1 Department of Biology and Molecular Biology Institute, San Diego State
University, San Diego, CA 92182-4614, USA
2 Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr
Way, Oakland, CA 94609, USA
* Authors for correspondence (e-mail: jsaba{at}chori.org and gharris{at}sunstroke.sdsu.edu)
Accepted 24 February 2003
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SUMMARY |
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Key words: Sphingosine-1-phosphate, Sphingolipids, Sphingosine phosphate lyase, Muscle, Drosophila, Serine palmitoyltransferase, Sphingolipidoses, Reproduction, Apoptosis
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INTRODUCTION |
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In addition to these findings, recent studies performed in
Drosophila S2 cells demonstrate that sphingolipid intermediates are
required for regulation of fatty acid biosynthesis through their ability to
inhibit the cleavage of Drosophila sterol regulatory element binding
protein (SREBP) (Dobrosotskaya et al.,
2002; Seegmiller et al.,
2002
). SPL and a product of the reaction catalyzed by SPL,
ethanolamine phosphate, appear to be required for regulation of SREBP
cleavage, possibly through conversion of ethanolamine phosphate to
phosphatidylethanolamine. Although the effect of SREBP dysregulation on
Drosophila development was not explored, these findings raise the
possibility that SPL could exert biological effects by modulating the levels
of upstream (sphingolipid) and/or downstream (phospholipid) intermediates.
In this study, we demonstrate that normal sphingolipid catabolism is required for Drosophila development. We identified the Drosophila SPL homolog Sply and showed that this gene is developmentally regulated. Furthermore, we demonstrated the biochemical and physiological characteristics of Sply05091 loss-of-function mutants. Sply05091 homozygotes exhibited pattern abnormalities in the dorsal longitudinal flight muscles (DLM) of the adult thorax and were flightless. Mutant larvae exhibited diminished survival, with the most pronounced effect occurring during the first larval instar. Egg-laying was diminished in homozygous Sply05091 adults, and enhanced apoptosis in the region of the embryonic genital disc was observed. Some improvement in flight performance and reduction in phosphorylated long chain bases (LCBPs) occurred after treatment with the sphingosine kinase inhibitor D,L-threo-dihydrosphingosine (D,L-threo-DHS). Normalization of muscle morphology, viability, flight and reproductive function were achieved by genetic Sply05091 reversion. Interestingly, introduction of a single hypomorphic allele of lace, resulting in diminished de novo synthesis of sphingolipids, could correct the Sply05091 mutant phenotype. Finally, we demonstrated that introduction of Sply05091 into a homozygous lace background greatly improves viability by blocking sphingolipid catabolism and increasing available sphingolipid intermediates. Taken together, our results show that the primary mechanism responsible for the Sply mutant phenotype is the accumulation of sphingolipid signaling molecules, and that tight regulation of sphingolipid metabolite levels is essential for Drosophila development.
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MATERIALS AND METHODS |
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Cloning of Drosophila SPL homologue
The Drosophila melanogaster genomic database
(http://flybase.bio.indiana.edu)
was searched for predicted proteins using mouse (Accession Number AAH26135)
and human (Accession Number XP_166113) SPL sequences. DNA homology searches
were performed via the Berkeley Drosophila Genome Project web site
(http://www.ncbi.nlm.nih.gov)
using the BLAST search program. One computed gene (CG8946) was identified that
corresponded to a predicted SPL gene. Subsequently, two ESTs were identified
which contained open reading frames that corresponded to the two predicted
splice variants. The open reading frame contained in LP04413 was amplified
using primers LPEcoRI5 (5'-TGGAATTCGATGCGTCCGTTCTCCGGCAGC-3') and
LPXhoI3' (5'-CTCCTCGAGTCTATTTCTGGCTGGGAGT-3') and was cloned
into the yeast expression vector, pYES2 (see below), at EcoRI and
XhoI restriction sites. This construct was transformed into a
dpl1 strain using the lithium acetate method
(Ito et al., 1983
).
Saccharomyces cerevisiae strains and growth conditions
Wild-type yeast used in this study were of strain JK9-3d (leu2-3,112
ura3-52 rme1 trp1 his4 HMLa) (Heitman
et al., 1991). The yeast strain JSK386 (dpl1
) is
an isogenic derivative of strain JK9-3d in which the DPL1 gene has
been replaced by a G418-resistant marker
(Kim et al., 2000
). Strains
JS204 and JS205 are derivatives of JSK386 which contain the
Drosophila ESTs LP04413 and GH13783 respectively in expression
vector, pYES2 (Invitrogen, Carlsbad, CA). pYES2 is a yeast expression vector
containing the URA3 gene (which provides transformants the ability to
grow in media without uracil), and an Ampicillin resistance marker and origin
of replication functional in Escherischia coli. Genes expressed using
this system are regulated under the control of the GAL1,10 promoter,
which allows expression in the presence of galactose and not in the presence
of glucose. Cells were grown in minimal or uracil- media containing
either 20 g glucose or galactose per liter, as indicated.
Functional complementation in yeast
Strains of interest were grown to saturation in liquid culture for 2-3
days. They were then resuspended in minimal medium, placed in the first row of
a 96-well plate and diluted serially from 1:2 to 1:4000 across the plate. The
cultures were normalized for OD600=2 and template inoculated onto a
control plate and a plate containing 50 µM sphingosine, obtained from Sigma
Chemical Company (St Louis, MO). Sphingosine enriched plates were made with
minimal media containing 0.0015% NP40 and 50 µM
D-erythro-sphingosine. At this concentration of NP40, no effects on
cell viability are observed. Plates were incubated at 30°C for 2 days and
assessed visually for differences in growth.
SPL assays
SPL assays of yeast extracts from strains expressing Drosophila
sequences LP04413 and GH13783 were performed as previously described using a
[3H] labeled C18-dihydrosphingosine-1-phosphate
substrate, obtained from American Radiolabeled Chemicals (St Louis, MO)
(Saba et al., 1997;
Van Veldhoven and Mannaerts,
1991
). In this method, SPL activity is measured by determining the
conversion of radiolabeled C18-dihydrosphingosine-1-phosphate
substrate to long chain aldehyde product. To assess the ability of homozygous
Sply05091 versus wild-type flies to degrade endogenous
LCBPs, an HPLC method was developed and employed to examine extracts of
wild-type and homozygous Sply05091 adults. Endogenous
LCBPs were first isolated as described under `Analysis of Drosophila
Sphingolipids', and the lipid extract from 15 mg of homozygous
Sply05091 flies were dried down using nitrogen gas. Lipids
were resuspended in SPL reaction buffer and incubated for various time points
at 37°C. Lipids were reisolated, derivatized with
o-phthalaldehyde and analyzed by HPLC, as described below. Activity
was determined by measuring the percent degradation of endogenous LCBPs in
comparison with standards incubated in the absence of protein extracts.
Expression of Sply
For northern analysis, full-length probes were labeled by random priming
with [-32P] dGTP. Hybridization was carried out under
standard conditions against an RNA blot prepared from total RNA of
Drosophila embryos. RpL32 is a constitutively expressed
ribosomal gene used as a loading control.
The ribonuclease (RNase) protection assay was performed by hybridizing a radiolabeled antisense riboprobe to 20 µg of total RNA obtained from staged Drosophila as indicated. Unhybridized probe was digested with 60 µg/ml RNase A and 150 u/ml RNase T1 for one hour at 37°C. Samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated and run on a 10% polyacrylamide gel.
In situ hybridization was performed with a digoxigenin-labeled probe (Roche
catalog number 1 175 025) and hybridized to fixed embryos at various stages
essentially as described (Tautz and
Pfeifle, 1989).
Analysis of Drosophila sphingolipids
Flies (100 mg) of were homogenized in 6 ml of ice-cold methanol/water 1:1
(vol:vol) with a Potter-Elvehjem homogenizer with a loose pestle followed by a
tight pestle until the pestle moved smoothly. Extract was further homogenized
by tip sonication for three times 20 seconds. Extract was spun at low speed
and supernatant was removed and dried down in speed vac. Extract was
resuspended in 500 µl of methanol containing 0.1 M potassium hydroxide and
incubated for 1 hour at 37°C. After incubation, the extract was dried down
in speed vac. Extract was resuspended in 500 µl of 50% methanol containing
0.1% glacial acetic acid and applied to a C18E STRATA solid phase extraction
column. C18E STRATA column was washed with 50% methanol containing 0.1%
glacial acetic acid followed by a wash with 100% methanol containing 0.1%
glacial acetic acid. Lipids of interest were eluted with methanol/10 mM
ammonium acetate, 9:1 (vol:vol). Lipids were dried down in speed vac. and
o-pthaladehyde labeled for HPLC analysis as previously described
(Kim et al., 2000).
Lethal phase analysis
One hundred embryos from the indicated lines were collected and observed at
each developmental stage. Viability is expressed as the percentage of flies
that survived through the indicated stage.
Adult flight performance
Two- to 7-day-old adult flies were released into a top-lit Plexiglas
chamber. Flight behavior was scored as follows: upward flight, 3; lateral
flight, 2; downward flight, 1; flightless, 0
(Vigoreaux et al., 1993).
Average flight scores were compared using a two-tailed Student's
t-test.
Adult and larval microscopy
Preparation of tissue, staining, mounting and visualization was performed
using standard techniques (Sullivan et
al., 2000). Thoraces from adult flies were dissected, fixed with
formaldehyde and osmium tetroxide, and embedded in EPON. These blocks were
then cut into 1 µm sections, stained with Methylene Blue and Azure II, and
viewed with a Lieca DMIRBE microscope.
Larvae were filleted during the third instar, pinned with the dorsal cuticle down, and eviscerated to allow an unobstructed view of the body wall muscles. The tissue was fixed with 4% formaldehyde, permeabilized in 100% acetone and stained with fluorescein-conjugated phalloidin. (Molecular Probes catalog number F-432.)
Electron microscopic analysis of DLMs was performed on adults essentially
as described (O'Donnell and Bernstein,
1988).
Hemithoraces were visualized essentially as described
(Fyrberg et al., 1994).
Briefly, adult flies were frozen in liquid nitrogen, bisected with a razor
blade and dehydrated in an ethanol series. The cuticles were then cleared with
methyl salicylate to allow visualization of the muscles with a Leica DMIRBE
microscope under polarized light.
Fluorescence microscopy
Embryos (0-24 hours) were prepared and fixed using standard techniques
(Rubin Manual) and stained with the indicated primary antibody or assayed for
apoptosis using a TUNEL-based staining method (in situ cell death detection
kit, Roche catalog number 1 684 795). Incorporation of fluorescein was
assessed with a Leica DMIRBE epifluorescence microscope and an upright Leica
TCS-NT confocal laser scanning microscope.
Antibodies and fluorescent reagents were as follows: polyclonal rabbit
anti-Drosophila myosin heavy chain
(Kiehart and Feghali, 1986)
1:1,000. Polyclonal rabbit anti-DMEF2
(Lilly et al., 1995
) 1:10,000.
Secondary antibody was a fluorescein-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories) 1:1,000.
Genetics
The precise excision of the ry+ PZ P-element was
performed by introducing transposase allele 2-3 into insertion line
BL-11393. In the subsequent generation, the transposase was removed and the
second chromosome was balanced over CyO. Offspring of these flies that lacked
the P-element were selected by scoring for loss of ry+.
Homozygous lines were generated, assayed for restoration of flight behavior,
and assessed for precise excision by PCR if indicated. Lines homozygous for
the Sply05091 allele and the
lacek05305 allele were generated by meiotic recombination.
Sply05091 and lacek05305 mutations
were introduced in trans and balanced in the next generation. Flies carrying
the lacek05305 allele were selected by presence of
w+. Presence of Sply05091 was verified
by PCR. The Sply27A allele was generated by mobilization
of the P-element in insertion line KG6148. Briefly, transposase was introduced
into KG6148 flies for one generation. Offspring were screened for loss of the
P-element, bred to homozygosity and assayed for reduced flight
performance.
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RESULTS |
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To determine whether loss of Sply expression affects the levels of Drosophila endogenous LCBs and corresponding LCBPs, the sphingolipid profile of homozygous Sply05091 flies was evaluated and compared with wild-type controls. Homozygous Sply05091 adults demonstrated an eightfold increase in LCBs and a 20-fold increase in LCBPs when compared with wild type (Table 1), indicating significant derangement of sphingolipid metabolism. This accumulation of LCBs and LCBPs was observed in homozygous Sply05091 mutants as early as hours 12-18 of embryogenesis, correlating with the onset of Sply expression.
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Sply05091 homozygotes demonstrate abnormal flight
muscle morphology
To investigate further the etiology of Sply05091 flight
defects, adult mutants were sectioned through the thorax, and muscles were
examined by light microscopy (Fig.
4). These studies revealed a reduction in the number of muscle
fibers comprising the DLMs required for flight. Whereas the thoraces of
wild-type flies invariably contained six symmetrical pairs of fibers,
Sply05091 homozygotes exhibited a general pattern of
missing fibers, asymmetry and hypertrophy of remaining fibers. Quantitative
analysis of DLM fibers revealed a reduction from six per hemithorax in wild
type to an average of 4.15 per hemithorax in the mutants
(Table 1). Microscopic analysis
of hemithoraces illuminated with polarized light confirmed the abnormal muscle
configuration while demonstrating that muscle insertions were not affected
(Fig. 5).
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Sply05091 homozygotes demonstrate decreased
fecundity, semi-lethality and increased apoptosis in embryos
The number of offspring resulting from homozygous
Sply05091 crosses was about 10% of the number observed in
wild-type crosses. This loss of progeny could result from diminished
egg-laying and/or diminished survival of embryos and larvae. Analysis of
egg-laying indicated that fecundity of the mutants was about one third that of
control flies (Table 1). This
outcome could be the result of diminished male and/or female fertility. To
distinguish between these possibilities, both male and female
Sply05091 homozygotes were mated to wild-type flies, and
egg-laying was measured in comparison with wild-type pairs and homozygous
mutant pairs. Numbers of eggs produced were significantly diminished in
crosses of both male and female mutant flies with wild-type mates (data not
shown), indicating that the effect on fecundity was not gender specific.
Additionally, crosses between Sply05091 homozygous males
and females resulted in progeny with an overall survival (from egg to
adulthood) of 33.5%, compared with an 80% survival rate in wild-type flies.
Lethality in the Sply05091 mutants was high during larval
stages (46%, compared with 3% in wild type), with the majority of larval death
occurring during the first larval instar. Less severe effects were observed
during metamorphosis (22% lethality, compared with 1% in wild type), and no
appreciable differences in survival were noted during embryogenesis.
Sply05091 mutant embryos were examined by in situ TUNEL
assay, and patterns of apoptosis were compared to those of wild-type controls
(Fig. 7).
Sply05091 mutant embryos demonstrated a pronounced
enhancement of apoptosis compared with wild-type controls, especially in a
specific region of the posterior pole near the developing genital disc.
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The Sply05091 muscle defect is suppressed by
reducing sphingolipid intermediates
To investigate the possibility that the Sply05091
muscle phenotype was caused by accumulation of LCBPs, an inhibitor of
sphingosine kinase, D,L-threo-DHS, was introduced to the growth media
of mutant and wild-type flies. Flies were grown on the supplemented media, and
F2 progeny were examined. When wild-type flies were grown on media
supplemented with 10 µM D,L-threo-DHS, no deleterious effects were
observed. Sply05091 mutants grown on this media
demonstrated a slight but significant improvement in flight performance. To
determine whether the flight improvement coincided with a restoration of LCBP
levels, LCB/LCBP levels were analyzed in mutants and controls grown on
D,L-threo-DHS. LCBP levels in Sply05091
homozygotes grown in the presence of sphingosine kinase inhibitor were reduced
by approximately 20% (Table 1).
Similarly, LCBP levels in wild-type flies were reduced by 20% from normal
levels (data not shown). Because it appeared that significant depletion of
LCBP intermediates would be difficult to achieve using pharmacological means,
a genetic approach was taken to more effectively block the accumulation of
sphingolipid intermediates in Sply05091 homozygotes.
Assuming that the mutant phenotypes are caused by an accumulation of LCB/LCBPs, we predicted that diminishing SPT activity in the Sply05091 homozygote would suppress the Sply05091 phenotype by reducing production of sphingolipid intermediates. Accordingly, a lacek05305 hypomorphic allele was introduced onto the Sply05091 chromosome by genetic recombination, thus generating a Sply05091, lacek05305/+ line. Sply05091, lacek05305/Sply05091, lace+ flies exhibited reversion of the abnormal muscle patterning (Figs 4 and 5), and flight performance was substantially improved (Table 1). Additionally, the pattern of embryonic apoptosis appeared similar to that of the wild type (Fig. 7). Phenotypic reversion correlated with a marked reduction of the LCBs and LCBPs (Table 1).
Loss of Sply expression suppresses the hypomorphic
lace phenotype
Inheritance of two lacek05305 hypomorphic alleles was
reported to be almost completely lethal, whereas a heterozygous allelic
combination (lacek05305/lace2) yields flies
that frequently survive but manifest severe developmental phenotypes leading
to eye, bristle and wing abnormalities
(Adachi-Yamada et al., 1999).
We predicted that the lace mutant phenotype is due to diminished
levels of sphingolipid intermediates. Furthermore, we reasoned that inhibiting
sphingolipid catabolism in lace mutants might allow sufficient
accumulation of trace sphingolipids obtained through the diet to ameliorate
developmental defects induced by the lack of crucial sphingolipid
intermediates. To address this possibility, a Drosophila line
homozygous for both the Sply05091 and
lacek05305 alleles was generated. Significantly, the
presence of the Sply05091 allele increased the recovery of
lace homozygotes from 9% to 39% of that expected by independent
assortment. Furthermore, the introduction of Sply05091
fully suppressed the eye, bristle and wing phenotypes in the resulting flies
(Fig. 8). In accordance,
sphingolipid intermediates were substantially increased in this line, in
comparison with lacek05305 mutants and
lace2/lacek05305 heterozygotes
(Table 1).
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DISCUSSION |
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In this study, we identify the Drosophila melanogaster Sply gene and demonstrate the importance of Sply expression in maintenance of Drosophila viability, reproduction and muscle development. Sply encodes a functional SPL that is capable of metabolizing dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate substrates, as shown by in vitro biochemical studies employing the former substrate and functional complementation in yeast strains that accumulate primarily the latter. Furthermore, this enzyme is responsible for catabolism of the endogenous LCBPs of Drosophila. This conclusion is supported by the finding that extracts from wild-type flies but not homozygous Sply05091 mutants are able to degrade this substrate in vitro, and by the lipid profile of homozygous Sply05091 mutants which demonstrate early, sustained and severe accumulation of endogenous LCBPs (and, to a lesser extent, accumulation of LCBs) in vivo.
Drosophila SPL expression is developmentally regulated, with
earliest expression occurring at 8-12 hours of embryogenesis (after loss of
maternal load), a time frame consistent with the accumulation of sphingolipid
intermediates in Sply05091 late embryos. Our data are in
agreement with a more extensive developmental analysis presented by the Yale
Drosophila Developmental Gene Expression Timecourse Database
(http://genome.med.yale.edu/Lifecycle/).
The LCBP levels observed in the homozygous Sply05091
mutants are on the same order of magnitude (10- to 20-fold higher than
wild-type levels) as those observed in the Saccharomyces cerevisiae
dpl1 strain, in which the sole endogenous SPL gene is deleted. The
temporal pattern of LCBP accumulation suggests that LCBP synthesis is minimal
in early embryogenesis.
We observed a significant defect in the DLM muscle configuration of adult
homozygous Sply05091 mutants. This pattern abnormality
appears to originate during metamorphosis and increases in severity with age.
During Drosophila metamorphosis, most larval muscles undergo
histolysis and are replaced by adult muscles formed entirely from myoblasts
derived from the imaginal discs. However, DLMs are unique in that they are
formed by a fusion event between myoblasts derived from the wing disc and
larval DOMs, which do not histolyse during pupal stages, but undergo a
splitting event, giving rise to six fusion-competent templates per hemithorax
(Roy and VijayRaghavan, 1998).
Embryonic muscle organization, larval muscle patterning, and persistence of
DOM templates remained intact in Sply05091 mutants. Our
preliminary analysis of pharate adults shows a similar but less severe pattern
of missing fibers than in older flies. Furthermore, shortly after eclosion a
small subset of fibers demonstrate hallmarks of degeneration, including
myofibrilar disorganization and mitochondrial disruption, coincident with the
appearance of large vacuoles in the myoplasm (D.R.H., H.F., V.P., K.H., R.G.,
G.L.H. and J.D.S., unpublished). In addition, Sply05091
larvae appear flaccid and have greatly decreased locomotion relative to
controls, suggesting that muscle function is compromised prior to the
patterning defect. This may be due to the effect that sphingosine has been
shown to have on calcium mobilization in muscle cells
(Sabbadini et al., 1992
).
Thus, there is a patterning defect in Sply mutant pupae, and the
muscle fibers that do form are susceptible to degeneration. There are a number
of cellular events downstream of these molecules that may be responsible for
the pathological sequela of this sphingolipidosis, including the disruption of
sphingolipid-regulated signaling pathways, loss of targeting cues for
migrating myoblasts (Spiegel et al.,
2002
) or a pathologic compromise in normal membrane architecture
(Fig. 9).
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The notable accumulation of LCBPs and LCBs in the Sply05091 homozygote was markedly reduced, and the muscle phenotype was greatly improved by replacing one wild-type lace allele with a lace05305 allele. This is consistent with a model in which the accumulation of sphingolipid intermediates is responsible for the observed muscle defect. However, not all features of the Sply05091 homozygote phenotype were completely abrogated by a partial block in de novo synthesis. This could be explained by the failure to normalize LCB/LCBP levels completely in the double mutant, or it is possible that LCB/LCBP accumulation is only partially responsible for the Sply05091 homozygote phenotype. We cannot discount the possibility that one or both of the products of the reaction catalyzed by Sply, ethanolamine phosphate and long-chain aldehyde, could influence Drosophila development, either through their influence on SREBP cleavage, or by some other mechanism which remains to be elucidated.
Conversely, we found that the semi-lethality of lace hypomorphs was abrogated by introduction of a block in sphingolipid degradation. This finding supports the notion that certain sphingolipid intermediates play a crucial role(s) in regulating Drosophila eye, limb and wing development. Furthermore, the finding of severe phenotypes that are corrected by normalization of LCBs/LCBPs in Sply and lace double mutants indicates that maintaining tight regulation of sphingolipid intermediates is crucial to the developing animal. The spatial expression of Sply during embryogenesis suggests that the phenotypes observed in Sply05091 mutants are due to the global derangement of sphingolipids in the developing organism rather than by localized mechanisms. Further studies are under way to determine what specific mechanisms are disrupted by this sphingolipidosis.
These pathologies are consistent with the roles that sphingolipid
intermediates play in signaling, and the toxicity induced by abnormal
accumulation of these lipids in mammalian cells
(Linn et al., 2001). The
future identification of additional genes and mutants of sphingolipid
metabolism in Drosophila will provide powerful genetic resources to
predictably manipulate specific sphingolipid intermediates and to elucidate
the roles of these signaling molecules during development and in cell
function.
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ACKNOWLEDGMENTS |
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