Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA
* Author for correspondence (e-mail: sibley{at}borcim.wustl.edu)
Accepted 3 April 2003
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Summary |
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Key words: Calcium, Invasion, Parasite, Secretion, Motility, Intracellular, Signaling
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Introduction |
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Artificially increasing intracellular calcium in the parasite through the
use of calcium ionophores A23187 or ionomycin triggers parasite microneme
secretion in the absence of host cells
(Carruthers and Sibley, 1999).
Conversely, chelation of intracellular calcium in the parasite blocks
microneme secretion and invasion
(Carruthers and Sibley, 1999
).
Studies showing that EGTA blocks invasion by T. gondii suggest that
extracellular calcium might also play a role in the host-parasite interaction
(Pezzella et al., 1997
).
Fluorescent imaging studies have revealed that parasites in association with
host cells show elevated levels of cytoplasmic calcium
(Vieira and Moreno, 2000
).
These studies have led to a model suggesting that contact with host cells
triggers a rise in cytoplasmic calcium in the parasite, thus triggering
exocytosis of adhesins that are needed for strengthening attachment and
promoting cell entry.
T. gondii possess several stores of calcium, including the
acidocalcisomes, mitochondria, and the endoplasmic reticulum
(Moreno and Zhong, 1996).
Whether all or some of these stores are released during invasion is unknown.
Recent studies have identified IP3 as a second messenger that mediates
increases in intracellular calcium in T. gondii following artificial
stimuli (Lovett et al., 2002
).
T. gondii also responds to caffeine and ryanodine, suggesting
apicomplexans also contain a ryanodine-like response channel for regulating
release of intracellular calcium (Lovett
et al., 2002
).
Although previous studies have indicated an important role for calcium during parasite invasion of host cells, the source of calcium and whether it acts within the parasite or the host cell remain unresolved. To resolve this important issue, we examined the contribution of intracellular and extracellular calcium sources during parasite motility and invasion into host cells. Additionally, we monitored dynamic calcium changes in parasites and host cells during invasion using the calcium sensitive indicator fluo-4.
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Materials and Methods |
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Chemicals and solutions
EGTA (Sigma, St Louis, MO) stock solutions (2x) were prepared in low
calcium Ringer's (155 mM NaCl, 3 mM KCl, 5 mM MgCl2, 3 mM
NaH2PO4, 10 mM HEPES, 10 mM glucose, 0.1 mM EGTA) and
adjusted to pH 6.0 or 7.2. BAPTA-AM [1,2-bis
(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid, sodium salt] and BAPTA were purchased from Calbiochem (San Diego, CA),
and fluo-4 AM was purchased from Molecular Probes (Eugene, OR). All other
reagents were analytical grade. Stock solutions of BAPTA were prepared in
distilled water, and BAPTA-AM solutions were made in DMSO.
SDS polyacrylamide gel electrophoresis and western blotting SDS-PAGE was
performed in 7% minigels under reducing conditions and transferred to
nitrocellulose as previously described
(Carruthers and Sibley, 1999).
Western blotting was performed with mouse anti-TgMIC2 monoclonal antibody 6D10
(ascites, 1:10,000) (Carruthers and
Sibley, 1999
) and rabbit polyclonal anti-TgACT1 actin antibody
(1:10,000) (Dobrowolski et al.,
1997
). Blots were detected using enzyme conjugated secondary
antibodies (Jackson ImmunoResearch Labs, West Grove, PA) combined with
SuperSignal ECL (Pierce, Rockford, IL).
Gliding assay
Glass coverslips (13 mm round) were incubated overnight with complete media
(Dulbecco's modified Eagle's medium, 10 mM HEPES, 44 µM sodium bicarbonate,
10% fetal bovine serum, 2 mM glutamine, 20 µg/ml gentamicin). Parasites
were resuspended in low calcium Ringer's. BAPTA-AM was loaded into host cells
or parasites for 10 minutes at 18°C, while BAPTA or EGTA were added to
parasites immediately prior to use to minimize leaching of intracellular
calcium stores. Parasites were added to coverslips previously washed with low
calcium Ringer's and incubated for 15 minutes in a 37°C water bath. After
removal from the water bath, coverslips were washed twice with warm low
calcium Ringer's and fixed with 2.5% formalin for 20 minutes at 4°C.
Coverslips were stained with Alexa Fluor® 488 (Molecular Probes)
conjugated monoclonal antibody DG52 against SAG1 (1:1000) and observed using a
Zeiss Axioskop (Carl Zeiss) equipped with epifluorescence and phase contrast
optics. Images were captured using an ORCA-ER digital cooled CCD camera
(Hamamatsu Photonics K.K., Hamamatsu City, Japan) at 63x magnification
controlled by Openlab v3.0.8 imaging software (Improvision, Lexington,
MA).
Microneme secretion assay
Parasites were resuspended at 108/ml in low calcium Ringer's.
BAPTA-AM was loaded for 10 minutes at 18°C, while BAPTA or EGTA were added
immediately prior to stimulation to minimize leaching of intracellular
Ca2+ stores. Secretion was stimulated by addition of ethanol to 1%
and incubation at 37°C for 2 minutes
(Carruthers et al., 1999b). An
unstimulated sample, that was not raised to 37°C and to which ethanol was
not added, served as a negative control for background. Parasite supernatants
were separated from pellets by centrifugation at 4°C and run on 7%
SDS-PAGE gels. Each gel contained standard dilutions of a parasite cell lysate
corresponding to percentages of the total number of parasites used.
Inadvertent lysis of parasites was monitored by the release of constitutively
expressed actin, which is 98% globular in T. gondii
(Wetzel et al., 2003
).
Attachment and invasion assays
Glass-bottom 10 mm microwells (MatTek Corp, Ashland, MA) were seeded with
host cells to a final density of 60 to 80% confluency. Parasites were
resuspended at 108 per ml in low calcium Ringer's. BAPTA-AM was
loaded into monolayers of host cells or parasites for 10 minutes at room
temperature, while BAPTA or EGTA were added to parasites immediately prior
placement on coverslips to minimize leaching of intracellular calcium stores.
Microwells were washed with low calcium Ringer's before the addition of
parasites and incubated for 15 minutes in a 37°C water bath. After removal
from the water bath, coverslips were washed twice with warm low calcium
Ringer's and fixed with 2.5% formalin for 20 minutes at 4°C. Coverslips
were blocked for 30 minutes with PBS containing 5% (v/v) fetal bovine serum,
and 5% (v/v) normal goat serum. Coverslips were sequentially stained with
Alexa Fluor® 594 (Molecular Probes) conjugated monoclonal antibody DG52
against SAG1 (1:1000) to visualize extracellular parasites, permeabilized with
0.01% saponin, then stained with Alexa Fluor® 488 conjugated monoclonal
antibody DG52 against SAG1 (1:1000)
(Håkansson et al., 1999)
to visualize intracellular parasites. Coverslips were observed with a Zeiss
Axioskop microscope and a 63x objective. Results of three independent
experiments were compiled. For each experimental condition, five fields with
approximately 20 parasites each were scored for the number of parasites
intracellular, extracellular but touching a fibroblast, or extracellular but
not in contact with host cells.
Video microscopy
Freshly egressed parasites were loaded with 300 nM Fluo-4 for 5 minutes at
37°C, centrifuged at 400 g for 5 minutes at room
temperature, and resuspended in 37°C Ringer's plus calcium (155 mM NaCl, 3
mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM
NaH2PO4, 10 mM HEPES, 10 mM glucose). Sub-confluent
monolayers of host cells were loaded by resuspending in Ringer's plus calcium
containing 2 µM fluo-4 and incubating at 37°C 15 minutes. After
incubation, parasites or cells were washed and resuspended in Ringer's plus
calcium. Parasites were added to the culture dishes containing host cells
dishes and observed on a Zeiss Axiovert equipped with phase contrast,
epifluorescence microcopy and a temperature-controlled stage (Medical Systems,
Greenvale, NY) held at 37°C. Parasite motility was observed within minutes
after placing the dish on the heated stage and images were recorded over a
period of up to 15 minutes. Time-lapse images were collected at two frames per
second under low-light illumination using an ORCA-ER digital cooled camera at
63x magnification and 640x480 pixels controlled by Openlab v3.0.8
imaging software (Improvision, Lexington, MA). Phase and fluorescent images
were cropped, merged, and saved as Quicktime movies (v5.0).
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Results |
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Previous reports indicate that chelation of calcium with high levels of
EGTA decreased parasite invasion (Pezzella
et al., 1997). However, our initial attempts to replicate this
finding indicated that dilution of a 20x stock solution of EGTA (pH 8.0)
into complete culture medium resulted in acidification. Nitrilo nitrogens in
EGTA bind protons with pKas of 8.96 and 9.58
(Martell and Smith, 1974
),
leading to pH-sensitive affinity for calcium. Consequently, the dilution of
EGTA stock solutions into calcium-containing medium releases 2 mol
H+ for each mol Ca2+ bound, thus resulting in
acidification. Previous studies have indicated that acidic pH is detrimental
to parasite invasion (Sibley et al.,
1985
). To determine the relative contribution of acidification
versus calcium chelation, we made 5 mM EGTA solutions in low calcium Ringer's
and adjusted them to pH 6.0 or pH 7.2. These solutions were used for
attachment and invasion assays shown in
Fig. 1. Incubation of parasites
in the presence of 5 mM EGTA at a medium pH of 7.2 did not affect attachment
or invasion into host cells. In contrast, parasite invasion in the presence of
5 mM EGTA at a medium pH of 6.0 was substantially inhibited, although
attachment appeared unchanged (Fig.
1). Collectively, these results indicate that although decreased
pH reduces invasion, chelation of extracellular calcium has no effect.
We also examined the effect of chelating intracellular calcium on the
attachment and entry of parasites into host cells. Loading of the
cell-permeant chelator BAPTA-AM into host cells prior to invasion had no
effect when compared to a diluent control
(Fig. 1). Thus, host cell
calcium does not appear necessary for invasion by T. gondii.
Alternatively, preloading parasites with BAPTA-AM prior to invasion inhibited
both attachment and invasion of host cells
(Fig. 1)
(Carruthers et al., 1999a).
Extracellular calcium is not required for trail formation
Parasite invasion into host cells is an active process that depends on
parasite motility (Dobrowolski and Sibley,
1996). Parasites exhibit contact-dependent gliding motility that
results in formation of membrane trails on the substratum
(Håkansson et al.,
1999
). We examined whether the presence or absence of
extracellular calcium affected motility as monitored by trail formation.
Parasites were treated with BAPTA-AM, BAPTA, or EGTA before addition to
coverslips where they were allowed to glide for 15 minutes. After washing and
fixation, trails were visualized by staining of the parasite surface protein
SAG1. BAPTA-AM pretreatment inhibited trail formation markedly versus a
diluent control (Fig. 2). In
contrast, BAPTA treatment had no apparent effect, and a similar density of
trails was observed as in the diluent treated control
(Fig. 2B,C). Addition of 5 mM
calcium did not alter motility compared to the diluent control
(Fig. 2D), yet trails appeared
discontinuous and punctuated upon close examination. Interestingly, treatment
of parasites with EGTA in either pH 6.0 or pH 7.2 media also did not affect
trail formation (Fig.
2E,F).
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Extracellular calcium is not required for microneme secretion
To determine the source of calcium important for influencing microneme
secretion, we treated parasites with 50 µM BAPTA-AM to chelate
intracellular calcium, or used 1 mM BAPTA or 5 mM EGTA to chelate
extracellular calcium. Parasites were then placed at 37°C to stimulate
secretion. Cells were removed by centrifugation and secretion was evaluated by
detecting MIC2 in the supernatants by western blotting
(Fig. 3). MIC2 is a convenient
marker for microneme secretion because the secreted 95-100 kDa form (sMIC2) is
released into the supernatant, whereas the cell-associated 115 kDa form
(cMIC2) remains in the parasite pellet
(Carruthers et al., 2000).
Constitutive expression of actin was used to control for inadvertent lysis of
the parasites during the experiment and was typically between 1% and 5%, as
indicated by comparison with dilutions of parasite cell standards (cell stds)
(Fig. 3).
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Secretion of MIC2 was inhibited by pre-loading of parasites with BAPTA-AM
in comparison with a DMSO diluent control, indicating an increase in
intracellular calcium is necessary for secretion
(Fig. 3). Treatment with
non-permeant BAPTA did not affect MIC2 secretion, indicating that
extracellular calcium is not required. Interestingly, addition of excess (5
mM) calcium did not provoke additional secretion, providing evidence that
extracellular calcium does not increase the efficiency of secretion
(Fig. 3). Secretion of MIC2
after treatment with EGTA at pH 7.2 did not compromise in the amount of
protein released (Fig. 3). In
contrast, treatment with EGTA at pH 6.0 led to a reduction in the amount of
MIC2 secreted when compared with the control
(Fig. 3). Addition of 1%
ethanol to stimulate secretion (Carruthers
et al., 1999b) was used as a positive control, whereas
unstimulated parasites that were not placed at 37°C were used as a
negative control (Fig. 3).
Collectively these results show that intracellular calcium in the parasite is
essential for microneme secretion, whereas extracellular calcium plays little
role in this process.
Host cell calcium does not change during parasite invasion
Although our studies using BAPTA-AM indicated that host cell calcium was
not required for parasite invasion, we were interested in determining whether
host cell calcium changed during the process of parasite invasion. We
monitored host cell calcium during parasite invasion using the indicator
fluo-4, which is a qualitative indicator of intracellular calcium in the range
of 0.1 to 1 µM (Gee et al.,
2000). Monolayers of human fibroblasts were loaded with 2 µM
fluo-4 AM and the excess dye washed away, leaving fluo-4 localized throughout
the cytoplasm. Images were taken at
0.5 second intervals, alternating
between phase contrast and fluorescence modes. Unstimulated fibroblasts showed
calcium increases lasting approximately six seconds, during which a wave of
increased fluorescence spread across the cell (Movie 1, available at
jcs.biologists.org/supplemental).
The magnitude of these changes was dramatic, with increased signals of fluo-4
being up to 250% higher than background levels (data not shown, n=3
examples monitored over a 15-30 second period). These events were rare,
occurring about once every 10 minutes (data not shown). Fibroblast calcium
fluxes were likely to be associated with normal signaling processes, as the
cells appeared healthy both before and after transients occurred.
To examine changes that occur during invasion, parasites were added to fluo-4-loaded fibroblasts, and phase and fluorescent images were acquired at 0.5 second intervals between each set of images. Host cell calcium levels remained relatively constant during invasion as judged by the fact that only small, localized increases in fluorescence were observed (Fig. 4B,C; Movie 2, available at jcs.biologists.org/supplemental). The magnitude of changes in the fluo-4 signal in host cells was ≤20% different from background (data not shown, n=3 separate examples monitored for 15-30 seconds), in contrast to the rather dramatic natural changes in host cell calcium that occasionally occurred in the absence of invasion (see Fig. 4A). These observations indicate that parasite invasion occurs without an active participation or response of calcium from the host cell.
|
Calcium fluxes occur during parasite gliding but are rapidly down
regulated during invasion
Previous studies have suggested that increases in parasite intracellular
calcium occur during contact with the host cell
(Vieira and Moreno, 2000);
however, the kinetics of calcium increase were not examined. We predicted that
parasites entering cells would show increases in calcium during attachment and
invasion. We used fluo-4 AM loaded tachyzoites to visualize qualitative
changes in parasite calcium during gliding motility and host cell
invasion.
Gliding was associated with brightly fluorescent parasites that underwent
periodic cycles of increase followed by dampening of the signal
(Fig. 5A; Movie 3, available at
jcs.biologists.org/supplemental).
Calcium fluxes correlated with parasites that were actively motile and often
preceded a burst of motility; whereas dimly stained cells remained immotile.
Kinetic changes in fluo-4 fluorescence were plotted over time by taking the
average pixel intensity of the parasite from successive frames of the video
recording (Fig. 6A). The cycles
of increased fluorescence were monitored from independent recordings to
determine the average time between successive cycles and the relative length
of the cycles (Table 1). Typically, individual cycles lasted 30 seconds and were closely spaced in
sequence. The initial cycle was the brightest with successive cycles becoming
dimmer. The drop in intensity correlated with the gradual loss of motility.
Irrespective of whether they are loaded with fluo-4, parasites that are
examined by phase contrast or epifluorescence illumination typically maintain
motility for only one to two minutes. It is uncertain whether this decrease is
because of photodamage or loss of essential factors such as energy stores that
are necessary to maintain activity.
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We also followed the calcium response of fluo-4-labeled parasites while they invaded fibroblasts. Surprisingly, the bright fluorescence exhibited by motile parasites rapidly diminished during the initial steps of invasion (Fig. 5B,C; Movie 4, available at jcs.biologists.org/supplemental). The drop in signal intensity following invasion occurred more rapidly than had been observed with fluxes occurring during gliding motility (Table 1 and Fig. 6B). The decrease in signal was not due to photobleaching, as other brightly labeled parasites in the same field did not display similar decreases in intensity (data not shown). Intracellular parasites remained dim even when analyzed for extended periods of time (up to five minutes, data not shown).
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Discussion |
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We addressed the role of host cell calcium by using the intracellular
chelator BAPTA-AM. Chelation of host cell calcium using BAPTA-AM had no effect
on parasite attachment or invasion. The concentration of BAPTA-AM used should
have effectively chelated intracellular calcium in the fibroblasts based on
other studies using lower concentrations of this compound on fibroblasts
(Scheidegger et al., 1999;
Shahrestanifar et al., 1999
),
and the observation that the cell monolayer began to detach from the substrate
if loading times were prolonged (data not shown). Thus, unlike the entry of
T. cruzi into mammalian cells
(Rodriguez et al., 1995
),
changes in host cell calcium do not appear to be required for efficient entry
of T. gondii.
Consistent with the inhibitor studies, direct monitoring of intracellular
calcium in host cells indicated that only weak local calcium transients occur
during parasite invasion. Such calcium transients may be associated with
membrane perturbation by the parasite that was previously observed as a spike
in capacitance of patch-clamped host cells
(Suss-Toby et al., 1996).
Alternatively, it could indicate activation of local host signaling pathways
required for membrane repair. In either case, the very low response observed
in the host cell parallels our finding that host cell calcium was not required
for parasite invasion. These findings support previous observations that
indicate a relatively passive role of the host cell during parasite invasion.
For example, unlike phagocytosis or entry of many bacterial pathogens,
invasion of mammalian cells by T. gondii does not evoke changes in
the host cell cytoskeleton, tyrosine phosphorylation or membrane ruffling
(Morisaki et al., 1995
;
Håkansson et al.,
1999
).
In contrast to the lack of an essential role for host cell calcium,
chelation of intracellular calcium in the parasite using BAPTA-AM treatment
prevented microneme secretion and dramatically reduced both motility and
invasion into host cells. Since levels of calcium in the parasite appeared
critical to the process of invasion, we sought to identify whether
extracellular calcium was necessary for parasite invasion into host cells. A
previous report that chelation of extracellular calcium by EGTA inhibited
T. gondii invasion hypothesized that a flux of calcium from outside
to inside the parasite was required during invasion
(Pezzella et al., 1997).
However, as we show here, this effect was likely to be as a result of the
acidifying affects of EGTA on the medium. When EGTA containing medium was
adjusted to pH 7.2, or when the non-acidifying chelator BAPTA was used, no
effect of chelating extracellular calcium was observed. Furthermore, adding
excess calcium had no influence on parasite invasion into host cells. We
therefore conclude that extracellular calcium is not required for invasion of
cells by T. gondii. These results indicate that a
calcium-induced-calcium-entry pathway, where initial increases in cytosolic
calcium result in influx of extracellular calcium from outside the cell
(Berridge et al., 2000a
;
Berridge et al., 2000b
), is not
essential for parasite invasion of host cells.
To explore the role of calcium on T. gondii secretion and
motility, we used a similar approach to look at the role of extracellular
versus intracellular calcium sources in mediating these events. Chelation of
intracellular calcium prevented microneme release, consistent with a previous
report (Carruthers and Sibley,
1999), whereas chelation of extracellular calcium had no effect.
Likewise, although chelation of intracellular calcium with BAPTA-AM prevented
motility, chelation of extracellular calcium had little effect on motility.
Collectively, these studies indicate that intracellular calcium in the
parasite controls microneme secretion and motility.
Monitoring of intracellular calcium in the parasite revealed oscillating
fluxes in motile parasites, either immediately preceding or during gliding
motility. Previous models suggested that calcium increases would correlate
with contact with the host cells immediately prior to invasion. Instead, our
observations indicate that oscillating increases in parasite intracellular
calcium occur during gliding, regardless of contact with host cells. These
changes in calcium may reflect an important role for this second messenger in
regulating motility and/or release of adhesins that are necessary for
attachment to the substratum. Calcium transients that were observed during
motility halted abruptly and the fluorescent calcium signal dramatically
decreased during invasion of the parasite into host cells. This finding was
unexpected, since previous studies have shown increased cytoplasmic calcium in
parasites that were associated with host cells
(Vieira and Moreno, 2000).
These previous findings may have reflected parasites moving while on top of
host cells, which could explain their increased calcium levels. Collectively
our findings suggest a more complex model wherein increases in intracellular
calcium are needed for motility, while cell entry does not require continued
elevation of intracellular calcium in the parasite. Furthermore, the rapid
shut off of intracellular calcium in invading parasites may be important for
downregulating cellular processes, such as microneme secretion and motility,
once invasion is complete.
Despite the central importance of calcium in parasite secretion and
motility, relatively little is known about the intracellular pools of calcium
or how the release of calcium is regulated. The oscillating patterns of
calcium in gliding parasites suggest that intracellular release channels and
re-uptake mechanisms are highly active in the parasite. In most cells, the
major pool of mobilizable calcium is the endoplasmic reticulum (ER) where
release is mediated by IP3 or ryanodine-type channels
(Berridge et al., 2000a;
Berridge et al., 2000b
). T.
gondii responds to agonists/antagonists of both IP3-type
release channels and ryanodine-type channels, suggesting both pathways could
operate in the parasite (Lovett et al.,
2002
). Typically, ER stores are refilled by calcium transporters
known as SERCA-type ATPases, which can be inhibited by thapsigargin
(Thastrup et al., 1989
).
Protozoan parasites, including T. gondii, are sensitive to this
inhibitor, and yet they also have thapsigargin-insensitive calcium pools
(Carruthers et al., 1999). SERCA-type ATPases have not been described in
T. gondii, although several putative orthologs are present in the
genome databases for T. gondii
(http://ToxoDB.org/ToxoDB.shtml).
T. gondii also contains plasma membrane type Ca-ATPases, one of which
has recently been shown to localize both to the plasma membrane and a unique
organelle called the acidocalcisome (Luo
et al., 2001
). These acidic, calcium-rich organelles appear to
play an important role in polyphosphate metabolism and serve as a storage site
for calcium in protozoan cells (Docampo
and Moreno, 2001
; Rodrigues et
al., 2002
).
Our investigations demonstrate that intracellular calcium plays a crucial role in controlling protein secretion and motility in the parasite T. gondii. The complete reliance of the parasite on its own intracellular calcium stores for motility presents a potential target for intervention. Further studies might reveal sufficient differences between parasite and host calcium homeostasis mechanisms that can be used to disrupt calcium-regulated secretion and/or motility, and thereby prevent infection.
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Acknowledgments |
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