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Applied and Environmental Microbiology, October 1998, p. 3707-3712, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Butyrolactone I on the Producing Fungus,
Aspergillus terreus
Timothy G.
Schimmel,1,2,*
Allen D.
Coffman,1 and
Sarah J.
Parsons2
Technical Operations, Merck and Co., Inc.,
Elkton, Virginia 22827,1 and
Department of Microbiology, University of Virginia,
Charlottesville, Virginia 229082
Received 5 August 1997/Accepted 22 July 1998
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ABSTRACT |
Butyrolactone I
[
-oxo-
-(p-hydroxyphenyl)-
-(p-hydroxy-m-3,3-dimethylallyl-benzyl)--methoxycarbonyl--butyrolactone]
is produced as a secondary metabolite by Aspergillus
terreus. Because small butyrolactone-containing molecules act as
self-regulating factors in some bacteria, the effects of butyrolactone
I on the producing organism were studied; specifically, changes in
morphology, sporulation, and secondary metabolism were studied.
Threefold or greater increases in hyphal branching (with concomitant
decreases in the average hyphal growth unit), submerged sporulation,
and secondary metabolism were observed when butyrolactone I was added to cultures of A. terreus. Among the secondary metabolites
whose production was increased by this treatment was the
therapeutically important compound lovastatin. These findings indicate
that butyrolactone I induces morphological and sporulation changes in
A. terreus and enhances secondary metabolite production in
a manner similar to that previously reported for filamentous bacteria.
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INTRODUCTION |
Like many filamentous fungi,
Aspergillus terreus undergoes morphological and
physiological changes when the abundance and complexity of nutrient
sources are altered. When readily utilizable nutrients are abundant,
the organism undergoes exponential growth, a phase known as primary
metabolism. Primary metabolism includes the metabolic processes that
are required for growth, maintenance, and survival of the organism and
are basically similar for all living cells. Under conditions of
nutrient limitation, such as deprivation of easily assimilated
carbon, nitrogen, or phosphorus sources, the organism enters a
period of slower growth, morphological alterations, and changes
in metabolism known as secondary metabolism (10). Secondary
metabolism is dispensable to normal metabolism, is widely variable in
occurrence, and may or may not have a readily apparent biological
function. A variety of clinically beneficial secondary metabolites are
produced by fungi, such as the beta-lactam antibiotics penicillin and
cephalosporin, the antifungal antibiotic griseofulvin, and the
pharmacologically active compounds known as the ergot alkaloids.
A. terreus is an especially prolific producer of
secondary metabolites. A few of the compounds that are
produced by A. terreus are aspulvinone
(30), asterric acid (8), asterriquinone
(17), butyrolactone I (26), citrinin
(27), emodin (7), geodin (20),
itaconate (5), lovastatin (2, 12), questrin
(8), sulochrin (33), and terrecyclic acid
(24). Lovastatin, also known as mevinolin or
monacolin K, is clinically useful for reducing serum cholesterol
(2) and slowing the progression of atherosclerosis (34). Lovastatin and related compounds inhibit
cholesterol synthesis by inhibiting the rate-limiting step in
cellular cholesterol biosynthesis, namely, the conversion of
hydroxymethylglutaryl coenzyme A to mevalonic acid by
3-hydroxy-3-methylglutaryl-coenzyme A reductase. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, such as
lovastatin, reversibly inhibit cell proliferation by inducing a block
in the G1 phase of the cell cycle in a wide variety of normal and
tumorigenic mammalian cell lines (1, 19).
Butyrolactone I, another A. terreus secondary
metabolite, inhibits eukaryotic cyclin-dependent kinases. The
cyclin-dependent kinases are protein kinases that control cell
cycle progression in all eukaryotes and are regulated by
phosphorylation and dephosphorylation of critical serine,
threonine, or tyrosine residues. Full kinase activity is dependent on
the interaction of each cyclin-dependent kinase with a specific cyclin.
Butyrolactone I is a selective inhibitor of cell cycle kinases
(18, 25) but has little effect on other protein
kinases, such as mitogen-activated protein kinase, protein kinase
C, cyclic AMP-dependent kinase, or casein kinases. Butyrolactone I
inhibits cyclin-dependent kinases cdk1 and cdk2 of mammalian
cells with 50% inhibitory concentrations of 2.6 and 0.8 µM,
respectively, while the 50% inhibitory concentrations for
non-cyclin-dependent kinases are more than 100 µM (21).
Small -butyrolactone-containing molecules act as diffusible
self-regulating factors in a number of bacteria and control many diverse functions, such as antibiotic production, biofilm
formation, bioluminescence, virulence factor production, and plasmid
conjugal transfer (4, 9). These compounds may provide a
common means of bacterial cell-to-cell communication or signaling
(16, 22). One well-studied compound, A-factor
(2-isocapryloyl-3R-hydroxmethyl--butyrolactone), regulates cellular differentiation and secondary metabolism in Streptomyces griseus (3, 14). Structurally
related -butyrolactones are present in a variety of other
Streptomyces species and are involved in morphological
differentiation (aerial mycelium and spore formation) or secondary
metabolism (4, 13). A number of
-butyrolactone-containing compounds are also produced by
different fungi; these compounds include aspulvinone in A. terreus (30), -decalactone in Sporobolomyces
odorus (32), lachnumlactone A in Lachnum
papyraceum (28), and multicolanic acid in
Penicillium multicolor (32). However, the
function of these compounds is not yet known. We undertook this study
to determine if butyrolactone I, a
-butyrolactone-containing secondary metabolite of the
filamentous fungus A. terreus, functions in a manner
analogous to the small -butyrolactone-containing compounds of
the filamentous bacteria belonging to the genus
Streptomyces. We show that as in prokaryotes, in
A. terreus butyrolactone I has the ability to induce
morphological changes, increases in spore formation, and increases in
the production of A. terreus secondary metabolites.
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MATERIALS AND METHODS |
Materials.
Glucose, lactose, buffers, and other reagents
were obtained from Sigma Chemical Co. (St. Louis, Mo.). Yeast extract
and malt extract were obtained from Difco Laboratories (Detroit,
Mich.); peptonized milk was obtained from Quest International (Norwich, N.Y.); and corn steep liquor was obtained from Grain Processing Corp.
(Muscatine, Iowa). Solvents were obtained from Fisher Scientific Co.
(Pittsburgh, Pa.). Purified butyrolactone I and authentic samples of
lovastatin and sulochrin were provided by William Saum (Chemistry
Section, Technical Operations, Merck and Co., Inc., Elkton, Va.), and
the butyrolactone I standard was provided by Henry Joshua (Merck
Research Laboratories, Rahway, N.J.).
Cultures and growth conditions.
A. terreus ATCC
20542 was obtained from the Merck Culture Collection (Rahway, N.J.). A
reisolate, M8, was used throughout this study as it exhibited less
variability in growth and secondary metabolism than the parent strain.
The culture was maintained on yeast extract-malt extract (YME) agar
slants containing 0.4% yeast extract, 1% malt extract, 0.4% glucose,
and 2% agar (pH 7.0). Freshly inoculated slants were incubated at
28°C for 5 days, after which they were stored at 4°C. Frozen spore
suspensions were prepared by washing the agar slants with 5 ml of a
sterile 5% (wt/vol) glycerol-10% (wt/vol) lactose solution,
combining the washes, and freezing (
70°C) 2-ml aliquots. Spores
were propagated from 1 ml of a frozen spore suspension by using the
inoculum medium (medium A) described by Alberts et al. (2).
Medium A contained (per liter) 5 g of corn steep liquor, 40 g
of tomato paste, 10 g of oat flour, 10 g of dextrose, and 10 ml of a trace element solution; the pH was 6.8. The trace element
solution contained (per liter) 1 g of FeSO4 · 7H2O, 1 g of MnSO4 · 4H2O, 25 mg of CuCl2 · 2H2O,
100 mg of CaCl2 · 2H2O, 56 mg of
H3BO3, 19 mg of (NH4)6Mo7O24 · 4H2O, and 200 mg of ZnSO4 · 7H2O. The secondary metabolite production medium contained
glucose, peptonized milk, and yeast extract, as well as additional
lactose (6). This medium, which was designated GPY-L,
contained (per liter) 25 g of glucose, 24 g of peptonized
milk, 2.5 g of yeast extract, 50 g of lactose, and 2.5 ml of
P2000 defoamer; the pH was 7.4. The inoculum used for secondary
metabolite production was prepared by inoculating 250-ml Erlenmeyer
flasks containing 40 ml of medium A with 1-ml portions of frozen spore
suspension (approximately 2 × 106 spores). The flasks
were incubated at 27°C on a rotatory shaker at 220 rpm for 25 h,
and then 2 ml of each inoculum was used to inoculate 20 ml of
production medium. The flasks were then incubated at 27°C on a
rotatory shaker at 220 rpm for up to 10 days.
Evaluation of morphological changes: branching.
Portions (1 ml) of the spore suspensions (approximately 2 × 106
spores) were used to inoculate flasks containing 40 ml of inoculum sporulation medium (medium A), which were incubated at 27°C on a
rotatory shaker at 220 rpm for 18 h. Butyrolactone I was added to
individual 18-h flasks in 500 µl of sterile ethanol so that final
butyrolactone I concentrations of 0, 63, 125, 250, 500, and 1,000 µM
were obtained. Each concentration was tested in triplicate. After
2 h of incubation at 27°C with agitation, the numbers of branches and hyphae were determined with a light microscope (Standard 16; Carl Zeiss, Inc., Thomwood, N.Y.). Three aliquots from three different samples at each concentration were taken, and a total of at
least 30 hyphae from three different fields for each aliquot were
counted. Controls were treated with sterile ethanol alone.
Evaluation of morphological changes: HGU.
Eighteen-hour-old
inoculum flasks were prepared as described above. Butyrolactone I or
lovastatin was added to each flask to a final concentration of 0, 50, 100, 200, or 500 µM. Duplicate flasks containing each concentration
were incubated for 4 h at 27°C with agitation. Two aliquots were
taken from each flask, and 8 to 12 fields were examined with a light
microscope and captured digitally by using Picture Publisher software
(Micrografx, Richardson, Tex.). The number of branches and the length
of approximately 10 hyphae in each field were determined. The results
were plotted as the average hyphal length divided by the number of
branches in each 10-hypha group in order to derive the hyphal growth
unit (HGU) (the average length of the hypha associated with each
branch). The mean and standard error of the mean for 8 to 13 groups at each concentration were determined.
Submerged sporulation studies.
For each concentration
tested, 1 ml of spore suspension (approximately 2 × 106 spores) was used to inoculate 40 ml of medium A. Butyrolactone I or lovastatin was added at different times to
individual flasks to provide the final concentration needed in each
experiment. The inoculum was incubated at 27°C with agitation at 220 rpm until day 8, and then the flasks were assayed to determine spore
production. The number of visible spores was determined by taking two
aliquots from each flask, diluting each aliquot 10-fold, and counting
the spores with a light microscope by using a hemacytometer. Two
samples from each aliquot were counted to provide four replicates for each concentration. The number of viable spores was determined by
plating diluted samples from each flask onto YME agar plates; four
replicates were examined at each concentration. The plates were
incubated at 27°C for 5 days.
Secondary metabolite production studies.
Flasks containing
20 ml of GPY-L were inoculated with 2 ml of 25-h-old inoculum (see
above) and incubated at 27°C with agitation at 220 rpm. Butyrolactone
I was added after 24 h to one-half of the flasks to a final
concentration of 500 µM. Two or more of the butyrolactone-containing
flasks were harvested along with two or more of the
non-butyrolactone-containing controls at various times to evaluate
lovastatin production. Lovastatin was quantified from methanol extracts
of culture broth by using reverse-phase chromatography on an octyldecyl
silane Hypersil column (length, 100 mm; particle size, 5 µm;
Hewlett-Packard, Palo Alto, Calif.). The mobile phase, 0.1% aqueous
phosphoric acid-acetonitrile (45:55, vol/vol), was added isocratically
at a flow rate of 1 ml/min, and detection was at 238 nm. Authentic
lovastatin was used to confirm the retention time and the quantities of
lovastatin in the culture extracts.
A full factorial experiment was performed by using Design Expert
software (Stat-Ease, Minneapolis, Minn.) to examine the effect of
addition of butyrolactone I or lovastatin on secondary metabolism. Flasks containing 20 ml of GPY-L were inoculated with 2 ml of inoculum
(as described above) and incubated at 27°C as described above.
Butyrolactone I or lovastatin was added to replicate flasks after 120, 145, and 170 h to a final concentration of 0, 10, 50, or 100 µM.
Each flask was harvested after 12 days. Each concentration at each time
point was tested by using at least four replicate flasks; 16 replicates
were used for the designated midpoint of the experiment, 50 µM at
145 h, in order to determine experimental variability. The
concentrations of lovastatin and sulochrin in methanol extracts of
culture broth were quantified by high-performance liquid chromatography
as described above.
Dry cell weight determinations.
The dry cell weight was
determined by filtering 20 ml of culture broth through filter paper
(Whatman 934-AH; Fisher Scientific), washing the preparation three
times with distilled water, and drying the remaining mycelial mat under
a vacuum overnight at 60°C.
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RESULTS AND DISCUSSION |
Morphological changes.
Several A-factor-related
compounds have an effect on aerial mycelium morphogenesis in a
number of filamentous Streptomyces species. A. terreus, a filamentous fungus, was therefore examined for
possible morphological changes in response to -butyrolactone treatment. In strain M8, no overt morphological changes in aerial hyphae and conidiophores were observed by microscopy when butyrolactone I was added exogenously. However, Fig. 1
shows that an increase in the overall hyphal branching was discerned
when butyrolactone I was added to 18-h-old submerged cultures and the
cultures were incubated for an additional 2 h. At that time, the
percentage of hyphae with branches was determined and compared to the
percentage in the control cultures. The results indicated that there
was a dose-dependent increase in the number of hyphae with branches, which peaked in response to approximately 500 µM butyrolactone I, as
shown by second-order regression analysis (Fig.
2A). Monitoring changes in branching
alone did not take into account any changes in hyphal extension that
may have been induced by butyrolactone I. For this reason, the effect
of butyrolactone I on the HGU, which was the average length of the
hypha associated with each branch (31), was determined.
Lovastatin was used as a control compound, as it has been found to be
active at concentrations ranging from 20 to 100 µM in inhibiting cell
cycle progression in a wide variety of mammalian cells (19)
and is produced as a secondary metabolite concurrently with
butyrolactone I in A. terreus M8. Figure 2B shows that
addition of 200 to 500 µM butyrolactone I resulted in a statistically
significant decrease in the HGU, while addition of lovastatin at the
same concentrations resulted in no change in the HGU. These changes in
fungal morphology are consistent with previous reports on antheridiol,
a butyrolactone-containing diffusible hormone of the saphrophytic water
mold Achlya ambisexualis, which causes cessation of growth
and profuse hyphal branching (11).
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FIG. 1.
A. terreus hyphal filaments without (A
and B) and with (C and D) butyrolactone I added. Butyrolactone I in
sterile ethanol was added to a final concentration of 200 µM to 18-h
cultures. Aliquots of the cultures were taken after 5 h, and
photomicrographs were made. Bars = 50 µm.
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FIG. 2.
Effect of butyrolactone I on morphology. (A) Percentage
of hyphae with branches after butyrolactone I was added to aliquots of
a 20-h culture. Each bar represents the mean obtained with at least 10 hyphae in three samples at each concentration. (B) Changes in HGU (the
average length of hypha associated with each branch) of fungal pellets
treated with butyrolactone I or lovastatin. The data for both 200 µM
butyrolactone and 500 µM butyrolactone I are significant at the
P < 0.001 level.
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Effect of butyrolactone I on submerged sporulation.
The effect
of butyrolactone I on submerged spore production in A. terreus was tested. Sporulation in submerged liquid cultures occurs when conidia are produced directly from hyphae without prior
differentiation into conidiophores and can be induced by carbon or
nitrogen starvation (23, 29). Different concentrations of
butyrolactone I were added to submerged spore-producing inoculum cultures in medium A. These cultures were examined after 8 days of
incubation both for visible spores as determined by microscopic examination and for viable spore production as determined by CFU on YME
agar plates. Figure 3A shows that
addition of butyrolactone I resulted in increases in the numbers of
both visible and viable spores, while addition of lovastatin resulted
in no changes in spore number at the concentrations tested. Addition of
butyrolactone I to a final concentration of 500 µM yielded
approximately threefold more spores (visible or viable) than the
number of spores in the control samples. Analysis of visible spores
provided a straightforward and quick assay whose results strongly
correlated with the number of viable spores detected on agar plates
after 5 days of incubation (R = 0.992). Therefore, the
visible spore assay was used to determine the effect of addition of
butyrolactone I on 8 days of spore production when it was added on days
1, 4, and 7. Figure 3B shows that there was a greater increase in the
day 8 spore number when butyrolactone I was added on day 1 than when it
was added on day 4 or 7. By day 7, addition of butyrolactone I had
little or no effect on spore production. Even at the highest
concentration used, 1,000 µM, addition on day 7 produced only 40%
more spores, while addition of the same concentration on day 1 resulted
in more than sixfold more spores than the number in the control.
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FIG. 3.
Effect of butyrolactone I on submerged spore production.
(A) Number of visible spores as determined with the hemacytometer and
number of viable spores as determined by CFU counts on agar plates
after butyrolactone I or lovastatin was added at different final
concentrations on day 2. Spores were counted on day 8. The data for
both 250 µM butyrolactone and 500 µM butyrolactone I are
significant at the P < 0.001 level. (B) Number of
visible spores produced when butyrolactone I was added on different
days and spores were counted on day 8.
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Effect of butyrolactone I on secondary metabolism.
Since
A. terreus produces many secondary metabolites,
including the clinically useful compound lovastatin, we wanted to
determine if addition of butyrolactone I had an effect on secondary
metabolism in the producing organism. A full factorial experiment was
designed to examine the combination of time of addition and
concentration in order to evaluate the effect of both metabolites on
secondary metabolite production. Butyrolactone I or lovastatin was
added to a final concentration of 0, 10, 50, or 100 µM during
secondary metabolism (at 120, 145, or 170 h postinoculation), and
each culture was incubated until day 12; then each culture was assayed
for metabolite production. As shown in the response surface map
generated from the results of the factorial experiment (Fig.
4A), addition of butyrolactone I
increased lovastatin production. The mean titer of lovastatin for six
replicates to which butyrolactone I (diluted in sterile ethanol) was
added to a final concentration of 100 µM at 120 h was 0.94 g/liter (standard error of the mean, 0.08 g/liter), while the mean for
the controls to which only sterile ethanol was added was 0.32 g of
lovastatin per liter (standard error of the mean, 0.06 g/liter). The
same factorial experiment was performed with lovastatin (rather than
butyrolactone I) added to a final concentration of 0, 50, or 100 µM
at 120, 145, or 170 h postinoculation. These levels of lovastatin
represent amounts that have been reported to influence cell cycle
activity in mammalian cells but are less than the amount of lovastatin
produced by A. terreus M8. Figure 4B shows that no
increase in the final lovastatin titer was detected at the time of
harvest when lovastatin was added between 120 and 170 h after
inoculation. Figure 4C shows that only a slight increase in cell mass
occurred when butyrolactone I was added, indicating that the increase
in lovastatin production upon addition of butyrolactone I was probably
not due to the small increase in cell mass. The dry cell weights
of the treated cultures were 93.6 to 108.5% of the untreated
control dry cell weights. The large change in secondary metabolism (an
increase of more than twofold for lovastatin) was probably not due to
the small change in cell mass (less than 9%). The final cell mass was
approximately the same whether butyrolactone I (Fig. 4C) or lovastatin
(Fig. 4D) was added. Butyrolactone I not only affected lovastatin
production but also increased the production of sulochrin, another
secondary metabolite of A. terreus produced
concurrently with lovastatin. Table 1
shows that addition of 100 µM butyrolactone I at 120 h
postinoculation increased sulochrin production by 86% compared with
the controls, while no increase was observed in the lovastatin-treated cultures.
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FIG. 4.
Effect of butyrolactone I on secondary metabolism. (A)
Response surface map showing the effects of different concentrations of
butyrolactone I added at different times. For the sum of squares (a
measure of variability between groups), P < 0.001. (B)
Response surface map showing the effect of lovastatin addition on
lovastatin production. (C and D) Response surface maps showing the
effect of butyrolactone I and lovastatin additions on cell mass,
respectively. DCW, dry cell weight.
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When added during the primary metabolism phase, butyrolactone I did not
have the dramatic effect on lovastatin production
seen when it was
added during secondary metabolism. Figure
5 shows
that adding butyrolactone I to
cultures during exponential growth,
even to a concentration fivefold
greater than the concentration
shown to be effective during secondary
metabolism (500 µM), shifted
lovastatin production ahead by
approximately 5 to 10 h, while
the rate of lovastatin production
remained approximately the same.
Addition of butyrolactone I during the
growth phase advanced the
timing of secondary metabolism by
approximately 5 to 10 h. This
is comparable to the advance in
secondary metabolite production
when the
-butyrolactone compound
virginiae butanolide-C was added
to
Streptomyces virginiae
(
35) or A-factor was added to
S. griseus during
growth (
14). These results suggest that butyrolactone
I may
play a role in regulating the amount of lovastatin produced
by
A. terreus.
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FIG. 5.
Effect of butyrolactone I addition on lovastatin
production when butyrolactone I was added to 24-h cultures of
A. terreus during primary metabolism. Each point
represents at least two replicates, and the error bars represent the
standard errors of the means for three replicates. The correlation
coefficient for both lines is >0.993. DCW, dry cell weight.
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Although further work is needed to determine if butyrolactone I belongs
to the class of compounds that constitute the microbial
hormones,
treatment with butyrolactone I induced changes in the
host organism
that are similar to those induced by the more completely
characterized
microbial hormones. A practical application of these
findings is
the possibility that butyrolactone I could be used
to increase or
promote the production of desired secondary metabolites
in
A. terreus. It has been proposed that the
A-factor-related
microbial signals might be useful in controlling
secondary metabolism
in streptomycetes (
4), and this has
been demonstrated by using
the autoregulator virginiae butanolide-C to
control the induction
time of virginiamycin production and the amount
of virginiamycin
produced. A twofold increase in the amount of
virginiamycin was
observed when virginiae butanolide-C was added during
secondary
metabolism just after growth had slowed (
35).
Similarly, in
this study, a threefold increase in the amount of
lovastatin and
an approximately twofold increase in sulochrin
production were
observed after butyrolactone I was added to
postexponentially
growing cultures.
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FOOTNOTES |
*
Corresponding author. Mailing address: Technical
Operations, Merck and Co., Inc., P.O. Box 7, Elkton, VA 22827. Phone:
(540) 298-4054. Fax: (540) 298-4817. E-mail:
schimmel{at}merck.com.
This article is dedicated to the memory of William Saum, whose
untimely passing was a great loss for family, colleagues, and friends.
He will be remembered for his great dedication to helping others.
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Applied and Environmental Microbiology, October 1998, p. 3707-3712, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
