Next Article 
Appl Environ Microbiol, May 1998, p. 1589-1593, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
High-Fidelity Translation of Recombinant Human
Hemoglobin in Escherichia coli
Michael J.
Weickert* and
Izydor
Apostol
Somatogen, Inc., Boulder, Colorado 80301
Received 9 December 1997/Accepted 13 February 1998
 |
ABSTRACT |
Coexpression of di-
-globin and 
-globin in Escherichia
coli in the presence of exogenous heme yielded high levels of
soluble, functional recombinant human hemoglobin (rHb1.1). High-level
expression of rHb1.1 provides a good model for measuring mistranslation
in heterologous proteins. rHb1.1 does not contain isoleucine;
therefore, any isoleucine present could be attributed to
mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino
acid analysis of highly purified rHb1.1 typically revealed 
0.2 mol of
isoleucine per mol of hemoglobin. This corresponds to a translation error rate of 0.001, which is not different from typical translation error rates found for E. coli proteins. Two different
expression systems that resulted in accumulation of globin proteins to
levels equivalent to ~20% of the level of E. coli
soluble proteins also resulted in equivalent translational fidelity.
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INTRODUCTION |
Protein synthesis in living
organisms, the ultimate step in biological information transfer,
is also the most error-prone step. In Escherichia coli, 2 to
20 translation errors typically occur in every 10,000 codons translated
(15). Errors can occur through incorrect charging of tRNAs
(misaminoacylation), incorrect pairing of noncognate tRNAs with codons
(missense errors), frameshifting and drop-offs (processivity errors),
misreading of sense codons as terminators (false termination), or
misreading of terminators as sense codons (termination readthrough)
(13; reviewed in references 2, 5,
8, and 15). Growth conditions,
particularly starvation for one or more amino acids, can significantly
increase the translation error rates (15, 16). The rates at
which many of these errors occur are also exacerbated by the presence
of codons whose cognate tRNAs are rare in E. coli (reviewed
in reference 6). For this reason, most heterologous
proteins are overexpressed in E. coli from genes whose codon
bias has been altered to conform to that typical of E. coli
(6).
Missense errors resulting from incorrect pairing of noncognate tRNAs
with mRNA codons are the most frequent translation errors under general
growth conditions (8). These errors are attributed to
recognition of a codon by a tRNA containing a less-than-perfect anticodon nucleotide match, as described by the wobble hypothesis and
the two-out-of-three hypothesis (reviewed in reference
2). Missense errors have been measured at specific
sites, including in recombinant methionyl human granulocyte
colony-stimulating factor, which contained Gln substitutions for His
(12), or in general throughout the protein, including in
mouse epidermal growth factor (mEGF) (18). The general
missense errors were measured by analyzing mEGF for the presence of
phenylalanine not encoded by the gene (18). Radiolabeled Phe
and Leu were incorporated into mEGF, and the ratio of these amino acids
indicated that there was a missense error rate of 2 × 10
2. This translation error rate is 1 to 2 orders of
magnitude higher than the rates typical for E. coli protein
synthesis. However, no other studies of missense errors in intact
overexpressed proteins have confirmed that this is a general phenomenon
for highly expressed heterologous proteins in E. coli.
Functional recombinant human hemoglobin (rHb1.1) is produced in
E. coli as a heterotrimer when there is concomitant
expression from a plasmid-borne operon of di- and subunits and
exogenous heme is provided (10). Di--globin is a pair of
-globin molecules that are genetically linked to prevent
dimerization and thereby reduce renal filtration and extend the
intravascular half-life (11). Low-level mistranslation of
rHb1.1 by misaminoacylation resulting in norvaline substitution for
leucine has been observed (1). In this study, we examined
the missense error rate by a technique which we call absent amino acid
analysis (AAAA). Using this technique, we detected trace amounts of
isoleucine, which is not encoded by the rHb1.1 genes. Therefore, the
isoleucine content of the purified hemoglobin reflected a specific
class of missense errors. The study described here had two advantages over previous studies: (i) very highly purified protein samples were
used, so contributions of contaminants were negligible; and (ii) a
sensitive direct method was used to measure traces of isoleucine by
AAAA rather than radiolabeling.
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MATERIALS AND METHODS |
Hemoglobin expression and purification.
E. coli
SGE1662 (19) contains pSGE705, a medium-copy-number
pBR-based plasmid with di-alpha- and beta-globin genes in which most
codons conform to the E. coli bias. These genes are in an operon whose transcription is dependent upon the tac
promoter (11) and express rHb1.1. A similar high-copy-number
plasmid containing the pUC origin of replication was used to express
rHb1.1 in SGE1464 (19).
Fermentations were performed at 28 to 30°C in defined medium in 15-, 600-, and 1,500-liter fermentors generally as described by Looker et
al. (10) and Weickert et al. (20). Expression was
induced by adding isopropyl--D-thiogalactopyranoside
(IPTG) to a concentration of either 55 µM (SGE1662) or 100 µM
(SGE1464). IPTG was added when the optical density at 600 nm was
approximately one-third the expected final cell density. Incubation was
continued for 10 to 16 h postinduction, and hemin was added to a
concentration of ~0.2 g/liter (SGE1662) or ~0.4 g/liter (SGE1464).
One-milliliter samples were withdrawn, placed in 1.7-ml Eppendorf
tubes, and centrifuged for 3 min, and the supernatants were removed.
The pellets were stored at 
80°C until they were assayed for soluble and/or insoluble rHb1.1, as described by Weickert and Curry
(19) and Weickert et al. (20). rHb1.1 was
purified as described by Plomer et al. (17). The
functionality of the purified rHb1.1 samples was determined as
described by Hoffman et al. (4). Purified protein was
characterized by trypsin mapping performed as described by Lippincott
et al. (9), and a liquid chromatography-mass spectrometry
analysis was performed as described by Apostol et al. (1).
Measurement of E. coli proteins.
The amounts of
E. coli protein contaminants in purified rHb1.1 samples were
determined by a proprietary enzyme-linked immunosorbent assay (ELISA)
by using antibodies developed at Somatogen. After identical
fermentation of an equivalent strain containing plasmids from which the
rHb1.1 genes were removed, E. coli proteins were recovered
by purification of a lysate. This produced antibodies that were more
sensitive than commercial anti-E. coli protein reagents and
that could be used to detect protein contaminants in an rHb1.1 sample
which were present at levels too low to be visualized by gel
electrophoresis.
Amino acid analysis.
Purified rHb1.1 samples were subjected
to gas phase hydrolysis at 165°C for 1.5 h in the presence of 6 N HCl containing 0.1% phenol with a Savant AminoPrep model AP100
hydrolyzer. Levels of norvaline substitution were determined by a
modified ortho-phthaldehyde method as described in Apostol
et al. (1). Other amino acids were analyzed by precolumn
derivatization with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC).
The standard AccQ-Tag method was used to derivatize amino acids. AQC
derivatives of amino acids were separated on a Zorbax XDB
C8 column (2 by 150 mm) by using a model HP 1090IIM chromatograph. Separation was monitored by examining fluorescent signals (excitation at 254 nm, emission at 395 nm) and UV signals (254 nm). The UV signal was used to determine the concentration of all amino
acids except Ile, and the amplified fluorescent signal was used to
determine the isoleucine content (Fig.
1). One level of calibration at 125 pmol/injection was used for the UV signal. Solvent A was diluted
AccQ-Tag eluant A from Waters (1,000 ml of water, 85 ml of concentrated
eluant A). Solvent B was 48% acetonitrile-12% isopropanol in
high-performance liquid chromatography grade water. Separation was
accomplished by using the following gradient conditions: 1% solvent B
at zero time, 4% solvent B at 1 min, 20% solvent B at 16 min, 37%
solvent B at 40 min, 100% solvent B from 42 to 46 min, and no solvent
B at 47 min. A hydrolysate of hemoglobin from sample 79 (Table
1) was used to calibrate the assay and to
determine the degree to which low levels of isoleucine added to the
sample were recovered. This hydrolysate was used as the matrix, and
different levels of amino acid standards, including Ile, were added.
The areas under Ile peaks (fluorescent signal) were plotted versus the
amounts of Ile added (Fig. 2). The levels of recovery of small amounts of added Ile (0.5 and 1.0 pmol) exceeded 100% (130 and 118%, respectively) because of the small amount of Ile
already present in the hydrolysate. This small amount of native
isoleucine was not significant when the larger amounts (2.0, 5.0, and
10.0 pmol) of Ile were added; under these conditions the average level
of recovery was 102%. The area under the Ile peak was proportional to
the amount added, with an R2 value of 0.9987 and
a P value of 1.1 × 106. The inverse of
the slope represented the color factor (extinction coefficient) used to
determine low levels of isoleucine in a hemoglobin matrix. The level of
Ile in an unknown sample was calculated by multiplying the area under
the Ile peak by the color factor.
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FIG. 1.
Magnified portion of chromatogram from an amino acid
analysis (sample 94-3A) (Table 2), showing resolution of Ile and
Val-Val dipeptide. The lines used to establish a baseline and to
separate the Val-Val and Ile peaks in order to enable peak integration
and Ile quantitation are shown.
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FIG. 2.
Calibration curve for Ile. Hydrolysate of sample 79 was
used as the matrix, and different levels of amino acid standards,
including Ile, were added. Areas under the Ile peak were plotted versus
the amount of added Ile. The inverse of the slope represents the color
factor for a low level of isoleucine in a hemoglobin matrix (R-squared = 0.9987 and P value = 1.1 × 106). Fl,
fluorescence.
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Sample 94 (Table
1) was used as a reference sample for determining the
precision and consistency of the Ile analyses. Aliquots
of sample 94 were hydrolyzed and analyzed multiple times. The
molar content of Ile
in hemoglobin was calculated by using the
three most stable amino
acids, Leu, Val, and Ala, as the references.
The value reported for
each experiment was the mean number of
moles of Ile per mole of rHb1.1
calculated by using these three
amino acids. On average, 0.102 mol of
Ile/mol of rHb1.1 was found
in sample 94, and the standard deviation
was 0.036 mol (relative
standard deviation [RSD], 35%) (Table
2). In low-level amino
acid analyses
factors such as foreign particles, cross contamination,
traces of glove
powder, etc. may contribute to relatively large
errors.
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RESULTS |
Expression and purification of rHb1.1.
rHb1.1 was produced in
E. coli with medium-copy-number (SGE1662) and
high-copy-number (SGE1464) plasmids containing the di-alpha-globin and
beta-globin genes in an operon under control of the tac
promoter (4, 10, 19, 20). In eight fermentations of SGE1662
approximately 5.4% of the soluble E. coli protein was
soluble rHb1.1 (Fig. 3A). In two
fermentations of SGE1464 the average level of soluble rHb1.1 accumulated was 13.4% of the soluble E. coli proteins
(Fig. 3A). Western blot assays of the insoluble and soluble fractions
of recombinant hemoglobins from SGE1464 lysates indicated that
approximately one-third (37% ± 3%, as determined by densitometry
scanning) of the globins were insoluble (Fig. 3B). In SGE1464, this
corresponded to a maximum accumulation of total hemoglobin equivalent
to approximately 20% of the E. coli proteins, which is
consistent with previous observations (20).
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FIG. 3.
rHb1.1 expression. (A) Accumulation of soluble rHb1.1
during fermentation in E. coli SGE1662 and SGE1464. N is the
number of fermentation preparations from which samples were taken.
Except for the last time point, most but not all preparations were
assayed for each time point. (B) Western blot of soluble (lane S) and
insoluble (lane I) globin from an SGE1464 fermentation cell lysate.
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Soluble rHb1.1 was recovered and rigorously purified and the final
yield was 20 to 36% of the starting material (Table
1).
The level of
E. coli proteins was measured by the ELISA, and in
the
majority of samples the levels of
E. coli proteins were
below
the level of detection (<0.16 ppm relative to rHb1.1) (Table
1).
The presence of very pure rHb1.1 was confirmed by trypsin mapping
and
reversed-phase chromatography along with mass spectrometry
(
9). The functionality of the purified rHb1.1 was also
assessed,
and the values for all samples were within the expected range
of values for oxygen affinity and cooperativity (Table
1). Rigorous
purification of rHb1.1 allowed us to assess the amino acid composition
without worrying about potential contamination of the signal by
E. coli proteins (Table
1).
Measurement of the isoleucine content of rHb1.1.
An analysis
to determine the absent amino acid was the analytical method used to
determine the isoleucine content in several samples of hemoglobin
produced by two different strains (Table 3). Highly purified rHb1.1 samples were
subjected to hydrolysis followed by amino acid analysis as described
above. AQC derivatives of amino acids were separated on a
reversed-phase column, and the concentrations of all amino acids except
isoleucine were determined from the UV
signal. The highly amplified fluorescent
signal was used only to determine isoleucine content (Fig. 1). Special
care was taken to resolve the residual Val-Val dipeptide from Ile to avoid interference. The Val-Val dipeptide results from incomplete hydrolysis of the peptide bond (3), which occurs twice in
-globin.
The number of moles of isoleucine in 1 mol of rHb1.1 was divided by the
number of codons in rHb1.1 that differed from the
Ile codons by one
base to calculate the frequency of this class
of mistranslation error.
A total of 200 of the 575 codons in the
sequence of recombinant
hemoglobin met this criterion. Three of
these codons encode initiating
methionines (one for di-
subunits
of rHb1.1, two for
subunits of
rHb1.1), and they typically could
be excluded, except that initiation
of heterologous protein translation
by isoleucine has been observed in
E. coli (
7). Exclusion of
these three amino acid
codons should have had little effect on
the calculated rate of
translation error. The calculated translation
error rate did not
exceeded 0.0011, and one-third of the samples
had a translation error
rate of
0.0002 (Table
2). It was estimated
that the lower limit of
quantitation for Ile determinations was
0.05 mol of Ile per mol
of rHb1.1, which corresponds to a translation
error rate of
0.0002. This is equivalent to two translation errors
in every
10,000 translations of rHb1.1 codons that differ from
isoleucine codons
by one nucleotide. The observed levels of translation
error did not
correlate with hemoglobin functionality measurements
for oxygen
affinity (P
50) and cooperativity (Hill max) (Tables
1 and
3). There was a considerable distribution of error rates
among the
various purification yields (Fig.
4).
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FIG. 4.
Relationship between purification yield and amino acid
misincorporation. Misaminoacylation produced norvaline (Nval), shown as
the percentage of norvaline substituted for leucine, and missense
substitutions (subst.) introduced isoleucine, shown as the error rate
per codon translated.
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Measurement of the norvaline content of rHb1.1.
Another type
of mistranslation has been observed in rHb1.1; this type of
mistranslation, substitution of norvaline for leucine, arises by
misaminoacylation and depends on conditions during cell growth (1). Norvaline substitutions in rHb1.1 occur
late in fermentations after most cell protein mass has already
accumulated (1); thus, norvaline is not typically found in
E. coli proteins. Highly purified rHb1.1 samples were
analyzed for the frequency of this substitution. The level of norvaline
measured in purified rHb1.1 varied by at least 1 order of magnitude
(Table 1). The frequency of this substitution was not correlated with
the purification yield, with the level of isoleucine misincorporation
(Fig. 4), or with hemoglobin functionality (P50, Hill max)
(Table 1).
Another advantage of examining norvaline substitution is that since
norvaline is not typically found in
E. coli proteins,
contaminants should not interfere with measurements of norvaline
in
rHb1.1 in crude samples. We studied norvaline substitution
throughout
purification to see if bias was introduced into the
final pure sample
by removal of the substituted population. Several
additional
fermentations were performed under conditions likely
to generate
relatively high levels of norvaline substitution (
1).
The
norvaline substitution levels in crude rHb1.1 from two early
purification steps were compared to norvaline levels in final,
highly
purified rHb1.1 (Table
4). Purification
did not significantly
influence the quantity of norvaline in the
samples. The norvaline
level in highly purified rHb1.1 was 104% ± 15% of the level found
in crude rHb1.1 pools.
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DISCUSSION |
A serious dilemma confronts investigators attempting to determine
translation error in specific proteins. A crude protein preparation
represents a large sample of the population of protein molecules of
interest, but contaminants can produce misleading signals; however, a
highly purified protein preparation could be biased if the purification
procedure selectively eliminates the mistranslated members of the
population. In addition, many of the proteins studied contain at least
one residue of each amino acid. Therefore, researchers analyzing the
inappropriate presence of absent amino acids frequently rely on only a
fragment of the protein and extrapolate mistranslation of the whole
protein from the measurement obtained for this fragment (2,
15). Our approach examines mistranslation in the whole protein
and avoids signals arising from contaminants. Several lines of evidence
indicate that rigorous sample purification is unlikely to bias our
results significantly. We examined translational fidelity during
overexpression of recombinant hemoglobin by taking advantage of the
absence of isoleucine in the amino acids of rHb1.1. Therefore, most of
the isoleucine found in purified rHb1.1 samples could be attributed to
mistranslation. Misacylation could also have accounted for some Ile in
rHb1.1 samples, but the fidelity of aminoacylation is generally greater
than ribosome fidelity by at least 1 order of magnitude (5),
so this class of translation errors probably contributed little to the
Ile detected in these experiments.
Many (33%) of our samples had no quantifiable translation error (error
rate, 0.0002). The average number of errors for rHb1.1 translation
was 6 in 10,000 codons (Table 3). This is within the normal range of
the error rates measured for homologous proteins in E. coli.
When the samples with error rates of 2 × 104 were
not included in the analysis, the upper estimate of the error rate was
8 × 104. There was no significant difference
between the two strains studied, even though the level of rHb1.1
accumulation was much higher in SGE1464. The average error rate
for SGE1662 was 0.0005 ± 0.0003 (n = 13), while
the average error rate for SGE1464 was 0.0007 ± 0.0005 (n = 2), suggesting that higher levels of soluble and/or total globin expression did not increase mistranslation in
highly purified samples. The average error rate for purified soluble
rHb1.1 (6 × 104) was at least 2 orders of
magnitude lower than the average error rate for another highly
expressed recombinant protein, recombinant mEGF (18).
Purification can affect the measurement of translation error in two
ways; first, contaminating E. coli protein can contribute an
isoleucine signal in AAAA, and second, the purified sample may be
biased by the purification techniques if they eliminate a higher
proportion of the mistranslated protein. Since the level of E. coli protein can result in an overestimate of Ile, we measured the
E. coli protein contents of our samples. Only one rHb1.1
sample contained more than 1 ppm of E. coli proteins, and
most E. coli protein levels were below the limit of
quantitation (<0.16 ppm). A sample containing 0.1 mol of Ile per mol
of rHb1.1 would require about 0.4% E. coli protein
contamination, assuming that E. coli proteins contain 5%
Ile, which is typical (Table 5). The levels of E. coli
proteins in our samples were at least 3 to 4 orders of magnitude lower
than this and therefore could not significantly contribute to the Ile
signal measured.
Purification bias might be introduced if it selectively removes
molecules containing mistranslations, resulting in a virtually error-free product. The scatter plot of the Ile mistranslation levels
distributed among the various purification yields (Fig. 4) did not
reveal any purification bias that affected our results. We
simultaneously measured a second, independent mistranslation event,
norvaline substitution for leucine, which is due to misaminoacylation (1). We reasoned that the levels of amino acid
misincorporation, whether occurring by mistranslation or
misaminoacylation, should be similarly affected by the protein
purification procedure. Again there was no correlation between the
purification yield and the level of norvaline substitution (Fig. 4) or
between norvaline misincorporation and isoleucine misincorporation
(Fig. 4). In addition, norvaline is not typically found in native
E. coli proteins, which allowed us to easily measure
norvaline substitution in upstream (crude) samples. The proportions of
this mistranslation were the same for crude rHb1.1 and for highly
purified rHb1.1, indicating that purification did not affect norvaline
mistranslation measurements. We also saw no evidence that the rate of
either type of mistranslation correlated with rHb1.1 functionality
(Tables 1 and 3).
Although highly purified, our samples represented a large portion of
the soluble rHb1.1, averaging about 30% of the starting material in
the experiments. Since the soluble rHb1.1 accounts for at least 60 to
65% of the total globin protein, purified samples included 20 to 30%
of the total globin. Therefore, the measured rate of translation error
was the average rate for a large fraction of the total globin
population. It may have represented the lower limit of mistranslation
for globin, but was unlikely to differ a great deal from the rate for
the entire globin population, assuming that translation errors are
normally distributed among proteins and Ile-substituted proteins behave
like norvaline-substituted proteins during purification. In rHb1.1,
most of the globin codons have been optimized for E. coli
expression (4, 10). Since translation errors are strongly
context dependent (15), codon optimization may have removed
the opportunities for mistranslation at a rate other than the rate
typified by E. coli proteins.
It has been proposed that high-level overexpression of a heterologous
protein having a distribution of amino acids atypical for E. coli could result in overutilization of some amino acids. This
could result in amino acid starvation or other perturbations in
E. coli amino acid availability. Starvation or depletion of particular amino acid pools could stimulate a compensatory increase in
the mistranslation error rate, especially for those codons involving
cognate tRNAs usually charged with amino acids made scarce by
overutilization (reviewed in reference 8). We
calculated the effect of rHb1.1 overexpression on E. coli
amino acid requirements and found it to be surprisingly small. Assuming
that accumulation of rHb1.1 accounted for about 25% of the E. coli protein, a value slightly greater than the value measured in
SGE1464 fermentations, the requirement for histidine increased by 68%,
but no other amino acid requirement increased by more than 13% (Table
5). It appears that the level of histidine utilization did not affect
E. coli growth to a degree indicative of amino acid
starvation, since no significant difference was observed in the growth
rates and cell densities of SGE1464 cultures in which rHb1.1 synthesis
was induced or not induced (18a). This suggests that the
amino acid requirements in E. coli do not appear to be
seriously perturbed by rHb1.1 overexpression. However, our method was
not suitable to address the potential for His substitutions.
We quantified translation missense errors in highly purified samples
representing 20 to 30% of the total population of globin molecules by
using AAAA. The fidelity of translation of this portion of rHb1.1 in
E. coli is indistinguishable from the fidelity of translation of homologous proteins. This may indicate that when native
E. coli codons are used, even expression of globin proteins accounting for up to ~20% of the total protein can be accommodated without seriously compromising the quality of the purified fraction of
this large, complex protein. It appears that overexpression of
heterologous proteins in E. coli is not necessarily
deleterious to the fidelity of translation, which is especially
reassuring for those proteins destined for therapeutic use, like
rHb1.1.
| |
ACKNOWLEDGMENTS |
We sincerely thank Maria Pagratis for technical assistance in the
insoluble globin analysis and Mary Simonson, Julie MacGregor, Chris
Tapparo, Bruce Whitesel, Jared Rowe, and Daryl Ogden for soluble
hemoglobin measurements and functionality testing. We also greatly
appreciate the assistance of a group, too large to name individually,
that was responsible for fermentation and purification of rHb1.1. We
also thank Doug Looker, Dan Doherty, Jeff Etter, and Spencer
Anthony-Cahill for their comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Present address: Ligand
Pharmaceuticals, Inc., 10275 Science Center Dr., San Diego, CA 92121. Phone: (619) 550-7664. Fax: (619) 550-7801. E-mail:
mweickert{at}ligand.com or weickert{at}aol.com.
| |
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
