From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232
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ABSTRACT |
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The relationship between glucokinase (GK) and
glucose-stimulated metabolism, and the potential for metabolic coupling
between Glucose-stimulated insulin secretion by pancreatic A second, less well studied feature of glucose-stimulated insulin
secretion from To elucidate the roles of GK and intercellular communication in glucose
metabolism within intact pancreatic islets, we utilized three recently
developed technologies. First, the Cre/loxP strategy for inducible gene
knockouts enables the elimination of specific genes in vitro
in tissues from mature animals (16). To use this strategy, mice have
been created with a conditional GK gene allele (gklox) in which exons 9 and 10 of the GK gene
are flanked by loxP sites, thereby allowing for its inactivation by Cre
recombinase (6). Second, specific inactivation of single genes in
isolated islets has been enhanced by the development of a recombinant
adenovirus expressing Cre (AdenoCre) (17). Adenoviruses have been shown previously to be an efficient means of introducing genes into The combination of these three recent methodological advances enabled
us to directly examine the role of GK in glucose-stimulated metabolism
in single Animals--
Mice that contain a conditional GK gene allele
(gklox) have been described previously (6). All
animals used in this study were homozygous at this allele
(gklox/lox) in a mixed C57Bl/6-129SvEvTac
genetic background. This conditional allele is identical to the
wild-type allele, but contains loxP sites flanking exons 9 and 10 of
the gene. The neomycin resistance cassette used to perform the gene
targeting is absent in the gklox allele. PCR
primer pairs for distinguishing the gklox and
gkdel alleles have been described previously
(6).
Preparation of AdenoCre--
293 cells (21) were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
AdenoCre (obtained from F. Graham, McMaster University, Ontario,
Canada) expresses Cre under the control of the cytomegalovirus
immediate early promoter (17). This virus was grown and purified
according to Becker et al. (22). High titer stocks of
adenovirus (3-7 × 1012 plaque-forming units/ml,
determined as 1 A260 Islet Isolation and Culture--
Islets were isolated from
gklox/lox mice by distention of the splenic
portion of the pancreas followed by collagenase digestion (23, 24).
Islets were cultured at 37 °C in RPMI 1640 medium containing 20 mM glucose, 10% fetal bovine serum, 100 IU/ml penicillin,
and 100 µg/ml streptomycin (Life Technologies, Inc.) with 5%
CO2 atmosphere. For each experiment islets were divided
into two groups. The first group was incubated in 2 ml of culture
medium containing recombinant adenovirus at a concentration of 1.5 × 109 plaque-forming units/ml for 1 h at 37 °C
prior to culturing (18). Higher concentrations of adenovirus were also
tried, but did not result in greater infection efficiency. The second
group was not exposed to adenovirus. After infection islets were washed
three times in culture medium. Both groups of islets were maintained in
culture for 4 days before imaging. For the AdenoCre treated islets,
culture in 20 mM glucose maintained their viability and morphological integrity better than those cultured at lower glucose levels.
Analysis of GK Protein Levels--
Western blot analysis
performed on preparations from isolated islets and GK activity was
measured in isolated islets as described previously (6).
Immunofluorescence staining of GK was also performed on isolated islets
as described previously (13), except that an affinity-purified sheep
anti-GK-IgG was used at a 1:10 dilution, and preimmune sheep serum was
used as the control. The primary antibody was detected by donkey
anti-sheep IgG-CY3 (Jackson ImmunoResearch, West Grove, PA) and imaged
using a Zeiss LSM410 laser scanning confocal microscope (Vanderbilt
Cell Imaging Resource).
Preparation of Islet DNA--
Pooled islet preparations (~500
islets each) were placed in a 0.5-ml Eppendorf tube with 50 µl of 50 mM Tris (pH 8), 100 mM EDTA, 0.5% SDS, and 2.5 µl of a 10 mg/ml solution of proteinase K, and this was incubated at
55 °C for 2 h. Samples were extracted once each with equal
volumes of phenol, phenol/chloroform, and chloroform, respectively.
Islet genomic DNA was precipitated with 3 M sodium acetate
and 100% ethanol and washed with 70% ethanol.
DNA Analysis--
PCR reactions were performed on DNA from
control and AdenoCre-treated islets. Two separate PCR reactions were
used to detect the gklox and
gkdel alleles respectively. Reaction mixtures
contained 1 × Perkin-Elmer PCR buffer, 0.2 mM each
dNTP, 0.4 µM amounts of each primer, and 0.5 µl of
Perkin-Elmer AmpliTaq Gold in 100 µl total volume. Reaction products
were visualized by agarose gel. DNA from control and AdenoCre-treated
islets were further analyzed by Southern blot. 4 µg of genomic DNA
from control and AdenoCre-treated islets was digested with
BglII in a 25-µl reaction for 4 h at 37 °C.
Digested samples were run in a 0.8% agarose gel, then blotted onto
Zeta-Probe membrane overnight. A 0.5-b
EcoRI/BamHI fragment of the gk gene located ~1.4 kilobases 3' of the targeted region was used as the hybridization probe to distinguish the gklox and
gkdel alleles (6). The resulting autoradiogram
was quantified by densitometry.
Measurement of NAD(P)H Autofluorescence--
NAD(P)H imaging was
performed by TPEM as described previously (13). For imaging, two islets
(one from each group) were adjacently attached to a coverslip bottom
dish (Mat-Tek Corp.). A 0.5-µl drop of Cell-Tak (Collaborative
Biomedical Products) was placed in the center of the dish and dried for
30 s at 42 °C; the dish was rinsed with Hanks' balanced salt
solution (Life Technologies, Inc.) and the islets placed on the
Cell-Tak. During imaging, the islets were perifused at 1 ml/min with
BMHH buffer (125 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2,
and 10 mM HEPES, and 0.1% bovine serum albumin (pH 7.4)).
The sample was held at 37 °C with a microincubator (TLC-MI, Adams & List Associates, Westbury, NY) that heated both the sample dish and
incoming perifusate. An air stream incubator (Nicholson Precision
Instruments, Gaithersburg, MD) heated the objective to eliminate heat
transfer through the glass-oil-objective interface. All NAD(P)H
autofluorescence measurements were made after a 15-min equilibration
period on the microscope stage at 1 mM (basal) glucose.
NAD(P)H glucose dose response images were acquired after a 5-min
equilibration at each glucose concentration. Three consecutive 3-s
scans were averaged to form the image for each concentration.
Image Analysis and Quantitation--
To quantify the glucose
response of NAD(P)H autofluorescence, digital image analysis was
performed on Macintosh Power PC computers running NIH Image 1.61 (Bethesda, MD). Single cell data were taken in 25-pixel circular
regions of interest that did not include the cell nucleus; the same
regions of interest was used for all measurements (in images acquired
with different glucose perifusion concentrations) on that cell. Only
cells that remained in the same location within the image and
maintained its same total area (to within 3%) were used in the
analysis. This excluded cells that might have shifted into or out of
the focal plane during the experimental procedures.
Both the ability and efficiency of AdenoCre to cause recombination
in islets from mice that were homozygous for the
gklox allele was determined. PCR analysis of
AdenoCre-treated islets revealed the conversion of the
gklox allele to the gkdel
allele only after virus treatment (Fig.
1A). To assess the efficiency of AdenoCre-mediated recombination, DNA was isolated from ~500 each
of AdenoCre-treated and control islets and analyzed by Southern blot
analysis. Densitometry of the resulting autoradiograms revealed that
AdenoCre had converted ~30% (shown in Fig. 1B) of the
gklox alleles to the
gkdel allele (lane 2). Similar
analysis of DNA from untreated gklox/lox islets
did not indicate any gkdel allele (lane
1).
cells, was examined in isolated mouse islets by using a
recombinant adenovirus that expresses Cre recombinase (AdenoCre) to
inactivate a conditional GK gene allele
(gklox). Analysis of AdenoCre-treated islets
indicated that the gklox allele in ~30% of
islet cells was converted to a nonexpressing variant
(gkdel). This resulted in a
heterogeneous population of
cells where GK was absent in some
cells. Quantitative two-photon excitation imaging of NAD(P)H
autofluorescence was then used to measure glucose-stimulated metabolic
responses of individual islet
cells from
gklox/lox mice. In AdenoCre-infected islets,
approximately one-third of the
cells showed markedly lower NAD(P)H
responses. These cells also exhibited glucose dose responses consistent
with the loss of GK. Glucose dose responses of the low-responding cells
were not sigmoidal and reached a maximum at ~5 mM
glucose. In contrast, the normal response cells showed a sigmoidal
response with an KcatS0.5 of ~8
mM. These data provide direct evidence that GK is essential
for glucose-stimulated metabolic responses in
cells within
intact islets and that intercellular coupling within the islet plays
little or no role in glucose-stimulated metabolic responses.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cells is a
multistep process that depends on increased metabolic flux (1). The
rate-determining step in
cell glucose metabolism is widely thought
to be glucose phosphorylation, which is catalyzed to a large extent by
glucokinase (GK)1 at
physiological glucose concentrations (2). Although many studies have
implied an essential role for GK in glucose metabolism, most of this
information has been correlative and has not precisely defined the role
of this particular hexokinase isoform in
cell glucose usage. GK
gene knockout studies in transgenic mice, which offer the most direct
and minimally ambiguous route to assessing the function of this enzyme
in
cells, have been performed by several different groups. Both
global gene knockout studies, and more recent
cell-specific gene
knockouts indicate that GK is indispensable for glucose-stimulated
insulin secretion (3-6). While these studies clearly demonstrate an
essential role for GK in glucose homeostasis, they have been unable to
determine the precise role of GK, since studies in
cells from adult
animals are prevented by the perinatal mortality that occurs in GK-null mice.
cells is its dependence on cell-cell interactions. The amount of insulin secreted from intact islets has long been known
to be greater than that secreted from an equivalent number of dispersed
cells (7). Models to explain this behavior generally include
cooperative phenomena between islet cells, as suggested by the presence
of synchronous electrical responses from clustered
cells and intact
islets (8, 9). The pharmacological blocking of gap junctions reduces
islet insulin secretion, thereby suggesting that conexins are involved
in the intercellular cooperation (10-12). Based on measurements of
NAD(P)H in both intact islets, we previously proposed that metabolic
uniformity arises from a uniform GK distribution and is not dependent
on intercellular coupling (13). The concept of uniform cell-to-cell GK
distributions is also supported by a recent immunofluorescence study of
intracellular expression patterns in intact islets (14). Given that the
role of cell-cell communication within intact islets during
glucose-stimulated metabolic flux has never been directly examined, and
that glucose-stimulated metabolism in islets does not exhibit the
NAD(P)H response heterogeneities observed in isolated
cells (15),
it remains possible that intercellular coupling may play a role in
generating the metabolic uniformity.
cells
in isolated islets (18). Third, we used two-photon excitation microscopy (TPEM) to study glucose-stimulated processes within intact
islets (19, 20). This quantitative optical sectioning technique has
been demonstrated to be useful for assessing glucose-stimulated metabolic responses in intact islets and allows simultaneous
determination of the glucose dose response in many individual
cells
(13).
cells within intact pancreatic islets. By exposing
cultured islets isolated from mice that are homozygous for the
conditional gklox allele to AdenoCre, we have
been able to eliminate GK in a sizable fraction of
cells and to
examine interactions between cells with normal GK levels and those that
are not expressing GK. These data provide direct evidence that GK is an
important component of the
cell glucose sensor. However, even cells
that are presumed to fully lack GK retain an attenuated glucose
response, thus suggesting that the glucose sensor is multicomponent and
likely involves hexokinases other than GK. In addition, these data
provide compelling evidence that individual
cells function as
independent glucose-sensing units at the metabolic level.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1012
particles/ml) were prepared by equilibrium centrifugation in CsCl,
stored in small aliquots at
80 °C, and used immediately after thawing.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Conversion of gklox
allele to gkdel allele by treatment of isolated
islets with AdenoCre as determined by PCR and Southern blot
analysis. A, PCR amplification shows that the deleted
GK gene (gkdel allele) was present only in
AdenoCre-treated islets. The intact GK gene
(gklox allele) was detected in both control and
AdenoCre-treated islets. B, representative Southern blot of
the GK gene shows that while no recombination occurs without AdenoCre
exposure (lane 1), ~30% (n = 2, 28 and
31%) of the islet gklox DNA has been converted
to gkdel by in vitro exposure to
AdenoCre (lane 2). C, Western blot showing
reduction of GK protein after in vitro exposure to AdenoCre
(60 islets per group). In this representative blot, AdenoCre treatment
caused a 34% decrease in GK protein level (lane 2) as
compared with untreated gklox/lox islets
(lane 1).
While the appearance of the gkdel allele predicted a partial loss of GK expression in AdenoCre-treated islets, the actual reduction of GK levels was assessed by Western blot, GK activity measurements, and GK immunofluorescence. Western blot analysis yielded a 29.7 ± 4.4% reduction in GK levels from AdenoCre-treated islets (n = 3; shown in Fig. 1C), and activity measurements (n = 3, data not shown) also showed reduced GK activity in AdenoCre-treated islets. Because adenovirus treatment disrupts islet integrity, GK immunofluorescence could not be accurately determined since none of the islets which were imaged for NAD(P)H autofluorescence responses survived the fixation and staining procedures (n = 28 islets). However, four GK immunostained islets each showed reduced GK levels and exhibited heterogeneous GK immunofluorescence, similar to the NAD(P)H results presented below (data not shown).
To determine whether AdenoCre-mediated elimination of GK caused differences in glucose-induced NAD(P)H responses, control and treated gklox/lox islets were examined side-by-side using TPEM. Representative NAD(P)H autofluorescence images of control and treated islet pairs are shown in Fig. 2. Control islets showed a very uniform autofluorescence signal during the 1 mM glucose perifusion, which was enhanced by perifusion with the higher glucose. In contrast, AdenoCre-treated islets exhibited significant heterogeneity of NAD(P)H response. Under low glucose perifusion, the latter group of islets showed low but fairly uniform autofluorescence patterns, but in response to high glucose, many cells failed to show the expected rise in NAD(P)H.
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To assess quantitatively the metabolic response of individual cells
within whole islets, NAD(P)H response ratios (defined as (NAD(P)H
signal at 25 mM glucose perifusion)/(NAD(P)H signal at 1 mM glucose perifusion) were determined for 220 nonperipheral
cells from 5 AdenoCre-infected islets. A histogram of
the resulting ratios (Fig. 3) shows two
separate distributions of cellular response. Approximately two-thirds
of the analyzed
cells showed a response similar to that observed in
wild-type mice. The other approximately one-third of the cells formed a
population with a lower NAD(P)H response. The solid line in
Fig. 3 indicates the response ratios of > 1000
cells in
islets isolated from wild-type mice. The right-hand peak of the
histogram shows that
cells from gklox/lox
islets are ~5% less responsive than cells from wild-type mice. The
one-third AdenoCre infection rate is consistent with the 35% of islet
cells that were infected with another adenovirus, which expresses
-galactosidase.
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Measurements of NAD(P)H levels over a range of different glucose
concentrations were performed to further define the metabolic responses
of single cells in AdenoCre-treated islets. Two resulting glucose
dose response curves are shown in Fig. 4.
The first curve (solid circles) was generated from 10 normal
response
cells in two islets (cells that fall near the
right-hand peak of the histogram in Fig. 3). This curve is
sigmoidal with an inflection point at ~8 mM glucose,
consistent with KcatS0.5 of GK, thus
indicating that GK is the main determinant of glucose metabolism in
these cells. The second curve (open circles) was generated
from eight low response
cells in the same two islets (these cells
fall near the left-hand peak of the histogram in Fig. 3).
This curve is not noticeably sigmoidal and reaches a maximal value at
~5 mM glucose. The kinetics of response in these cells is
consistent with glucose usage being determined by one or more other
hexokinase gene family members with lower Km values
and not by GK.
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DISCUSSION |
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Many previous studies have pointed to a rate-determining role for
GK in glucose-stimulated responses of cells (2, 25-28). While the
evidence for GK as the
cell glucose sensor is compelling, it has
been difficult to functionally distinguish the role GK from other
hexokinases in
cells. Mannoheptulose, a competitive inhibitor of
hexokinases, has been used to inhibit glucose phosphorylation in
cells (e.g. Ref. 27), but it is not specific for GK. In fact, application of mannoheptulose to AdenoCre-treated islets caused a
uniform decrease in autofluorescence from all
cells (not shown).
Thus, to distinguish the specific role of GK apart from other
hexokinase isoforms, we have made use of the highly specific Cre/loxP
system to inducibly eliminate GK within
cells in intact islets.
This approach allows GK gene expression to remain normal in the adult
animal prior to islet isolation, and avoids the lethality caused by
either a global or
cell-specific deficiency in GK (3-6). By using
a recombinant adenovirus to express Cre, conversion of the
gklox allele to gkdel
allele was delayed until after islets are isolated from the adult animal. While an infection efficiency of only ~30% was achieved using this approach, it offered the advantage in this instance of
creating heterogeneities of glucose sensing within the islet. These
heterogeneities allowed us to examine the role of cell-cell communication during glucose-stimulated metabolic activity, in addition
to determining the specific functional role of GK.
The results from these studies are important in at least two regards.
First, they provide strong additional evidence that GK does indeed
function as the glucose sensor in cells. TPEM analysis showed that
approximately one-third of the cells in AdenoCre-treated islets lacked
a normal metabolic response to glucose (Figs. 2 and 3). This percentage
was closely correlated to the amount of Cre-mediated recombination
determined by Southern blot. Because AdenoCre uses the potent immediate
early cytomegalovirus promoter to drive Cre expression, infection of a
cell probably results in efficient Cre expression and thus
recombination of both copies of the gklox allele
into the gkdel allele. Because of this,
intermediate levels of GK gene expression within infected cells
(i.e. recombination of only a single allele) are unlikely,
and the amount of recombination probably reflects the percentage of
cells without GK. Similar percentages were observed in Western blots
for GK. Unfortunately, it was not possible to examine the
AdenoCre-treated islets by immunofluorescence after NAD(P)H imaging
because, unlike normal islets (13), they did not remain immobilized on
the Cell-Tak during fixation (despite over 20 attempts). We have found
this to be a limitation regardless of the recombinant adenovirus used.
Consequently, we were unable to correlate NAD(P)H responses with either
Cre infection or GK expression on a cell-by-cell basis in the islets.
Nonetheless, a few AdenoCre-treated islets that did survive the
immunostaining process in parallel preparations showed increased
heterogeneity and an overall reduction in GK immunofluorescence
compared with untreated gklox/lox islets.
Furthermore, cells that were presumed to lack GK showed a muted NAD(P)H
response to glucose that saturated at a lower glucose concentration
than the more highly responding cells, consistent with glucose
phosphorylation by other hexokinases.
It is well established that other hexokinases are expressed within cells and that glucose responsiveness requires a high GK/hexokinase
ratio (29). Indeed, transformed
cells maintained in culture for
extended times demonstrate a lower GK/hexokinase ratio and exhibit
diminished glucose-stimulated responses (30). Thus, the significant
NAD(P)H response observed at the lower glucose levels in the
AdenoCre-infected islet cells is consistent with the activity of
hexokinases other than GK. The absence of GK may cause an up-regulation
of these other hexokinases, all of which exhibit a more pronounced
inhibition by glucose 6-phosphate than does GK (29). Because of this
differential inhibition, however, it is difficult to assess precisely
the contribution of other hexokinases to glucose sensing in a normal
cell. To rigorously determine the role of each hexokinase isoform,
mice with conditional knockouts of each gene would have to be created
and examined.
Second, these studies demonstrate that individual cells within
intact pancreatic islets show independent metabolic responses. Because
gradients in NAD(P)H levels between adjacent
cells persist even
after 30 min, there does not appear to be any mechanism within the
islet to generate metabolic uniformity among heterogeneous cells. Thus,
we conclude that intercellular communication is not involved in
glucose-stimulated metabolic flux and that each
cell senses glucose
independently at the metabolic level. Exactly how cell-cell
communication, which clearly plays a significant role in augmenting
insulin secretion, is involved in more distal signaling events
(e.g. membrane depolarization, Ca2+ influx, and
insulin exocytosis) remains to be determined.
The detection of artificial heterogeneities among single cells
within AdenoCre-treated islets helps to further validate the use of
TPEM for high-resolution measurements of cellular metabolism in thick
samples. Because the optical section in TPEM is ~1 µm thick,
information from single cells should be uncontaminated by background
fluorescence that arises from other cells. In fact, TPEM has proved
useful for measuring activity of single synapses in brain slices using
exogenous fluorescent probes (31). Here we have shown that TPEM
measurements of NAD(P)H accurately report the metabolic flux in
individual cells. Since TPEM offers submicron resolution, it also opens
the possibility to perform subcellular metabolic measurements, such as
differentiating the NAD(P)H signals between the cytoplasm and mitochondria.
It may be important to note that islets from gklox/lox mice are slightly less responsive than islets from wild-type mice (~5% difference between the line and the right-hand peak of the histogram in Fig. 3). This was not unexpected because the blood glucose levels in the gklox/lox mice are slightly elevated (<10%) from those in wild-type mice (6). Both of these findings indicate that the gklox allele may express slightly less GK than the wild-type gene GK gene. This slight difference in responsiveness is not likely to be a limitation of the present study, since the difference in NAD(P)H response is less than we have observed between different mouse strains.2
The use of in vitro gene knockouts offers great potential
for investigations of cellular signal transduction. Because the Cre/loxP approach allows normal gene expression to continue while the
animal develops, any deleterious effects of the knockout in the whole
animal are avoided. Another advantage of targeted gene knockouts is
that, unlike pharmacological approaches, they are not, at least in
principle, prone to nonspecific effects in the cells. Although we were
able to take advantage of the heterogeneity introduced by the
adenovirus infection in this study, complete in vitro gene
knockouts could often be advantageous. To obtain 100% efficient
recombination of the loxP sites, methods other than adenovirus-mediated
transfections could be used for the introduction of Cre. One such
alternative is to use mice that contain inducible Cre transgenes
(32).
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ACKNOWLEDGEMENT |
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We thank Dr. Richard Whitesell for his help with the GK activity measurements.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK53434 (to D. W. P.) and DK42612 and DK42502 (to M. A. M.). Some image processing and analysis was performed in the Vanderbilt Cell Imaging Resource (supported by National Institutes of Health Grants CA68485 and DK20593).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.
To whom correspondence should be addressed (to Dr. Piston or Dr.
Magnuson): Dept. of Molecular Physiology and Biophysics, 702 Light
Hall, Vanderbilt University, Nashville, TN 37232. E-mail: dave.piston{at}mcmail.vanderbilt.edu or
mark.magnuson{at}mcmail.vanderbilt.edu.
The abbreviations used are: GK, glucokinase; AdenoCre, recombinant adenovirus that expresses Cre recombinase; TPEM, two-photon excitation microscopy; PCR, polymerase chain reaction.
2 S. M. Knobel, J. S. Salmon, and D. W. Piston, unpublished observations.
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REFERENCES |
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