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Proc Natl Acad Sci U S A. 2003 August 5; 100(16): 9330–9335. Published online 2003 July 22. doi: 10.1073/pnas.1633513100. | PMCID: PMC170918 |
Copyright © 2003, The National Academy of
Sciences Cell Biology Profiling receptor tyrosine kinase activation by using Ab
microarrays Ulrik B. Nielsen,*† Mike H. Cardone,*† Anthony J. Sinskey,* Gavin MacBeath,‡§ and Peter K. Sorger*§ *Department of Biology 68-371, Massachusetts Institute of Technology, Cambridge, MA 02139; ‡Bauer Center for Genomics Research, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and †Merrimack Pharmaceuticals, Inc., Cambridge, MA 02142 Received December 17, 2002. Signal transduction in mammalian cells is mediated by complex networks of
interacting proteins. Understanding these networks at a circuit level requires
devices to measure the amounts and activities of multiple proteins in a rapid
and accurate manner. Ab microarrays have previously been applied to the
quantification of labeled recombinant proteins and proteins in serum. The
development of methods to analyze intracellular signaling molecules on
microarrays would make Ab arrays widely useful in systems biology. Here we
describe the fabrication of multiplex Ab arrays sensitive to the amounts and
modification states of signal transduction proteins in crude cell lysates and
the integration of these arrays with 96-well microtiter plate technology to
create microarrays in microplates. We apply the Ab arrays to monitoring the
activation, uptake, and signaling of ErbB receptor tyrosine kinases in human
tumor cell lines. Data obtained from multicolor ratiometric microarrays
correlate well with data obtained by using traditional approaches, but the
arrays are faster and simpler to use. The integration of microplate and
microarray methods for crude cell lysates should make it possible to identify
and analyze small molecule inhibitors of signal transduction processes with
unprecedented speed and precision. We demonstrate the future potential of this
approach by characterizing the action of the epidermal growth factor receptor
inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based
screening in 96- and 384-well plates should allow small molecules to be
identified with specific inhibitory profiles against a signaling network. The signal transduction systems that control cellular physiology are
comprised of biochemical networks with shared components, common inputs, and
overlapping outputs. Understanding how signals flow through these pathways,
how the pathways vary among cell types, and how normal and diseased tissues
differ requires information on signaling networks as a whole rather than
simply on one or two components. To make network (or systems) biology
possible, we need devices that can probe the activities of signaling proteins
in a parallel and reliable manner. We envision these as a biological analog of
the multiprobe “bed of nails” testers that are a mainstay of the
electronics industry. Bed of nails testers can monitor printed circuit boards
at enough locations to fully trace and test a circuit. In this paper we describe the development of an Ab microarray integrated
with 96-well microtiter plates that can quantify the amounts and modification
states of ErbB receptors in crude cell lysates. Ab microarrays are an
extension of DNA microarrays. In both cases, ratiometric comparisons derived
from differentially labeled control and experimental samples are an effective
way to standardize measurements among and within experiments
( 1). Ab arrays have the
potential to reveal the amounts and modification states of proteins and also,
when integrated with fractionation steps, subcellular protein
compartmentalization. The use of Ab arrays has previously been described to
quantify proteins in serum and to measure the levels of fluorescently labeled
recombinant proteins
( 2– 6).
It might be assumed that building arrays for cell signaling processes
represents a direct extension of this technology. However, we and others
( 7) have discovered that
reducing array-based analysis of signaling proteins to practice has required
new fabrication and experimental methods. To determine the critical steps in fabricating Ab arrays for signal
transduction, we have focused on early events in ErbB receptor activation
( 8). The epidermal growth
factor receptor (EGFR or ErbB1) is a prototypical receptor tyrosine kinase
whose intracellular domain becomes phosphorylated on a series of tyrosine
residues after activation by EGF
( 9). ErbB2 (also known as HER2)
is a structurally related protein that does not appear to bind extracellular
ligands but is a potent oncogene
( 10,
11). ErbB2 is phosphorylated
in response to EGFR activation
( 12), and EGFR and ErbB2 act
together to regulate cellular proliferation. Misregulation of EGFR and ErbB2
is implicated in a wide variety of cancers, and a humanized mAb against ErbB2,
Herceptin, is effective for the treatment of metastatic breast cancer
( 13). We show here that Abs
specific for EGFR, ErbB2, and their tyrosine-phosphorylated forms can be used
to monitor the levels and activities of receptor tyrosine kinases in a
multiplexed, ratiometric microarray format. We use Ab microarrays and a panel
of tumor cell lines to demonstrate five applications of microarrays to the
study of ErbB signaling: ( i) profiling protein abundance,
( ii) profiling the functional state of a signaling system,
( iii) analyzing the kinetics of ligand-activated signaling,
( iv) measuring the in vivo inhibitory constant of a small
molecule EGFR inhibitor, and ( v) array-based profiling in 96-well
plates for screening. We find that cell lines differ markedly not only in the
levels of ErbB proteins that they express, as expected, but also in their
responsiveness to EGF activation. A direct scale-up of the Ab microarrays
described here to 10–20 independent elements will permit the systematic
analysis of a complete signal transduction system in normal and diseased
tissues in a rapid and parallel fashion. Ab Array Methods. BSA- N-hydroxysuccinimide slides were
prepared as described ( 14).
Anti-ErbB2 mAb clone 3B5, anti-pY1248-ErbB2 mAb clone PN2A, anti-EGFR mAb
clones 199.12, and anti-EGFR 111.6 were purchased from Lab Vision (Fremont,
CA). Anti-transferrin receptor (TfR) mAb clones 7F8 and 11F5 were purchased
from Research Diagnostics (Flanders, NJ). Polyclonal rabbit anti-pY1068-EGFR
was purchased from BioSource International (Camarillo, CA). Recombinant
humanized mAb Herceptin was obtained from the pharmacy. Anti-ErbB2 scFv F5 was
produced as described ( 15) (a
kind gift from James D. Marks, University of California, San Francisco).
BSA- N-hydroxysuccinimide slides were prepared as described
( 14) and affixed to bottomless
96-well plates (Greiner, Nurtingen, Germany) by using adherent precut gaskets
(Grace BioLabs, Bend, OR). Abs clones 111.6, 11F5, and Herceptin were spotted
at 0.5 mg/ml in PBS containing 40% glycerol by using a GMS 417 Arrayer
(Affymetrix, Santa Clara, CA), and the resulting Ab microarrays were stored at
4°C. Typically, 12 Ab arrays (or 96 for plates) were spotted per slide and
separated with a hydrophobic pen or silicone gasket. Slides were blocked with
glycine and BSA immediately before use
( 14). Detection Abs 3B5,
199.12, 7F8, anti-pY1068, and F5 scFv were labeled with Cy3, Cy5 (Amersham
Biosciences), or Alexa 488 (Molecular Probes) as recommended by the
manufacturer. Recombinant extracellular domain (ECD) of ErbB2 and EGFR was
expressed in Chinese hamster ovary cells [kind gifts of James D. Marks and
Greg Adams (Fox Chase Cancer Center, Philadelphia), respectively]. Purified
transferrin receptor was obtained from Research Diagnostics. Recombinant human
EGF was obtained from PeproTech (Rocky Hill, NJ). The small molecule inhibitor
PD153035 (4-[(3′-bromophenyl)amino]-6,7-dimethoxyquinazoline) of EGF
receptor kinase was purchased from Calbiochem and dissolved at 2 mM in DMSO
before use. All cell lines were obtained from American Type Culture Collection
and cultured in the recommended media. Extracts of cells grown in 6- or
12-well tissue culture plates were prepared by passing cells five times
through a 27-gauge needle in lysis buffer (20 mM Tris, pH 7.5/150 mM NaCl/1 mM
EDTA/1 mM EGTA/1% Triton X-100/0.5% Nonidet P-40/10 mM; alternatively, the
lysis buffer contained 0.25% SDS in place of Triton X-100 and Nonidet P-40)
containing phosphatase inhibitors (β-glycerolphosphate/10 mM NaF/1 mM
Na 3VO 4) to minimize changes in the phosphorylation state
after lysis. In addition, protease inhibitors were added (1 mM PMSF/1 μg/ml
leupeptin/1 μg/ml pepstatin) to reduce protein degradation, and incubations
were carried out on ice. For fluorescent cell surface labeling, cells were
washed five times in cold PBS, incubated with 10 mg/ml of
fluorescein-polyethylene glycol 2000- N-hydroxysuccinimide (Shearwater
Polymers, Huntsville, AL) on ice for 2 h, and then washed five times with cold
PBS before blocking with 100 mM glycine in PBS. Before lysis, cells were
washed twice in cold PBS. Arrays were incubated with lysates mixed 1:1 with 2%
BSA in PBS containing 0.1% Tween for 3 h at 4°C before washing three times
in PBS containing 0.1% Tween and three times in PBS. Arrays in 96-well plates
were washed with an automated plate washer (BioTek). When the sandwich assay
was performed, the arrays were further incubated with fluorescently labeled
detection Abs for 1 h at room temperature and washed again. Arrays were
quantified by using an Applied Precision (Issaquah, WA) ArrayWoRx scanner or
on a Tecan (Durham, NC) LS400 (in the case of 96-well plates). Spots were
quantified by using arrayworx software (Applied Precision) and
fluorescent signals corrected for local background. Immunoblotting and Flow Cytometry. Abs PN2A and anti-pY1068 were
used for immunoblotting at 1:1,000 dilution and detected with
goat–anti-mouse-horseradish peroxidase or
goat–anti-rabbit-horseradish peroxidase, respectively. Ab clones 111.6,
11F5, and Herceptin were used for flow cytometry at 2 μg/ml and detected
with goat–anti-mouse FITC or goat–anti-human FITC (Sigma). Developing an Ab Microarray for Cell Signaling Molecules. To build
an Ab array, mAbs specific to EGFR, ErbB2, and TfR were printed onto
BSA-coated glass slides at a density of ≈1,600 spots per cm 2 by
using a contact printing robot
( 14). The activated BSA on the
slides serves to passivate the surface and to covalently link the Abs. We
first tested the sensitivity and linearity of the Ab arrays by using
recombinant proteins labeled directly with dye
().
Extracellular domains of the three receptors were conjugated to fluorophores
as follows: ErbB2 to Alexa 488 (blue), EGFR to Cy5 (red), and TfR to Cy3
(green; TfR serves as a control for a receptor that is essentially unaffected
by EGF stimulation). Specific binding of labeled antigens to arrays could be
detected over a range of 0.1–100 ng/ml
(). Next, we tested an indirect detection method based on
sandwich assays. Unlabeled antigens, individually or in a mixture, were bound
to “capture” Abs linked to BSA-coated slides. The slides were then
washed and the captured antigens detected by the addition of a mixture of
labeled secondary Abs (detection Abs; ). One detection Ab is required for each antigen, and
it must bind to a different epitope than that recognized by the capture Ab,
permitting an Ab sandwich to form. When we used this indirect microsandwich
detection, we could quantify purified antigens over a 1,000-fold range and
down to ≈1 ng/ml, making indirect detection ≈10-fold less sensitive than
direct labeling (). We conclude from these data that our high-density
Abs microarrays, using either direct or indirect detection, are linear over a
wide-range of antigen concentrations and at least as sensitive as standard
ELISA methods ( 6). Next, we tried to measure the levels of EGFR, ErbB2, and TfR in A-431
squamous carcinoma and SK-BR-3 breast cancer cell lysates
(). A-431 cells
have high EGFR and low ErbB2 levels, whereas the opposite is true of SK-BR-3
cells. TfR serves as a control protein whose levels should not vary among
experiments. We first attempted direct detection because it appeared to be
more sensitive and because it represents a direct extension of previous work.
After considerable testing and optimization, we determined that our Ab arrays
worked best when cells were labeled with fluorescent dyes conjugated to
polyethylene glycol and extracts prepared by lysis in dilute SDS buffer. When
A-431 and SK-BR-3 cells were labeled with activated fluorescein-polyethylene
glycol and then analyzed on Ab arrays, ErbB2 and EGFR could be detected in the
expected relative amounts (). Sample-to-sample variation was high, however, and
TfR was undetectable. In contrast, when unlabeled cell extracts were analyzed
by using microsandwich assays, all three cell surface proteins could be
detected, the coefficient of variation among repeat determinations was
typically <10%, and a very good correlation (r > 0.99 for ErbB2
and EGFR) was observed between receptor levels measured by using microarrays
and the receptor levels measured by using conventional flow cytometry
(). Thus, although both direct labeling and microsandwich
methods can be used with Ab arrays to detect cell surface receptors expressed
at endogenous levels, the microsandwich method appears to work better with
cells and cell extracts and, given the variability of cell-surface labeling
reactions, is better suited to high-throughput biology. Experiments with
purified proteins suggest that direct labeling has a higher ultimate
sensitivity. We suspect low dye-to-receptor stoichiometry to be the primary
problem with direct labeling of cell lysates. It is also possible that
coupling receptors to dyes via lysine residues interferes with their binding
to Abs (although this did not appear to be a problem in experiments with
recombinant proteins). Ratiometric Profiling ErbB-Signaling Activity. For microarray-based
profiling to provide useful data on cell signaling proteins, it is important
to monitor their state of activation. We therefore asked whether levels of
phosphotyrosine 1068 (pY1068) on EGFR and phosphotyrosine 1248 (pY1248) on
ErbB2 could be detected on microarrays by using phospho-specific Abs. The
phosphorylations at EGFR-Y1068 and ErbB2-Y1248 are excellent measures of
receptor activation because the sites are modified, in trans, by
receptor autophosphorylation and are binding sites for the Grb2 adapter
protein that initiates downstream signaling via Ras
( 16,
17). To monitor activation, receptors in cell lysates were captured on arrays by
using pan-specific capture Abs that are insensitive to the state of receptor
tyrosine phosphorylation. The arrays were then probed with a mixture of two
detection Abs, a Cy3-labeled phospho-specific Ab and a Cy5-labeled
pan-specific Ab different from the capture Ab
(). No crossreactivity
was observed when the detection Abs were used individually (results not
shown). The ratio of Cy3 to Cy5 fluorescence at each spot is proportional to
the fraction of receptor that is phosphorylated at Y1068 (EGFR) or Y1248
(ErbB2). The amount of TfR (detected by using an Alexa 488 conjugate) controls
for variations in extract preparation. In most experiments with a single cell
line, TfR signals were within 15% of each other when a constant amount of cell
extract was applied to arrays, as judged by total cell protein (data not
shown). Thus, both total protein and TfR intensities can be used for
normalization, with similar results. To activate signaling, cells were treated with EGF for 5 min and extracts
analyzed for the amount of total and phosphorylated EGFR and ErbB2. Three
tumor lines were compared to uncover cell-type specific differences. In
SK-BR-3 cells, tyrosine phosphorylation was stimulated on EGFR but not on
ErbB2; in A-431 cells, the levels of ErbB2 and EGFR tyrosine phosphorylation
increased 3- to 4-fold on EGF treatment; and in ErbB2-transfected MCF-7 cells,
the levels of pY1248-ErbB2 decreased on EGF stimulation
(; EGFR levels are
undetectable in ErbB2-transfected MCF-7 cells by flow cytometry and on arrays;
data not shown). In all cases, microarray data correlated well with data from
the same extracts obtained by immunoblotting
( and data not
shown, r > 0.98). It is possible to view the microarray data in
several different ways. For example, when normalized to TfR (or total
protein), the levels of tyrosine-phosphorylated EGFR and ErbB2 (5 min after
EGF stimulation) were observed to vary 8-fold or more among different cell
types. The most reproducible measurements of protein phosphorylation from
experiment to experiment (with a coefficient of variation typically ≤5%)
were obtained by calculating the ratio of signals for phospho-dependent and
phospho-independent Abs to generate ratiometric data that correlate with the
fraction of receptor activated. To demonstrate that ratiometric measurements on receptor phosphorylation
lie within the linear range of the microsandwich method, lysates from
ErbB2-transfected MCF-7 cells were diluted into lysates from nontransfected
MCF-7 cells, which have very low endogenous levels of ErbB2
(). When the
diluted lysates were analyzed on arrays, Cy5 and Cy3 fluorescence measures of
ErbB2 abundance and Y1248 phosphorylation were observed to vary linearly over
at least the 16-fold range that spans our assay conditions. The ratio of Cy3
to Cy5 fluorescence, which is proportional to the fraction of phosphorylated
receptor, varied <40% over the same 16-fold range of receptor
concentrations (). Thus, ratiometric measurements from Ab microarrays
are relatively insensitive to variables such as sample loading and constitute
reliable measures of receptor phosphorylation (with an error of approximately
±25%). In our hands, this level of accuracy is at least as good as that
obtained with Western blots. We conclude from these data that Ab microarrays represent a simple,
accurate, and rapid method to assay the protein phosphorylation events
associated with cell signaling. The rapidity and accuracy make it possible to
detect clear differences in ErbB signaling systems among different cell types.
Extending such analysis to human breast cancer samples would make it possible
to characterize ErbB signaling systems in patients before treatment with
therapeutics directed against the ErbB family of receptors. Kinetics of ErbB Signaling. Cell signaling is a highly dynamic
process in which time-dependent changes in protein activities are critical. To
explore the utility of Ab arrays in analyzing the kinetics of ErbB activation,
we analyzed extracts from SK-BR-3 cells over the course of 2 h after treatment
with EGF (). We observed
that TfR levels remained steady throughout the experiment and that the
coefficient of variation of these measurements was <5% (over three
determinations of six time points; ). The total amounts of detectable EGFR and ErbB2
dropped steadily over the course of the EGF treatment
(), reflecting
receptor internalization and degradation. The half-life of EGFR as measured on
microarrays was ≈1.5 h, identical to the value reported previously, and the
half-life of ErbB2 was ≈6–7 h, also consistent with previous data
( 18). The amount of
phospho-Y1248 ErbB2 was highest at t = 0 and then decreased steadily,
whereas phospho-Y1068 EGFR rose 5-fold by 1 min and then fell steadily. The
fraction (rather than absolute amount) of EGFR that was phosphorylated
remained above basal levels throughout the experiment, consistent with the
idea that EGF signaling has an early acute phase and then a subsequent
sustained phase ( 19). Overall,
these data reveal that Ab microarrays can be used to monitor the earliest
steps of ErbB receptor signaling. Microarray Analyses After Perturbation with Small Molecule
Inhibitors. The connectivity of signaling networks is often investigated
by selectively perturbing the networks by using small molecules. Moreover,
these small molecules can be of considerable pharmaceutical interest. To
investigate whether Ab microarrays might be used in combination with small
molecule inhibitors for this purpose, we treated A-431 cells with varying
amounts of the EGFR tyrosine kinase inhibitor PD153035
( 20)
(). As
expected, the phosphorylation of EGFR on Y1068 decreased with increasing
inhibitor concentration, as did ErbB2 PY1248, consistent with the idea that
ErbB2 is activated by EGFR and that both are inhibited in vivo by
PD153035 ( 12,
21). The apparent in vivo
Ki for PD153035 as estimated by the array method was ≈20
nM, well above the reported in vitro Ki for EGFR of 5 pM
but consistent with the IC 50 of 14 nM
( 20). It is thought that
PD153035 is much less potent on cells than on purified receptors as a
consequence of competition between the drug and endogenous ATP. Integrating Microarrays in Microtiter Plates (MIMs). High-throughput
biological analysis requires efficient handling of multiple samples. This is
accomplished most effectively by using 96- and 384-well microtiter plates. To
determine whether our Ab microarrays can produce reliable data from samples
prepared in parallel in microtiter plates, we printed 18 spots at the bottom
of each well in a 96-well plate, added a common stock of unfractionated cell
lysate by using a liquid handling robot, and then washed arrays by using
automated plate washers. Plates were then imaged in reader capable of scanning
96-well plates (). When ratiometric measurements were performed on
receptor abundance and phosphorylation the accuracy of the measurements was
high (the well-to-well coefficient of variation was ≈20%). Similarly high
accuracy was observed when cells were grown in 24-well plates, treated with
various cytokines and small molecules, lysed in situ, and then
transferred to arrays. By way of illustration, we have determined the
IC 50 of PD153035 by the MIM method to be 18 nM. These and similar
data demonstrate that MIMs will be accurate enough for cell profiling and
screening of small molecules. The importance of ratiometric measurements for
MIMs emphasizes the value of multispectral fluorescence detection as opposed
to chemiluminescence or single-color fluorescence methods
( 5,
22). In this paper we demonstrate the fabrication and use of Ab microarrays to
study ErbB signal transduction in human cells. The application of Ab
microarrays to the detection of recombinant proteins and proteins in serum has
been described ( 2,
5,
22), and it might be assumed
that fabricating arrays that can monitor intracellular signal transduction is
a simple extension of these methods. However, we are not aware of other
reports using Ab microarrays to monitor signaling in cells. Assaying
endogenous proteins in crude lysates by using microarrays requires effective
surface passivation, ratiometric measurements (necessitating multiwavelength
fluorescence, as opposed to chemiluminescence)
( 5), and Abs with high
selectivity and affinity. Here we show that suitably fabricated Ab microarrays
can be used to determine the abundance of receptors in cells, profile
signaling in different cells types, study small molecule inhibitors, and
monitor the kinetics of signal transduction. When compared to DNA microarrays,
Ab microarrays provide much more data per element. They also appear to be
about as accurate. The overall coefficient of variation for repeat
measurements of protein abundance and phosphorylation levels for the data in
this paper was ≈15%. Both direct fluorescent labeling and indirect sandwich detection can be
used for quantifying proteins bound to microarrays. Each method has strengths
and weaknesses. The most obvious advantage of direct labeling is that it
requires only one Ab per antigen. However, our data suggest that it may be
difficult to incorporate sufficient label into low abundance proteins to make
direct labeling routinely useful. Presumably, this problem will be overcome in
the future through the use of instruments designed for label-free detection
( 23). In our hands, indirect
microsandwich assays are clearly superior to direct labeling in sensitivity
and accuracy (as determined by the variation between repeated experiments).
The use of detection Abs specific for different states of an antigen also
makes it possible to determine the abundance and modification states of a
protein simultaneously. It might be argued that Ab arrays based on
fluorescence microsandwich assays will be useful in the short term but will
eventually be replaced by direct label-free detection. However, the use of two
Abs to generate a signal has important advantages in selectivity that cannot
be matched by direct detection by using even the most exotic instrumentation.
Very few Abs, including those used in this paper, are truly monospecific when
used to probe cell extracts; the vast majority also bind to at least one other
cellular antigen. The microsandwich assay achieves exquisite selectivity
without the size fractionation afforded by immunoblotting because the
specificities of two different Abs are exploited. Very rarely will the capture
and detection Abs bind to the same extraneous protein. Unless affinity
reagents more effective than Abs can be developed, sandwich methods will enjoy
fundamental advantages for the analysis of very complex protein mixtures. Although polyclonal, monoclonal, and recombinant Abs can be used for
detection in microsandwich assays, we are disappointed to be describing arrays
with so few elements. In very recent work, we have managed to add additional
elements for several other kinases and signaling proteins. However, fewer than
one in 20 of the commercial Abs we have tested are suitable for
microarray-based analysis of cell lysates, and it appears that the
requirements for the capture Ab are the most demanding (the detection Ab
appears to be less critical). Thus, we are currently exploring conventional
and recombinant methods to isolated new capture Abs by using MIMs for
automated screening (). If these efforts succeed, then MIMs capable of a few
dozen measurements will become as useful for intracellular signaling as
cytokine arrays have become from serum profiling. We do not believe that the
reliance of microsandwich methods on solution-phase detection will prove a
significant hurdle. When MIMs are used, microsandwich-based Ab microarrays can
be split among several wells, each of which contains perhaps 20 immobilized
capture Abs in triplicate and 20–40 detection Abs
( 22), thereby avoiding
background problems introduced by Ab mixtures. In conclusion, we have successfully used a prototype Ab microarray to
analyze signal transduction in mammalian cells. Arrays containing a modest
increase in the number of independent elements are easily within reach. Future
Ab microarrays with several dozen elements should be able to monitor
information flow within a signal transduction system with unprecedented
precision. Such a network view of cell signaling will be invaluable for cell
biologists, for drug discovery, and for investigating the differences between
normal and diseased tissues. We thank D. Lauffenburger, Thomas Joos, D. Kirpotin, B. Hendriks, S.
Gaudet, J. Marks, and C. Shamu for comments and discussion. This work was
supported by Defense Advanced Research Planning Agency
“Bio-Info-Micro” Program Grant MDA972-00-1-0030, the National
Institutes of Health, J. P. Moreau of Biomeasure, Inc., and the Bauer Center
for Genomics Research at Harvard University. U.B.N. was a research fellow of
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