1 Program in Molecular Biology
2 Department of Cellular and Structural Biology, University of Colorado Health
Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262, USA
* Author for correspondence (e-mail: tom.evans{at}uchsc.edu)
Accepted 28 February 2003
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SUMMARY |
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Key words: Translation, Notch, STAR, GLD-1, Caenorhabdtis elegans
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INTRODUCTION |
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In the C. elegans embryo, post-transcriptional regulation is
crucial for the specification of early cell fates
(Goodwin and Evans, 1997;
Rose and Kemphues, 1998
).
During the first cell division, polarity within the zygote leads to the
localization of cell fate regulators to specific embryonic cells
(Fig. 1). For example, the
Notch membrane receptor, GLP-1, is localized to anterior cells, while one of
its ligands, the Delta-like APX-1, is localized to a single posterior cell
(Evans et al., 1994
;
Mickey et al., 1996
). The
localization of GLP-1 and APX-1 is probably crucial for spatially constraining
cell signaling that regulates anterior cell fates
(Mello et al., 1994
;
Priess et al., 1987
). In
addition, several transcription factors, including the caudal homolog PAL-1,
are localized to posterior cells where they control posterior cell development
(Bowerman et al., 1993
;
Hunter and Kenyon, 1996
;
Lin et al., 1998
). The mRNAs
of most of these factors are made in the germline during oogenesis and
delivered to all cells of the embryo after fertilization. Thus, translational
and/or post-translational controls are critical for distinct patterns of
protein localization in the C. elegans embryo.
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The factors that directly control glp-1 translation are not known.
Several genes required for different aspects of glp-1 regulation have
been identified in mutant screens. Genes that control early cell polarity are
necessary for GLP-1 localization in the embryo, presumably because they
regulate the activities or localization of glp-1 regulators
(Crittenden et al., 1997;
Rose and Kemphues, 1998
). In
addition, two functionally redundant proteins, MEX-5 and MEX-6, act downstream
of polarity genes and promote GLP-1 expression in anterior embryonic cells
(Schubert et al., 2000
). MEX-5
and 6 contain Zn-finger like domains and could be RNA-binding proteins, but it
is not known how these factors affect glp-1 mRNA regulation.
In the germline, the KH-domain protein GLD-1 is required to restrict germ
cell mitosis and GLP-1 expression to the distal tip region of the gonad
(Crittenden et al., 1994;
Francis et al., 1995a
). GLD-1
is a member of the STAR family of RNA-binding proteins, several members of
which have been linked to regulation of various signaling pathways
(Vernet and Artzt, 1997
). Germ
cells in gld-1 null mutants fail to progress from early meiotic
prophase to oogenesis, and instead they proliferate inappropriately, forming
germline tumors (Francis et al.,
1995a
). These tumorous germlines express GLP-1 throughout the
gonad (Crittenden et al.,
1994
). Because mutations in other genes that cause excessive
proliferation also cause ectopic GLP-1 expression
(Berry et al., 1997
;
Crittenden et al., 1994
), the
increased GLP-1 expression in gld-1 mutants could be an indirect
result of excessive germ cell mitosis. Alternatively, it could be due to a
more direct loss of glp-1 mRNA repression. GLD-1 has been implicated
as a direct RNA-binding regulator of translation of other C. elegans
mRNAs (Jan et al., 1999
;
Lee and Schedl, 2001
;
Xu et al., 2001
). Moreover,
GLD-1 has multiple functions in germ cell development, and has multiple mRNA
targets (Francis et al.,
1995a
; Francis et al.,
1995b
; Lee and Schedl,
2001
). Interestingly, GLD-1 is also expressed in the early embryo
and is localized to posterior blastomeres of the embryo
(Jones et al., 1996
). The
embryonic functions of GLD-1 are not known. Thus GLD-1 may control a variety
of germ cell and embryonic functions by specific interactions with numerous
mRNAs.
In this paper, we show that two distinct types of translational control elements reside within the glp-1 SCR. One element is required for repression of translation in posterior cells of the embryo, and also contributes to translational repression in the germline. A second element is required for derepression of translation in anterior cells of the early embryo. Furthermore, we show that GLD-1 binds directly and specifically to the repressor element of the SCR, and is required in vivo for repressing GLP-1 expression at two distinct developmental stages. The results suggest a new function for GLD-1 in regulating early embryonic asymmetry, and that functional interactions between GLD-1 and other factors contribute to the localization of this Notch receptor. These interactions are probably important for spatial organization of Notch signaling in both the gonad and early C. elegans embryo.
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MATERIALS AND METHODS |
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RNA precipitations
All steps were performed at 4°C unless otherwise noted. Synchronized
adult hermaphrodites were grown as described previously
(Barbee et al., 2002), and were
homogenized using a French Press in homogenization buffer (10 mM Hepes pH 7.2,
75 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 0.1 mM EGTA, 50 mM
sucrose, 5% glycerol, 1 mM DTT, EDTA-free protease inhibitors; Roche).
Homogenates containing 50-100 mg of protein were brought to 600 mM KCl, and
then spun at 10,000 g for 10 minutes. Samples were diluted to
150 mM KCl, with buffer containing 10 mM Hepes pH 7.2, 2 mM MgCl2,
0.1 mM CaCl2, 1 mM DTT. Micrococcal nuclease (15 U/ml) (Amersham
Pharmacia) was added and the extracts were incubated at room temperature for
20 minutes. EGTA was added to 2 mM to inhibit the nuclease, and the extract
was spun at 100,000 g for 1 hour. Digoxigenin-RNAs (20 nM) and
heparin (1 mg/ml) were incubated with the supernatants for 1 hour.
Antidigoxigenin magnetic particles (1 mg magnetic particles per 35 pmol of
RNA; Roche), were pre-washed twice in wash buffer (20 mM HEPES pH 7, 75 mM
KCl, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.05% Nonidet P-40), and were
then incubated with extracts for 1 hour. The magnetic particles were separated
for 5 minutes using a magnetic particle separator (Roche) and the unbound
extract was removed. The magnetic beads were washed three times with wash
buffer for 10 minutes. Finally, the proteins were eluted twice for 10 minutes
using wash buffer with 1 M KCl. The proteins eluted from the RNA
precipitations were concentrated and dialyzed with wash buffer using centricon
filters (Amicon). Digoxigenin RNA (dig-RNA) was made using T7 Megascript
transcription reagents (Ambion) with ATP, GTP and CTP at 6.7 mM, UTP at 5.2
mM, and digoxigenin-11-UTP (Roche) at 1.7 mM. After 4-6 hours of
transcription, DNase I was then added and unincorporated nucleotides were
removed by gel filtration.
RNA-binding assays
UV cross-linking
Radiolabeled probes were transcribed with T7 polymerase (Gibco) using
-32P-labeled UTP (2.6 µM), with unlabeled UTP (10 µM),
and GTP, ATP and CTP at 0.5 mM. Binding reactions (20 µl) contained 2 µl
concentrated eluted proteins with wild-type SCR probe (0.5 nM to 2 nM) in
buffer consisting of 10 mM Hepes pH 7, 2 mM MgCl2, 80 mM KCl, 2 mM
EGTA, and 1 mM DTT. Binding reactions were incubated on ice for 45 minutes,
and then were UV irradiated in 0.5 ml Eppendorf tubes for 5 minutes using 254
nm bulbs in a Stratalinker (Stratagene). RNAse A and RNAse T1 were used to
digest the RNA, and the proteins were separated by SDS-PAGE and visualized by
phosphoimager.
GST GLD-1 pulldown
GST-GLD-1 fusion protein used for some experiments was a generous gift of
E. B. Goodwin, and for other experiments was made and purified as described
(Jan et al., 1999). Similar
results were obtained with both preparations. 32P-labeled probes
were incubated with 200 nM GST-GLD-1 fusion protein in 10 mM Hepes pH 7, 2 mM
MgCl2, 80 mM KCl, 2 mM EGTA, 0.57 mg/ml heparin, 1 mM DTT, at
4°C for 1 hour. Equal amounts of 50% slurry of glutathione beads were
added to each binding reaction. The beads were allowed to bind for 30 minutes,
then they were spun at 500 g for 5 minutes. The unbound
fraction was removed and the beads washed five times for 10 minutes with wash
buffer. The labeled RNA remaining with the beads was then counted using a
scintillation counter.
Filter binding assay
32P-labeled RNA probe was made as described above except with a
10:1 ratio of UTP to -32P-labeled UTP. Binding reactions
containing buffer (10 mM Hepes pH 7, 2 mM MgCl2, 80 mM KCl, 2 mM
EGTA, 1 mM DTT), recombinant GST-GLD-1, 60 nM labeled RNA with or without
excess amounts of unlabeled RNA (made using a Megascript transcription kit;
Ambion) were incubated for 1 hour at 4°C. The reaction mixtures were then
filtered through nitrocellulose and DE81 membranes to collect protein/RNA
complexes and unbound RNA, respectively
(Wong and Lohman, 1993
). The
RNA bound to each membrane was determined by phosphoimaging.
For western blots, total extracts or concentrated eluates from
digoxigenin-RNA pull downs were separated on 10% SDS-PAGE, transferred to
nitrocellulose and detected with rabbit antibodies against GLD-1 (generously
provided by Elizabeth Goodwin) as described previously
(Jan et al., 1999). To compare
immunoblots of eluates pulled down by different RNAs, an equal percentage of
total eluates were loaded on SDS gels.
Reporter mRNA assays and in situ hybridization
Reporter mRNAs were made and injected as described previously
(Evans et al., 1994). All
mRNAs were tested for integrity by gel electrophoresis, and by injection into
adult somatic cells. Only mRNAs that ran as a tight single band and that
produced strong ß-gal expression in somatic cells were assayed, and 3-5
separate mRNA preparations were used for each mRNA. Reporter mRNAs were
injected at 50 nM unless otherwise specified. In situ hybridizations were done
as described using an antisense lacZ probe
(Evans et al., 1994
).
RNAi and immunofluorescence
RNAi was performed by microinjection of dsRNA essentially as described
(Montgomery and Fire, 1998).
PCR products that contained T7 and T3 promoters were amplified from a
gld-1 cDNA plasmid (generously provided by E. B. Goodwin), or from
cDNA encoding the GLD-1-related genes T21G5.5 or K07H8.9, and were gel
purified, extracted with phenol/chloroform, and ethanol precipitated. Sense
and anti-sense RNA strands were made separately using Megascript (Ambion), and
were annealed by heating and slow cooling. One-day old N2 adult hermaphrodites
were injected into one gonad with dsRNA (0.5-1 mg/ml), and then incubated at
20°C, or 25°C for various times. Essentially the same
gld-1(RNAi) phenotypes were seen at all temperatures, and included
the following classes in temporal sequence: F1 sterile animals with
tumorous gonads, F1 embryonic arrest (up to 25%), and then Po
sterility with oogenesis and germ cell hyper-proliferation defects. For the
experiments shown in Fig. 5,
gld-1(RNAi) and control animals were incubated at 25°C for 15
hours, but the same effects on GLP-1 expression were detected at 20°C
after 24 hours of incubation (data not shown). For T21G5.5 or K07H8.9, no RNAi
phenotypes were detected through 48 hours of incubation at 20°C (data not
shown). For GLP-1 staining, gonads were dissected from injected animals,
fixed, and stained with antibodies and DAPI as described previously
(Barbee et al., 2002
;
Evans et al., 1994
). GLP-1 was
detected using rabbit antibodies against the LNG region (a gift from Judith
Kimble), and P-granules using the K76 monoclonal (a gift from Susan Strome,
through the Developmental Studies Hybridoma Bank, the NICHD, and the
University of Iowa, Dept. of Biological Sciences, Iowa City, IA 52242).
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RESULTS |
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Surprisingly, base substitutions adjacent to the GRE strongly or partially
inhibited ß-gal expression in the embryo (lacglp(LS2) in
Fig. 2D, lacglp(M8) and
lacglp(LS2-LS5) in Table 1). In
contrast, all of these mutant mRNAs produced strong ß-gal expression when
injected into intestinal cells (data not shown). The loss of embryonic
expression from these mutant mRNAs could be due to defective activation of
translation or by decreased mRNA stability in the embryo. When injected
animals were examined by in situ hybridization with a lacZ RNA probe,
lacglp mRNAs with the LS2 or LS4 mutations were detected at similar
intensities to wild-type or LS1 mRNAs in both gonads and all cells of early
embryos (Fig.
2E,F,
data not shown). These results suggest that the LS2 and LS4 mutations do not
dramatically reduce mRNA stability. Furthermore, when both the GRE and these
neighboring sequences were simultaneously disrupted by base substitution or
deletion, strong but un-localized translation was detected in early embryos
(lacglp(LS1LS2) and lacglp(M7) in Table
1) (Evans et al.,
1994). Therefore, an RNA element defined by mutants LS2-LS5
promotes translation in the embryo by inhibiting GRE-mediated repression. We
call this element the GDE, for glp-1 Derepressor Element
(Fig. 2A).
We noticed that GRE mutation or SCR deletion in lacglp mRNAs also caused a
small but reproducible increase in the number of gonads with ß-gal
expression (data not shown) (Evans et al.,
1994). However, full repression of germline translation requires
RNA elements outside of the SCR (Evans et
al., 1994
). To examine GRE function in the germline independently
of other glp-1 elements, we compared the translation of lacunc mRNA
carrying a wild-type 34 nt SCR fragment [lacunc(34WT)] to one with the LS1
mutation in the GRE [lacunc(34LS1)]. Lacunc(34WT) mRNA translation was
strongly inhibited in the germline; ß-gal expression was restricted to
embryos in 75% of the injected gonads that stained (n=20)
(Fig. 1D). None of these gonads
expressed ß-gal in early meiotic germ cells of the distal arm or near the
gonad bend (Fig. 1D), while 25%
expressed ß-gal within later stage oocytes that were closest to the
proximal end of the gonad (not shown). In contrast, lacunc(34LS1) mRNA was
strongly translated in germ cells (compare Fig.
1D to
1F). Of the gonads injected
with lacunc(34LS1) that produced ß-gal, 100% had staining within the
distal arm and 96% in the oocytes (n=23). These results suggest that
the GRE not only functions in embryos, but also contributes to repression of
glp-1 translation in the gonad. GRE activity was strongest within
more distal regions of the gonad that contain germ cells in early stages of
oogenesis. Because the GRE is not essential for germline repression within the
intact glp-1 3' UTR, it functions redundantly with other
glp-1 RNA elements (see Discussion)
(Evans et al., 1994
).
GLD-1 directly binds the glp-1 GRE
To identify proteins that bind to glp-1 RNA elements of the SCR,
RN-binding factors were affinity precipitated from crude adult extracts
using tagged RNAs (see Materials and Methods). RN
-binding proteins in
enriched fractions were detected by UV cross-linking to 32P-labeled RNA
probes. Wild-type tagged SCR RNA pulled out polypeptides of 58 kDa (p58) and
30 kDa (p30) that could be cross-linked to an RNA probe containing both the
GRE and GDE (Fig. 3A, lane 1).
In contrast, tagged RNA that contained the M7 mutation failed to pull out
either p58 or p30 (Fig. 3A,
lane 2). To examine the specificity of p58 and p30 further, RNAs containing
GRE or GDE mutations were tested for their ability to bind these proteins. UV
cross-linking of p58 and p30 was strongly disrupted by the LS1 mutation in the
GRE, but not by the LS2 or LS5 mutations within the GDE
(Fig. 3B). The LS3 and LS4
mutations partially attenuated cross-linking to both p58 and p30. Excess
amounts of unlabeled RNA containing the LS1 mutation was a poor competitor of
32P-labeled wild-type probes for cross-linking to p58 and p30
(Fig. 3C). In contrast, excess
wild-type RNA, or RNAs with any of the GDE mutations, displaced the labeled
wild-type probe (Fig. 3C, data
not shown). Together, these experiments demonstrate that both p58 and p30
specifically require the GRE for binding to the glp-1 SCR. Sequences
that overlap with the GDE also appear to promote but are not essential for
their binding to glp-1 RNA (Fig.
3).
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To determine if GLD-1 can bind the GRE directly, a recombinant purified GST-GLD-1 protein fusion was tested for its ability to bind glp-1 RNAs in vitro. Using a GST pull-down assay, we found that 32P-labeled RNA containing the GRE and the GDE was pulled down by GST-GLD-1, but RNA with the LS1 mutation in the GRE was not (Fig. 4A). Neither wild-type nor mutant RNAs were pulled down using GST alone (data not shown). To more carefully examine the specificity of GLD-1 binding, we used a filter binding assay to test the abilities of unlabeled mutant RNAs to compete with labeled wild-type probe for binding to GLD-1 (Fig. 4B). Unlabeled wild-type RNA and RNAs with mutations in the GDE were effective competitors for binding to GLD-1. In contrast, the LS1 mutation within the GRE greatly inhibited competition for GLD-1 binding. We conclude that GLD-1 binds directly to the glp-1 3' UTR and binding depends specifically on the GRE, suggesting that GLD-1 could be a repressor of glp-1 mRNA translation.
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RNAi of gld-1 also affected the regulation of GLP-1 expression in early embryos. At the 4 to 8-cell stages, 36% of gld-1(RNAi) embryos (n=36) had GLP-1 staining in posterior blastomeres that was equivalent or nearly equivalent to staining in anterior blastomeres (Fig. 5E). In contrast, none of 4- to 8-cell embryos (n=62) from non-injected mothers had high GLP-1 staining in posterior cells, and all showed enrichment of GLP-1 in anterior cells (Fig. 5D). The posterior expression of GLP-1 seen in gld-1(RNAi) embryos was higher than the very low levels detected in all 1-cell zygotes examined, suggesting that posterior expression of GLP-1 was not due to perdurance of protein translated in the germline (data not shown). Therefore, these data suggest that GLD-1 is required for normal translational repression of glp-1 mRNA in posterior cells of the embryo. The incomplete penetrance of GLP-1 mis-localization could indicate that GLD-1 only partially contributes to glp-1 mRNA regulation in the embryo. Alternatively, GLD-1 may be only partially depleted in these RNAi experiments. Using immunofluorescence, we found that GLD-1 staining in gld-1(RNAi) animals was reduced but not eliminated in both gonads and embryos under the conditions used for these assays (data not shown). By the time GLD-1 staining was severely reduced, gonads from injected animals produced no embryo progeny. Thus, GLD-1 could be an essential repressor of glp-1 translation in the early embryo.
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DISCUSSION |
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GLD-1 is a conserved member of the STAR/Quaking/GSG family of proteins that
have been linked to various signal transduction and developmental events. In
C. elegans, GLD-1 has been shown to control sex determination and
oogenesis in the germline by regulation of several germline mRNAs
(Francis et al., 1995a;
Jan et al., 1999
;
Lee and Schedl, 2001
;
Xu et al., 2001
). Our studies
suggest a new function for GLD-1 in the control of early embryonic asymmetry.
This function contributes to the localization of a Notch receptor to anterior
cells of the embryo. In other organisms, STAR family members have been
connected to various protein kinase signaling cascades
(Vernet and Artzt, 1997
). In
C. elegans, GLD-1 regulates the translation of tr
-2,
which encodes a key membrane receptor for sex determination
(Jan et al., 1999
;
Kuwabara et al., 1992
). GLD-1
also associates with mRNA encoding a Raf protein kinase in the germline
(Lee and Schedl, 2001
). Our
results link GLD-1 function to the control of another signaling system, the
Notch pathway. Therefore, this family of RNA binding proteins may be generally
important for regulation of a variety of different signaling events that
control important developmental decisions in metazoans.
These results suggest that GLD-1 contributes to the spatial organization of
Notch signaling in both the embryo and germline. In the embryo, localization
of GLP-1/Notch ligands and the receptor is probably important for the spatial
patterning of anterior cell fates (Mello
et al., 1994; Priess and
Thomson, 1987
). We suggest that GLD-1 participates in this process
by repression of glp-1 translation in posterior cells of the embryo.
In the germline, GLD-1 repression of glp-1 could be part of a
negative feedback system of RNA regulation that controls spatial organization
of Notch signaling and germ cell development. GLP-1 is expressed in mitotic
germ cells of the distal tip, where it maintains the mitotic stem cell
population (Austin and Kimble,
1987
; Crittenden et al.,
1994
). Interestingly, GLD-1 expression is inhibited in the mitotic
distal tip region by a process dependent on the RN
-binding proteins
FBF-1 and FBF-2, which can directly bind the gld-1 3' UTR
(Crittenden et al., 2002
;
Jones et al., 1996
). This may
be important to permit full expression of the GLP-1 receptor in these cells.
As germ cells move away from the distal tip and the source of the ligand for
GLP-1 signaling, they enter meiosis and begin to express GLD-1 and probably
other factors (see below), which then repress the translation of
glp-1 mRNA. GLD-1 is essential to promote exit from mitosis and
female gamete differentiation (Francis et
al., 1995a
; Francis et al.,
1995b
). However, genetic studies suggest that GLP-1 down
regulation by GLD-1 only weakly contributes to inhibition of germ cell mitosis
(Francis et al., 1995b
). It
could be that GLD-1 repression of glp-1 mRNA and other mRNAs, coupled
with localization of the GLP-1/Notch ligand to the somatic distal tip cell
(Henderson et al., 1994
),
provide redundant systems to spatially constrain GLP-1 signaling and organize
mitotic proliferation of germline stem cells in the gonad.
Our studies and previous observations suggest that the precise localization
of glp-1 translation in the embryo requires regulation of GLD-1 by
other factors. In the early embryo, localized translation requires not only
the GRE but also the GDE that promotes translation. Because the GDE is only
needed to regulate the GRE, we suggest it functions as a binding site for a
derepression factor that inhibits GLD-1 binding to the GRE or its activity
(Fig. 6). Localization of
translation could be most simply achieved by localization of the derepressor
to anterior cells. However, GLD-1 is greatly enriched in posterior cells
(Jones et al., 1996). It could
be that a derepressor is necessary to inhibit low residual levels of GLD-1
that persist in anterior cells. Perhaps, localization of both GLD-1 and
derepressor factors ensures tight localization of GLP-1 and other cell fate
regulators. Alternatively, another un-localized repressor may also contribute
to glp-1 mRNA repression through the GRE.
|
GLD-1 regulates a variety of different mRNAs, but how it specifically
controls these mRNAs is not well understood. Although some sequence similarity
exists among the GLD-1 binding regions of other mRNAs, the GRE region of
glp-1 has only limited similarity to these other elements in primary
sequence (Goodwin et al.,
1993; Jan et al.,
1999
; Lee and Schedl,
2001
; Xu et al.,
2001
). In contrast, the nucleotides within and surrounding the GRE
are mostly conserved in glp-1 homologs of different nematode species
(Rudel and Kimble, 2001
). The
binding site in glp-1 may fold into a structure related to these
other RNA targets, or glp-1 RNA may bind a distinct binding site in
GLD-1. Alternatively, distinct binding partners for GLD-1 may promote
recognition, and specific regulation, of different mRNAs. The F-box protein
FOG-2 is a GLD-1 binding protein that is specifically required for a sex
determination function of GLD-1 (Clifford
et al., 2000
). Perhaps other specific factors may interact with
GLD-1 to control glp-1 RNA, and distinct complexes may function in
the germline and early embryo. It will be important to identify these
interactions, and the interactions with more general translation factors, to
understand the molecular mechanisms that lead to distinct but precise patterns
of mRNA regulation.
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ACKNOWLEDGMENTS |
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