1 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, Guy's Campus, London SE1 1UL, UK
2 Oxford BioMedica (UK) Limited, Medawar Centre, Robert Robinson Avenue, The
Oxford Science Park, Oxford OX4 4GA, UK
Author for correspondence (e-mail:
malcolm.maden{at}kcl.ac.uk)
Accepted 8 July 2002
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Summary |
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Key words: Retinoic acid receptor ß, Neurite outgrowth, Viral vectors, Mouse, Spinal cord
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Introduction |
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Neurite growth inhibitors such as Nogo
(GrandPre et al., 2000;
Chen et al., 2000
;
Prinjha et al., 2000
) are
present in myelin and when, in young rats, these inhibitors were neutralized
with antibodies, longer axonal extension was observed compared with control
animals (Schwab, 1991
). A
similar treatment has led to the recovery of specific reflex and locomotor
functions after spinal cord injury (Bregman
et al., 1995
). A combination of neurotrophin-3 and these
neutralizing antibodies was successful in inducing long distance regeneration
of corticospinal tract axons (Schnell et
al., 1994
), and neurotrophins alone have induced the regeneration
of dorsal root sensory axons back into the spinal cord
(Ramer et al., 2000
). The
glial scar is also a component of the inhibitory environment and is composed
of extracellular matrix molecules, including chondroitin sulphate
proteoglycans. Such molecules are inhibitory to axon growth in vitro
(Niederost et al., 1999
), and
when chondroitinase ABC was administered to the lesioned dorsal columns of
adult rats regeneration of both the ascending sensory and descending
corticospinal tract axons was promoted
(Bradbury et al., 2002
).
It is possible, therefore, that neurotrophins may act simply to keep
axotomized neurons alive (Kobayashi et
al., 1997) and that CNS axons are capable of regenerating, but are
normally prevented from doing so by an adverse environment in vivo.
Consequently, the environment surrounding the neurons has been a focus of
attention in these regenerative studies. However, it is also possible that a
combination of approaches, including additional activation of transcription
within the nucleus of a damaged neuron, may be required and may ultimately
prove successful.
Retinoic acid (RA) can induce nuclear transcription. It is the biologically
active metabolite of vitamin A and is present in various tissues of the
developing embryo and adult animal, especially the nervous system
(Wagner et al., 1992;
Horton and Maden, 1995
;
McCaffery and Drager, 1994
;
McCaffery and Drager, 1995
;
Yamamoto et al., 1996
;
Maden et al., 1998a
). In the
absence of RA, developing neurons of the CNS do not extend neurites into the
periphery (Maden et al., 1996
;
Maden et al., 1998b
).
Conversely, many experiments have shown that when applied to cultured neurons,
RA induces both a greater number of neurites as well as increased neurite
length (for a review, see Maden,
2001
) as well as being capable of dictating their direction of
growth (Maden et al., 1998c
).
RA acts at the level of gene transcription because it is a ligand for two
classes of nuclear transcription factors, the retinoic acid receptors (RARs)
and the retinoid X receptors (RXRs) (for reviews, see
Kastner et al., 1994
;
Kliewer et al., 1994
). There
are three members of each class of retinoid receptor
, ß and
as well as several isoforms of each member, and this diversity may be
responsible for the pleitropic effects of RA on cells.
In our previous studies to determine the molecular mechanism of action of
RA in neurons, we showed that one of these retinoic acid receptors, namely
RARß2, is the crucial transducer of the RA signal. This conclusion is
based on the observations that RARß2 is upregulated after RA
treatment of embryonic mouse dorsal root ganglia (DRG) neurons and adult mouse
DRG neurons and in both of these situations neurite outgrowth is stimulated
(Corcoran and Maden, 1999;
Corcoran et al., 2000
). In
addition, a RARß agonist rather than a RAR
or a RAR
agonist
specifically induces neurite outgrowth from embryonic DRG neurons
(Corcoran et al., 2000
). As a
result we hypothesized that the absence or below-threshold level of this
nuclear receptor in the adult spinal cord may contribute to the failure of
this tissue to regenerate axonal projections.
The experiments described here test this hypothesis and demonstrate that by manipulating the genome of the neurons themselves, stimulation of neurite outgrowth can be obtained from non-regenerative adult mouse and rat spinal cord in vitro. Adult spinal cord was transduced with a viral vector expressing the nuclear transcription factor RARß2, and as a result, the normally inert spinal cord was transformed into one that can extend neurites. No neurites are extended in spinal cords transfected with the empty vector, with a mutated vector, with a different RAR isoform or after treatment with nerve growth factor, thereby confirming the specificity of the RARß2 effect.
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Materials and Methods |
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RT-PCR analysis
RNA was extracted (Trizol, Gibco, RNeasy Mini Kit, Qiagen) and cDNA
prepared by the use of a Pharmacia kit as described in the manufacturer's
instructions. The primers used were specific for GAPDH, RARß2
and RARß4 (for details, see
Corcoran et al., 2000).
Polymerase chain reaction (PCR) was carried out for 30 cycles for embryonic
spinal cord and 40 cycles for adult spinal cord. Amplification was carried out
as follows: denaturation for 30 seconds at 95°C, annealing for 30 seconds
at 55°C and extension for 1 minute at 72°C. One fifth of the resultant
product was then run on an agarose gel.
Real-time PCR was carried out using the Lightcycler SYBR Green Dye kit and Lightcycler machine (Roche), using GADPH and RARß2 primers as described above. PCR was carried out for 40 cycles for both RARß2- and LacZ-transduced adult spinal cord explants. Amplification was carried out as follows: denaturation for 0 seconds at 95°C, annealing for 5 seconds at 55°C and extension for 10 seconds at 72°C. Levels of RARß2 transcription were calculated as a ratio of RARß2 expression to GAPDH expression.
Viral vector construction and production
Minimal lentiviral vectors have been described recently
(Mitrophanous et al., 1999;
Mazarakis et al., 2001
) and
are repeated briefly here. The pONY8.0 series of EIAV vectors were derived
from pONY4.0Z by introducing mutations that prevented accessory gene (tat, S2
and rev) expression and prevented expression of the N-terminal portion of gag
by insertion of T in the first two ATG codons. pONY8.0G was derived from
pONY8.0Z by exchanging the LacZ reporter gene for the enhanced green
fluorescent protein (GFP) gene. The central polypurine tract (cPPT), which
enhances RNA processing and consequently proviral integration
(Charneau et al., 1992
;
Charneau et al., 1994
;
Zennou et al., 2000
), was
inserted into the vector 5' to the genome to make pONY8.0cZ. The
RARß2 gene was amplified from pBluescriptRARß2 and the Flag
epitope inserted at the N-terminus in a single polymerase chain reaction using
the primers
5'-ACTGCCGCGGGCCACCATGGACTACAAGGACGACGATGAACAAGTTTGACTGTATGGATGTTCTGTC-3'
and 5'-ACTGGCGGCCGCTCACTGCAGCAGTGGTG-3'. Underlined
bases indicate restriction enzyme sites (SacII and NotI
respectively), and the bold type indicates bases encoding the Flag epitope.
The resulting 1399 base pair product was cloned into
SacII/NotI-digested pONY8.0c to make pONY8.0cRARß2.
Vector stocks were generated by calcium-phosphate transfection of human embryonic kidney 293T cells plated on 10 cm dishes using a three plasmid co-transfection with vector (16 µg), gag/pol (pONY3.1, 16 µg) and envelope (VSV-G, pRSV67, 8 µg) plasmids. DNA for use in these transfections was obtained using Qiagen Maxi-Preps (genomes) or from commercial sources (gag/pol and envelope plasmids). After transfection (36-48 hours), supernatants were filtered (0.45 µm), aliquoted and stored at -70°C. Concentrated vector preparations were made by initial low speed centrifugation 6,000 g for 16 hours at 4°C followed by ultracentrifugation at 50,000 g for 90 minutes at 4°C. The virus was resuspended in formulation buffer consisting of sodium chloride (37.5 mM), Tris, pH 7.0 (19.75 mM), lactose (40 mg/ml), human serum albumin (1 mg/ml) and protamine sulfate (5 µg/ml), for 3-4 hours, aliquoted and stored at -70°C. Viral titers were at least 3x108 t/u/ml.
Herpes viral vector stocks were prepared exactly as described previously
(Lim et al., 1997). The titres
used for pHSVRARß2, pHSVRARß4 and pHSVLacZ were
5x104, 4x104 and 5x10 t/u/µl,
respectively.
Immunohistochemistry
Explants were fixed in 4% paraformaldehyde in phosphate-buffered saline
(PBS) for 20 minutes and permeabilized with 0.1% TritonX-100 (20 minutes).
Blocking was carried out in PBS containing 10% v/v normal donkey serum as
appropriate for the secondary antibodies. Primary antibodies were used as
follows: anti-neurofilament NF200 (1:5000, Sigma), anti-Flag (1:500 Sigma) and
anti-NeuN, (Neuronal nuclei, 1:100, Chemicon). Antibodies were incubated
overnight at 4°C in PBS-10% v/v serum. Samples were washed three times
with PBS and then incubated with secondary antibodies: Texas-Red-coupled
donkey anti-rabbit IgG and FITC-coupled donkey anti-mouse IgG (1:100 and 1:50,
respectively, both from Jackson Immunoresearch) were incubated at room
temperature for 2-3 hours. After washing, samples were examined under a
confocal microscope.
Western blotting
Protein was isolated as previously described
(Corcoran and Ferretti, 1999)
from embryonic and adult mouse spinal cord. 10 µg of protein was run on a
5% polyacrylamide stacking gel, then a 10% separating gel. The gel was then
semi-dry blotted onto nitrocellulose and processed for antibody staining and
visualization (Corcoran and Ferrettti, 1999). The antibody used was a rabbit
polyclonal RARß antibody, a gift of P. Chambon.
HPLC
Retinoids were extracted from approximately 300 mg of adult mouse spinal
cord tissue according to the method of Thaller and Eichele
(Thaller and Eichele, 1987) by
homogenizing the tissue in 1 ml of stabilizing solution (5 mg/ml ascorbic
acid, Na3EDTA in PBS, pH 7.3). The homogenate was extracted twice
with 2 volumes of 1:8 methyl acetate/ethyl acetate, with butylated
hydroxytoluene as an anti-oxidant, and then dried down over nitrogen. The
extract was resuspended in 100 µl methanol, centrifuged at high speed to
remove any particulate matter and placed into an autosampler vial for
analysis.
Reverse phase HPLC was performed using a Beckman System Gold Hardware with
a photodiode array detector and a 5 µm C18 LiChrocart column
(Merck) with an equivalent precolumn. The mobile phases used were those of
Achkar et al. (Achkar et al.,
1996), which allows a good separation of the retinoic acids and
the retinols. The flow rate was 1.5 ml/min using a gradient of
acetonitrile/ammonium acetate (15 mM, pH 6.5) from 40% to 67% acetonitrile in
35 minutes followed by 100% acetonitrile for an additional 25 minutes.
Individual retinoids could be identified according to their UV absorption
spectra.
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Results |
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To demonstrate that the induction of neurite outgrowth involved the upregulation of RARß2, reverse transcription followed by PCR (RT-PCR) was performed on cultures after 5 days using the same range of RA treatments as described above (Fig. 1C). This revealed that RARß2 is normally expressed in embryonic spinal cord and that it is strongly up regulated after 1x10-6 M RA treatment (Fig. 1C, lane 4); the same concentration that gives maximal neurite outgrowth.
Lack of effect of RA on adult mouse spinal cord in vitro
An identical series of experiments was performed using 10-month-old adult
mouse spinal cord rather than the embryonic cord. In contrast to the embryonic
cord, RA had no effect on neurite outgrowth at any concentration tested, and
like the untreated controls (Fig.
1D), these RA-treated adult cords failed to extend any neurites at
all (Fig. 1E). Examining the
involvement of RARß2 by RT-PCR revealed that control adult
spinal cord had little or no detectable endogenous levels of this receptor
(Fig. 1F, lane 1) and that,
unlike the embryonic cord, there was no change in RARß2 level in
response to RA treatment at any concentration
(Fig. 1F, lanes 2-4).
It was hypothesized that the lack of endogenous RARß2 expression or the absence of RA-induced RARß2 upregulation may be responsible for the inert behaviour of the adult spinal cord. As this observation was central to our hypothesis, we further verified the lack of RARß2 expression in the adult cord by the more sensitive light cycler PCR methodology (Fig. 3C) and also by western blotting (Fig. 1G). The former shows the complete absence of any detectable RARß2 transcripts in three individual cultured pieces of cord (Fig. 3C, lanes 4-6) and the latter shows that RARß2 protein is expressed in the embryonic cord (Fig. 1G, lane 1) but not in the adult cord (Fig. 1G, lane 2).
|
In further support of this hypothesis, we have previously shown that adult
DRG that respond to RA by extending neurites also upregulate
RARß2 (Corcoran and Maden,
1999). To test this RARß2 hypothesis, we transduced adult
mouse and rat spinal cords in vitro with the RARß2 gene using
both an equine infectious anaemia virus (EIAV) and a herpes viral vector
system.
Neuronal transduction and expression of RARß2 from EIAV
vectors
EIAV is a lentivirus, which is a non-human pathogen and has the simplest
genome of all lentiviruses, having only three accessory genes. Minimal vectors
based on EIAV have been described and transduce both dividing and non-dividing
cells (Mitrophanous et al.,
1999; Mazarakis et al.,
2001
). EIAV-based vectors (e.g. pONY8.0Z) cause widespread and
efficient transduction in the rat CNS (e.g. striatum, hippocampus, substantia
nigra) and in spinal cord in vivo
(Mazarakis et al., 2001
). To
demonstrate that effective transduction can also be obtained in vitro, we used
dissociated cultures of rat DRG. Using a pONY8.0GFP virus at a multiplicity of
infection (MOI) of 10 our results revealed that approximately 45% of the cells
in such a dissociated culture were transduced
(Fig. 2A,B). When the mouse
RARß2 gene, tagged with the Flag epitope, was inserted into the
EIAV vector genome (pONY8.0cRARß2.Flag), expression of the Flag epitope
can be detected in both heterologous dividing cell lines (data not shown) and
in dissociated primary rat striatal cultures. Colocalization studies with the
anti-Flag antibody and an RARß antibody in striatal neurons indicate that
staining is specific for the RARß2 protein expressed from the
pONY8.0cRARß2.Flag virus (Fig.
2C-E) and that it is expressed specifically in the nuclei of these
cells. Like dissociated DRG cells, about 45% of the cells in striatal cultures
were transfected.
|
Induction of neurites in adult spinal cord
The pONY8.0cRARß2.Flag virus was used next to transduce adult rat and
mouse spinal cord explants. Virus (1 µl, minimum titer
5x108 transducing units/ml) was injected directly into the
explants, and immunohistochemistry with the anti-Flag antibody demonstrated
efficient RARß2 gene transfer (up to 60% transduction) of both
mouse and rat spinal cords in vitro (Fig.
3A, also Fig. 3F).
Individual explants were tested to ensure that they were expressing the
RARß2 gene using the sensitive light cycler PCR technique.
Fig. 3C shows three examples of
RARß2-transduced cord explants expressing high levels of
RARß2 compared with GAPDH (lanes 1-3) and three control
explants showing no RARß2 expression (lanes 4-6).
Following transduction of adult spinal cords with the pONY8.0cRARß2.Flag, many neurites throughout the cellagen matrix were observed (Fig. 3B). The cells that extended neurites were those that had been transduced, as demonstrated by colocalizing the Flag marker with neurofilament staining (Fig. 3D,E). Fig. 3E shows a high power magnification of an explant, with the transduced cells in green, neurofilament staining in red (Fig. 3D) and coexpressing cells in yellow. Although a sizeable region of the explant had been transduced, as shown by the green region in Fig. 3D, neurites only extended from one corner of the explant (red neurites in Fig. 3D). We estimate that about 50% of the transfected cells showed colocalized neurofilament staining (yellow or orange cells in Fig. 3E). Thus all transfected cells do not extend neurites. Perhaps this reflects the variation in levels of RARß2 expressed in individual cells in the transfected region, which is likely to occur since individual explants varied in RARß2 levels as a whole (Fig. 3C).
Explants were harvested at 5, 8 and 10 days in vitro. At 5 days, neurites first appeared and at 8 days and 10 days, they extended further into the cellagen matrix and now resembled embryonic cords (cf. Fig. 1D) in both the number and the extent of neurite outgrowth. In Table 1, the data for 5, 8 and 10 days are pooled since we have not quantified the lengths of neurites, and it emphasizes that all explants that were successfully transduced (70% success rate) induced neurite outgrowth from at least part of the explant (Fig. 3D). The typical number of neurites that grew into the cellagen matrix was 30-50 neurites per explant.
|
The cords that had been transduced with pONY8.0cRARß2 extended
neurites whether or not RA was added to the medium. This could mean that no
ligand was required to activate the RARß2. Assuming a RXR heterodimeric
partner is required for RARß2 activation, then this could potentially
occur by the phantom ligand effect (Schulman et al., 1977) in which RAR is
activated by a conformational change in its RXR partner. Alternatively it
could be that there is enough endogenous RA already in the explant to activate
the newly synthesized RARß2. RA is certainly found throughout the brain
and spinal cord of the adult rat and comprises a proportionately higher level
of the retinoid pool compared with other tissues
(Werner and DeLuca, 2002). In
addition, we have detected endogenous retinoids, including RA, in the adult
mouse spinal cord by high-pressure liquid chromatography
(Fig. 4), although we do not
know what happens to these lipophilic compounds during the culturing period.
Nevertheless, the potential is there for the RARß2 to be liganded by
RA.
|
Two control experiments were performed. In one, pONY8.0cZ, a virus encoding
the bacterial ß-galactosidase gene LacZ, was used to transduce
parallel cultures. No neurite outgrowth resulted
(Table 1), although after
exposure to X-gal, clear blue staining was observed
(Fig. 3F). Individual explants
were taken for light cycler PCR analysis to ensure that no RARß2
could be detected in these controls (Fig.
3C, lanes 4-6). In the second control experiment, a virus encoding
RARß2 with a mutation in the Integrase gene,
(pONY8.0cRARß2.Flag.Int), which renders the provirus
unable to integrate into transduced cells, was used
(Mitrophanous et al., 1999).
Again, no expression of the RARß2 gene or neurite outgrowth was
observed (Table 1).
These data have been repeated in another series of experiments in which a
defective herpes simplex virus type 1 (HSV-1) vector containing another
isoform of the RARß gene, RARß4 (pHSVRARß4),
LacZ (pHSVlacZ) or RARß2 (pHSVRARß2) were transduced into
adult spinal cord explants. In these experiments, the RARß4
isoform provides an additional control as it has been clearly shown not to be
involved in neurite outgrowth (Corcoran et
al., 2000). Transduction with the pHSVRARß4 failed to change
the behaviour of the cultured adult cord, which remained completely
unresponsive in terms of neurite outgrowth (n=12,
Fig. 3G). Similarly, the
transductions with pHSVlacZ produced no response in the cultured cord, which
remained inert (n=12, data not shown). However, neurite outgrowth
(eight out of 12 explants) was observed following transduction with the
pHSVRARß2 vector (Fig.
3H). RT-PCR demonstrated that transduction with the
RARß2 or the RARß4 vectors resulted in specific
expression of each transcript (RARß2;
Fig. 3I, lane 2 and
RARß4, lane 7) but not of the other gene (controls in lanes 3
and 6, respectively). In the non-transduced cord neither RARß2
nor RARß4 were detected (Fig.
3I, lanes 1 and 5).
Finally, since neurotrophins have been used in attempts to induce nerve
regeneration in vivo (see Introduction) we wanted to examine whether the same
might occur in our explant system. Nerve growth factor (NGF) was chosen
because it specifically activates the RARß2 gene
(Corcoran and Maden, 1999;
Cosgaya and Aranda, 2001
).
However, in the absence of the RARß2 gene we expected it to have
no effect. Indeed, this was the case, as addition of NGF to our spinal cord
cultures did not stimulate neurite outgrowth or extension
(Table 1).
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Discussion |
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Therefore we hypothesized that if the non-regenerating adult spinal cord
cannot upregulate RARß2, and this is the cause of a lack of
neurite outgrowth, then introducing the RARß2 gene into the
adult spinal cord should induce neurite outgrowth. To test this, we transduced
adult mouse and rat spinal cords in vitro with the RARß2 gene
using an EIAV-based vector or a herpes simplex virus vector. With the
pONY8.0cRARß2.Flag virus, a high level of transduction was obtained and a
large number of neurites were extended from the explanted adult cords in both
mouse and rat. Double immunostaining showed that cells in which the Flag tag
(and by implication, RARß2) was expressed were those that extended
neurites. No neurites were observed in several control experiments, which
included transfecting with the pONY8.0cZ construct (the virus with no
RARß2 gene) or the pONY8.0cRARß2.FlagInt
(the virus with the RARß2 gene but a defective
Integrase gene). Experiments with HSV-based vectors gave similar
results: neurites were observed from spinal cords transduced with
RARß2 but not from those transduced with either LacZ or the
RARß4 isoform. The RARß4 provides an additional
control for the RARß2 transductions, as it is unaffected by RA
in embryonic mouse dorsal root ganglia
(Corcoran et al., 2000). In
further experiments, NGF did not induce neurite outgrowth from adult mouse
spinal cords, in contrast to adult DRG
(Corcoran and Maden, 1999
;
Ramer et al., 2000
). These
results strongly suggest that RARß2 is required for neurite
outgrowth and that its absence in the adult CNS explains, at least in part,
the lack of CNS regenerative capacity. Of course, the experiments were all
performed in vitro, and it is not necessarily the case that they are relevant
to the in vivo situation. With this major proviso, however, it is conceivable
that by introducing the RARß2 gene into the adult spinal cord it
would be possible to reawaken the regenerative potential of the CNS in
vivo.
It will be of great interest to discover the downstream targets of the
RARß2 gene and therefore the mechanism of genetic induction of
neurite outgrowth. It is possible that the targets concerned act purely within
the neuron itself to stimulate the formation and outgrowth of a growth cone
and ultimately stabilize the new neurite. However, this mechanism being only
concerned with events within the neuron itself ignores the data concerning the
growth-inhibiting properties of the glial scar and the inhibitory molecules
present in the CNS environment surrounding the axon (see Introduction), both
of which would serve to inhibit neurite regeneration. So it would be a much
more likely possibility that the targets of RARß2 in the neuron include
the induction of genes that signal to the oligodendrocyte to downregulate
molecules such as Nogo; the downregulation of genes encoding neuronal cell
surface receptors for inhibitory molecules such as the Nogo receptor
(Fournier et al., 2001); the
induction of genes encoding extracellular enzymes such as chondroitinase
(Bradbury et al., 2002
) or
perhaps matrix metalloproteases, which are known to be induced by RA.
It would also be of interest to examine the relationship between
RARß2-mediated induction of neurite outgrowth and the methods described
previously for inducing regeneration in vivo to see whether there are any
common mechanisms. For example, is RARß2 involved in neuronal responses
to olfactory ensheathing cells (Li et al.,
1997)? With regard to neurotrophin-induced regeneration, it is
known that NGF acts via the RA pathway in the adult PNS
(Corcoran and Maden, 1999
;
Cosgaya and Aranda, 2001
) as
NGF upregulates an enzyme that synthesizes RA, and this RA in turn upregulates
RARß2. Although we have shown that NGF does not upreguate
RARß2 in the adult CNS, it would be interesting to know whether
other neurotrophins could act via the RA pathway as a potential general
mechanism for neurotrophin action in neurite regeneration. In conclusion,
these data support a role for RARß2 in the regeneration of
neurites in the adult CNS in vitro and indicate that experiments involving in
vivo gene therapy with this transcript, perhaps in combination with other
treatments, would be worthwhile to test whether any functional recovery of the
injured spinal cord could be obtained.
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Acknowledgments |
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Footnotes |
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References |
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