They demonstrated that pICln is localized primarily to the cytoplasm and that it binds to a number of other cytoplasmic proteins. Although these findings were interesting, they did not take into account, and could not be readily reconciled with, the earlier mutagenesis experiments of Paulmichl et al. (19). We therefore proposed the hypothesis that pICln is a channel activated by translocation from the cytoplasm and reversible, swelling-induced insertion into the plasma membrane (23).

The results of the present studies do not support the translocation-insertion hypothesis. Cell fractionation revealed that the bulk of pICln in C6 glioma cells is confined to the cytoplasm. Furthermore, cell fractionation, immunofluorescence microscopy, and transfection of cells with GFP-pICln failed to reveal postulated swelling-induced translocation of the protein from cytoplasm to the plasma membrane. Our findings are consistent with studies carried out on Madin-Darby canine kidney cells (14) and endothelial cells (3). However, other investigations have reported an apparent membrane localization of pICln. Schwartz et al. (21) concluded that pICln was, "concentrated in the membrane" of red blood cells. Fractionation studies carried out on Madin-Darby canine kidney cells (15), embryonic skate heart cells (17), and neonatal rat myocytes (7) have revealed that swelling induces an apparent increase of pICln content in the plasma membrane fraction.

How can these discrepant results be explained? Our inability, as well as that of other investigators (3, 14), to demonstrate significant membrane localization of pICln does not rule out the possibility that pICln is the VSOAC channel. Very small amounts of pICln present in the plasma membrane may escape detection and yet may be sufficient to account for the observed channel activity. In addition, if pICln does insert into the plasma membrane in response to swelling, the insertion event may be very labile and difficult to detect with cell fractionation techniques and microscopy.

It is equally possible that the apparent association of pICln with the plasma membrane observed by others is simply an epiphenomenon unrelated to VSOAC activation and cell volume regulation. The fractionation studies carried out by Laich et al. (15), Musch et al. (17), and Goldstein et al. (7) do not demonstrate that pICln inserts into the plasma membrane in response to swelling. The apparent swelling-induced association with the plasma membrane-containing fraction may represent an association of pICln with the membrane-attached cytoskeleton rather than with the lipid bilayer. Immunoprecipitation (14, 20), in vitro protein binding assays (14, 20), and yeast two-hybrid studies (21) have suggested that pICln interacts with cytoskeletal components including actin and the myosin light chain. Because cell swelling alters the structure of the cytoskeleton, particularly F-actin (reviewed in Ref. 18), it is quite possible that apparent changes in the interaction of pICln with the cytoskeleton may reflect events entirely unrelated to cell volume homeostasis. The apparent association of pICln with the plasma membrane of red blood cells reported by Schwartz et al. (21) also likely represents a pICln-cytoskeleton interaction (reviewed in Ref. 22).

If the results of the present investigation are taken at face value, does this indicate that pICln is a regulator of VSOAC as argued by Krapivinsky et al. (14)? Recent work by Eggermont, Nilius, and co-workers (3, 25, 26) has suggested very strongly that the purported connection between pICln and VSOAC may be an artifact related to the use of the oocyte expression system (reviewed in Ref. 22).

The role of pICln in volume homeostasis, if any, remains unclear, and extensive additional studies are required to define the physiological role of this protein. In this regard, it is worth noting recent work on ClC-3, a member of the ClC family of anion channels (11). When expressed in NIH/3T3 cells, ClC-3 cloned from guinea pig heart gives rise to an outwardly rectifying anion current that is activated by cell swelling (5). This current has characteristics remarkably similar to those of VSOAC, with one major exception. The ClC-3 current is inhibited by activation of protein kinase C (PKC) (Ref. 5; see also Ref. 12). In rabbit atrial myocytes, ICl,swell is also inhibited by PKC activation (4). This is very different from ICl,swell in other cells, in which PKC has no inhibitory effect on ICl,swell (e.g., Refs. 18, 24) and may actually stimulate channel activity (10). If the findings of Duan et al. (5) can be confirmed, it will be interesting and important to determine whether a non-PKC-regulated ClC-3 isoform or a ClC-3 heteromultimer (16) accounts for ICl,swell in other cells types.

    ACKNOWLEDGEMENTS

We thank Dr. Dennis Brown for helpful advice.

    FOOTNOTES

This work was supported by National Institutes of Health (NIH) Grants NS-30591 and DK-51610 (to K. Strange). F. Emma was supported in part by a National Kidney Foundation Volunteer/Donor award. S. Breton was supported by NIH Grant DK-38452, by a Hoechst Marion Roussel fellowship from the National Kidney foundation, and by a Claflin Distinguished Scholar Award from the Massachusetts General Hospital. S. Wright was supported by National Science Foundation Grant IBN-9407997.

Address for reprint request: K. Strange, Vanderbilt University Medical Center, Dept. of Anesthesiology, 504 Oxford House, 1313 21st Ave. South, Nashville, TN 37232-4125.

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.

Received 6 January 1998; accepted in final form 25 February 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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