Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan
* Author for correspondence (e-mail: ymaeda{at}mail.cc.tohoku.ac.jp)
Accepted 13 March 2003
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Summary |
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Key words: Dictyostelium discoideum, EF-2, Cell cycle, Growth, Differentiation, Cytokinesis, Mitochondria
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Introduction |
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We have identified several genes (car1, caf1, quit3, dia1, dia2,
dia3) that are specifically or predominantly expressed in response to
differentiation of starved Ax-2 cells from the PS-point and analyzed their
functions (Abe and Maeda.,
1994; Abe and Maeda.,
1995
; Okafuji et al.,
1997
; Itoh et al.,
1998
; Chae and Maeda,
1998a
; Chae and Maeda,
1998b
; Chae et al.,
1998
; Inazu et al.,
1999
; Hirose et al.,
2000
). We have also demonstrated that the phosphorylation levels
of 90 kDa and 101 kDa phosphoproteins are specifically reduced during early
cellular differentiation from the PS-point
(Akiyama and Maeda, 1992
). The
90 kDa phosphoprotein is a homologue of GRP94 (glucose-regulated protein 94;
the endoplasmic reticulum HSP90) in D. discoideum (Dd-GRP94)
(Morita et al., 2000
). The
expression of grp94 is induced by a variety of stress conditions,
such as glucose-depletion (Pouyssegur et
al., 1977
; Shiu et al.,
1977
) and Ca2+ depletion in the ER
(Drummond et al., 1987
).
Differentiation and morphogenesis of Dictyostelium cells is actually
impaired by the overexpression of Dd-GRP94
(Morita et al., 2000
). Since
another protein (the 101 kDa phosphoprotein) remained to be identified, we
sequenced the protein and analyzed its function in Dictyostelium
development.
As presented here, a partial amino acid sequence `VNFTIDQIRA' of the 101
kDa phosphoprotein purified by 2D-SDS-PAGE was found to be identical with the
polypeptide chain elongation factor 2 (EF-2) in D. discoideum
(Dd-EF2) that was originally reported by Toda et al.
(Toda et al., 1989). Ef-2 is
believed to be indispensable for the polypeptide chain elongation step in
eukaryotic protein synthesis. EF-2 translocates a peptidyl-tRNA from the
aminoacyl site to the peptidyl site on a ribosome
(Weissbach and Ochoa, 1976
),
thus being essential for cell proliferation
(Perentesis et al., 1992
;
Mendoza et al., 1999
). The
N-terminus of Dd-EF2 is a GDP-binding domain and the C-terminal half interacts
with ribosomes. Both show homology to hamster EF-2. The amino acid sequence of
the carboxy half includes the site of ADP-ribosylation by diphtheria toxin. In
mammalian cells, the activity of mammalian EF-2 for translocation is regulated
by its state of phosphorylation. The dephosphorylated state is the active form
(Ryazanov et al., 1988
). EF-2
is specifically phosphorylated by Ca2+/calmodulin-dependent protein
kinase III, known as EF-2 kinase (Nairn
and Palfrey, 1987
), and dephosphorylated by PP2A (phosphatase 2A)
(Michael et al., 1989
). EF-2
is also known to be a GTP-binding protein
(Kohno et al., 1986
) and is
colocalized with actin (Shestakova et al.,
1991
; Bektas et al.,
1994
). Surprisingly, the present work using Dd-EF2null
cells has revealed that the 101 kDa phosphoprotein is not required for protein
synthesis and cell proliferation but it is involved in cytokinesis and the
growth to differentiation transition.
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Materials and Methods |
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Transformation of cells
For the overexpression of Dd-ef2h, the full-length cDNA (clone
SLE406) was inserted into the original vector (pDNeo2), using SalI
and BamHI. To create a vector bearing the antisense Dd-ef2h,
a 815-bp fragment (99-+716) of the cDNA clone was inserted into the
BamHI and BglII sites of pDNeo2 in antisense direction. The
vector constructs with sence or antisense Dd-ef2 were separately
introduced into Ax-2 cells by electroporation, as described
(Howard et al., 1988).
Transformed cells were cloned and selected in PS-medium containing 30 µg/ml
of G418 in 96- or 384-well titer plates (Falcon). Five to six days after the
appearance of colonies of transformed cells, the colonies were transferred to
24-well plates. Dd-ef2h-overexpressing (ef-2OE)
and -underexpressing cells (ef-2AS) were incubated by
shake-culture in PS-medium containing 30 µg/ml of G418. To disrupt the
Dd-ef2h gene, the blasticidin S (bsr) gene cassette (1.3 kb)
was inserted into the vector for Dd-ef2h overexpression in which
nucleotides +494-+808 of the Dd-ef2h cDNA had been deleted using
SalI and EcoR1. This plasmid was amplified and the
linearized SalI-NotI fragment was introduced into Ax-2 cells
by electroporation. After 15 minutes at room temperature, the cells were
dispensed into three 9 cm culture dishes and growth medium (PS-medium) added.
Selection at 10 µg/ml blasticidin S was started 10-20 hours later in
PS-medium, and bsr-resistant cells were cloned by axenic culture.
Assay of protein synthesis
To examine protein synthesis in various transformants and parental Ax-2
cells, Trans 35S-label (35S-methionine-cysteine; ICN)
was applied to exponentially growing cells (5x106 cells/ml in
PS-medium) at a concentration of 3.7 MBq/ml and shaken for 2 hours at 150 rpm.
The radio-labeled cells thus obtained were washed twice in 20 mM
Na/K-phosphate buffer (PB; pH 6.5) and suspended at 1x107
cells/ml in 20 mM PB. After 10 µl of 20 µg/ml BSA was added to the same
volume (10 µl) of cell suspension, aliquots (5 µl) of the mixed
suspension were plated on GF/A filters (Whatman) and then dried. The filters
were treated with 5% TCA twice for 10 minutes on ice, 10 minutes at 100°C,
and 10 minutes at room temperature to remove free
35S-methionine-cysteine. Subsequently, the filter was washed twice
in absolute ethanol, twice in diethylether, and dried. The radioactivity
levels of the filters were measured by liquid scintillation counting. The
kinds of proteins synthesized during the pulse (2 hours)-label of cells with
35S-methionine-cysteine were determined by isoelectric focusing
(IEF), subsequent 2D-SDS-PAGE and autoradiography of the samples, as
previously described (Akiyama and Maeda,
1992).
Preparation of the anti-Dd-EF2H antibody and western blot
analysis
Chemically synthesized oligopeptide (RKRKGLAPEIPALDK; from amino acids
799-813 of Dd-EF2H) with an additional cysteine residue at the C-terminus was
conjugated with KLH (keyhole limpet hemocyanin) as a carrier protein (Research
Genetics, Huntsville, AL). The KLH-conjugated oligopeptide was injected
4x1 ml subcutaneously (s.c.) into the foot pads of rabbits with complete
Freund's adjuvant. The total amount of the antigen was 5 mg per animal. Five
weeks later, a total amount of 1 mg KLH-conjugated oligopeptide per animal
with adjuvant was injected s.c. Samples of blood (about 40 ml) were collected
10 days after the final injection, and aliquoted serum containing the
polyclonal anti-Dd-EF2H antibody was stored at -80°C.
Following SDS-PAGE, the gels were transferred to a PVDF membrane, and the membrane was gently shaken in TBS-T containing 5% BSA or 5% nonfat milk, overnight at 4°C. Subsequently, the membrane was probed with the primary antibody diluted 1:5000 in TBS-T containing 5% BSA or 5% nonfat milk and 0.15% Tween 20, overnight at 4°C. After washing in TBS-T for 20 minutes, the blots were probed for 1 hour with a HRP-conjugated anti-rabbit secondary antibody (Amersham Pharmacia Biotechnology) diluted 1:30,000 in TBS-T. The blots were developed with ECL detection reagents (Amersham Bioscience) for 1 minute and exposed to X-ray film (Amersham Hyperfilm-MP) for 25-120 seconds.
Double staining of cells with the anti-Dd-EF2H antibody and DAPI
Vegetative cells were harvested at the exponential growth phase, prefixed
in ice-cold 50% methanol for 10 minutes and then fixed in absolute methanol
for 10 minutes on an ice-bath. After the fixed cells were dried on cleaned
coverslips, they were dipped in PBS (140 mM NaCl, 3 mM KCl, 10 mM
Na2HPO4, 2 mM K2HPO4, pH 7.2) for
10 minutes. The anti-Dd-EF2H antibody or preimmune rabbit serum, both of which
were diluted 1:50 in PBS containing 1% BSA, was placed as a droplet on the
coverslip and incubated for 2-3 hours at room temperature. The samples were
washed in three changes of PBS (10 minutes for each). Subsequently,
FITC-conjugated anti-rabbit IgG (Amersham) diluted 1:50 in PBS containing 1%
BSA and 10% DAPI (4'-6-diamidino-2-phenylindole) was placed as a droplet
on the coverslip and incubated for 2-3 hours at room temperature. After five
washes in PBS (5 minutes for each), the samples were mounted in PBS containing
20% glycerol and observed under a fluorescence microscope. The FITC- and
DAPI-stains in the same optical field were visualized using BV- and
UV-excitation, respectively.
Staining of cells with a mitochondrion-specific dye, MitoTracker
Orange
Dd-ef2-null cells and Dd-ef-2AS cells that had
been starved for 2 hours at 22°C were stained with the anti-Dd-EF2H
antibody and 0.5 µM MitoTracker Orange CMTMRos (Molecular Probes) for 15
minutes. The stained cells were washed twice in BSS and then fixed for
staining with the anti-Dd-EF2 antibody, as described above. The preparations
were observed under scanning confocal fluorescence microscope.
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Results |
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Changes of the Dd-EF2H expression during the progress of cell cycle
and starvation
The northern blot analyses of Dd-ef2h showed that an mRNA of about
2.6 kb was strongly expressed during the vegetative growth phase and also
during the early stage of starvation, followed by a decrease at about 6 hours
of starvation (Fig. 1A). This
is consistent with previous results (Toda
et al., 1989). The developmental kinetics of the Dd-EF2H protein
were qualitatively the same as those of the Dd-ef2h mRNA, except that
the decrease in the amount of the protein during 4-6 hours of starvation was
scarcely recognized (Fig.
1B).
|
Fig. 2A shows temporal changes in the amount of Dd-EF2H during the progression of the cell cycle and the starvation of synchronized cells. No significant differences in the amount were observed among T0, T1, T3, T5, T7 and T9 cells synchronized by the temperature shift method. Comparison of the Dd-EF2 levels in just-differentiating T7+2 cells with that in starved but not differentiated cells (T1+2 and T3+2 cells) also indicated no significant difference between them (Fig. 2A).
|
101 kDa Dd-EF2H is not required for protein synthesis and cell
proliferation in Dictyostelium
Since EF-2 is believed to be indispensable for protein synthesis and cell
proliferation, it was a surprise that we could obtain transformants
(Dd-ef2AS cells) in which the expression of
Dd-ef2h mRNA was supressed by antisense-mediated gene inactivation.
As shown in Fig. 2B, both of
the Dd-ef2h mRNA (2.6 kb) and Dd-EF2H protein (101 kDa) were scarcely
detected in Dd-ef2AS cells. The Dd-ef2h gene is
known to be unique in the Dictyostelium genome
(Toda et al., 1989). An
exhaustive search of the Dictyostelium genome databases has also
demonstrated that the Dd-ef2h gene is located as a single copy on
Chromosome 2, and thus far there have been no similar nucleotide sequences
found in the databases. Only EF-G (Dictyostelium EF-G; Dd-EF-G),
which shows little homology (22.5% similarity in aa) to Dd-EF2H, has been
mapped on Chromosome 6. This raised the possibility that Dd-ef2 null
cells could be isolated by homologous recombination, which was later
accomplished (Fig. 1B).
However, enforced expression of Dd-EF2H in Dd-ef20E cells
was not as striking as shown in Fig.
1C, though the reason for such an incomplete overexpression is
presently unknown.
Fig. 3 shows the growth kinetics of the several transformed cells and parental Ax-2 cells in growth medium. The data indicate that Dd-ef20E cells grew normally with almost the same growth rate as that of Ax-2 cells, and that both Dd-ef2AS and Dd-ef2 null cells exhibited slightly delayed growth. Here it is noteworthy that Dd-ef2AS cells as well as Dd-ef2 null cells are larger than Ax-2 and Dd-ef20E cells.
|
To know if the overexpression or underexpression of Dd-ef2h affects protein synthesis, incorporation of 35S-labelled methionine-cysteine into proteins was compared in transformed cells and parental Ax-2 cells. As a result, there was no significant difference in protein synthetic activity among ef-2OE, ef-2AS, ef2-null and Ax-2 cells, in spite of altered levels of 101 kDa Dd-EF2H in the respective cells (Table 1). Autoradiography of 2D-SDS-PAGE also showed no significant differences in quantity and quality of proteins pulse-labeled with 35S-methionine and 35S-cysteine among ef-2OE, ef-2AS, ef2-null and Ax-2 cells, with the exception of the enhanced synthesis of a 43 kDa protein in ef-2AS and ef2-null cells compared with that in ef-2OE and Ax-2 cells (data not shown).
|
Dd-EF2 is required for normal cytokinesis during growth
Although ef-2AS and ef2-null cells during
shake culture in growth medium (PS medium) exhibited almost normal growth
kinetics, they were found to be considerably larger than Ax-2 cells.
DAPI-staining of fixed cells has revealed that most of the large cells are
multinucleate (Table 2).
Seventy to eighty percent of Ax-2 cells are known to be mononucleate with one
nucleus per cell, and about 20% binucleate with two nuclei per cell
(Maeda et al., 1989). In
contrast, it is clear that the ratio of multinucleate cells (with three or
more nuclei per cell; about 2% in Ax-2 cells) is significantly increased up to
5% and 15% in ef-2AS and ef2-null cells,
respectively, with a decrease in the numbers of mononucleate cells
(Table 2). Taken together,
these results suggest that the Dd-EF2H protein might be involved in
cytokinesis, also that slightly delayed cell proliferation
(Fig. 3), as observed in
ef-2AS and ef2-null cells, might be due to
augmented formation of multinucleate cells during growth. However, when
ef2-null cells that had been shake-cultured in growth medium were
transferred into a 24-well plastic plate and incubated without shaking, many
large (multinucleate) cells were found to become smaller mononucleate cells by
division within 3 hours of incubation at 22°C. This seems to indicate that
impaired cytokinesis in ef2-null cells may be restored by cell
adhesion to the substratum.
|
Involvement of Dd-EF2H in cellular differentiation and
morphogenesis
When ef2-null cells and parental Ax-2 cells were separately
starved and incubated in BSS under submerged conditions, the former exhibited
delayed differentiation compared with the latter. Ax-2 cells acquired
aggregation competence at 6 hours of incubation
(Fig. 4A), whereas
ef2-null cells showed no sign of cell aggregation
(Fig. 4B). The early
morphogenesis including aggregation in ef2-null cells was also
delayed compared with that in Ax-2 cells: although Ax-2 cells formed tight
aggregation streams at 10 hours (Fig.
4E), whereas ef2-null cells still remained as
aggregation-competent cells (Fig.
4F). ef2-null cells were just able to form aggregation
streams after 12 hours of incubation. Essentially the same results were
obtained using Ax-2 and ef2-null cells, both of which had been grown
without shaking and starved in a 24-well plastic plate.
|
On agar, starving ef2-null cells showed no sign of cell aggregation after 5.5 hours of incubation, whereas Ax-2 cells formed aggregation streams. Subsequently, ef2-null cells formed early aggregation streams after 7.0 hours of incubation. Just as in submerged conditions, ef2-null cells delayed the initial step of differentiation, as realized by the elapsed time for cell aggregation. In spite of the delay in development, ef2-null cells eventually formed fruiting bodies with normal morphology. Essentially the same result was obtained using ef-2AS cells underexpressing the Dd-ef2 gene under the control of actin 6 promoter.
In contrast to ef2-null cells and ef-2AS cells, ef-2OE cells displayed more rapid aggregation than Ax-2 cells under submerged conditions (Fig. 5). ef-2OE cells acquired aggregation competence after 5 hours of incubation (Fig. 5B) and formed early aggregation streams at 6 hours (Fig. 5D), while Ax-2 cells were just able to become aggregation competent after 6 hours of incubation (Fig. 5C) and then formed early aggregation streams at 7.5 hours (Fig. 5E). A similar result was obtained on agar using starving ef-2OE cells and Ax-2 cells. Just as in submerged conditions, the progression of morphogenesis in ef-2OE cells on agar was enhanced by about 1.5 hours compared with that in parental Ax-2 cells.
|
Intracellular localization of 101 kDa Dd-EF2H and the existence of a
70 kDa mitochondrial protein sharing the antigenicity with Dd-EF2H
In general, EF-2 is located in the cytoplasm. Immunostaining using the
anti-Dd-EF2H antibody has confirmed that in Ax-2 and
ef-2OE cells the Dd-EF2H protein is located in the
cytoplasm (Fig. 6B). As was
expected, ef-2OE cells exhibited slightly stronger
staining than that in Ax-2 cells (data not shown). Surprisingly, however,
cytoplasmic granules were found to be stained in ef-2AS
and ef2-null cells devoid of 101 kDa Dd-EF2H
(Fig. 6E,G,I). The stained
pattern in ef-2AS cells was almost the same as that in
ef2-null cells. Double-staining of ef2-null cells with the
anti-Dd-EF2H antibody and DAPI revealed that both of the cytoplasmic stains
are completely merged with each other (Fig.
6G,H). This was also confirmed by double-staining of the cells
with the anti-Dd-EF2H antibody and MitoTracker Orange
(Fig. 6I-K), thus indicating
that the cytoplasmic granules stained with the anti-Dd-EF2H antibody are
mitochondria.
|
Western blot analysis using the anti-Dd-EF2H antibody gave a single band at the position of 101 kDa Dd-EF2H when proteins extracted from a relatively small number of Ax-2 cells were applied to SDS-PAGE, whereas no positive band was detected in ef-2AS and ef2-null cells under this condition (Fig. 7A). However, when loaded protein concentrations were increased by 100-times, the antibody recognized several bands even in the protein samples from ef-2AS and ef2-null cells (Fig. 7B). Among these bands are 71 kDa and 41 kDa proteins. Considering the results of immunostaining, it is possible that the two proteins are predominantly localized in mitochondria. To test this possibility, cytosolic and mitochondria-rich fractions derived from Ax-2 cells were processed for western blot analysis using the anti-Dd-EF2H antibody. As a result, the antibody detected the 71 kDa protein mainly in the mitochondria-rich fraction, while it recognized the 101 kDa Dd-EF2H protein predominantly in the cytosolic fraction (Fig. 7C). Taken together, these results seemed to indicate that the 41 kDa protein of Fig. 7B might be a hydrolysed product of the 71 kDa mitochondrial protein, and that the 58 kDa and 36 kDa proteins of Fig. 7B might be hydrolysed products of 101 kDa Dd-EF2H. Again, it is of importance to note that the 71 kDa mitochondrial protein shares the antigenicity with 101 kDa Dd-EF2H.
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Discussion |
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EF-2 is believed to be indispensable for cell proliferation as well as
eukaryotic protein synthesis, and therefore its knock-out caused a lethal
effect on cells (Livingston and Bodley,
1992). Surprisingly, however, the results presented here have
demonstrated that the 101 kDa Dd-EF2H of Dictyostelium cells is not
required for protein synthesis, and that Dd-EF2H-null cells as well as
ef-2AS cells are able to grow almost normally, except for
formation of multinucleate cells during growth. Protein synthesis in
ef-2ASand ef2-null cells was almost the same as
that in parental Ax-2 cells. This seems to indicate that in
Dictyostelium cells there must be a molecule(s) other than 101 kDa
Dd-EF2H that is involved in protein synthesis and therefore capable of
compensating for the function of EF-2. Proteins containing the same or a
similar epitope that would crossreact with the anti-Dd-EF2H antibody have not
yet been identified in the Dictyostelium mitochondrial genome or the
genome sequencing project. After northern analysis, no RNA transcripts, as
probed by the full-length Dd-ef2h cDNA, were detected in
Dd-ef2-null cells. Therefore, it is unlikely that smaller Dd-EF2H
fragments may work to compensate the EF-2 function. The lack of RNA
transcripts and Dd-EF2H protein in Dd-ef2hAS cells also
suggests that molecules structurally similar to the 101 kDa Dd-EF2H protein
cannot compensate for EF-2 function. EF-G (Dd-EF-G), which is devoid of the
epitope used for preparation of the anti-Dd-EF2H antibody, might be a
candidate capable of compensating for EF-2 function, although its homology to
Dd-EF2H is not very high (22.5% similarity in aa). Alternatively, it is
possible that the 43 kDa protein and/or 71 kDa mitochondrial protein are
responsible for protein synthesis instead of 101 kDa Dd-EF2H, because the
former is predominantly synthesized in Dd-ef2AS and
Dd-ef2-null cells compared with parental Ax-2 cells, and the latter
shares the antigenicity with the 101 kDa Dd-EF2H.
As noticed particularly in ef2-null cells, the knock-out of 101
kDa Dd-EF2H brought about impaired cytokinesis, thus resulting in the
appearance of multinucleate cells. Positive participation of 101 kDa Dd-EF2H
in the process of cytokinesis would be a novel function. In connection with
this, some of eukaryotic EF-2 is known to be colocalized with actin
microfilament bundles in mouse embryo fibroblasts, which suggests a possible
link between the protein synthetic machinery and the cytoskeleton
(Shestakova et al., 1991;
Bektas et al., 1994
).
The 101 kDa protein was originally marked as a phosphoprotein involved in
the growth/differentiation transition at the PS-point, and was now identified
as Dd-EF2H. This protein is known to be strongly labeled with 32Pi
in growing and starving Ax-2 cells at most cell-cycle phases other than the
PS-point, and was never phosphorylated around the PS-point under conditions of
nutritional deprivation (Akiyama and Maeda,
1992). This suggested that a low phosphorylation level of Dd-EF2H
might favor the entry of Ax-2 cells into differentiation from the PS-point. In
this connection, it has been demonstrated that the activity of EF-2 in
translation is regulated by its phosphorylation levels, and that the
dephosphorylated state is generally the active form
(Ryazanov et al., 1988
). EF-2
is specifically phosphorylated by Ca2+/calmodulin-dependent protein
kinase III, known as EF-2 kinase (Nairn
and Palfrey, 1987
), and dephosphorylated by PP2A
(polycation-stimulated serine/threonine-specific protein phophatase;
phosphatase 2A) (Michael et al.,
1989
). It has been shown in Ax-2 cells that the phosphorylation
level of Dd-EF2H around the PS-point is low in starvation medium, even in the
presence of 0.5 µM calyculin A, a potent and specific inhibitor of the PP2A
and PP1 (ATP-Mg2+-dependent serine/threonine-specific protein
phosphatase) that is capable of completely inhibiting entry of cells (located
just before the PS-point) into differentiation in response to starvation, and
that dephosphorylation of a 32 kDa protein is perfectly inhibited by 0.5 µM
calyculin A (Akiyama and Maeda,
1992
). Thus, in addition to low activity of the serine/threonine
protein kinases including EF-2 kinase under starvation conditions around the
PS-point, pronounced dephosphorylation of the 32 kDa protein might be required
for transition of Dictyostelium cells from growth to differentiation.
Although the overexpression of Dd-ef2h is not striking
(Fig. 1C), more rapid
aggregation was achieved in Dd-ef2h-overexpressing cells
(ef-2OE) than in parental Ax-2 cells. In contrast, gene
inactivation of Dd-ef2h by homologous recombination or antisense RNA
considerably impaired the progression of differentiation. Again, these results
seem to indicate that the presence of dephosphorylated Dd-EF2H may be involved
in the initiation of differentiation.
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Acknowledgments |
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