Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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It has been reported that increased Shh expression in the notochord results in the increased expression of HNF3ß in the floor plate and the subsequent appearance of other ventral markers in the neural tube (6). This results in the suppression of the dorsalizing signals, induced by BMP-2/4 originating from the neuroectoderm (7). The appearance of ventral markers within the neural tube establishes a dorso-ventral boundary, which is broadly described as dorso-ventral patterning. The induction of motor neuron markers within the ventro-lateral region is coincident with this Shh-mediated activity in the neural tube. Jessell and co-workers (8) have demonstrated that conditioned media from COS-1 cells that overexpress and secrete Shh can mediate motor neuron differentiation in naive neural plate explants.
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are members of the nuclear receptor superfamily and belong to the orphan receptor subclass (9, 10). Two genes called COUP-TFI and COUP-TFII have been identified in mammals. COUP-TFs are capable of dimerizing with the retinoid X receptor and are known to affect the regulation of retinoic acid receptor, retinoid X receptor, and vitamin D receptor responsive genes (9). COUP-TFs in general are expressed during embryonic development and are proposed to mediate essential mesenchymal-epithelial interactions that are crucial for proper organogenesis (10). To that effect, mRNA of COUP-TFII has been detected in the mesenchymal compartment of several developing organs such as the kidney, breast, and prostate (10).
COUP-TFs are expressed in the developing murine central and peripheral nervous systems. It is hypothesized that these two genes play a role in regulating the developmental fates of diencephalic neuromeres by altering the expression of some of the key factors involved in cell fate determination (11). Most importantly, COUP-TFII mRNA is highly expressed in differentiating motor neurons within the neural tube of a developing chick embryo. Notochord transplantation experiments suggest that this increase in COUP-TFII mRNA within the ventro-lateral region of the chick neural tube is mediated by signals derived from the notochord (12). Hence, we hypothesize that Shh, a secreted morphogen derived from the notochord, can elicit this increase in COUP-TFII expression in differentiating motor neurons.
For our studies we used the P19 mouse embryocarcinoma cells that were previously used to study the retinoid-mediated regulation of COUP-TFs (13, 14). Also, these cells exhibit some characteristics of neuronal cells when treated for extended periods with all-trans retinoic acid (15). For our initial experiments, conditioned medium derived from COS-1 cells transfected with a mouse Shh expression construct was applied to P19 cells. Later, an Escherichia coli-expressed N-terminal 20-kDa Shh protein fragment was used in our studies. In this report we provide evidence that Shh can induce COUP-TFII transcripts in P19 cells and evidence to implicate a novel target element on the COUP-TFII promoter that mediates this Shh-induced activity.
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RESULTS |
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Induction of COUP-TFII Promoter Activity by Conditioned Media
Containing Shh
To identify the mechanism of this Shh-mediated increase in
COUP-TFII mRNA, we analyzed the effect of the Shh-containing
conditioned media on the COUP-TFII promoter. A 1.6-kb promoter fragment
that includes the transcription initiation site of COUP-TFII was cloned
upstream of the chloramphenicol acetyltransferase (CAT) reporter gene
(p1.6CII-CAT) and transfected into P19 cells (13). As shown in Fig. 2 the conditioned media containing Shh
(lanes 2 and 3) can increase COUP-TFII promoter activity. In a separate
experiment we show that this increase can be elicited by adding 1
nM N-terminal 20-kDa Shh protein directly to COS-1 cells
transfected with the p1.6CII-CAT plasmid (Fig.
A; lanes 1 and 2).
Hence, it can be inferred that P19 and COS-1 cells possess the
necessary factors to transduce the Shh signal. These results indicate
that the 1.6-kb fragment of the COUP-TFII promoter harbors the target
element(s) that mediates Shh activation.
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Additional deletions were made within this region of the promoter to
localize the target element that mediates Shh activation. These
fragments were subsequently cloned upstream of a tkCAT reporter. COS-1
cells were transfected with these plasmids and treated with 1
nM Shh. As shown in Fig. 3C, deletion of 27 nt from -1343
does not affect Shh activation (compare lanes 4 and 6). However,
additional removal of 18 nt (from -1316 to -1298) abolishes Shh
activation (compare lanes 6 and 8). These results indicate that the
19-bp region between nucleotides -1316 and -1298 harbors most of this
activity. Collectively, our results indicate that a region (-1316 to
-1298) within the COUP-TFII promoter harbors a ShhRE that mediates the
Shh-induced increase in COUP-TFII promoter activity that leads to
increased steady-state levels of COUP-TFII mRNA.
Characterization of the ShhRE in P19 Cells
To further confirm that the region between -1316 and -1298 is
sufficient to confer the Shh response, we used this sequence in a
heterologous promoter function assay. A 42-bp oligonucleotide (ShhRE)
that includes this region and the flanking sequences was synthesized,
and three copies of this oligo were placed upstream of a tkLUC reporter
gene to determine whether this element confers Shh inducibility through
a heterologous promoter. In addition, four specific mutations, M1, M2,
M3, M4, and M5, were introduced along this element and three copies of
each of these mutant oligos were cloned upstream of a tkLUC reporter
(Fig. 4A). The resulting plasmids were
then transfected into P19 cells, and reporter gene activity in response
to 1 nM Shh was analyzed. As seen in Fig. 4B
, three copies
of ShhRE can provide a 5- to 7-fold increase in reporter gene activity
in response to Shh (compare C and Shh). Also, as shown in Fig. 4B
, the
M2, M3, and M4 mutations abolish the Shh response (compare S and S M1,
S M2, S M3). In contrast the M1 and M5 mutants were still capable of
mediating this Shh-induced reporter gene activity (compare S, S M1, and
S M5). These results indicate that the nucleotide sequences defined by
the mutant oligos M2, M3, and M4 are important for mediating the Shh
response. Hence, the point mutations introduced within the ShhRE that
abolish transactivation must affect the binding of a putative
transcription factor to this DNA element.
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DISCUSSION |
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The identification of this ShhRE provides us with a valuable tool to study the various signaling pathways that are induced by Shh. Previously, it was reported by Ingham and his colleagues (17) that a binding site for the Ci (cubidus interruptus) protein is a consensus HhRE (GACCACCCA), since it mediates the activation of the ptc (patched) promoter by Shh. Ptc is a downstream target gene of Hh and is part of the putative membrane-bound Shh receptor complex (18, 19, 20). However, the exact mechanism of ptc activation via this Ci element remains to be determined. Interestingly, the ShhRE found on the COUP-TFII promoter bears very little similarity to that of the Drosophila Ci binding site. In our oligo competition experiments, we demonstrate that any changes in the core sequence (TACATAATGCGCCG) are deleterious to Shh-induced binding. Also, a transcription factor data bank analysis revealed very little information as to the exact identity of the factor(s) that may bind this element. The identification of this novel response element suggests that in higher vertebrates the pleiotropic effects of Shh can be mediated by multiple pathways, some of which may target the Gli (mammalian homolog of Ci) binding site and others may target novel elements such as the one in the COUP-TFII promoter.
As shown in Fig. 5, the DNA-binding complexes in P19 nuclear extracts
are induced by Shh. The precise mechanism for this increase in binding
is not known. It is possible that Shh may simply modulate the activity
of this factor(s) that binds to the ShhRE. In agreement with this
possible modulatory role for Shh, the catalytic subunit of protein
kinase A (PKA) has been implicated in the Hh-signaling pathway. In
Drosophila, it is believed that PKA agonists can suppress
signaling mediated by Hh. Also, localized loss of PKA -/-
activity in a Drosophila imaginal disc can mimic Hh activity
even in the absence of Hh (21, 22). Similar results have been obtained
using a dominant negative form of PKA in the mouse dorsal central
nervous system (23, 24). These observations have led to the popular
belief that reversal of a PKA-repressed pathway is one of the major
mechanisms of Hh activity in Drosophila and in mammals.
However, we do not observe a significant decrease in our transient
transfection assays when COS-1 cells are treated with both Shh and
forskolin (90 µM) using the 3X ShhRE-tkCAT. Also,
forskolin fails to decrease Shh-induced binding in an EMSA using
radiolabeled ShhRE (data not shown). These results suggest that in
higher vertebrates additional pathways that are independent of PKA may
also be induced by Shh. The identification of COUP-TFII as a novel
target gene and the characterization of a ShhRE within its promoter may
allow for the unraveling of this complex pathway.
Our results provide a novel target gene for Shh, namely COUP-TFII, which can be exploited to study some of the intermediate steps in the Shh-signaling pathway. In particular, COUP-TFII is a very well characterized transcription factor whose ability to affect the function of several other nuclear receptors provides an attractive target for the pleiotropic effects of Shh signaling. However, the expression of COUP-TFII transcripts and areas in which Shh exerts its signaling activity may be limited to a few regions within the developing embryo (1, 10).
The Shh homozygous null mice display the predicted phenotype based on the expression profile of this protein. The Shh null mice display a cyclopia phenotype along with a loss of maintenance and organization along the axial midline structures of the developing embryo (25). However, in the Shh null mice we can detect some COUP-TFII transcripts (F. Pereira, and M-J. Tsai, personal communication). This observation suggests that COUP-TFII is not exclusively regulated by Shh. These results are not surprising since COUP-TFII is expressed throughout mouse embryonic development in a wide variety of tissues (10, 11). Also, we and others have shown that COUP-TFs are downstream target genes of retinoic acid and that their expression is increased by treatment with both 9-cis and all-trans retinoic acid (13, 14).
We have generated COUP-TFII homozygous null mice to study the possible involvement of COUP-TFII in motor-neuron differentiation. However, the COUP-TFII null mice die before 10.0 days postcoitum (dpc). Interestingly, no obvious change in the overall morphology within the ventro-lateral region of the neural tube is observed (F. Pereira and M-J. Tsai, personal communication). We believe that the presence of COUP-TFI transcripts in the neural tube may allow for some functional compensation of COUP-TFII, which may obscure any obvious defects in motor neuron differentiation. We are currently attempting a localized knock out of both COUP-TFI and COUP-TFII in the ventro-lateral region of the neural tube to study the possible involvement of these genes in motor neuron differentiation.
Our results have provided a novel target gene for Shh signaling in P19 mouse embryocarcinoma cells. Activation of COUP-TFII, which is known to harbor a trans-repressor domain in its C-terminal 15 amino acids, may have important implications in neural tube patterning (26). Shh has been implicated in enabling the notochord-derived signals to repress the dorsalizing signals induced by BMP-2/4 from the roof plate and or the neural epithelium of the neural tube (7, 27). It has been reported that COUP-TFI can inhibit the BMP-4 promoter in fetal rat calvarial osteoblasts (28). It is possible that temporal induction of COUP-TFII expression by Shh may serve to mediate this very repression in specific regions that are fated to become motor neurons in the ventro-lateral region of the neural tube. In conclusion, our results provide a novel target gene (COUP-TFII) and a novel target element (ShhRE) that seem to mediate Shh-induced COUP-TFII expression in P19 mouse embryocarcinoma cells. This finding will allow for the elucidation of the numerous signaling pathways induced by Shh.
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MATERIALS AND METHODS |
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Oligonucleotide Sequences
ShhRE: CGGGATCC AGT GGG TTC TAC ATA ATG CGC CCG GGA
AGATCTTCC
M1: CGGGATCC AGT GAA TTC TAC ATA ATG CGC CCG GGA AGATCTTCC
M2: CGGGATCC AGT GGG TTC CGC ATA ATG CGC CCG GGA AGATCTTCC
M3: CGGGATCC AGT GGG TTC TAC GTG ATG CGC CCG GGA AGATCTTCC
M4: CGGGATCC AGT GGG TTC TAC ATA ATG CGC ATT GGA AGATCTTCC
M5: CGGGATCC AGT GGG TTC TAC ATA ATG CGC CCG GAG AGATCTTCC
The ShhRE, M1, M2, M3, M4, and M5 oligos were cut with BamHI and BglII and ligated to generate a 3-copy insert. Later this multimer was excised from a gel and subcloned into the BamHI site of the ptk109 plasmid, which contains a luciferase reporter gene driven by a minimal tk promoter.
PCR Primers
-808 forward primer: GCT CTA GAG CAG AGA GCT CAG TGA
GCT
+117 reverse primer: TGA AGA TCT GAG TGT GCA GC
-1343 forward primer: GCT CTA GAG CGG AGA GCA TTA TTC AGT
-1267 forward primer: GCT CTA GAG CAA ACC ACT GTG CCC GAT
-1150 forward primer: GCT CTA GAG CAT TTA GAG GCA TCG CCA
-1316 forward primer: GCT CTA GAG CGT TCT ACA TAA TGC GCC
-1298 forward primer: GCT CTA GAG CAT GCG CCG GGA GTC CCG GGT GGA C
-1070 reverse primer: GAA GAT CTT CCT GGC CAC AGA GAG G
The above mentioned primers were used to PCR amplify specific regions within the COUP-TFII promoter. One microgram of the 1.6-kb region was used as template, and 100 ng/ml of each of the forward and reverse primers were used in a 50-µl reaction along with 0.5 mM deoxynucleoside triphosphates (final concentration) and 1 µl of Taq polymerase. Ten percent dimethylsulfoxide was included in some reactions when the regions to be amplified had a GC content that was greater than 50%. The amplified DNA fragments were phenol- and chloroform-purified followed by an overnight restriction enzyme digest with the appropriate enzymes. All forward primers were digested with XbaI, and all reverse primers were digested with BglII. Later these fragments were subcloned into a pBluescript II KS plasmid vector at the XbaI and BglII sites. T7 and T3 primers were used to sequence these PCR-amplified fragments and after verification, these fragments were subcloned into the pBLCAT2 (tkCAT) or pBLCAT3 (CAT alone) plasmid at the XbaI/BamHI site. The resultant plasmids were sequenced using the PCR primers and were subsequently used in transient transfection assays.
COS-1 Conditioned Media
COS-1 cells were plated at a density of 2 x
106 cells per 10-cm plate. Cells were transiently
transfected using the diethylaminoethyl-Dextran method as described
previously (29). Briefly, 1 µg of pBKCMV-Shh or pBKCMV plasmid was
dissolved in 60 µl Tris-borate-NaCl (TBS) buffer and was incubated
along with 120 µl diethylaminoethyl-Dextran (Sigma, St. Louis, MO; 5
mg/ml in TBS) for 30 min at room temperature. Later, this DNA solution
was mixed with 4 ml TBS buffer containing 10% NuSerum (Collaborative
Biomedical Products, Bedford, MA). This mixture was then applied to the
10-cm plate, and the cells were incubated in this mixture for 6 h.
Cells were shocked with 10% dimethylsulfoxide for 2 min followed by a
4-h incubation with DMEM (no serum) containing 100 µM
Chloroquine (Sigma, St. Louis, MO). Cells were washed and fresh medium
was added containing 5% FBS. Conditioned medium was obtained after 24,
36, and 48 h and assayed by Western blot after the medium had been
concentrated to 1/20 of the volume in an Amicon (Beverly, MA)
concentrator. The 48-h time point was found to be optimal for use in
other assays.
Northern Blot Analysis
P19 cells were grown to a density of 5 x 105
cells per 10-cm plate. Cells were used between passage numbers 5 and
20. Late passage cells displayed significant variations in our
experiments and hence were not used in these reported studies.
Conditioned media obtained from the appropriately transfected COS-1
cells were applied at a specific time point, and 40 h later total
RNA was obtained using the Trizol (Life Technologies, Gaithersburg, MD)
reagent. This method is a modification of the guanidinium-HCl method of
RNA isolation (30). RNA was dissolved in 100% deionized formamide and
used within a short period or stored in 70% ethanol for extended
periods at -70 C. Ten micrograms of total RNA were reprecipitated and
washed with 70% ethanol and redissolved in diethyl
pyrocarbonate-treated water. This RNA was heated at 55 C for 15 min in
loading buffer (35% formamide, 10% formaldehyde, 20 mM
Na2HPO4, pH 6.8, and loading dye containing
0.25% xylene cyanol and 0.25% bromophenol blue). RNA was quick cooled
on ice and loaded onto a 1% agarose gel containing 1 M
formaldehyde in an SPC buffer (20 mM
Na2HPO4, pH 6.8, and 2 mM EDTA).
The gel was run in 1x SPC buffer and stained with ethidium bromide
(0.01% solution in 1x SPC) for 20 min followed by destaining for
1 h. The separated RNA was blotted onto a Hybond N nylon membrane
(Amersham, Arlington Heights, IL). A RNA ladder (6 µg) was run along
with each gel and was used to estimate the size of the transcript. The
membrane was UV-cross-linked (Stratalinker, Stratagene, La Jolla, CA)
for 2 min and was dried in a gel dryer at 80 C. The membrane was
prehybridized using the QuikHyb solution from Stratagene for 1 h
and was hybridized with a radiolabeled 500-bp 5'-untranslated region
fragment (31) of COUP-TFII for 2 h. Later, the membrane was washed
twice with 2x saline sodium citrate (SSC) buffer and 0.1% (wt/vol)
SDS. This was followed by two high-stringency washes using 0.1 x
SSC and 0.1% SDS at 65 C. The residual SDS was removed using a quick
2x SSC wash, and the membrane was exposed to a XOMAT-AR film (Eastman
Kodak, Rochester, NY) for 6 to 12 h. The membrane was then
stripped using 0.1x SSPE (sodium phosphate-EDTA), 1% SDS solution at
85 C for 15 min. It was then rehybridized with a specific 5' 1.2-kb
GAPDH probe (32).
Transient Transfection Assays
COS-1 cells were transiently transfected as described earlier,
using 1 µg reporter plasmid. P19 cells were transfected using a
replication-deficient adenovirus as described earlier (33). Briefly,
adenovirus (2 x 108 particles) and 100 ng reporter
plasmid were incubated at room temperature for 30 min in a buffer
containing 20 mM HEPES, pH 7.3, and 150 mM
NaCl. This was followed by infection of P19 cells (400,000 cells per
well) with this viral-DNA mixture for 2 h in a 37 C incubator in
DMEM containing 5% FBS. E. coli-expressed Shh (1
nM) was added along with the fresh media supplemented with
10% FBS after transfection and incubated for 24 h, before the
cells were harvested for reporter gene activity. A Bio-Rad (Hercules,
CA) protein assay reagent was used to quantitate the amount of protein.
Five micrograms of cell lysate were used in a CAT assay, which was
performed as described previously (34). For the luciferase reporter
gene assays, 10 µg cell lysate were incubated with the enzyme
substrate in a 60-µl reaction mix according to the instructions
provided in the Promega luciferase assay system kit (Promega, Madison,
WI). Enzyme activity was measured in a Monolight analytical luminometer
(Analytical/Luminescence Laboratory, Ann Arbor, MI) for 5 sec and was
plotted as relative luciferase units per 20 µg lysate.
Alternately in Fig. 2, P19 cells were transfected using 1 µg
p1.6CII-CAT plasmid along with 6 µg Lipofectamine (Life Technologies)
reagent in 4 ml DMEM without serum for 10 h. Later cells were
washed and 12 ml fresh DMEM containing 10% FBS were added along with
200 µl or 400 µl concentrated COS-1-conditioned media containing
Shh. In parallel, 400 µl concentrated COS-1-conditioned media without
Shh, along with 12 ml DMEM, were also added to P19 cells. After 36
h cells were harvested and cell lysate was obtained. Protein
concentration was determined using the Bio-Rad protein assay reagent.
Twenty micrograms of cell lysate were used to analyze CAT activity as
described earlier.
EMSAs
P19 cells were seeded at 5 x 105 cells
per 10-cm plate in DME-F12 media with or without 1 nM Shh.
After 36 h cells were harvested and washed in PBS. Cells were
incubated in 200 µl hypotonic buffer containing 10 mM
KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT),
0.5 mM phenylmethylsulfonylfluoride, and 10 mM
HEPES, pH 7.9, for 15 min at 4 C. Twenty-five microliters of 10% NP-40
were added, and the cells were vortexed for 30 sec. Later these cells
were centrifuged at 5000 rpm at 4 C to spin down the nuclei. Nuclei
were then incubated in 30 µl hypertonic buffer containing 0.4
M NaCl, 1 mM EDTA, 1 mM DTT, 1
mM phenylmethylsulfonylfluoride, 10% glycerol, and 20
mM HEPES, pH 7.9, for 30 min in a gentle shaker at 4 C.
This mixture was centrifuged at 13,000 rpm at 4 C for 10 min, and the
supernatant was immediately dialyzed for 4 h in 500 ml buffer
containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1
mM EDTA, 1 mM DTT, and 5% glycerol at 4 C. A
10 kDa molecular mass cut-off membrane was used for this dialysis. A
Bio-Rad protein assay reagent was used to quantitate the amount of
protein. Five micrograms of nuclear extract were incubated along with
50,000 cpm of radiolabeled ShhRE for 15 min, in a buffer containing 10
mM Tris, pH 7.5, 80 mM NaCl, 1 mM
EDTA, 1 mM DTT, 0.1 mg/ml BSA, 200 ng salmon sperm DNA, 3
µg poly dG-dC, 1 µg poly dI-dC, and 5% glycerol. One hundred molar
excess of competitor oligos were incubated in the appropriate reaction
mix along with the nuclear extract. The samples were then loaded onto a
5% native polyacrylamide gel, and the protein-DNA complexes formed
were separated and analyzed by autoradiography.
Western Blot Analysis
Conditioned media from the appropriately transfected COS-1 cells
were concentrated to 1/20 the volume using an Amicon concentrator and
applied onto a 12% SDS-polyacrylamide gel. The separating gel was
immediately transferred onto a nitrocellulose membrane in a buffer
containing 50 mM Tris, 380 mM glycine, 0.1%
(wt/vol) SDS, and 20% methanol. The transfer was performed at 65 V for
16 h in a cold room. To confirm complete transfer, the gel was
stained with a Coomassie stain for 20 min followed by destaining in a
40% methanol-10% glacial acetic acid solution for 12 h. The
resultant membrane was subjected to the standard enhanced
chemiluminescence (ECL) Western blotting kit protocol (Amersham, Life
Science). A rabbit polyclonal antibody that specifically recognized the
N-terminal 20 kDa Shh protein (3) was used to detect the levels of
N-terminal 20-kDa protein in the conditioned media. The primary
antibody was divided into two groups and was blocked with BSA or 4
µg/ml N-terminal Shh protein fragment for 2 h at 4 C with gentle
shaking. The membrane divided into two halves and was incubated with
the primary antibody or the preblocked antibody at a 1:3000 dilution in
20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6
(TBS-T) for 1 h. The membrane was washed and then incubated with a
horseradish peroxidase-labeled anti-rabbit secondary antibody (1:1000
dilution) for 1 h. This was followed by another wash and
incubation with the detection mix provided in the ECL kit, for 1 min.
The membrane was immediately drained and exposed to film for 30 sec. A
prestained marker (Bio-Rad) was used to estimate the apparent molecular
masses of the resultant bands.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant DK-44988 (to S.Y.T.) and NIH Grant DK- 45641 (to M.J.T.).
Received for publication February 20, 1997. Revision received June 3, 1997. Accepted for publication June 9, 1997.
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REFERENCES |
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