1 School of Life Sciences, Wellcome Trust Biocentre, University of Dundee,
Dundee DD1 5EH, UK
2 MRC Laboratory for Molecular Cell Biology and Department of Biology,
University College, Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: j.g.williams{at}dundee.ac.uk)
Accepted 22 June 2004
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SUMMARY |
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Key words: GSK-3, Dictyostelium, cAMP, DIF, DIF-1, 2C gene
![]() |
Introduction |
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Dictyostelium has a single GSK3 homologue, GskA, that has 78%
amino acid identity to mammalian GSK3ß
(Harwood et al., 1995). Upon
starvation, neighbouring Dictyostelium amoebae aggregate, using cAMP
as chemoattractant. The cells then differentiate into prestalk and prespore
cells, which coordinately move and terminally differentiate to form the stalk
and spore head of the fruiting body. Cell fate is determined by diffusible
signalling molecules; extracellular cAMP induces prespore differentiation
(Kay et al., 1978
;
Mehdy et al., 1983
;
Oyama et al., 1988
), while
differentiation inducing factor 1 (DIF-1) directs prestalk-specific gene
expression (Williams et al.,
1987
). Prestalk cells comprise a number of subpopulations and one
of these, the pstB cells, ultimately forms the basal disc; the structure that
anchors the stalk to the substratum
(Jermyn et al., 1996
).
The gskA-null mutant, which is generated in the auxotrophic strain
DH1, develops to form a fruiting body with a greatly enlarged basal disc and
tiny spore head (Harwood et al.,
1995). The strain displays a highly aberrant pattern of expression
of both the prestalk/stalk-specific gene ecmB and the
prespore-specific gene psA. ecmB mRNA is detected 4 hours earlier and
at a much higher level in the mutant than in DH1 parental cells. By contrast,
expression of the prespore marker gene psA is reduced to 5% of
control levels in the gskA-null mutant. Consistent with this gene
expression pattern, prespore cell numbers are severely reduced and the mound
is almost entirely filled with cells that, based upon their patterns of
expression of the ecmA and ecmB genes, are pstB cells. In
monolayer culture experiments, DH1-derived gskA-null cells do not
display the cAMP repression of stalk cell induction, which is normally seen in
wild-type cells, and are only poorly induced to form spores.
Consistent with a role in regulating cell fate, GskA activity is
upregulated about twofold at the mound stage and the cAMP-receptor cAR3
mediates this activation (Plyte et al.,
1999). The dual specificity tyrosine kinase ZAK1 tyrosine
phosphorylates GskA and this activates it
(Kim et al., 1999
;
Kim et al., 2002
). The
activity of ZAK1 is developmentally regulated, with kinetics that are
approximately coincident with the activation profile of GskA. Furthermore,
there is no peak of GskA kinase activity in a zakA-null strain
(Kim et al., 1999
). Likewise,
the GskA activation peak during development is missing in the
cAR3-null strain (Plyte et al.,
1999
). All three strains, the gskA-null, the
zakA-null and the cAR3-null, are insensitive to the
inhibitory effect that cAMP exerts on the induction of stalk cell
differentiation by DIF-1 (Harwood et al.,
1995
; Kim et al.,
1999
).
The only known substrate for GskA is the STAT transcription factor Dd-STATa
(Kawata et al., 1997).
Phosphorylation of Dd-STATa by GskA enhances nuclear export of Dd-STATa
(Ginger et al., 2000
).
Although upstream regulators of GskA, and a target of its action, have been
identified, it is unclear precisely how GskA exerts its effects on cell fate.
We report an analysis of a gskA mutant strain, made in a different
genetic background, that significantly modifies understanding of the
developmental roles played by GskA.
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Materials and methods |
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Cell culture and development
Axenic Dictyostelium strains were grown at 22°C in HL5 medium
(Watts and Ashworth, 1970).
For selection of transfected strains expressing dominant markers, the medium
was supplemented with 10 µg/ml Blasticidin S (Cayla) or 20-200 µg/ml
G418 (Sigma). For development on a bacterial lawn, cells were spread together
with a suspension of Klebsiella aerogenes on a 160 mm culture dish
with SM-agar (0.5% Bacto-peptone (Difco), 0.05% yeast extract (Oxoid), 0.5%
glucose, 0.23% KH2PO4, 0.13% K2HPO4, 1.5% bacto-agar (pH 6.4) and
allowed to develop at 22°C.
In order to obtain synchronous development, cells were harvested during
logarithmic growth, washed twice in 16.5 mM KH2PO4, 3.8 mM
K2HPO4 (pH 6.2; KK2), resuspended to 1x109
cells/ml in H2O and spread at a density of 2.5x106
cells/cm2 on 1.5% water agar plates. If the formation of slugs was
desired, the cell pellet was resuspended in an equal volume of H2O
to give 1x109 cells/ml. Spots of cell suspension were
placed on 1.5% water agar plates. The agar plates were incubated for 16 hours
in the dark with a unidirectional light source. Slime trails and developing
structures were transferred to overhead projector films (Punchline) and
stained with Coomassie staining solution. For development on filters, the
washed cells were spotted onto nitrocellulose filters sitting on a
KK2-soaked filter pad (Whatman). The development of
Dictyostelium in monolayer culture, and stalk and spore cell
inductions were as described previously
(Harwood et al., 1995
).
Spore viability assay
Spore viability was determined essentially as described
(Dynes et al., 1994). In
brief, spores were suspended in 10 mM EDTA/0.1% Nonidet P-40 in
KK2, incubated at 42°C for 45 minutes, washed three times in
KK2, serially diluted plated on Klebsiella aerogenes. The
number of colonies was scored after 3-4 days.
Immunostaining of prespore vesicles
Slugs were dissociated by trituration through a syringe, fixed in methanol
and immunostained with a polyclonal rabbit, anti-prespore vesicle antibody.
Immunofluorescence was generated using an Alexaflour 488-coupled anti-rabbit
antibody (Molecular Probes).
lacZ reporter gene expression
Clones of an Ax2/gskA mutant and a control random integrant clone
were transformed by electroporation
(Howard et al., 1988) with
lacZ fusion constructs and selected at 20 µg/ml G418. Slugs were
developed on 1.5% water agar, transferred onto glass cover slips and then
processed for lacZ gene expression as described
(Dingermann et al., 1989
).
Induction of prespore cell differentiation in suspension culture
Logarithmically growing cultures were harvested, washed twice with
KK2, plated at 1x108 cells per 9 cm plate on 1.5%
water agar and allowed to develop until aggregation centres formed. Cells were
harvested, resuspended to a density of 1x107 cells/ml in KK2
and shaken in suspension at 300 rpm with pulses of 300 µM cAMP every hour.
Every 2 hours, 0.5 ml of cell suspension were harvested and frozen. Cells were
lysed by repeated freeze-thawing and 100 µl aliquots of cell lysate were
incubated in microtitre plates with 30 µl 2.5x Z buffer
(Dingermann et al., 1989). The
reaction was started by addition of 20 µl ONPG (10 mg/ml), incubated at
22°C until the colour changed and the increase in absorption was measured
at 420 nm. ß-galactosidase activity was normalised against protein
content of the samples and is expressed as gain in absorption per hour per mg
of protein.
Western transfer and northern transfer analyses
Western Transfer analysis was performed as described previously
(Araki et al., 1998). For
detection of GskA the anti-GSK-3 antibody 4G-1E (Upstate Technology) was used.
Northern transfer analysis was performed as described previously
(Fukuzawa et al., 1997
).
Expression profiling
Microarray analysis was performed as described previously
(Araki et al., 2003) but using
the PCR products from 5568 cDNA clones
(Morio et al., 1998
)
(http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html).
The results were normalised for signal intensities, signal-to-noise ratios
(>3), and Lowess-normalised for different dye intensity and analysed using
`GeneSpring' (Silicon Genetics).
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Results |
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|
|
Development of the AX2-derived gskA-null strain on non-nutrient agar is clonally variable but the migratory slug stage is always greatly curtailed
Although many Ax2/gskA cells complete development relatively
normally when allowed to develop asynchronously, on a bacterial lawn, their
development is significantly more aberrant when developing synchronously.
Thus, when Ax2/gskA cells growing in axenic medium are removed from
their food source and allowed to develop on non-nutrient agar, mounds appear
2 hours earlier than in parallel random integrant controls (data not
shown). Furthermore, many of the Ax2/gskA structures terminate
development as mound-shaped structures or very small fruiting bodies
(Fig. 3A). In the
Ax2/gskA mutant migration of slugs, away from the point of aggregation,
is also greatly decreased relative to controls
(Fig. 3B).
|
Ax2/gskA cells precociously express cell-type specific markers
An axenically grown Ax2/gskA clone and a control random integrant
clone were developed on non-nutrient agar and the expression levels of
ecmA, ecmB and psA were compared by northern transfer. In
the Ax2/gskA mutant there is a lower apparent expression level of all
three genes (Fig. 4). This
general reduction in gene expression levels varies from experiment to
experiment, and probably reflects the fact that a variable proportion of the
mutant cells are left behind as mounds
(Fig. 3A); such cells never
enter late development and consequently do not express late developmental
genes. Although there is a lower peak level of psA and ecmB
gene expression in the Ax2/gskA mutant, both genes start to be
expressed about 2 hours earlier in the mutant than in the random integrant.
This also holds true for the ecmA gene. The fact that developmental
gene expression is brought forward by about 2 hours in the Ax2/gskA
strain is consistent with the precocious mound formation mentioned above.
|
|
GskA is not essential for the induction of prespore or spore differentiation
Prespore cell differentiation can be induced in isolated cells by exposure
to extracellular cAMP and the DH1-derived gskA null strain is highly
defective in this response (Harwood et
al., 1995). We therefore determined the cAMP-responsiveness of the
psA promoter in the Ax2/gskA mutant, by monitoring the
induction of ß-galactosidase activity in cells transformed with
psA-lacZ. Exposure to pulses of cAMP induces expression of
lacZ in the mutant and random integrant cells to comparable extents
(Fig. 6A). Induction of the
endogenous psA gene by extracellular cAMP was also monitored, by
northern transfer analysis, and it too is fully cAMP inducible in the
Ax2/gskA mutant (data not shown).
|
At the slug stage ecmB expression is aberrant in the Ax2/gskA strain
We next investigated prestalk differentiation, again using lacZ
reporter gene constructs. The slug contains several different prestalk
populations that are defined by their patterns of expression of reporters
driven by ecmA and ecmB promoter fragments. PstA cells
comprise the front 1/3 of the prestalk region, pstO cells occupy the remainder
of the prestalk region (Early et al.,
1993) and there are scattered cells in the prespore region that
show a similar gene expression pattern to pstO cells. These are termed the
anterior-like cells or ALC (Sternfeld and
David, 1982
). The ecmA-derived prestalk markers,
ecmAO-lacZ (a marker for pstA cells, pstO cells and ALC),
ecmA-lacZ (a marker for pstA cells) and ecmO-lacZ (a marker
for pstO cells and ALC), are expressed in an apparently identical manner in
mutant and control random integrant cells
(Fig. 5A).
The ecmB gene is expressed at a relatively low level at the slug
stage, but its expression increases when culmination begins
(Jermyn et al., 1987). In
marked contrast to the DH1-derived gskA-null strain,
ecmB-lacZ is not overexpressed at the mound stage in Ax2/gskA
cells (data not shown). However, there is a very significant difference in
ecmB-lacZ expression at the slug stage. In the random integrant,
ecmB-lacZ is expressed normally, i.e. in the small cone of pstAB
cells in the slug tip (Fig.
5A), but in the Ax2/gskA strain ecmB-lacZ is
expressed throughout the prestalk region.
The Ax2-derived gskA-null strain is hypersensitive to DIF and shows a greatly reduced sensitivity to extracellular cAMP as an inhibitor of stalk cell differentiation
The above results show that ecmB is mis-expressed at the slug
stage in the Ax2/gskA strain. Therefore, we analysed the response of
the AX2-derived null mutant to the extracellular signals that control
ecmB gene expression. In a monolayer assay, stalk cell
differentiation and ecmB gene expression are induced by the addition
of DIF-1 (Berks and Kay, 1990).
Hence, we first compared the DIF inducibility of parental AX2 cells and the
Ax2/gskA strain. There is a major difference in sensitivity; the
concentration that induces half-maximal stalk cell differentiation is about
10-fold lower in the Ax2/gskA strain
(Fig. 7A). In addition, the
maximal extent of stalk cell differentiation is approximately halved in the
parental strain.
|
As the gskA-null mutant is hypersensitive to DIF-1 (Fig. 7A), it seemed possible that loss of cAMP repression of stalk cell formation may result from saturating concentrations of DIF-1 overriding the inhibition by cAMP. We therefore determined the effect of extracellular cAMP at subsaturating levels of DIF-1. There is a slight increase in the relative inhibitory effect of cAMP in the gskA-null cells as DIF-1 levels are lowered, but this is a very small effect in comparison with the dramatic inhibitory effect that cAMP exerts on control cells at low DIF-1 concentrations (Fig. 7C). We conclude, therefore, that the cAMP repression pathway is defective in the gskA-null strain, but that this not a consequence of elevated DIF sensitivity.
Gene expression profiling of random integrant and Ax2/gskA strains at the slug stage identifies potential activation targets
In order to search for other genes that are regulated by GskA during
multicellular development, we performed microarray analysis. A profile of gene
expression differences in the gskA-null strain was obtained using RNA
samples isolated at the slug stage. The cDNAs were prepared using RNA from
either random integrant or Ax2/gskA slugs and they were labelled with
either Cy3 or Cy5. A mixture of the two differently labelled cDNA preparations
was hybridised to an array bearing PCR products derived from 5568 ESTs
(Morio et al., 1998). Based
upon the estimate of VanDriessche et al.
(VanDriessche et al., 2002
),
there should be no more than a 20% redundancy in this EST set. Hence, the
array potentially accesses
40% of the estimated 11,000 Dictyostelium
genes (Glockner et al.,
2002
).
We are not, unfortunately, able to interpret those ESTs that appear to be over-expressed in the null mutant. This limitation arises because, when developing on non-nutrient agar, a significant proportion of the null strain cells do not reach the slug stage (Fig. 3A). Therefore, those genes that are expressed at a high level during early development, and are then switched off, will appear to be overexpressed in the mutant. We can, however, interpret the data for those genes that are underexpressed in the null strain. This was achieved using psA as a standard to correct for the `dilution effect' that results from the presence of cells arrested as mounds. From the composite microarray data for psA, we estimate this `dilution' effect causes an apparent underexpression of less than twofold [average value for the null/random integrant signal (±s.d.) for psA=0.60±0.085 (n=15)]. As psA is barely expressed at the mound stage but is highly expressed at the slug stage, this will be the maximum extent of the dilution effect; i.e. any gene that is significantly expressed at the mound stage will cause a smaller dilution effect than is observed for psA. As the dilution effect creates an apparent approximate twofold underexpression, we placed a relatively severe significance cut off for the primary analysis of fourfold (Fig. 8).
|
Identifying those ESTs that are strongly overexpressed on all slides automatically excludes ESTs where just one hybridisation failed or gave a high background. Finally, therefore, the entire data set was reanalysed for all ten 2C-related ESTs on the array. The ratio of the 2C signal for null/random integrant is 0.19±0.088. Correction for the dilution factor, 0.60±0.085 (legend to Fig. 8), indicates that 2C is expressed at approximately one-third the wild-type level in the gskA-null strain.
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Discussion |
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Paradoxically, the use of the DH1 strain in the original gskA
study may have actually been fortuitous
(Harwood et al., 1995),
because the milder phenotypes seen with the AX2-derived gskA-null mutant could
have been overlooked. This was the original fate of the cAR3 cAMP receptor
mutant. Initial characterisation of the mutant suggested that it was
morphologically normal but more detailed analysis showed that it is defective
in GskA signalling (Plyte et al.,
1999
). The difference in severity of phenotype between the
gskA- and cAR3-null mutants was originally thought to be due
to receptor redundancy (Plyte et al.,
1999
). However, our present analysis suggests that the
gskA and car3 phenotypes are much more similar than
previously believed.
The rate of early development is accelerated in AX2G/gskA-null cells but the slug phase is curtailed
Dictyostelium cells enter development when their food source, more
specifically their amino acid supply, becomes limiting
(Marin, 1976) and growth
conditions are an important modulator of subsequent development. GSK3 is a
metabolic enzyme and this provides one possible explanation for the fact that
early development is accelerated in AX2G/gskA cells. As the
transition from slug migration to culmination is regulated by endogenous
ammonia produced by cellular catabolism
(Schindler and Sussman, 1977
),
a metabolic defect may also explain the greatly curtailed migratory slug phase
in the Ax2/gskA strain.
Loss of gskA has weak effects on spore differentiation
When the DH1-derived gskA null strain is allowed to develop
clonally on a bacterial lawn, it exhibits a severe reduction in prespore and
spore differentiation. By contrast, the AX2-derived gskA-null cells
express prespore genes and form fruiting bodies. Although inactivation of the
gskA gene in AX2 cells is permissive for prespore and spore
differentiation, analysis under conditions of synchronous development revealed
a heterogeneity in the response. In some clones of the AX2-derived null
strain, pre-growth in axenic medium and development on non-nutrient agar
caused a proportion of the aggregates to arrest as mounds. One possible
explanation for the variable penetrance of the gskA-null mutation,
under these particular developmental conditions, is a cryptic heterogeneity in
the AX2 parental cells used to generate the null strains.
Analysis of the signalling pathways that direct prespore and spore differentiation
The fact that prespore and spore differentiation occur in the
Ax2/gskA strain led us to study cellular responses to known agonists of
the two processes. Extracellular cAMP acts, via an unknown intracellular
signalling pathway, to induce prespore gene expression
(Gomer et al., 1986;
Kay et al., 1978
). In the
DH1-derived gskA-null strain, extracellular cAMP activates prespore
gene expression very poorly, but in AX2G/gskA cells, cAMP is a potent
activator. The membrane-permeant cAMP analogue 8-bromo cAMP induces spore
differentiation (Kay, 1989
).
This and much other evidence (reviewed by
Williams et al., 1993
)
suggests that activation of PKA, via an elevation in intracellular cAMP, is
both necessary and sufficient to trigger terminal spore differentiation. This
induction is greatly impaired in DH1-derived gskA-null cells. The
induction of spore cell formation by 8-bromo cAMP is also reduced in
AX2G/gskA cells, to about 30% of control levels. However, this is a
much less severe effect than in the DH1-derived strain
(Harwood et al., 1995
) or in
the gskA-null derived in strain JH10
(Kim et al., 1999
), where
spores are formed at only 10% of control levels. One possible explanation for
this difference, for the DH1 strain, is that it is defective in prespore
differentiation and that this enhances a subsequent effect of the
gskA mutation on spore formation. Consistent with this, the DH1
strain produces very few viable spores, during normal development (A.H.,
unpublished) while AX2G/gskA cells forms completely viable spores
(Fig. 2D).
Temporally and spatially regulated ecmB expression is dependent upon GskA
PstA cell and pstO cell differentiation occur normally in the
AX2G/gskA strain but one aspect of prestalk/stalk-specific gene
expression is significantly aberrant; in slugs of the AX2G/gskA strain,
the ecmB gene is activated, precociously and ectopically, in all
cells within the prestalk region. Normally, cells within the prestalk region
of migrating slugs undergo a movement and differentiation cycle that presages
events at culmination (Abe et al.,
1994). A subset of the pstA cells, located very near the slug tip,
activate expression of ecmB and are then termed pstAB cells. These
cells are periodically shed from the back of the slug, where they rapidly
differentiate into stalk cells (Sternfeld,
1992
). During culmination, the differentiation of prestalk cells
into pstAB cells becomes continuous rather than sporadic. However, very
similar processes of movement and ecmB activation occur; after first
expressing ecmB, at the entrance to the stalk tube, the newly formed
pstAB cells move down the tube and terminally differentiate as vacuolated
stalk cells (Jermyn and Williams,
1991
).
In the ZAK1-null and, as we have now shown, in the gskA-null, the ecmB gene is expressed throughout the prestalk region. In addition, in slugs of the original DH1-derived gskA-null strain, in the Ax2-derived gskA-null strain and in ZAK1-null slugs inhibition of stalk cell differentiation by cAMP is greatly attenuated. In combination, these data suggest that extracellular cAMP signalling activates ZAK1 and GskA to repress ecmB expression in pstA cells and prevent their premature differentiation into pstAB cells (Fig. 9).
|
The extracellular cAMP repression pathway functions independently of Dd-STATa
Dd-STATa acts as a repressor of ecmB gene transcription by binding
to two, mutually redundant sites within the promoter
(Fig. 9)
(Mohanty et al., 1999). In
addition, Dd-STATa is a direct target of GskA; the kinase modifies a region
near the N terminus of Dd-STATa and triggers its export from the nucleus
(Ginger et al., 2000
).
Moreover, just as for the gskA-null, the Dd-STATa-null strain is
hypersensitive to DIF-1 (Mohanty et al.,
1999
). Despite these congruencies, however, Dd-STATa cannot be the
mediator of extracellular cAMP repression; because the Dd-STATa-null strain is
hypersensitive to the repressive effect of cAMP on ecmB gene
expression (Mohanty et al.,
1999
). In addition, an ecmB promoter construct lacking the
repressor elements remains subject to extracellular cAMP repression (Y. Yamada
and J.G.W., unpublished). It would seem, therefore, that GskA exerts its
effects via the ecmB activator (Fig.
9), an as yet unidentified factor that interacts with a directly
repeated sequence in the ecmB promoter
(Ceccarelli et al., 2000
).
Evidence that GskA also activates gene expression
To determine whether GskA has a more general role in regulating gene
expression, we used expression profiling to analyse the gskA-null
mutant. Using a microarray that monitors almost one-half of expressed genes,
we discovered a gene family that is significantly underexpressed in the
Ax2-GskA-strain. 2C is a very small gene, of less than 0.5 kb, that has the
potential to encode an 8.8 kDa protein of unknown function
(Richards et al., 1990). The
2C gene is strongly developmentally regulated, with a peak during
mid-culmination (Corney et al.,
1990
). Analysis of the genome sequence shows that there are
multiple copies of the 2C gene and their level of relatedness is so high we
assume we are measuring their composite behaviour. Given the role of cAMP in
GskA regulation, it is perhaps significant that the intracellular
concentration of 2C mRNA is regulated by the extracellular cAMP concentration
(Richards et al., 1990
).
Relationship to other GSK3 regulated signalling pathways
In animal cells, GSK3 represses gene expression by causing the degradation
of ß-catenin. Wnt stimulation alleviates this repression by preventing
GSK3 phosphorylation and subsequent degradation of ß-catenin, allowing it
to activate gene expression. Superficially, the results presented here mirror
the metazoan pathway; however, there are many differences.
Dictyostelium expresses a homologue of ß-catenin, known as
Aardvark (Aar) (Grimson et al.,
2000). In the aar-null mutant at culmination,
ecmB expression expands outside the stalk tube and throughout the tip
(Coates et al., 2002
). However,
this is an indirect structural effect; it arises from a loss of adherens
junctions, which require Aar protein for their formation
(Coates et al., 2002
).
Although we conclude that the Dictyostelium pathway does not
conform to the canonical Wnt signalling pathway, even among the metazoa, the
canonical pathway is not the only route whereby Wnt signals and GSK3 can
regulate cell fate (Veeman et al.,
2003). For example, during early nematode development, the Wnt
signal MOM-2 acts positively on GSK3 to regulate the function of the
ß-catenin homologue WRM-1 (Thorpe et
al., 2000
). This is an interesting potential parallel with the
positive effect that gskA has on expression of the 2C gene family. However,
much of the mechanism of these GSK3-mediated pathways has yet to be resolved
and it remains to be determined whether there is any functional overlap with
the pathways used by Dictyostelium.
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ACKNOWLEDGMENTS |
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