Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water
Department of Biology, St Francis Xavier University, Antigonish, NS, Canada B2G 2W5
* Author for correspondence (e-mail: bmarshal{at}stfx.ca )
Accepted 22 February 2002
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Summary |
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Key words: epithelial ion transport, cystic fibrosis transmembrane conductanctance regulator, protein trafficking, Na+,K+,2Cl- cotransporter, osmoregulation, euryhaline teleost, killifish, Fundulus heteroclitus
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Introduction |
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Salt secretion by MR cells involves furosemide- and bumetanide-sensitive
Na+,K+,2Cl- cotransport at the basolateral
membrane (for reviews, see Karnaky,
1998; Marshall and Bryson,
1998
), a function mediated by the protein NKCC
(Haas and Forbush, 1998
).
Na+,K+,2Cl- cotransport is present in
basolateral membrane vesicles from gills of FW-adapted rainbow trout
Oncorhynchus mykiss and at higher levels in 70% SW-adapted animals
(Flik et al., 1997
). The
cotransport is inhibited by furosemide and bumetanide and shows appropriate
kinetics with apparent control by the transmembrane K+ activity
gradient (Flik et al., 1997
).
NKCC cotransporter is immunocytochemically localized to the basolateral
membranes of MR cells of euryhaline fish in SW
(Wilson et al., 2000a
) and SW
salmon smolts (Pelis et al.,
2001
). NKCC is essential to NaCl secretion by SW animals but the
gene has not yet been cloned from teleost fish. NKCC distribution in MR cells
of FW-adapted Atlantic salmon parr (Salmo salar) colocalizes with
Na+K+ATPase (Pelis
et al., 2001
), suggesting a basolateral membrane location. The
function of NKCC in FW teleost fish gills remains a matter of speculation
(Flik et al., 1997
).
Ion transporters are known to be trafficked from staging areas in Golgi
apparatus to the apical or basolateral membranes of epithelial cells. CFTR is
thought to be directed to the apical membrane of cells via syntaxin,
which binds to the carboxy terminus of human CFTR
(Lehrich et al., 1998;
Moyer et al., 1998
;
Naren et al., 2000
; reviewed
by Kleizen et al., 2000
).
Killifish CFTR has the same carboxy-terminal sequence (-dtrl) as human CFTR
and thus is identifiable using a monoclonal antibody directed against this
epitope; CFTR could be similarly trafficked in teleost MR cells. The aim of
this study was to determine if kfCFTR distribution in MR cells was altered
during adaptation to SW when Cl- secretion rate is rising and
expression of kfCFTR is high (at 24 and 48h;
Marshall et al., 1999
), with
the hypothesis that kfCFTR should appear in the apical crypt during this
period. If kfCFTR were already in the apical crypt, it would suggest
activation of kfCFTR already in situ in the membrane. We also
predicted a redistribution during acclimation to SW of NKCC to the basolateral
membrane in SW MR cells from another location.
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Materials and methods |
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Immunocytochemistry
The primary antibody used to detect kfCFTR was mouse monoclonal anti-hCFTR
(R&D Systems, Minneapolis, MN, USA) with the known epitope of (-dtrl), the
carboxy terminus of hCFTR. Killifish CFTR has the same carboxy terminus
(Singer et al., 1998), thus is
selective for this protein. The primary antibody for detection of the
Na+,K+,2Cl- cotransporter (NKCC) was T4
(Lytle et al., 1992
), an
antibody to the carboxy region of NKCC that has been shown to bind to several
isoforms of NKCC across several species
(Haas and Forbush, 1998
;
Wilson et al., 2000a
). The
secondary antibody was goat polyclonal anti-mouse IgG conjugated to an Oregon
Green 488 fluorophore (Molecular Probes, Eugene, OR, USA), chosen because of
its stability and reliability. Opercular epithelia were dissected without the
dermal chromatophore layer and pinned to modeler's wax. They were incubated in
100 nmoll-1 (final concentration) Mitotracker Red (Molecular
Probes, Eugene, OR, USA) in phosphate-buffered saline (PBS, composition in
mmoll-1: NaCl 137, KCl 2.7, Na2HPO4 4.3,
KH2PO4 1.4, pH 7.4) for 2h at room temperature in the
dark. Preparations were then rinsed three times in rinsing buffer comprising
0.1% bovine serum albumin (BSA), 0.05% Tween 20 in PBS (TPBS). The membranes
were then fixed for 3h at -20°C in a formaldehyde-free, 80% methanol/20%
dimethyl sulfoxide (DMSO) fixative. The methanol was used as a dehydrating
agent and the DMSO as a cryoprotective agent. After three rinses the membranes
were blocked with 5% normal goat serum (NGS), 0.1% BSA, 0.05% TPBS, pH 7.4,
for 30 min at room temperature in the dark and incubated in the primary
antibody (8 µg ml-1 in PBS+0.5% BSA) overnight at 4°C. They
were then rinsed three times and exposed to the secondary antibody (diluted
1:50 in PBS+0.5% BSA) for 5h at 4°C. After three final rinses the
membranes were mounted in mounting medium (Geltol; Immunon Thermo Shandon,
Pittsburgh, PA, USA). Slides were viewed in single blind fashion and images
collected with a laser confocal microscope (Olympus FV300). In each opercular
membrane, randomly selected Z-stack series were collected at 40x, zoom
3.0, with optical sections of 1.5±0.1 µm. The surface was identified
by the microridge pattern of pavement cells and depths were measured by
reference to this surface. Punctate fluorescence in MR cells was positioned
for at least ten cells from at least three Z-stacks for each membrane, with
membranes coming from at least four animals for each salinity treatment group
(FW control, N=9; FW to SW for 24 h, N= 4; FW to SW for 48
h, N=6; SW, N=5).
Western immunoblots
Opercular epithelium (dissected from opercular bone), heart (dissected
ventricle without conus arteriosus) and gill filaments (scraped from the arch
with a razor blade) were homogenized in ice-cold SEI buffer (300 mmol
l-1 sucrose, 20 mmol l-1 EDTA, 100 mmol l-1
imidazole, pH 7.4) using a homogenizer. Three SW-adapted animals were used and
three separate runs performed. Homogenates were centrifuged at 2000g
for 6 min. The pellet was resuspended in 2.4 mmol l-1 deoxycholate
in SEI buffer and centrifuged a second time at 2000g. The total
protein content of the resulting supernatant was determined using the Bradford
method (Bradford, 1976).
Proteins were separated on a 7 % polyacrylamide gel using a Mini-Protean 3
Cell system (Bio-Rad, Mississauga, ON, Canada). A total of 20 µg of protein
for the opercular epithelium, heart and gill was loaded and run for 30 min at
200 V. Proteins were then transferred to a Immobilon-P membrane (Millipore,
Bedford, MA, Canada) for 2.0 h using a Mini-Trans-Blot Cell (Bio-Rad,
Mississauga, ON, USA). Blots were dried at 37°C for 1.0 h, stained with
Ponceau S, then visualized by destaining with 90 % methanol/2.0 % acetic acid.
Blots were then blocked in 3 % bovine serum albumin (BSA)/TTBS (0.05 % Tween
20 in Tris-buffered saline: 20 mmol l-1 Tris-HCl, 500 mmol
l-1 NaCl, 5.0 mmol l-1 KCl, pH 7.4) for 2.0 h at room
temperature on a shaker. The blocking buffer was poured off and the blots
incubated with the primary antibody solution 1.0 µg ml-1 in 1.0
% BSA/TTBS) for 2.0 h at room temperature. The primary antibody used to detect
CFTR was mouse anti-human CFTR antibody (R&D Systems, Minneapolis, MN,
USA). The antibody used against the
Na+,K+,2Cl- cotransporter was mouse anti
human NKCC (antibody T4; Iowa Hybridoma Bank, University of Iowa, IA, USA) and
the primary antibody for Na+,K+-ATPase was mouse
anti-Na+,K+-ATPase
-subunit from chicken
(antibody
5; Iowa Hybridoma Bank, University of Iowa, IA, USA).
Following a 5.0 min wash in TTBS buffer, the membranes were incubated with the
secondary antibody solution (biotin-SP-conjugated AffiniPure Goat anti-mouse
IgG (Biochem Scientific, Mississauga, ON, Canada), diluted 1:8000 in 1.0 %
BSA/TTBS, for 1.0 h at room temperature. The blots were washed in TTBS and
incubated for 1.0 h with an alkaline phosphatase-conjugated Streptavidin
solution (Biochem Scientific, Mississauga, ON, Canada) diluted 1:1000 in 1 %
BSA/TTBS. Bands were visualized by incubating the blots in a BCIP/NBT Blue
substrate development solution (Sigma, Oakville, ON, Canada).
Statistics
Values are given as means ± 1 S.E.M. The minimum level of
significance is P<0.05 from a two-tailed test. Ratio data of
fluorescence scores were analyzed by 2 test for k
independent samples. Depth-measurement data were analyzed by single
classification analysis of variance (ANOVA) with a Bonferroni post-test.
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Results |
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FW control fish (9 animals) had kfCFTR immunofluorescence in MR cells that was diffuse and present throughout the basal portions of the cells but outside the nucleus (Fig. 1D,E). In a minority of MR cells (8.1 %, Fig. 3) there was also strong apical crypt staining of the pattern, typical of SW (i.e. similar to Fig. 1A). However, all the cells that had apical membrane kfCFTR staining also had diffuse cytosolic kfCFTR immunostaining (unlike SW MR cells). Surprisingly, there was kfCFTR-positive immunostaining in pavement cells of FW-adapted tissues (Fig. 1D) that was completely absent from pavement cells of SW tissues (Fig. 1A). At this point it appears that the kfCFTR immunostaining is not exclusively in the apical membrane of pavement cells; rather the staining occurs at the same plane as the nucleus, indicative of cytosolic or basolateral membrane distribution. Anti-NKCC immunofluorescence was present outside the nucleus in eccentric portions of the cytosol where MR cell pairs meet (Fig. 2B). In FW opercular epithelia, MR cells usually existed in pairs or groups of three with the cells sharing a flat area of closely juxtaposed membrane. The NKCC immunofluorescence was usually localized to these zones if the MR cells were in groups.
|
Transitional stages of MR cells were examined in animals transferred to SW for 24 h (4 animals) and 48 h (6 animals). In the 24 h transfer group there was an increasing proportion of MR cells that had punctate kfCFTR immunofluorescence in addition to the diffuse CFTR immunofluorescence (Fig. 3), but the condensed kfCFTR distribution was not superficial, but deeper into the cell, averaging 14 µm below the surface, which was significantly deeper than in FW controls, SW controls and FW to SW for 48 h groups (P<0.001, Bonferroni post-test following ANOVA; Fig. 4). At 48 h after transfer to SW, the majority of MR cells had both diffuse and punctate kfCFTR distribution and a distance to the surface that was intermediate but still significantly deeper (P<0.001; Bonferroni post-test following ANOVA) than in fully adapted SW animals. An example of this type of kfCFTR immunofluorescence distribution is seen in Fig. 1F,G. FW controls had few cells with punctate kfCFTR immunofluorescence compared to the fully adapted SW group (Fig. 3), but the depth from the surface was similar between the FW and SW groups (P>0.05; Bonferroni post-test following ANOVA; Fig. 4).
|
Western blots
The CFTR antibody was specific for a protein at 147.7±1.8 kDa
molecular mass (N=3 separate animals in 3 separate runs for all bands
reported) in the opercular epithelium and a 151.3±2.0 kDa protein in
the gills (Fig. 5). However,
two lower molecular mass additional bands were also visible, at
92.7±0.3 kDa and 88.7±0.3 kDa, respectively, in the opercular
epithelium and 93.3±0.3 kDa and 89.3±0.9 kDa in the gills.
|
The heart (negative control) did not show any CFTR immunoreactivity. With the anti-NKCC antibody, immunoreactive bands appeared at 148.0±2.0, 92.7±0.3 and 87.8±0.3 kDa in the opercular epithelium, and at 150.0±2.0, 92.7±0.3 and 87.8±0.3 kDa in the gills. With anti-NKCC the heart also displayed three bands of similar size to those in the other tissues (149.0±1.7, 91.7±0.3 and 87.7±0.3 kDa). The Na+K+ATPase antibody reacted prominently with a high molecular mass protein at 119.0±1.5 kDa in the opercular epithelium and a 119.7±0.9 kDa protein in the gills. However, three additional fainter bands were also visible with lower molecular mass namely 99.7±0.7, 72.0±0.6 and 67.3±0.7 kDa in opercular epithelium and 99.3±0.3, 71.7±0.3 and 66.7±0.7 kDa in gill tissue. The heart showed four additional bands at 118.3±1.5, 102.3±3.9, 72.7±0.3 and 66.3±0.9 kDa).
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Discussion |
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The apical crypt localization is similar to that seen in another euryhaline
teleost species adapted to SW, the mudskipper, and using a different antibody
to CFTR (Wilson et al.,
2000a). Given that two different anti-CFTR antibodies, one with a
known epitope exactly the same as for the endogenous protein in killifish,
yield similar localizations in two different SW-adapted teleosts, we can
conclude that kfCFTR is concentrated at the apical membrane of SW MR cells.
Furthermore, because kfCFTR expressed in amphibian oocytes imparts anion
conductance in this system (Singer et al.,
1998
), and as anion channels similar in properties to hCFTR anion
channels are present in the apical membrane of killifish opercular epithelium
MR cells (Marshall et al.,
1995
), the immunofluorescence seen represents the cAMP-stimulated
apical anion conductance of MR cells. The presence of the protein does not, of
course, guarantee that the channels are all activated.
In opercular membranes from FW-adapted killifish, the diffuse distribution
of kfCFTR immunofluorescence in the basal portion of MR cells could represent
kfCFTR in the basolateral membrane (on the extensive infoldings of basolateral
membrane in the tubular system of these cells), but equally possible it could
be kfCFTR expressed in vesicles throughout the cytosol. A functional
interpretation of the former is that basal kfCFTR could serve in anion uptake
as well as transfer of acid equivalents, since CFTR anion channels are
permeable to HCO3-
(Linsdell et al., 1997) and
associated with HCO3- secretion in mammalian duodenum
(Hogan et al., 1997
). The
latter possibility implies expression and storage of CFTR in `anticipation' of
future function, once inserted into the apical membrane during SW adaptation.
Other recent immunocytochemical examinations of teleost gills, did not test
for CFTR antibodies in FW animals (Wilson et al., 2001a,b). Overall, the
possible role of CFTR in FW ion transport is an intriguing possibility that
has not been examined.
The small percentage (8.1 % of the total) of MR cells in FW-adapted animals
that displayed apical crypts containing kfCFTR immunofluorescence may
represent a functional subgroup of MR cells that are either involved in
HCO3- permeation in acidbase balance (as
suggested by Wilson et al.,
2000a) or are preadapted for Cl- secretion, should the
animal encounter high salinity (implying that the kfCFTR is present in the
apical membrane but normally inactivated). The shallow depth (as in SW
membranes) of the kfCFTR distribution suggests that it is exposed to the
environment rather than being covered over by pavement cells. The latter is
probable because stimulation of FW opercular epithelia with cAMP produces an
immediate initiation of serosa positive transepithelial potential and an
Isc of approximately 25 µamp cm-2, 10 % of
the full SW capacity of the opercular epithelium, to generate cAMP mediated
Cl- secretion (Marshall et al.,
1999
).
In the 24 h transfer group the kfCFTR immunofluorescence is punctate in a
larger portion of MR cells (18.8%), but the larger distance from the surface
suggests that either some of the kfCFTR is in cells without apical exposure or
that the apical crypts are deeper than in SW-adapted animals. Diffuse kfCFTR
immunofluorescence persists in most cells and, at this same time, the
generation of cAMP-stimulatable Cl- secretion is lower than that
seen in fully adapted marine killifish (approximately 23 % of SW levels;
Marshall et al., 1999). At 48
h, 76.3 % of the MR cells had punctate kfCFTR immunofluorescence at a time
when the cAMP-stimulatable Cl- secretion by the opercular membrane
is 71 % of that in fully adapted SW killifish
(Marshall et al., 1999
). There
appears to be good agreement between the percentage of MR cells with punctate,
SW-like, kfCFTR immunofluorescence and the capacity of the epithelium at each
stage to generate cAMP-stimulatable Cl- secretion.
The change in distribution of kfCFTR from a diffuse cytosolic pattern
progressively to one of punctate apical localization suggests that kfCFTR is
trafficked from subapical locations to concentrated points and inserted in the
apical membrane, coinciding with the generation of cAMP-stimulatable
Cl- secretion. The process probably includes de novo
expression of kfCFTR, as indicated by the increased level of expression,
detected by northern analysis, starting at 8 h, reaching a maximum at
approximately 24 h and remaining elevated during the balance of SW adaptation
(Singer et al., 1998). The
mechanism and regulation of this phenomenon are unknown, but presumably
resemble that of higher vertebrates. In any case, it would seem to be a
relatively slow process occurring over a period of days, not minutes.
Previous data reported on CFTR redistribution indicate a rapid process of
exocytosis from areas immediately below the apical membrane of shark
Squalus acanthias rectal gland salt-secreting cells
(Lehrich et al., 1998). The
response is rapid and mediated by vasoactive intestinal polypeptide (VIP).
Such redistribution is strongly associated with elevated NaCl and fluid
secretion by the rectal gland. This phenomenon has been confirmed in rat
duodenum, and is also in response to VIP
(Ameen et al., 1999
). Hence,
activation of quiescent NaCl-secreting epithelia is strongly associated with
trafficking of CFTR into the apical membrane. In another system, CALU-3 airway
epithelial cells, rapid CFTR activation does not appear to require exocytosis,
as the channel is already present in the apical membrane
(Loffing et al., 1998
). The
heat stress protein HSP70, which in animals is activated by a variety of
stressors, promotes trafficking of CFTR, even with the
F508 error that
in CF impedes normal trafficking (Chou-Kang
and Zeitlin, 2001
). Salinity change is an adaptation involving
cortisol responses that occur several hours before the increase in CFTR
expression (Jacob and Taylor,
1983
; Marshall et al.,
1999
), so in teleosts we also see the association of a `stressor'
with augmented CFTR trafficking. The common feature shared by airway epithelia
and MR cells is that the resting secretion rate is well above zero, implying
that CFTR is already present in the apical membrane, in contrast to shark
rectal gland and rat duodenum, where resting secretory rates are zero,
obliging the cells to remove CFTR from the apical membrane in the absence of
hormone.
CFTR distribution in pavement cells
Whereas kfCFTR immunostaining is absent from pavement cells of opercular
epithelia from SW-adapted fish, there is strong immunostaining of pavement
cells in membranes from FW-adapted fish and in the 24 and 48 h SW-transferred
animals. The exact distribution is uncertain, because of the thinness of the
cells, but clear immunostaining at the plane of the nucleus indicates a
cytosolic or basolateral membrane localization. The exact localization will
require TEM and immunogold experiments. The presence of kfCFTR in the
basolateral membranes of pavement cells in FW is consistent with the uptake of
Cl- and/or translocation of acid equivalents across the basolateral
membrane in the form of HCO3-. It is well known that
CFTR in mammalian systems has significant conductance to
HCO3- (Linsdell et
al., 1997; Hogan et al.,
1997
) and has been used this way in a model for fish by Wilson et
al. (2000a
); apical CFTR is
thought to aid ammonia fluxes by moving base equivalents out across the apical
membrane. When killifish are acid-loaded with injected HCl, they appear to
secrete acid equivalents efficiently along with Cl-, the acid load
significantly augmenting the Cl- efflux. In contrast, the standard
FW trout response to similar treatment is to increase Na+ efflux
and inhibit Cl- efflux while augmenting acid secretion (e.g.
Wood, 1991
). The killifish
response might therefore be connected to activation of apical CFTR channels in
FW pavement cells. Clearly closer examination of pavement cell transporters
may illuminate their function in ion transport.
NKCC distribution in MR cells
NKCC immunofluorescence in Cl--secreting epithelia is
basolateral, as measured in vesicle experiments and as detected by
immunocytochemistry and immunoelectron microscopy. Basolateral membrane
vesicles from rainbow trout gill tissue demonstrated a bumetanide- and
furosemide-sensitive Na+,K+,2Cl- cotransport
that was particularly sensitive to extracellular K+
(Flik et al., 1997). Further,
there was obvious expression of the cotransporter in FW-acclimated animals and
an upregulation of the cotransporter on acclimation to 70 % and 100 % SW
(Flik et al., 1997
). NKCC has
been identified immunocytochemically in basolateral surfaces of epithelial
cells of shark rectal gland (Lytle et al.,
1992
), MR cells of mudskipper gill
(Wilson et al., 2000a
), MR
cells of juvenile salmonid gill (Pelis et
al., 2001
) and human colonic epithelial cell line T84
(D'Andrea et al., 1996
). In T84
cells, agonists that operate in part via cAMP evoke a rapid,
actin-dependent, activation of NKCC and an increase in cell-surface expression
of the cotransporter (D'Andrea et al.,
1996
). In epithelia undergoing NaCl uptake, such as the kidney,
NKCC localizes to the apical membrane instead
(Biemesderfer et al.,
1996
).
Our work suggests that a change in salinity is associated with a relatively
slow redistribution of NKCC from an eccentric localized position in FW MR
cells to an evenly distributed diffuse pattern in the basal portion of MR
cells in SW-adapted animals. The FW immunoreactivity distribution pattern of
NKCC confirms the cotransporter operation in basolateral membrane vesicles
from FW rainbow trout gill (Flik et al.,
1997) and extends the finding by demonstrating that NKCC is indeed
restricted to MR cells of FW-adapted fish, as predicted by Flik et al.
(1997
). Because NKCC is
apparently not present in the apical membrane area of MR cells, ion uptake
observed in opercular epithelia from FW-adapted killifish
(Marshall et al., 1997
)
probably does not involve this transporter. It is not clear how killifish
differ in the handling of NKCC in FW, but the eccentric localization of NKCC
in ion-transporting cells is a new finding and may be significant. Basolateral
NKCC in FW could be involved in cell volume regulation of MR cells
(Flik et al., 1997
), but this
suggestion is currently speculative. The links between MR cell volume and
transport rate in SW clearly connect cell swelling with decreased salt
secretion (e.g. Daborn et al.,
2001
), but equivalent investigations have not yet been performed
in FW systems. In any case, as the NKCC operation is strongly dependent on the
degree of phosphorylation of the protein
(Haas and Forbush, 1998
), NKCC
may potentially be involved in regulation of cell volume and, secondarily, ion
transport rates.
In contrast to the images seen in FW cells, the diffuse distribution in SW
MR cells is similar to those seen in elasmobranch rectal gland
(Lytle et al., 1992),
mudskipper gill MR cells (Wilson et al.,
2000a
) and salmon smolt gill MR cells
(Pelis et al., 2001
), and is
fully consistent with localization of the cotransporter to infoldings of the
basolateral membrane, the tubular system of MR cells.
Na+,K+,2Cl- cotransport increases almost
fivefold with adaptation of rainbow trout to 70 % and full-strength SW
(Flik et al., 1997
). NKCC
abundance and the number of MR cells increased with smolting, and apparently
colocalized in all cases with Na+,K+-ATPase
immunofluorescence (Pelis et al.,
2001
). In FW salmon, the basal NKCC is presumably not hormonally
activated because the MR cells are not actively involved in ion secretion
until the animal enters SW. MR cells can rapidly close the apical crypt,
allowing pavement cells to cover over MR cells, thus effectively removing the
cells from contributing to NaCl secretion. This dynamic covering of MR cells
has been observed in mudskipper gill when the animals are transferred to FW
(Sakamoto et al., 2000
) and in
isolated killifish opercular epithelium exposed to basolateral hypotonic shock
(Daborn and Marshall, 1999
;
Daborn et al., 2001
).
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Acknowledgments |
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