1 Life Sciences Division, NASA Ames Research Center, Moffett Field, California 94035-1000; and 2 National Research Center for Hematology, Moscow, 125167 Russia
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ABSTRACT |
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To evaluate the
relationship between osteoblast differentiation and bioenergetics,
cultured primary osteoblasts from fetal rat calvaria were grown in
medium supplemented with ascorbate to induce differentiation. Before
ascorbate treatment, the rate of glucose consumption was 320 nmol · h1 · 106
cells
1, respiration was 40 nmol · h
1 · 106
cells
1, and the ratio of lactate production to glucose
consumption was ~2, indicating that glycolysis was the main energy
source for immature osteoblasts. Ascorbate treatment for 14 days led to
a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that
shown in immature cells. Confocal imaging of mitochondria stained with
a transmembrane potential-sensitive vital dye showed that mature cells
possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the
cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.
ATP; respiration; glycolysis; bone
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INTRODUCTION |
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ROBUST DEMANDS FOR ENERGY are placed on osteoblasts during production of a mineralized matrix, which provides both structural support and a calcium reservoir. Although oxidative phosphorylation produces 17 times more ATP per mole of glucose than glycolysis, both pathways participate in the response of bone cells to calciotropic stimuli (31). Treatment of young rats with parathyroid hormone (PTH) increases glycolytic activity of bone tissue (4). Furthermore, gonadal steroids stimulate the enzymatic activity of creatine kinase, which transfers phosphate from creatine to generate ATP (32). Mechanical loading affects blood flow in bone and, consequently, oxygenation; conversely, skeletal disuse induces hypoxia in osteocytes (8). Thus the activities of bioenergetic pathways in osteoblasts are likely to play key roles in the physiology of skeletal tissue.
Mitochondria are diverse in both form and function, and they
participate in various critical cell functions in addition to bioenergetics, including apoptosis and calcium signaling (12, 15). Oxidative ATP synthesis is driven by an electrochemical gradient across the inner mitochondrial membrane, which comprises differences in both pH and transmembrane potential ().
There is a direct correlation between the energized state of
mitochondria and the
when analyzed in isolated mitochondria
(28). Recent studies that used the fluorescent vital dye
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodine (JC-1), which preferentially accumulates in mitochondria by a
-dependent mechanism, revealed that individual mitochondria undergo large changes in
and in subcellular distribution in response to a variety of stimuli, including apoptosis, receptor activation, and hypoxia (5).
Remarkably little is known about how mitochondrial activity and the
principal pathways of energy metabolism are regulated during osteoblast
differentiation. Previous studies on energy metabolism used bone slices
or bone marrow stromal cultures, which contain multiple cell types, or
cultures of bone cells, which are not yet fully mature (4, 9, 20,
21, 30). However, the techniques of primary bone cell culture
have advanced sufficiently to assess biochemical and molecular changes
in osteoblasts at various well-defined stages of differentiation once
isolated from the complexities of intact bone. In primary cultures of
bone cells, treatment with ascorbic acid (AA) and -glycerophosphate
(
GP) leads to production of a mineralized matrix and expression of a
phenotype characteristic of mature osteoblasts. This process entails a
progressive sequence of morphogenic events, including proliferation and
multilayering, synthesis of an abundant extracellular matrix,
mineralization of that matrix, and, finally, differentiation into
osteocyte-like cells or apoptosis (11, 23, 24, 26, 27).
The aim of this study was to identify changes in bioenergetic pathways
(glycolysis, oxidative phosphorylation) and mitochondrial activity
() that occur at different stages of osteoblast differentiation and then to evaluate the contribution of these pathways to the maintenance of the ATP content of mature osteoblasts at steady state.
We found that cellular bioenergetics and
were differentially regulated during differentiation, perhaps functioning to maintain the
relatively high ATP content observed in mature osteoblasts compared
with immature cells.
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MATERIALS AND METHODS |
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Cell culture.
Cells were isolated from 21-day-old fetal rat calvaria as previously
described (26). All animal procedures were reviewed and
approved by the Institutional Animal Care and Use Committee at
NASA Ames Research Center. Briefly, cells were plated at a density of
36,000 cells/cm2 on eight-well chamber slides (0.81 cm2 per well; Permanox, Nunc, Naperville, IL) coated with
0.2% gelatin (Sigma, St. Louis, MO) and then cross-linked with
carbodiimide. Cells were grown in -MEM (GIBCO, Grand Island, NY)
supplemented with 10% serum (GIBCO) in 5% CO2 at 37°C
for the indicated times. After the cultures achieved confluence
(day 3), cells were induced to differentiate by the addition
to the culture medium of freshly prepared AA (50 µg/ml), which is
needed for matrix formation, and
GP (3 mM), which is needed for
mineralization. To maintain cells in an immature state, cultures were
grown continuously without these additives. The media were changed
every 2-3 days. Samples were recovered on day 3 (before
the addition of AA and
GP) or at the indicated times after growth in
the presence or absence of AA and
GP and then were stored at
20°C until later analysis.
Cell number. Cultures were treated sequentially with PBS, pH 7.4, containing 10 mM EGTA and 20 mM HEPES, pH 7.4, for 20 min and then for 60 min at 37°C with 572 U/ml collagenase in 115 mM NaCl, 5.3 mM KCl, 3 mM K2HPO4, 1 mM CaCl2, 30 mM mannitol, 10 mM glucose, 2 g/l BSA, and 24 mM HEPES, pH 7.4. An equal volume of 0.25% trypsin in EDTA (1 mM) in Hanks' balanced salt solution without Ca2+ and Mg2+ (GIBCO) was added to the collagenase, and cells were incubated for another 30 min. Dispersed cells were counted using a hemocytometer. Exclusion of 0.1% trypan blue in saline was used to determine cell viability, which was typically 80-90% of the total cell population.
Glucose consumption and lactate production.
Concentrations of glucose and lactate in media were measured using a
model 2700 select analyzer from Yellow Springs Instruments (Yellow
Springs, OH). The rates of glucose consumption (glu) and lactate production (
lac) were shown to be linear
over 6 h of incubation (data not shown). Medium samples (0.05 ml)
were recovered between 2 and 6 h of incubation with cells.
glu or
lac were calculated as the
difference between initial and final concentration divided by
incubation time.
O2 consumption and ATP production.
O2 consumption (O2) was
measured in culture medium at 37°C with a polarographic
O2 electrode (Radiometer, Copenhagen, Denmark) installed
into one well of an eight-well chamber slide to form an air-tight seal.
At the indicated times in culture, the concentration of O2
was measured in medium containing HEPES (10 mM), and
O2 was determined over 60 min.
O2 decreased to zero when cells were
treated with sodium azide (NaN3) or potassium cyanide
(KCN), showing that changes in
O2
measured in culture medium were due to mitochondrial respiration. The
addition of HEPES did not affect the rates of
glu
and
lac or ATP concentration in osteoblasts during
4 h of incubation (data not shown). Total ATP production from
glucose in mature osteoblasts was estimated on the basis of the
assumption that anaerobic glycolysis produces 2 mol of ATP from 1 mol
of glucose and reduction of 1 mol of O2 is coupled to the
production of 6 mol of ATP.
Alkaline phosphatase and ATP content.
To measure alkaline phosphatase activity and ATP, cultures were
extracted in 1% Triton X-100 in HEPES buffer, pH 7.4, and then
sonicated and centrifuged at 10,000 g for 3 min.
Supernatants were stored at 80°C until analysis. Alkaline
phosphatase activity and ATP content were measured
spectrophotometrically (Spectronic 1001 plus, Milton Roy) with
commercial kits (Sigma). The alkaline phosphatase assay measured the
hydrolysis of p-nitrophenol phosphate, and the ATP assay
used the phosphoglycerate phosphokinase and phosphate dehydrogenase
reactions, which couple the utilization of ATP with the oxidation of
NADH to NAD.
Flow cytometry. Flow cytometry was used to estimate cell size in mature and immature populations of osteoblasts. Cells were dispersed as described in Cell number and then prepared for cytometric analysis as described in detail by Ilic et al. (16). The viable and apoptotic cell populations were distinguished according to a cytometric method (14). In brief, dispersed cells were incubated with propidium iodide (PI; 5 µg/ml) on ice for 15 min before the addition of Hoechst 33342 dye (Molecular Probes, Eugene, OR). After a further 6-min incubation at room temperature, samples were acquired with the use of a dual-laser FACStar cell sorter (Becton-Dickinson, San Jose, CA) until 20,000 cells were analyzed. Cells that stained weakly for PI and Hoechst dye (viable cells) comprised 75-85% of the total cell population recovered. Cell size was assessed by forward light scatter. The flow cytometry data shown are representative of two separate experiments.
Staining and confocal microscopy. Mineralized nodules in mature cultures (day 14) were demonstrated by alizarin red S staining of calcium salts. Cells were fixed in ethanol, stained for 60 min in 1% alizarin red S in distilled water, pH 6.4, and then washed with distilled water. Images of osteoblasts at different stages of differentiation were acquired at the indicated times using an inverted scanning confocal microscope (Zeiss LSM 510) equipped with differential interference contrast optics.
MitochondrialMetabolic inhibitors.
Cells were grown in medium containing AA and GP for 14 days; the
medium was then supplemented with sodium fluoride (NaF) (5-30 mM),
NaN3 (1-8 mM), or KCN (2-4 mM) for a 1-h
preincubation period. Fresh medium was added with the addition of
inhibitor, and changes in O2 were measured for 2-4 h.
Samples were recovered from cultures treated in parallel to measure
glucose and lactate. ATP was measured after 4 h of incubation with inhibitor.
Statistics. The data shown are representative of four to six separate experiments performed in duplicate, and values are expressed as means ± SE. Statistical evaluation was made with ANOVA, using the software SuperANOVA (Abacus Concepts, Berkeley, CA).
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RESULTS |
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Temporal sequence of osteoblast differentiation. Experimental protocols for the differentiation of primary osteoblasts typically entail growth for 3-4 wk of culture before nodule formation and mineralization (24, 27), whereas our protocol (see MATERIALS AND METHODS) results in a more rapid differentiation (26). Because both proliferation and maturation can influence cellular bioenergetic pathways, we therefore first carefully defined the temporal sequence of changes in cell number and differentiation, as assessed by nodule formation and alkaline phosphatase activity.
Osteoblasts were plated to achieve confluence by day 3 in culture (Fig. 1A), at which time AA (50 µg/ml) and
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Glycolytic and oxidative pathways.
We measured glu,
lac, and
O2 to assess glycolytic and oxidative
components of energy metabolism at various times in culture (Fig.
3).
glu in
AA-untreated cultures decreased from 320 nmol · h
1 · 106
cells
1 on day 3 to 240 nmol · h
1 · 106
cells
1 on day 7 and remained at this level
until the end of the experiment (day 14). AA treatment
caused a transient decrease in
glu
(days 3-7) and subsequent increase to levels twofold
higher than shown in AA-untreated cultures on day 14 (Fig. 3A). In contrast,
lac transiently
decreased (days 3-7) in both AA-treated and
AA-untreated cells (Fig. 3B) and was 2-fold lower on
day 7 and 1.4-fold higher on day 14 in AA-treated
compared with that in AA-untreated cells.
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Mitochondrial transmembrane potential.
We assessed the influence of differentiation on mitochondrial in
live cells by confocal microscopy using the fluorescent dye JC-1. When
stained with JC-1, mitochondria with
below ~120 mV emit light
in green wavelengths, whereas those with high
emit light in red
wavelengths (see MATERIALS AND METHODS). Figure 4 shows images captured from AA-untreated
cultures and nodules of AA-treated cultures on day 10 in
culture (Fig. 4, A-F). Images collected from the most
central regions of nodules, which consist of dense, mineralized
extracellular matrix (26), demonstrated a low level of
autofluorescence that obscured the fluorescent signal generated by JC-1
(data not shown). Therefore, we selected for analysis specific sites
within nodules in which the cells display a distinctive cuboidal
morphology characteristic of mature osteoblasts (a representative site
is marked with an asterisk on Fig. 1C). AA-untreated
cultures demonstrated predominantly low-
mitochondria, with few
high-
mitochondria on day 10 (Fig. 4,
A-C). In contrast, nodule cells in AA-treated cultures
possessed abundant high-
mitochondria (Fig. 4, D-F).
To quantitate these differences in numbers of high- and low-
mitochondria, the ratio of the number of pixels in each channel
throughout the entire image stack was determined. The ratio of red
signal (high-
mitochondria) to green signal (low-
mitochondria) was 0.1 in AA-untreated cells, whereas the ratio was 0.6 in AA-treated cells.
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Cellular ATP content.
Because respiration and mitochondrial activity changed markedly during
differentiation, we measured cellular ATP content, which is normally
maintained within narrow limits. In AA-untreated cells, the ATP levels
in osteoblasts declined between day 3 and day 7 and then remained constant through the remaining 14-day period (Fig.
6). Surprisingly, treatment with AA
caused ATP content per cell to rise threefold between day 7 and day 10. On day 14, ATP levels in AA-treated
cells were fivefold higher than in AA-untreated cells. To determine if
the rise in cellular ATP content of AA-treated cells could be
attributed to differences in volume, cell size was estimated by flow
cytometry (Fig. 7). Osteoblasts grown for 10 days were dispersed and stained with PI and Hoechst dye to assess
cell viability and then analyzed by flow cytometry. Forward light
scatter by the cells provides an estimate of cell size and, hence, cell
volume. The distribution of sizes in the viable cell population was
found to be similar in AA-treated and AA-untreated cultures (Fig. 6).
Therefore, the increase in total ATP per cell in AA-treated cells is
unlikely to be caused by increased cells volume; rather, it reflects a
higher concentration of ATP in individual cells.
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Contribution of glycolysis vs. oxidative phosphorylation to ATP
content in mature osteoblasts.
To evaluate the contribution of glycolysis and respiration to
maintaining the high ATP content of mature osteoblasts, metabolic inhibitors were added to AA-treated cells on day
14 for 2-4 h. The addition of NaF, which inhibits
glycolysis (13), caused a dose-dependent decrease in
glu and
lac, effectively
depleting cellular ATP content, whereas
O2 did not change significantly (Fig.
8). In contrast, the addition of an
inhibitor of cytochrome c oxidase, NaN3, caused
a decrease in
O2 and an increase in the
ratio of
lac to
glu to two at all
concentrations of NaN3 tested, showing that respiration was
effectively abolished. Under these conditions, NaN3 caused
a 2-fold increase in
glu and a 2.5-fold increase in
lac. Notably, the cellular levels of ATP were not
significantly affected after 4 h of incubation with
NaN3. Similar results were obtained with KCN, another
inhibitor of cytochrome c oxidase. Thus, in mature
osteoblasts, ATP levels were maintained by an apparent increase in
glu and
lac, despite complete
inhibition of mitochondrial respiration.
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DISCUSSION |
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Results from this study revealed that the progression of cultured
osteoblasts through sequential stages of differentiation coincided with
marked changes in metabolic and mitochondrial activity. The sequence of
major changes evident during differentiation were as follows: first,
the rate of respiration increased during active growth of the cells to
form multilayers and initiate differentiation (days
3-7); second, the ATP content of maturing cultures increased during nodule morphogenesis (days 7-10); and, third,
the rate of glycolysis rose during nodule mineralization (days
10-14). Mature cells also demonstrated increased numbers of
high- mitochondria relative to immature cells. Thus both
glycolytic and mitochondrial changes appear to be integral components
of the differentiation sequence in cultured osteoblasts.
During the first stage of differentiation that we studied (days
3-7), the rate of cellular respiration in maturing
osteoblasts rose markedly. During this period, the addition of AA
caused a doubling in cell number and the formation of multilayers,
alkaline phosphatase activity rose sevenfold, and morphologically
distinct nodules first appeared. These changes coincided with a 3-fold increase in O2, a 2-fold decrease in
lac, and a 1.4-fold decrease in the ratio of
lac to
glu. Together, these
results suggest that, by day 7, more of the pyruvate
produced by glycolysis was consumed in the Krebs cycle in mature
osteoblasts compared with immature cells, and oxidative phosphorylation
increased. Cells that were grown without AA failed to differentiate and
did not increase in
O2, whereas
lac declined, although to a lesser extent than in
maturing osteoblasts. Thus cellular aging, as well as differentiation,
induced changes in glycolysis over time in cultured osteoblasts. The
early and sustained rise in the respiratory rate of maturing
osteoblasts is likely to reflect the high metabolic demands placed on
cells when they are dividing and differentiating and is consistent with
the finding that
O2 is high in intact bone tissue (30). Similarly, oxidative phosphorylation
increases during differentiation of other cell types in vitro,
including, for example, placental trophoblasts (3), nerve
cells (6), and colon adenocarcinoma cells
(10).
The most notable feature of the second stage of differentiation (days 7-10) was an increase in cellular ATP content. During this stage, cell growth slowed, alkaline phosphatase activity continued to rise, and nodules further matured as more extracellular matrix was produced. We showed previously that expression of protein for the late osteoblast marker, osteocalcin, is restricted to nodules on day 8 and mRNA transcripts are detectable with the use of Northern blotting on day 10 (11, 26); thus this period of culture corresponds to a stage of advancing maturation. These changes in differentiation were accompanied by an unexpected fivefold increase in ATP content, compared with cells grown without AA, when corrected on a per cell basis. Typically, cellular ATP content does not change markedly in response to nonpathological stimuli (e.g., Ref. 6). Differentiation of erythroid precursor cells leads to a decline in cellular ATP content (corrected for cell number), although this difference can be attributed to a reduction in cell volume as these cells mature such that the intracellular concentration of ATP remains constant (19).
To address the possibility that cytosolic volume increases during osteoblast differentiation, which could conceivably account for the observed rise in cellular ATP content, we examined in detail the distribution of cell sizes within cultured osteoblasts using a flow cytometry method that distinguishes viable cells from dead cells. Results from these experiments demonstrated that the size distributions of viable cells from AA-treated and untreated cultures were comparable, indicating that the intracellular concentration of ATP was in fact higher in mature osteoblasts than in immature cells.
The functional consequence(s) of the increased ATP pool size in maturating osteoblasts is unknown. One possibility is that adenylate metabolism regulates intracellular ATP and the total adenylate pool to control ATP-dependent processes, such as the activity of ion pumps (1, 2). In addition, ATP produced by osteoblasts may function as a paracrine agonist. ATP binds to purinergic receptors on osteoblasts, stimulating calcium signaling (22) and affecting osteogenesis (18). Thus an enlarged pool of ATP may be stored in mature osteoblasts for later secretion in response to appropriate stimuli, such as mechanical forces (7).
Once differentiation progressed to the stage in which abundant
extracellular matrix was evident in the nodules (day 10), a marked difference in mitochondrial activity was also observed. Three-dimensional confocal imaging using the mitochondrial
-sensitive dye JC-1 demonstrated that mature osteoblasts
contained a larger number of high-
mitochondria than immature
osteoblasts. These results are consistent with studies of Klein et al.
(20, 21) who showed that bone marrow stromal cell
populations accumulate more rhodamine 123 dye when treated with medium
that promotes the differentiation of osteoprogenitors.
High- mitochondria were preferentially distributed at the cell
culture medium surface of nodule cells, whereas the low-
mitochondria were more evenly distributed throughout the cell. On the
basis of the preferential distribution of enveloped viral glycoproteins, the cell culture-facing surface of polarized osteoblasts corresponds to the basolateral surface of epithelial cells
(17). The polar distribution of mitochondria in these
primary cells contrasts with the results obtained using cell lines, in
which mitochondria are localized to the edges of cells
(5). The precise consequence of localized regions of
high-
mitochondria is not yet known, although Reers et al.
(28) suggest that they are hot spots for ATP generation or
calcium signaling; in contrast, the low-
mitochondria may be
metabolically less active. Treatment of immature osteoblasts in culture
with PTH causes a modest decline in
as shown by JC-1 staining
(33); results obtained from this study indicate that the
stage of cellular differentiation is likely to affect changes in
mitochondrial
responses to calciotropic stimuli.
The most notable metabolic feature of the final stage of
differentiation (days 10-14) was an abrupt rise in
lac. During this stage, mineralization of nodules
occurred and alkaline phosphatase peaked on day 14.
Metabolically,
O2 and ATP content
remained constant, whereas
glu rose.
Together with the increase in
lac, these results
suggest that there is a marked increase in the rate of glycolysis at
this latter stage of differentiation. The total rate of ATP production
from glucose was estimated at 900 nmol · h
1 · 106
cells
1 by glycolysis and 800 nmol · h
1 · 106
cells
1 by oxidative phosphorylation. Thus glycolysis
provided ~50% of the energy requirements of mature osteoblasts and
is likely to be important for the function of mature cells. In fact,
inhibition of oxidative phosphorylation using NaN3 or KCN
for 2-4 h led to a rapid increase in
lac
and
glu by osteoblasts, and cellular ATP content was
thereby maintained at high levels for at least 4 h. In contrast,
acute inhibition of glycolysis using NaF did not affect
O2, and ATP levels declined sharply,
indicating that respiration did not compensate for the loss of
glycolytic activity. Interestingly, the maximum capacity of the
respiratory inhibitors to stimulate glycolytic activity in mature cells
was of the same magnitude (2- to 2.5-fold) as that which occurs when
bone tissue is exposed to hypoxic conditions in vitro (4).
Thus the glycolytic component of energy generation at this mature stage
of osteoblast differentiation may play a key role in adapting to
transient challenges such as changes in either O2 supply to
bone or increases in the acute and transient demands for energy.
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ACKNOWLEDGEMENTS |
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We thank Eduardo Almeida at the University of California, San Francisco, and Paul Dazin at the Howard Hughes Medical Center at the University of California, San Francisco, for help with the flow cytometry analyses. We also thank Nancy Searby and Sigrid Reinsch for helpful advice during the study and Charles Wade and Sigrid Reinsch for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by NASA Grant NAGS5-6374, a DDF award for the Core Confocal Microscopy facility at Ames Research Center, and a National Research Council Postdoctoral Fellowship (to S. V. Komarova).
Current address of S. V. Komarova: Dept. of Physiology, University of Western Ontario, London, Ontario, Canada.
Address for reprint requests and other correspondence: R. K. Globus, MS 236-7, Life Sciences Division, NASA Ames Research Center, Moffett Field, CA 94035-1000 (E-mail: rglobus{at}mail.arc.nasa.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 January 2000; accepted in final form 2 May 2000.
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