Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole
Department of Biomedical Sciences, Iowa State University, Ames, Iowa, IA 50011, USA
* Author for correspondence (e-mail: rjmartin{at}iastate.edu)
Accepted 16 September 2002
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Summary |
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Key words: levamisole, kinase antagonist, H-7, KN-93, genistein, staurosporine, Ascaris summ, nematode, CaM kinase, tyrosine kinase, membrane potential response
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Introduction |
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We are interested in the mechanisms by which parasitic nematodes become
resistant to anthelmintic drugs. The mechanisms that underlie resistance may
be more complex in helminths than in viruses and bacteria, in part because of
the larger genome of these pathogens. We have focused our attention on the
drug levamisole, which acts as a selective agonist on a sub-set of nicotinic
acetylcholine receptors (nAChRs) in nematodes
(Robertson et al., 1999;
Richmond and Jorgensen, 1999
).
At therapeutic concentrations, levamisole produces depolarization and
contraction of nematode somatic muscle, which leads to paralysis and
elimination of the parasite without affecting the host nicotinic receptors. We
have observed levamisole-activated single-channel currents in
levamisole-sensitive (SENS) and levamisole-resistant (LEV-R) nematode isolates
(Robertson et al., 1999
). One
of the striking differences between resistant and sensitive nAChR receptors is
that, in LEV-R, the average probability of the channel being in the open
state, Popen, is some ten times less than in SENS, while
the average channel conductance is the same. We have also observed in SENS and
LEV-R isolates that the Popen for individual channels
varies dramatically between patches. For example, in the presence of 30
µmol l-1 levamisole, Popen values in SENS
varied between 0.090 and 0.003 at a holding potential of -50 mV, which is a
30-fold difference between the highest and lowest Popen
value. These observations give rise to the question of what causes the wide
variation in Popen values: could it be that opening of
nAChRs in nematodes is regulated?
The levamisole receptors of nematodes, like those in vertebrates, are
understood to be composed of five subunits that surround a central
non-selective cation pore. In Caenorhabditis elegans, the subunits
that have been described include UNC-38 (an -subunit), UNC-29 and LEV-1
(ß-subunits; Fleming et al.,
1997
). It is anticipated that other subunits are also involved in
forming nAChRs on nematode muscle. A common feature of all signal-transduction
proteins, including ion channels, is regulation of their activity by kinase
enzymes. The addition of a bulky phosphate group to a serine, threonine or
tyrosine residue in the protein introduces a highly charged group into a
region that was only moderately polar before and, in the case of an ion
channel, affects its opening. We have examined the amino acid sequence of the
-subunit of the Ascaris levamisole receptor, ASAR-1 (GenBank
Accession No. AJ011382), and have identified consensus phosphorylation sites
(Kennely and Krebs, 1991
) for
protein kinase C (PKC), protein kinase A (PKA), protein kinase G (PKG),
Ca2+/calmodulin-dependent (CaM) kinase and tyrosine kinase. These
sites are all on the cytoplasmic domain of the subunit between the
transmembrane regions TM3 and TM4 (Fig.
1A). Some of these sites are overlapping, suggesting that
differing kinases could produce the same effects. Phosphorylation of the
nAChRs in nematodes, as in mammalian receptors, is expected to lead to changes
in Popen (Colledge and
Froehner, 1997
; Hopfield et
al., 1988
; Reuhl et al.,
1992
) and may contribute to the variation in values we have
observed in single-channel experiments.
|
In experiments on the large parasitic nematode of swine, Ascaris
suum, Trim et al. (1999)
described inhibitory effects of tamoxifen on the membrane potential responses
to levamisole and acetylcholine and suggested the involvement of PKC in
modulating the activity of nAChRs. However, a more selective PKC antagonist,
chelerythine had no effect. In view of the difficulty in interpreting the
effects of tamoxifen, we have examined the effects of a number of other
protein kinase inhibitors on A. suum muscle, to test the hypothesis
that kinase activity is required to support the activity of nAChRs and is
involved in regulating the electrical response to the anthelmintic levamisole
and acetylcholine. We tested the effects of both levamisole and acetylcholine,
because they may select for a different subset of nAChRs in A. suum,
and found that responses to these compounds are labile and inhibited by CaM
kinase II antagonists and a tyrosine kinase antagonist. These observations
suggest that the responses to acetylcholine and levamisole are not fixed but
vary, and this plasticity depends on the phosphorylation state of the nAChRs.
They also suggest that resistance to the anthelmintic levamisole may involve
changes in the phosphorylation state of the receptor and may not require amino
acid changes at the drug's binding site. The work has relevance to the
understanding of mechanisms of anthelmintic resistance.
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Materials and methods |
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Preparation
A body-wall muscle-flap preparation was prepared from a 1 cm section, 3 cm
caudal to the head of adult Ascaris and pinned, cuticle-side down,
onto a SylgardTM-lined chamber, where the intestine was removed. The
preparation was microperfused continuously with Ascaris peri-enteric
fluid [APF: NaCl (23 mmol l-1), Na acetate (110 mmol
l-1), KCl (24 mmol l-1), CaCl2 (6 mmol
l-1), MgCl2 (5 mmol l-1), glucose (11 mmol
l-1) and Hepes (5 mmol l-1), pH 7.6 with NaOH]. In
experiments to test the effect of low-Ca2+, APF was prepared by
replacing Ca2+ in APF on an equimolar basis with Mg2+.
Application of the perfusate was via a fine microtube placed with a
micromanipulator (approximately 500 µm) over the muscle cell bag. The rate
of perfusion was 1.5 ml min-1 and this allowed rapid change of the
solution bathing the cell being recorded from. The temperature in the chamber
was maintained at 32-33°C.
Electrophysiology
A two-microelectrode current-clamp technique was used for measuring the
membrane potential and input conductance changes of the Ascaris
muscle cell bags. Micropipettes made from borosilicate glass (Clarke
Electromedical, Reading, UK) with resistances in the range of 20-40 M
when filled with 2 mol l-1 potassium acetate were used for
recording. Two microelectrodes were carefully inserted into one muscle cell
bag with minimum damage. An Axoclamp 2B amplifier, 1320A Digidata Interface,
pClamp 8.0 software (all from Axon Instruments, Union City, CA, USA) and
Pentium III PC were used to display, record and analyze the membrane potential
and injected current. One micropipette was used for recording of membrane
potential, while the second was used for injection of current pulses
(hyperpolarizing: 40 nA, 500 ms filtered at 0.3 kHz).
In the present study, we measured the first fast peak membrane potential
change response to acetylcholine or levamisole as the nicotinic response to
these drugs. In approximately one-third of the responses, we also observed a
second component or tail to the response (arrows,
Fig. 1B). This component has
not been described previously, perhaps because it is more labile than the
first component. It may have arisen as a secondary effect of entry of
Ca2+ following stimulation of the nicotinic receptors, or following
stimulation of metabotrophic (muscarinic) receptors
(Hwang et al., 1999). In the
present study, we focused exclusively on the effects of the kinase antagonists
on the peak of the first component because it results from activation of
extrasynaptic nicotinic receptors on the bag region of the muscle
(Martin, 1982
), and these
receptors are accessible to direct stimulation by perfused drugs and to
recording by standard two-electrode current-clamp technique.
Drug solutions
H-7, staurosporine, genistein and KN-93 were purchased from Calbiochem (San
Diego, CA, USA). Levamisole hydrochloride and acetylcholine chloride were
purchased from Sigma (St Louis, MO, USA). Stock solutions for staurosporine,
genistein and KN-93 were prepared in dimethyl sulfoxide (DMSO) and frozen for
use following dilution in APF. The maximum concentration of DMSO used (0.1%)
did not affect membrane potential or responses to levamisole and
acetylcholine. Stock solutions of H-7 and levamisole were prepared each week
in APF and stored at 4°C. Stock and working solutions of acetylcholine
were prepared freshly every day in APF.
Application of drugs
In all experiments, acetylcholine was added to the bag region of the cell
via the microcatheter in the perfusate for 20 s; levamisole was
applied for 10 s. Peak membrane potential changes and input conductance were
measured. All kinase antagonists were added to the preparation in the
perfusate for at least 2 min before the application of acetylcholine or
levamisole.
Statistics
All results are presented as means ± S.E.M. Significance was tested
using paired t-test (for antagonist influences) or one-way analysis
of variance (ANOVA; for influence of Ca2+ on the levamisole
concentrationresponse relationship). The influences of Ca2+
on the effects of increasing levamisole concentrations on membrane potential
were estimated using the Hill equation: % response =
1/(1+[EC50/XA]n), where
EC50 is the concentration of agonist (XA)
producing 50% of the maximum response, and n is the Hill coefficient
(slope). Prism Pad software (Version 3.0, San Diego, CA, USA) was used for all
statistical determinations.
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Results |
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Fig. 2A shows a
representative example of the effect of a 10 s superfusion of the bag region
of the muscle with 10 µmol l-1 levamisole. The first response
recorded was a 7 mV depolarization peak associated with a change in input
conductance from 2.26 µS to 2.86 µS. The membrane potential recovery
after the levamisole pulse was slow, with a t1/2 of 94 s.
Fig. 2B shows a representative
trace from another preparation of the effect of 20 s of 10 µmol
l-1 acetylcholine at a resting membrane potential of -23.8 mV. The
peak depolarization of the first response was 3.4 mV associated with a
conductance change of 0.31 µS. Note, however, that the recovery from
acetylcholine was faster (t1/2=13 s) than with levamisole.
The slower recovery time of levamisole, when compared with acetylcholine, was
a consistent feature, making the measurement of the effect of kinase
antagonists on levamisole responses more difficult to determine because of the
longer stable recording conditions that were required. Several factors may
have contributed to the slow recovery with levamisole, including the more
lipophilic nature of levamisole (Robertson
and Martin, 1993), its resistance to degradation by cholinesterase
(Eyre, 1970
) or its ability to
antagonize phosphatases (Schnieden,
1981
). However, we did not investigate this phenomenon
further.
|
Effect of H-7 on levamisole and acetylcholine responses
H-7 is a broad-spectrum serinethreonine kinase inhibitor with
activities against the AGC kinase group of PKA, PKC and PKG, and inhibitor
constants (Kis) in the range of 0.1-6.0 µmol
l-1 (Kawamoto and Hidaka,
1984; Boulis and Davis,
1990
; Hoffman and Newlands,
1991
; Li et al.,
1992
; Reuhl et al.,
1992
; Hu and Li,
1997
; Ku et al.,
1997
; Calbiochem 1998 data sheet CN: 371955). We superfused H-7 at
a concentration of 30 µmol l-1 over the bag region of the muscle
cell to determine the effect of H-7 on responses to 10 µmol l-1
levamisole and 10 µmol l-1 acetylcholine
(Figs 2A,B). The mean control
peak levamisole response was 7.6±2.0 mV. In the presence of 30 µmol
l-1 H-7, the mean response was 6.0±1.0 mV (N=5) or
79% of the control. The mean control peak acetylcholine response was
4.1±0.3 mV and, in the presence of 30 µmoll-1 H-7, it was
3.5±0.6 mV (N=5) or 85% of the control. H-7 did not induce a
statistically significant effect on the response to either compound
(Table 1). These observations
suggest that PKA, PKC and PKG do not have a modulatory effect on nematode
nAChRs.
|
Effect of staurosporine on levamisole and acetylcholine
responses
Staurosporine is a broad-spectrum kinase inhibitor with activities against
PKA, PKG, PKC, myosin light chain kinase (MLCK), CaM kinase and tyrosine
kinase and Kis in the range of 0.7-70 nmoll-1
(Bergstrand et al., 1992;
Coultrap et al., 1999
;
Li et al., 1992
;
Nishimura and Simpson, 1994
;
Calbiochem 1999 data sheet CN: 569397). It combines with the ATP-binding site
of the kinase enzyme to inhibit its function. We perfused 1
µmoll-1 staurosporine over the muscle bag to determine its
effect on responses to levamisole and acetylcholine.
Fig. 3A shows the inhibitory
effect of staurosporine on representative recordings of levamisole responses.
Table 1 shows that the mean
control peak levamisole depolarization was 6.8±1.9 mV (N=6),
and, in the presence of 1 µmoll-1 staurosporine, the mean peak
levamisole response was 3.9±1.1 mV (N=6) or 57% of the
control. The difference was statistically significant
(P<0.0001).
|
Fig. 3B shows the effect of staurosporine on representative recordings of acetylcholine responses. Table 1 shows that, as with levamisole, there was a depressant effect on the acetylcholine responses. The mean control acetylcholine response was 5.4±0.6 mV (N=7) and this was reduced in the presence of 1 µmoll-1 staurosporine to 2.8±0.3 mV (N=7) or to 51% of the control (P=0.0016).
As we observed a significant effect with the broad-spectrum kinase antagonist staurosporine, but not with H-7, we can suggest that the difference is due to the effect of staurosporine on a kinase that is not affected by H-7. One of the protein kinase enzymes not affected by H-7, but affected by staurosporine, is CaM kinase II. A selective CaM kinase II antagonist was therefore used to investigate the role of this enzyme.
Effect of KN-93 on levamisole and acetylcholine responses
To follow up the above studies, we tested KN-93, a selective CaM kinase II
inhibitor with a Ki of 0.4 µmoll-1
(Mamiya et al., 1993;
Ishida et al., 1995
;
Anderson et al., 1998
;
Ledoux et al., 1999
;
Nakayama et al., 2001
;
Calbiochem 2001 data sheet CN: 422708) on levamisole and acetylcholine
responses (Fig. 4). 10
µmoll-1 KN-93 reduced the control mean peak levamisole response
from 6.2±1.0 mV (N=8) to 2.7±0.3 mV (N=8) or
44% of the control (Table 1;
P=0.035). An inhibitory effect on the acetylcholine responses was
also observed; the control response of 4.7±0.6 mV (N=9) was
reduced to 2.0±0.4 mV (N=9) by 10 µmoll-1 KN-93
(Table 1, P=0.0004).
The effect of KN-93 on levamisole and acetylcholine responses suggests that
CaM kinase II is involved in phosphorylating nAChRs on the muscle bag and
promoting the opening of the ion-channel receptor.
|
Reversal on washing
We tried routinely to wash off the effects of the kinase inhibitors, but
reversal of the effects of the antagonists was not always apparent. This was
probably due to the lipophilic nature of the antagonists and the large size of
the bag muscle region. However, we did occasionally observe reversal with
staurosporine and KN-93 when we used acetylcholine as an agonist.
Fig. 3B illustrates the
reversal on washing with acetylcholine as the agonist and staurosporine as the
kinase antagonist. Fig. 4B
illustrates reversal of the effect of KN-93 on washing: here, 20 min was
required to observe definitive recovery towards the control response
values.
Levamisole responses are sensitive to Ca2+
An effect of CaM kinase II in modulating levamisole receptors implies that
the response will also be sensitive to Ca2+. We tested the effect
of levamisole at different concentrations in preparations bathed for longer
than 30 min in low-Ca2+ APF and in normal Ca2+ APF.
Elimination of Ca2+ from the bathing solution is known to reduce
cytoplasmic Ca2+ concentrations because low-Ca2+
extracellular solutions reduce the opening of Ca-activated Cl-channels
(Thorn and Martin, 1987). In
normal Ca2+ APF, the resting membrane potential was
-22.9±3.5 mV and input conductance was 2.63±0.38 µS
(N=4). These values are similar to those recorded in
low-Ca2+ APF, where membrane potential was -21.7±0.8 mV and
input conductance was 2.16±0.12 µS (N=4, P=0.11,
t-test).
Fig. 5 shows the effect of different concentrations of levamisole on a preparation bathed in normal Ca2+ APF Fig. 5A) and one bathed in low-Ca2+ APF solution (Fig. 5B). Notice that the responses to levamisole at lower concentrations are bigger in the presence of Ca2+. Fig. 5C shows a plot of the mean (N=4) peak depolarizations in the presence and absence of Ca2+. The best fit to the observed responses in the presence of Ca2+ had an EC50 of 1.2 µmoll-1, with a Hill slope of 1.21 and a maximum response of 7.1 mV. In low-Ca2+ APF, the best-fit EC50 was 3.1 µmoll-1, the Hill slope was 3.9 and the maximum response was 6.8 mV. Note again that the presence of Ca2+ is associated with an increase in the size of the response (P=0.045, ANOVA).
|
Effect of genistein
We pointed out earlier that staurosporine, but not H-7, has significant
effects on the amplitude of the response to levamisole and acetylcholine. As
staurosporine at higher concentrations may also antagonize the effects of
tyrosine kinases, and there are consensus tyrosine kinase sites present on the
ASAR-1 -subunit of A. suum
(Fig. 1), we tested the effects
of genistein on the levamisole and acetylcholine response. Genistein competes
with ATP non-competitively, as a substrate for tyrosine kinases, to inhibit
phosphorylation, with Kis of
1 µmoll-1
(Huang et al., 1992
;
Young et al., 1993
;
Wang et al., 1997
;
Saxena and Henderson, 1997
;
Neye and Verspohl, 1998
;
Calbiochem 2001 data sheet, CN: 345834). We tested the effect of 90
µmoll-1 genistein on the levamisole and acetylcholine responses
(Fig. 6).
|
Genistein reduced the amplitude of the levamisole response (Fig. 6). Table 1 shows that the mean control peak levamisole response was 6.4±0.7 mV (N=8), and the mean response in the presence of 90 µmoll-1 genistein was 3.3±0.3 mV (N=8) or 51% of the control (P=0.001).
Fig. 6B shows a representative trace of the effect of 90 µmol l-1 genistein on the response to acetylcholine. The effect was small (Table 1), with the mean control acetylcholine peak response reaching 5.6±0.8 mV (N=6) and the acetylcholine response in the presence of 90 µmol l-1 genistein reaching 5.0±0.6 mV (N=6) or 89% of the control. Although the change in response was small, it did reach statistical significance (P=0.03). The effect of genistein on the levamisole and acetylcholine responses suggests that tyrosine kinase is involved in maintaining the nematode nicotinic receptor.
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Discussion |
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Development of resistance to levamisole therapy in parasitic nematodes has
not been linked to changes in amino acid sequences of nAChR subunits
(Wiley et al., 1997;
Hoekstra et al., 1997
). In
T. colubriformis, levamisole resistance has a recessive, sex-linked
single gene component and a small polygenic component on autosomes. In H.
contortus, resistance is polygenic and associated with a 4-25-fold
increase in the concentration of levamisole required to produce paralysis
(Sangster et al., 1991
,
1998
).
Role for CaM kinase II and tyrosine kinase in modulation of the
nAChRs
In the present study, we examined the effects of selected kinase
antagonists to test the hypothesis that phosphorylation of nAChRs is involved
in regulating the response to levamisole. We observed no significant effects
on levamisole and acetylcholine responses with H-7, an antagonist of PKA, PKC
and PKG (Kawamoto and Hidaka,
1984; Boulis and Davis,
1990
; Hoffman and Newlands,
1991
; Li et al.,
1992
; Reuhl et al.,
1992
; Hu and Li,
1997
; Ku et al.,
1997
). The broad-spectrum antagonist staurosporine, which inhibits
PKA, PKC, PKG, CaM kinase and tyrosine kinase
(Bergstrand et al., 1992
;
Coultrap et al., 1999
;
Li et al., 1992
;
Nishimura and Simpson, 1994
),
reduced the response to both acetylcholine and levamisole in A. suum
muscle. Reductions in response were also recorded in the presence of KN-93, a
selective CaM kinase II antagonist (Mamiya
et al., 1993
; Ishida et al.,
1995
; Anderson et al.,
1998
; Ledoux et al.,
1999
; Nakayama et al.,
2001
), and genistein, a selective tyrosine kinase antagonist
(Huang et al., 1992
;
Young et al., 1993
;
Wang et al., 1997
;
Saxena and Henderson, 1997
;
Neye and Verspohl, 1998
).
These effects suggest the involvement of CaM kinase II and tyrosine kinase in
supporting the opening of the ion channels. The lack of effect of H-7, a
protein kinase antagonist that is not active on CaM kinase II or tyrosine
kinase, and the antagonistic effects of staurosporine, a broad-spectrum
protein kinase antagonist, support this interpretation. The potentiating
effects of Ca2+ on the levamisole responses may be explained by the
involvement of CaM kinase II. Observations with H-7 and staurosporine, which
depress muscle contraction to acetylcholine in the filarid
Acanthocheilonema viteae (Minardi
et al., 1995
), are consistent with the view that CaM kinase II
regulates the response to acetylcholine in parasitic nematodes.
Trim et al. (1999) have
observed inhibitory effects of tamoxifen on the depolarizing responses to
levamisole and acetylcholine and suggested that a PKC-dependent
phosphorylation of A. suum nAChRs is necessary to maintain the
sensitivity of the receptor to acetylcholine. In this model, blockade of PKC
results in inhibition and reduction of the response. However, Trim et al.
(1999
) also tested the
selective PKC antagonist chelerythrine and did not observe an effect on the
membrane-potential responses to acetylcholine. In our experiments, we did not
observe a significant effect with H-7, another kinase antagonist that has
inhibitory effects on PKC. As tamoxifen also has an inhibitory effect on
calmodulin (Edwards, 2002), we can explain the inhibitory effects of tamoxifen
by inhibition of calmodulin and depression of the activity of CaM kinase II. A
role for CaM kinase II in regulating nematode body wall muscle activity has
been demonstrated in C. elegans, where a single gene,
unc-43, codes for CaM kinase II
(Reiner et al., 1999
). The
unc-43 gene was also shown to interact with unc-103, a gene
encoding a voltage-gated K+ channel, so a clear connection between
phosphorylation of ion channels, muscle excitability and CaM kinase II in
nematodes has been demonstrated. CaM kinase II may mediate cell excitability
through modulation of channel activity or modeling of the synapse, as
demonstrated in the vertebrate central nervous system
(Rongo and Kaplan, 1999
). We
do not rule out the possibility of PKC involvement, but our evidence with H-7
suggests that, over the time-span of the experiment (approximately 3 h), it is
less important than CaM kinase II. Over a longer time span (12-24 h), where
receptor synthesis can play a role, evidence from C. elegans does
indicate a role for PKC (tpa-1) in long-term adaptation of nAChRs
(Waggoner et al., 2000
). In
mammalian muscle, PKC and CaM kinase II can regulate nAChR expression, but it
is CaM kinase II rather than PKC that is required for Ca2+ or
activity-dependent control of nAChR gene expression
(Macpherson et al., 2002
).
In summary, our studies indicate that CaM kinase II and tyrosine kinases are involved in supporting the opening of acetylcholine/levamisole receptors on A. suum somatic muscle. In previous publications, we have observed differences in opening of nAChRs in SENS and LEV-R isolates of the parasitic nematode Oesophagostomum dentatum and hypothesized that phosphorylation may contribute to the drug-resistant phenotype by modulating the opening of levamisole receptors. In the present study, we have used selective kinase antagonists to test the importance of the kinases and phosphorylation process in modulating the receptors. These observations demonstrate that the responses to acetylcholine and the anthelmintic levamisole are not fixed but vary, and this plasticity depends on the phosphorylation state of the nAChRs. The observations emphasize the possibility that resistance to the anthelmintic levamisole does not necessarily involve amino acid changes at the levamisole-binding site but may involve regulation of the activity to the levamisole receptor via changes in its phosphorylation state.
The mechanisms used by nematodes to modulate their responses to acetylcholine and nicotinic anthelmintics is worthy of further study; it seems increasingly likely that modulation of the receptors may contribute to at least some of the observed drug resistance. Further study necessitates the use of Ascaris because it is the only readily available nematode parasite suitable for electrophysiological study. To date, there are no anthelmintic-resistant isolates of Ascaris, but information derived from Ascaris will inform work on parasitic nematodes where anthelmintic isolates are available.
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Acknowledgments |
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