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Introduction
The hepatitis B virus (HBV) is regarded as one of the
most fatal human pathogens, with an estimated 350 million
individuals chronically infected worldwide and approximately
one million deaths annually. Many chronically-infected
patients gradually develop severe liver cirrhosis that may
eventually progress to hepatocellular carcinoma (HCC). It has
been reported that chronic HBV infection is associated with
a 100-fold-higher risk of developing
HCC[1], thus the HBV has been classified by the International Agency for Research
on Cancer as "carcinogenic to humans". In order to reduce
the morbidity and mortality of the HBV-related disease, it is
of utmost importance to understand the mechanism of HBV
replication in order to identify novel targets for anti-HBV
therapeutic intervention.
The HBV is a DNA virus that undergoes reverse
transcription during its replication cycle. It has a unique
double-stranded, circular DNA genome that is capsuled within the
viral capsid and enveloped[2]. Four open reading frames,
including the viral envelope, core, reverse
transcriptase-polymerase, and the hepatitis B X (HBx) gene were encoded
by the viral genome. The product of the X gene, termed the
HBx, protein is multifunctional. In HBV replication, the HBx
protein interacts with the mitochondrial permeability
transition pore (MPTP), causing its opening and subsequent
outflow of intra-mitochondrial calcium into the cytoplasm,
followed by the activation of the cytoplasmic,
calcium-dependent, proline-rich tyrosine kinase-2 and downstream
Src kinase pathway to promote HBV replication. HBV
replication can be significantly inhibited by MPTP blockers,
cytosolic calcium chelators, and Src inhibitors, and can be
rescued by calcium-mobilizing
regents[3_5]. The HepG2.2.15 cell line, a HepG2 human hepatoma cell line derivative which was
stably transfected with a plasmid containing 2 head-to-tail
dimers of the HBV genome, can not only release high levels
of hepatitis B surface antigens (HBsAg) and hepatitis B e
antigens (HBeAg) into supernatants, but also supports the
assembly and secretion of replicative intermediates of HBV
DNA and Dane particles during
culture[6,7]. This provides us with a useful
in vitro model to study the impact on HBV replication by various reagents, as well as the mechanisms
that may be involved.
Recent studies have demonstrated that cyclosporine A
(CsA) can inhibit HBV replication in
vitro[4,5]. It is hypothesized that CsA can predominantly bind to mitochondrial
cyclophilin, inhibit the MPTP opening, and disrupt the
mitochondrial calcium signaling to inhibit HBV
replication[8]. CsA targets the mitochondria, which is located upstream in the
cellular signaling pathways and influence HBV replication.
In the current study, we examined the effect of CsA on HBV
replication in vitro and the mechanisms involved. In addition,
a proteomic analysis was used to assess the large-scale
protein expression profile of HepG2.2.15 cells after CsA
treatment in order to identify some key proteins that may play
critical roles in HBV replication and also discover some
potential targets for novel anti-HBV agents.
Materials and methods
Cell culture HepG2.2.15 cells were cultured in
RPMI-1640 medium (Hyclone, Logan, USA) supplemented with 10%
fetal bovine serum (FBS; Gibco, Carlsbad, CA,USA) and 200
mg/L G418 (Sigma, St Louis, MO, USA) at an atmosphere of
5% CO2 at 37 °C. Subconfluent monolayer HepG2.2.15 cells
were harvested from the culture dishes by trypsin treatment
and then plated onto 6-well flat bottom plates at a density of
1×106 cells per well. 24 h later, the cells were treated by CsA
at different concentrations (0, 0.6, 1.3, 2.5, 5.0, and 10.0
µg/mL). The culture supernatants were collected every 24 h
for ELISA analysis, and fresh mediums containing CsA at
the indicated concentration were added to the culture. After
CsA treatment for 4 d, the cells were harvested and
intracellular DNA was isolated for slot blot hybridization using the
DNA extraction kit (QIAGEN, Hilden, Germany) according to
the manufacturer's instructions.
MTT analysis of cytotoxicity A methyl thiazolyl
tetrazolium (MTT) test was used to evaluate the cytotoxicity of
CsA. HepG2.2.15 cells were first cultured in a 96-well
microplate (1×104 cells/well) in 100 µL complete RPMI-1640
for 12 h. The cells were then treated with the indicated
concentration of CsA in FBS-free Minimum Essential medium
(MEM) for 96 h. By the end of the incubation, 25 µL MTT (5
mg/mL) was added to each well and incubation was allowed
to continue for another 4 h. Finally, 100 µL DMSO was added
to each well. The plate was read using a microplate reader
(Bio-Rad, Hercules, CA, USA) at a wavelength of 570 nm.
ELISA analysis of HBsAg and HBeAg The HBsAg and
HBeAg levels in the supernatant were detected by an ELISA
detecting kit (Abbott, Abbot Park, IL, USA) according to the
manufacturer's instructions. The suppression rate of the
expression of HBsAg and HBeAg in the CsA-treated groups
was calculated using the following formula:
Suppression rate=[A value (control)/A value (negative
control) A value (CsA)/A value (negative
control)]/[A value (control)/A value (negative control) 2.1]
Slot blot hybridization The HBV DNA replication level
was evaluated by slot blot hybridization. Briefly, 5 µg
extracted DNA was boiled to denature for 10 min and filtered
with a Hoefer slot blot apparatus (San Francisco, CA, USA)
on a nylon membrane according to the supplier's protocol.
For the HBV DNA probes preparation, cloned HBV
complementary DNA and β-actin complementary DNA were
amplified by PCR. The following primers were used in this study:
HBV probe, forward: 5'-AGACTCGTGGTGGACTTCTCT-3', reverse: 5'-GGGTTCAAATGTATACCCAAAGAC-3';
b-
actin probe, forward: 5'-CGCTGCGCTGGTCGTCGACA-3', and reverse: 5'-GTCACGCACGATTTCCGGCT-3'. The
amplified probes were purified by electrophoresis from
agarose gels and labeled with digoxigenin-11-dUTP using the
DIG high prime kit (Roche, Mannheim, Germany).
Prehy-bridization, hybridization, washes after hybridization, and
antidigo-xigenin-alkaline phosphatase (AP) incubation were
performed as described by the manufacturers. After the
membrane was visualized by chemiluminescent substrate CSPD
solution with Kodak X-ray film (Kodak, Rochester, NY, USA),
the same membrane was rehybridized with the other probes
and exposed again. The slot blot result was quantitated by
densitometry using Kodak 1D 3.6 software and the HBV DNA
signal was normalized to β-actin.
2-D electrophoresis (2-DE) The cells were treated with
CsA at a concentration of 5 µg/mL for 4 d and harvested by
centrifugation for the protein sample preparation. Lysis buffer
containing 7 mol/L urea, 2 mol/L thiourea, 4%
(3-[(3-cholami-dopropyl)dimethyl-ammonio]-1-propanesulfonate) (CHAPS),
1% DTT, 2% immobilized pH gradient (IPG) buffer (pH 3~10
liner, Amersham Biosciences,Uppsala, Sweden), and a
protease inhibitor cocktail (Complete tablets, Roche,
Mannheim,Germany) were mixed with the harvested cells and incubated
for 20 min at room temperature. Nucleic acids were removed
by centrifugation at 50 000×g for 30 min. 2-D electrophoresis
(2-DE;Amersham Biosciences,Uppsala,Sweden). was
performed according to the manufacturer's instructions. In brief,
the samples containing the lysis buffer were mixed with the
rehydration solution [8 mol/L urea, 2% CHAPS, 20 mmol/L
DTT, 0.5% IPG buffer (pH 3~10 non-linear), and 0.002%
bromophenol blue] to a total volume of 450 µL, and subjected to
Isoelectric focusing (IEF) on an IPG strip. Rehydration and
IEF were carried out on the Amersham Biosciences IPGphor
as follows: rehydation with 30 V for 12 h, 500 V for 1 h, 1000 V
for 1 h, 1000_8000 V for 30 min, and 8000 V was applied until
the total volt hours reached 72.0 kVh at 20 °C. After IEF
separation, the strips were equilibrated for 15 min in SDS
equilibration buffer (50 mmol/L Tris-HCl, 6 mol/L urea, 30%
glycerol, 2% SDS, and 0.002% bromophenol blue)
containing 1% DTT, followed by SDS equilibration buffer
containing 2.5% iodoacetamide for another 15 min. After
equili-bration, the strips were loaded onto vertical SDS-PAGE
(12.5% T constant). The 2-D SDS electrophoresis was run
using the Ettan DALTsix electrophoresis unit (Amersham
Biosciences,Uppsala,Sweden). Three independent
experiments were carried out in this study. The experiments of
each treatment group (control or CsA-treated) were repeated
3 times.
Silver staining and image analysis Silver staining was
done according to the protocol of Yan et
al[9] with minor modifications (substitution of ethanol for methanol in
fixation). All chemicals used in silver staining were purchased
from Sigma (USA). Silver-stained gels were scanned using
an ImageScanner (Amersham Biosciences,Uppsala,Sweden)
and analyzed with the ImageMaster 2D Elite software. Six
gels were divided into 2 groups, 3 for the controls and 3 for
the CsA-treated cells. Image spots were initially detected;
the background was subtracted, followed by volume normalization, and spots matched by manual assistance.
Three gel images of the control group were averaged and set
as the reference gel. The protein spots were quantitated
based upon their relative volume. Only the spots with
expression levels greater than 2-fold were considered
significant and selected for the mass spectrometry analysis.
In-gel digestion, matrix-assisted laser
desorption/ionization-time of flight mass spectrometry analysis and
protein identification The protein spots were excised from
different gels and transferred into Eppendorf tubes (Gilson,
Villiers, Le Bel, France). Each spot was washed twice in
milli-Q water, destained by washing with a 1:1 solution of 30
mmol/L potassium ferricyanide and 100 mmol/L sodium thiosulfate,
and equilibrated in 200 mmol/L ammonium bicarbonate for 20
min to a pH of 8.0. The gels were then washed twice in
milli-Q water, dehydrated by the addition of acetonitrile, and dried
in a SpeedVac (Thermo Savant, Holbrook, NY, USA).
Subse-quently, the gels were rehydrated in 10 µL proteomics grade
trypsin (Sigma, St Louis, MO, USA) solution (20 ng/µL in 40
mmol/L NH4HCO3 in 9% acetonitrile, ACN) and incubated at
37 °C for 10 h. The peptides were extracted by adding 50 µL
of solution containing 50% ACN and 5% trifluoroacetic acid
(TFA). The extracted solutions were dried in a lyophilizer
and reconstituted in 5 µL 0.1% TFA solution (Virtis, Gardiner,
NY, USA).
One µL of peptide mixture was mixed with an equal
volume of 10 mg/mL alpha-Cyano-4-hydroxycinnamic acid
(Sigma, USA) saturated with 50% ACN in 0.05% TFA and
analyzed with a Voyager-DE STR matrix-assisted laser
desorption/ionization-time of flight mass
spectrometry (MALDI-TOF MS) using a delayed ion extraction and ion mirror
reflector mass spectrometer (Applied Biosystems, Foster
City, CA, USA). The instrument setting was set at reflector
mode with a 160 ns delay extraction time, positive polarity,
63.5% grid voltage, and 20 000 V accelerating voltage. Laser
shots at 200 per spectrum were used to acquire the spectra
with mass range from 1000~4000 Da. External calibration
was carried out using insulin chain B oxidized and P14R
(Sigma, St Louis, MO, USA) and the internal calibration was
performed using the autolytic peaks of trypsin.
For the interpretation of the mass spectra data, the
monoisotopic peptide masses were input into Mascot
(http://www.Matrixscience.com). A database search (NCBInr)
was performed using the following words: homo
sapiens/human, trypsin digest (we allowed for up to 1 missed
cleavage), cysteines modified by carbamidomethylation, and
100 ppm mass tolerance using internal calibration.
Western blot analysis After SDS-PAGE, the gels were
transferred electrophoretically onto a nitrocellulose
membrane (Amersham Biosciences,Uppsala, Sweden) and
blocked for 2 h with Tris-buffered saline (TBS) containing
5% skim milk. The primary antibody used was goat
anti-human eukaryotic translation initiation
factor (eIF)-5A (Santa Cruz, CA, USA; 1:500 dilution). The membranes were
incubated with the primary antibody overnight at 4 °C, washed 3
times with TBS containing 0.1% Tween-20, and then the
membrane was incubated with horseradish
peroxidase-conjugated anti-goat secondary antibody
(Chemicom, Temecula, CA, USA) 1:2000 for 1 h at room temperature, and developed
with a chemiluminescence reagent (EZ-ECL
chemiluminescence detection kit, Beit-Haemek, Israel). Detecting actin
(Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) acted
as the loading control. Images were acquired on an image
station 2000R (Kodak, Rochester, NY,USA) and analyzed
with the software supplied by the manufacturer.
Statistics The data of the ELISA analysis and slot blot
hybridization were expressed as mean±SD. One-way ANOVA
of Dunnett's test was used to analyze the significance
between the control and the CsA-treated groups A bivariate
correlate analysis was used to analyze the correlation
between the CsA concentrations and HBV replication levels.
The statistical significance in terms of the expression
profiles of HepG2.2.15 cells with or without CsA treatment was
estimated by the Student's t-test. All calculations were done
with SPSS 11.5 software (SPSS, Chicago, IL, USA).
P<0.05 was considered statistically significant.
Results
No apparent cytotoxicity of CsA on the HepG2.2.15 cell
line at low concentrations Before we studied the effect of
CsA on HBV replication in vitro, we first wanted to
determine whether CsA had direct toxicity on the HepG2.2.15 cell
line. The cells were incubated with CsA at graded
concentrations of 0 (vehicle control), 0.25, 0.5, 1, 2, 4, 8, 16, 20, 40,
80, and 100 µg/mL. The cytotoxicity of CsA on HepG2.2.15
cells was evaluated by MTT test. As shown in Figure 1,
HepG2.2.15 remained viable in the presence of 0_80 µg/mL
CsA treatment. When the final concentration of CsA was
increased to 100 µg/mL, there was a mild decrease in the
optical density value, indicating that CsA causes little
toxicity on HepG2.2.15 cells at low concentrations (<80 µg/mL),
but has a direct toxic effect with a concentration higher than
100 µg/mL. Thus, we used low concentrations of CsA to
study its effect on HBV replication using this in
vitro model system.
CsA inhibits the expression of HBsAg and
HBeAg To investigate the effect of CsA on the production of HBeAg
and HBeAb by HepG2.2.15 cells, the supernatant was
collected after treatment with different concentrations of CsA
at different times, and the titer of HBsAg and HBeAg was
determined by ELISA. Our results showed that CsA could
inhibit the expression of HBsAg and HBeAg in a
dose-dependent manner; a further bivariate correlation analysis
showed positive correlation between the suppression rates
of viral antigens and CsA concentration. After the CsA
treatment, the correlation index was 0.401, 0.816, 0.940, and
0.938, respectively, for HBsAg, and the P-value was 0.058,
0.000, 0.000, and 0.000, respectively; the correlation index
was 0.405, 0.983, 0.963, and 0.979, respectively, for HBeAg,
and the P-value was 0.069, 0.000, 0.000, and 0.000,
respectively (Figure 2). These results indicate that CsA is able to
dose-dependently inhibit HBsAg and HBeAg production
in vitro.
CsA inhibits HBV DNA replication
We were also interested in whether CsA has a direct effect on HBV DNA
replication in vitro. After treatment with CsA at different
concentrations for 4 d, the HepG2.2.15 cells were harvested and
intracellular DNA was extracted for the slot blot
hybridization to evaluate the HBV DNA replication level. As shown in
Figure 3, CsA significantly inhibited the HBV DNA replication. The replication levels in 2.5, 5.0, and 10.0 µg/mL
CsA-treated groups were significantly low compared to the
control groups (P=0.020, 0.014, and 0.048, respectively), and
there was a negative correlation between the HBV DNA
replication level and the different concentrations of CsA
(correlation index -0.770, P=0.000).
Differential analysis of 2-DE protein profiles in the
control and CsA-treated HepG2.2.15 cells To further
investigate the alteration of the protein expression in HepG2.2.15
after CsA treatment in vitro, protein lysates from the control
and CsA-treated HepG2.2.15 cells were subjected to 2-DE.
Following silver staining, the gels were digitized prior to
computer-based matching and quantitative analysis with image
analysis software. We found that the intensity of 17 protein
spots were altered after CsA treatment, eleven of which were
identified. Among them, 6 proteins were upregulated, 4 were
downregulated, and 1 was shifted (Figure 4).
Identification of proteins by MALDI-TOF MS and
database searching In order to identify key proteins that may
play a critical role in HBV replication and also to explore
some potential targets for novel anti-HBV agents, the 17
protein spots with altered intensity as a result of CsA
treatment were excised from the gels, digested with trypsin, and
subsequently analyzed by MALDI-TOF MS using the
MS-Fit software to search the nrNCBI database. Eleven of the
protein spots were identified. Among them, 6 were
upregul-ated, 4 were downregulated, and 1 was shifted after
treatment (Table 1). The identified proteins represented a
heterogeneous group that included several important molecules
relevant to gene transcription regulation, such as eIF,
proteins involved in signal transduction such as the 14.3.3
protein (Figure 5), stathmin 1/oncoprotein 18, and
ubiquitin-specific protease otubain 1. The functions of the remaining
proteins are still not clear.
Detection of identified proteins by Western blot
analysis From the identified candidates, eIF-5A was detected by
Western blot analysis. The results are shown in Figure 6.
The expression changes of eIF-5A were basically consistent
with the 2-DE results. eIF-5A was upregulated in 5 µg/mL
CsA treatment (P=0.008).
Discussion
In our study, an ELISA analysis was used to evaluate the
expression of HBsAg and HBeAg in the supernatants of
HepG2.2.15 cells treated by CsA, while a slot blot analysis of
HBV DNA was performed to evaluate the HBV replication
level. As mentioned earlier, the expression level of HBsAg,
HBeAg, as well as HBV DNA replication dramatically
decreased in the presence of CsA in a dose-dependent manner,
suggesting that CsA was able to significantly inhibit HBV
replication in this in vitro culture system.
HBV replication is a multi-step process requiring
fine-tuning of numerous viral and cellular proteins. During
replication, the viral HBx protein can mobilize the
intra-mitochondrial calcium into the cytoplasm, activate intracellular
calcium signaling, followed by the activation of the Src
kinase and downstream unknown signal transduction messengers, thus promoting HBV DNA
replication[3,4]. The specific blocker of MPTP, CsA, can impair the interaction of
the HBx protein with the
mitochondria[8], inhibit subsequent MPTP openings, and block the intracellular calcium
signaling pathway[10,11]. Based on the fact that CsA is involved in
the interaction between HBx and the mitochondria, a key
point lies upstream of the HBV replicative signaling pathway.
The different protein expression profiles between the
CsA-treated and untreated groups may provide some clues to
probe the mechanisms of the inhibitory action of CsA on
HBV replication, and possibly identify some key factors
involved in HBV replication and novel targets for anti-HBV
therapy.Among these proteins with altered expression
levels after CsA treatment, 11 protein spots were identified with
high confidence by peptide mass fingerprinting. The
remaining proteins failed to be identified mainly because the
low protein abundance was not enough to produce a good
signal to the noise spectrum, or the result scores of the
database search was too low to achieve significant results.
Among the identified proteins, we were particularly
interested in protein kinase 14-3-3, signal transduction
messenger stathmin 1/oncoprotein 18, ubiquitin-specific protease
otubain 1, and eIF.
14-3-3 kinases are highly conserved, ubiquitously
expressed[12], and multifunctional in controlling intracellular
signaling pathways via binding to various ligands such as
the Bad protein and p53 in
apoptosis[13,14], Cdc 25 and telomerase in cell life
cycle [15,16] . In virology studies, it has
been identified that 14-3-3 facilitates the complex formation
of the Vpr protein of HIV-1 with Cdc25 to regulate the host
cell life cycle[17]. In addition, the HCV core protein can
enhance Raf-1 kinase activity via interaction with 14-3-3 to
regulate hepatocyte growth[18].
Analyses of the HBx protein sequence reveal that HBx contains a 14-3-3 binding do-
main[19,20]. This binding is essential for the formation of a
complex composed of Mitogen-activated protein kinase
kinase 1(MEKK1), stress kinase 1 (SEK1), stress-activated
protein kinases (SAPK), 14-3-3 proteins and HBx, which can
subsequently upregulate SAPK
activity[21]. CsA not only blocks the activation of the MEKK1-SAPK
pathway[22], but also interferes in the intracellular calcium signaling pathway
which is essential for the interaction between 14-3-3
proteins and their partners[23]. Since calcium signaling is
involved not only in the HBx activation of HBV replication,
but also in the mechanisms of the inhibition of CsA on HBV
replication and the interaction of 14-3-3 kinases with their
partners, we speculate that 14-3-3 may play a pivotal role in
HBV replication and become a potential target for new
anti-HBV intervention. Stathmin 1/oncoprotein 18 has dual
meanings: "oncoprotein 18" means that it is highly expressed
in numerous malignant tumors[24_27], and "stathmin 1" is
derived from the Greek word "relay", indicating its intermediate
role in signal transduction. As an intracellular relay for
signal transduction, stathmin 1/oncoprotein18 is the target for
numerous cellular kinases, such as the mitogen-activated
protein kinase family members including extracellular
signal-regulated kinase (ERK) and p38 and the
Ca++/calmodulin dependent kinase, all of which be regulated in response to
intracellular calcium signaling. Both of p38 and the
Ca++/calmodulin dependent kinase can also be regulated by
CsA[22], and the latter can be regulated by 14-3-3
kinases[28]. Thus, we hypothesize that the stathmin 1/oncoprotein 18
may play an important role in the anti-HBV action of CsA via
interaction with 14-3-3 and the calcium signaling
pathway.The ubiquitin-specific protease otubain 1 was also over-
expressed after CsA treatment. Otubain 1 belongs to the
deubiquitylating enzyme (DUB)
family[31]. Like protein phosphorylation, protein ubiquitylation is critical for diverse
biological processes, including signal
transduction[29], and the protein ubiquitylation process can be reversed by a group
of proteases known as DUB[30]. Recent studies have
indicated that otubain 1 and ubiquitin are both involved in the
T-cell activation process[32,33],
and that ubiquitin can slightly bind to CsA as minor
immunophilin[34]. So we believe there is complicated interaction between CsA, otubain 1, and
ubiquitin in T-cell priming stage, but whether similar
interaction exists in HBV replication remains unknown and needs
to be further studied.
eIF are essential for the formation of a ribosomal
initiation complex during the process of translation
initiation[35]. eIF2 has been reported to mediate the host cells' antiviral
response during viral infection[36]. eIF5 is a shuttle protein
mediating unspliced and incompletely-spliced RNA from the
nucleus to the cytoplasm during reverse transcription, a
process essential for some viruses, including
HBV[37_39]. In our study, as the members of the eIF protein family, eIF2, eIF3,
and eIF5 were overexpressed in 2-D maps and identified by
MALDI-TOF MS. Western blotting revealed the consistent
result by detecting eIF-5A (Figure 6), suggesting that these
factors may participate in the cellular antiviral response;
however the molecular mechanism concerning their exact role
in HBV replication and the relevance to anti-HBV activity of
CsA remains to be elucidated.
The translationally-controlled tumor protein (TCTP) is a
highly-conserved protein that is ubiquitously expressed in
all eukaryotic organisms[40]. This protein plays an important
role in cell growth, cell cycle progression, malignant
trans-formation, and anti-apoptosis activity. TCTP synthesis can
be rapidly regulated by calcium stress, growth signals,
certain cytokines, and pro-apoptotic signals; this regulation
was done at both the transcriptional and translational
levels[41]. In addition, 3 isoforms of TCTP have been detected,
which might be caused by posttranslational modifica-
tions[42]. In our study, the location of TCTP was shifted after
CsA treatment, We hypothesize that this displacement may
be caused by TCTP isoform formation because CsA can
interfere with the intracellular calcium signal by interacting
with the mitochondria as described earlier.
In conclusion, we have found that CsA was able to
significantly inhibit HBV replication in
vitro. We also identified a group of proteins with altered expression profiles in
HepG2.2.15 cells after CsA treatment. These proteins may
provide new insights to the molecular events of HBV
replication and the inhibitory effect of CsA on HBV. However,
more work needs to be done in order to evaluate the detailed
function of particular proteins in HBV replication, as well as
the potential of these proteins as novel targets for anti-HBV
therapy in the future.
Acknowledgement
We thank Dr Ran TAO (Transplant Immunology,
Department of Pathology and Laboratory Medicine, Children's
Hospital of Philadelphia and University of Pennsylvania,
Philadelphia, PA, USA) for critical reading of the manuscript
and helpful discussion.
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