Download Nanotech Meets Microbiology

Nanotech Meets Microbiology
New tools for probing molecules, molecular assemblies, and cells are
advancing our understanding of how bacteria work
Viola Vogel and Wendy E. Thomas
ased on gene microarray analyses, reing the top-down controls that operate in living
searchers now realize that transcripcells entails studying how regulatory modules
tional controls regulate only a fracare coupled to one another and to gene trantion of cellular activities. While the
scription. The complexity of this problem goes
tools of biochemistry and molecular
further than many researchers realize. For exbiology have provided impressive knowledge
ample, Stanislas Leibler and colleagues at Rockabout the molecular components of cells, the
efeller University in New York, N.Y., recently
main challenge in biology is to decipher the
found that the connectivity within a biological
hierarchical architecture of molecular networks
network does not necessarily control its behavand how they work from a bottom-up as well as
ior in a deterministic manner. Instead, a given
a top-down perspective.
network might perform several different logical
Going from molecules to the next higher levoperations. Hence, novel technologies and exels of biological organization (bottom-up) repertise will be needed to establish how such
quires a quantitative understanding of how mocellular functions can be quantitatively underlecular components in cells interact with each
stood.
other and how they are organized into funcSeveral novel tools being developed by retional modules performing discrete tasks that
searchers in nanotechnology (NT) could help to
any single class of molecules cannot accomplish.
address some of these analytic challenges. NT
In a first step, high-throughput technologies
tools are already advancing our understanding
have been used to probe the concentrations,
of how bacteria work, while providing new opfunctional states, or interactions of components
portunities to probe the dynamic and physical
within such modules. However, these techaspects of molecules, molecular assemblies, and
niques are suited to probing moleintact microbial cells, whether in
cules and molecular complexes
isolation or under in vivo condionly after they are isolated. While
tions. Considering the reduced
Ultimately, we
such approaches can yield a wealth
complexity of bacterial compared
seek to
to eukaryotic cells, NT tools apof new information, they have
plied to microbiology are likely
their limitations since the moleunderstand the
also to have a major impact on the
cules are removed from their naorchestrated
emerging fields of proteomics and
tive environment. Furthermore,
interplay of
systems biology.
short-lived metabolic intermedimolecules in
Ultimately, we seek to underates and associations might be
regulating
cell
stand
the orchestrated interplay of
missed.
functions
molecules
in regulating cell funcNew technologies are also
tions—a
task
that will require us
needed to develop a quantitative
learning
how
to
reconstitute funcunderstanding of how individual
tional
modules
ex
vivo.
While
we use NT tools
cells process and integrate a myriad of spatioto understand biology in all its complexity, we
temporal stimuli. This approach of inferring
may also develop some of the expertise needed
function from whole system behavior has been
to design novel synthetic materials and technolreferred to as a “top-down” approach. Analyz-
B
Viola Vogel is a Professor of Bioengineering and the
founding Director of
the Center for
Nanotechnology
and Wendy E.
Thomas is a postdoctoral fellow, Department of Bioengineering,
University of Washington, Seattle.
Volume 70, Number 3, 2004 / ASM News Y 113
ogies that will be applicable to nonbiological
systems.
From Single Molecules to Cooperating
Systems
A quantitative understanding of signaling and
metabolic pathways requires an understanding
of how the molecules involved in such cascades
cooperate synergistically. This requires much
more than knowing intermolecular binding
strengths and the overall molecular concentrations. Pieces to the puzzle of how cells work will
be derived from a range of techniques that probe
biophysical and chemical characteristics of molecules and their assemblies. This includes tracing single molecules as they move through the
cell with high spatiotemporal resolution from
their synthesis to degradation, and potentially
probing the local forces that they encounter.
Furthermore, the kinetics of molecular assembly
processes need to be probed in living systems.
Equally important is to obtain quantitative information of the kinetics of chemical modifications of all molecular constituents associated
with a given pathway, and how pathways crosstalk. Moreover, many regulatory molecules such
as transcription factors are often present in vanishingly small quantities, so that the number and
location of individual molecules is important
and cannot be approximated by an average measurement over time or space. Addressing these
issues requires NT tools that can probe the location and behavior of single molecules. Interesting results are beginning to emerge from the application of nanotools to the study of bacteria.
Spying on Single Molecules
Imaging single molecules rather than averaging
the behavior of larger ensembles of molecules
has striking advantages. For instance, bacteria
are equipped with effective means for withstanding damage from hazardous chemicals—
namely, actively extruding such chemicals to
lower their intracellular concentrations. By following single fluorescent-labeled toxins as they
enter and leave a bacterial cell, Nancy Xu and
collaborators at Old Dominion University in
Norfolk, Va., observed the kinetics, efficiency,
and regulation of the cell’s extrusion pump machinery. In contrast, averaged ensemble measurements cannot distinguish between extru-
114 Y ASM News / Volume 70, Number 3, 2004
sion-mediated efflux rates and the influx rate
arising from passive diffusion into cells.
The behavior of single particles can also be
tracked by attaching particles of interest to a
bead, which serves as a handle for trapping the
particles within optical tweezers and then tracking both the position of and forces acting on the
bead and its attached particles with microsecond resolution. For instance, this method can
be used to determine whether molecules are
subject to random Brownian motion, diffusion, or active motion, thereby learning more
about their immediate environment. In one such
study, Lene Oddershede and colleagues at the
Niels Bohr Institute in Copenhagen, Denmark,
found that the lateral mobility of the ␭-receptor within the outer membrane of a living
Escherichia coli bacterium is restricted. They
speculate that receptor movement of this transmembrane protein is impeded because its
periplasmic domain is interacting with the underlying peptidoglycan layer.
Poking and Pulling Nanoscale Objects
Mechanical properties of single molecules or
molecular assemblies can be determined by
studying their response to controlled perturbations. Optical tweezers (Fig. 1) can be used to
apply loading forces to nano- and microscale
objects in a controlled manner and then to
record their responses. The tip of an atomic
force microscope (AFM; see Fig. 2) probe, while
often used to measure the topology of an object,
can operate in a similar way to measure mechanical force response (see ASM News, September
2003, p. 438).
For example, Daniel Muller and colleagues
now at the Max-Planck-Institute of Molecular
Cell Biology in Dresden, Germany, are using
AFM to image proteins that are embedded in
membranes. He and many other researchers are
also probing the interactions among receptor
proteins and various ligands. AFM and optical
tweezers thus provide sensitive new means for
studying the physical properties and dynamics
of such biomolecules and their assemblies.
Studies with optical tweezers are also providing new insights into twitching motility, a flagellum-independent mechanism by which some
bacteria move across surfaces and that also is
involved in colonizing surfaces during vegetative growth as well as in forming complex
FIGURE 1
A Atomic force microscope (AFM)
B Lower resolution in liquid
1. The sample is
C Higher resolution in air
Flagella
moved back
and forth…
Laser
Photodiode
2 µm
Cantilever/mirror
Rhodopseudomonas palustris
2 µm
D Sub-nanometer resolution
ATP-synthase rotors
AFM tip
z
x
2. …and is moved up
y
Sample
and down at a constant
distance from the tip.
5 nm
Optical tweezers, or laser traps. The focal point of a laser is used to trap a micron-scale particle. A tiny particle can either be steered
by moving it in the laser trap, or the forces acting on the particle can be recorded by measuring the displacement of the particle from
the center of the trap. Here we show how this method was used to measure the retraction of type IV pili in Neisseria gonorrhoea. The
double arrowheads show the position where a single diplococcus is trapped by the laser before it is pulled out of the trap and towards
the rest of the colony by type IV pili. The forces generated during this retraction were measured by trapping a small bead in the optical
tweezers while individual type IV pili covering the surface of an anchored diplococcus pull on the bead. The trace shows how several
attachment events pull the bead hundreds of nanometers and generate forces of up to 100 pN. (From Merz et al., Nature 407:98,
2000.)
colonial structures in biofilms and fruiting
bodies. Specifically, twitching entails extending, tethering, and then retracting type IV pili,
according to optical tweezer studies done by
Michael Sheetz, now at Columbia University in
New York, N.Y., and colleagues. Each pilus
retracts at a constant speed unless there is a
resisting force, in which case it slows down or
even stalls. Although individual pili likely depolymerize into subunits within the membrane,
how these changes generate force remains to be
determined.
While nanotechnology is providing new tools
to perturb and manipulate single molecules, our
current understanding of how proteins work is
derived from knowing their equilibrium structures. How do mechanical forces acting on
molecules and their assemblies, for example,
change their functional states? Since no experimental techniques are available to determine the
structures of proteins in nonequilibrium states
at high spatial resolution, computational tech-
niques will be essential to establish hypotheses
about how forces affect molecular functions.
For example, steered molecular dynamics
(SMD; see Fig. 3) simulations have been used to
predict the effect of mechanical force on the
structure of biomolecules with Ångstrom resolution. Two recent studies illustrate the power of
SMD simulations for determining how bacteria
sense mechanical force at the submolecular
level.
In one set of studies, we collaborated with
Evgeni Sokurenko at the University of Washington in Seattle, using SMD to determine how
shear flow strengthens instead of weakens E.
coli bacterial adhesion to surfaces. We found
that when mechanical forces stretch the adhesion protein FimH, which is at the outer tip of
type I fimbriae, it switches from low to a high
affinity for its target mannose.
In other SMD studies, Klaus Schulten and
colleagues at the Beckmann Institute in UrbanaChampaign, Ill., showed that membrane tension
Volume 70, Number 3, 2004 / ASM News Y 115
FIGURE 2
A Optical tweezers, or laser trap
Imaging Device
B
1 µm bead with
anti-pilin antibody
held in laser trap.
Colony
Diplococcus
Laser focus
Pilus
3 µm bead with
immobilized diplococcus
anchored to cover slip.
300
120
200
80
100
40
0
Laser
Force (pN)
Cover slip
Displacement (nm)
C
0
0
5
10
Seconds
15
20
The atomic force microscope (AFM) has a fine pointed tip attached to a cantilever that moves over or touches the sample. The cantilever
deflects as the tip is pulled toward or pushed away from the surface. A laser light is bounced off the mirrored backside of the cantilever onto
a photodiode to measure this deflection. The AFM can be used to obtain surface topography images by scanning the surface area, to
measure forces between molecules, or to dissect nano-objects by touching them with the tip. Live bacteria can be imaged this way in liquid,
as shown on the image on the left upper, of Rhodopseudomonas palustris bacteria (taken from Doktycz et al. Ultramicroscopy, 197:209,
2003). However, higher resolution is possible in air, where for example the flagella can be clearly seen (left bottom). The resolution of images
of soft samples is still limited in part because the sample moves. In contrast, sub-nanometer resolution is possible in isolated structures,
as seen in the image on the right of ATP synthase rotors (taken from Muller et al., J. Mol. Biol., 327:925–930, 2003.)
leads to tilting of the transmembrane helices of
the large conductance (MscL) protein, thereby
opening the pores of the mechanosensitive channel within the E. coli membrane. Indeed, in
taking into account data from patch-clamp experiments and X-ray crystallography structural
data, the SMD simulations predict which residues and forces are critical during each step of
channel opening.
Beyond Single Molecules:
Molecular Cooperation
Beyond determining how individual molecules
behave, it is important to understand how they
cooperate synergistically. In the cellular environment, molecules and functional modules are
densely packed, which has profound implications on how they work. Proximity mediates
cooperative effects at all levels of functional
organization.
Transcription rates, for example, depend on
cooperative interactions. Take the case of single
RNA polymerase molecules. They can pause
116 Y ASM News / Volume 70, Number 3, 2004
intermittently while transcribing messages, according to optical tweezer experiments with isolated molecules by Michele Wang at Cornell
University in Ithaca, N.Y., and her collaborators. Moreover, the backward motion that
causes these pauses can be prevented by cooperative interactions between several trailing polymerases, possibly explaining why transcriptional elongation is fast and processive in vivo
even without antiarrest factors, according to
Vitaly Epshtein and Evgeny Nudler from New
York University Medical Center in New York
City.
In another example, the conformation of
RNA and the supercoiling of DNA may accelerate the rate at which proteins that interact with
specific sequences find their targets among many
alternative sites. Thus, Stephen Halford at the
University of Bristol in Bristol, United Kingdom,
found that DNA-binding proteins move from
random to specific sites by repeatedly dissociating and reassociating at different sites along the
DNA, rather than by sliding or diffusing along
the double helix along a single dimension. This
FIGURE 3
A Adhesion to surface via fimbriae
Fimbriae
Fimbriabinding domain
Mannosebinding domain
Mannose
Type I fimbria tip
Surface
E. coli
Bacterial fimbriae bind to a surface
mannose via FimH adhesin at their tip.
Mannoseexpressing protein
B Steered molecular dynamics (SMD)
Mannose-binding
domain in water
Surface
Mannosebinding site
C
Stretching force
Conformational
change strengthens
FimH–mannose binding.
C
Stretching force
N
Surface
finding could mean that supercoiling
of DNA helps such proteins to rapidly
find particular DNA sites because the
probability of them reassociating with
the DNA is inversely proportional to
the distance between sites in threedimensional space. These studies
point to the importance of employing
novel tools to probe cooperative phenomena in vitro and in vivo with high
spatiotemporal resolution.
Yet another example comes from
studying how bacteria regulate chemotactic behavior— ultimately by
changing flagellar motion through the
transmembrane receptor (Tar). The
Tar extracellular binding site for aspartate dimerizes when aspartate is
present, enabling a cytoplasmic protein complex to associate with the
dimer and, in turn, catalyzing phosphorylation of a small molecule that
diffuses to and changes the rotation of
the flagella motor. Despite a wealth of
information gathered by many researchers about the constituents of
this self-contained signaling network,
we do not understand how these molecules interact. For instance, how can
a single E. coli receptor sense aspartate over a range of at least five orders
of concentrations?
As computational approaches are
being developed to simulate the responses of molecular networks,
physiochemical parameter sets derived from equilibrium data are likely
to be insufficient for predicting the
behavior of a cooperative system—yet
equilibrium data, often obtained under dilute conditions, are often all that
are currently available.
Linker chain of
binding domain
Steered molecular dynamics (SMD) simulates all the atoms in a molecule as they move
in response to internal and external forces. For example, this method was used to mimic
how mechanical force stretches the bacterial adhesin firmly located at the tip of type I
fimbriae, when bacteria bind to mannosylated surfaces as shown in panel A. In the
simulations, a molecule with its structure known from X-ray crystallography or NMR
experiments is hydrated in a box of water molecules. Once the molecule is equilibrated
an external force is applied to selected atoms pulling in directions along which the
molecule would experience force under in vivo or experimental conditions as shown in
panel B. The binding domain of FimH undergoes a conformational change switching it
from low to high affinity as the terminal chain that connects the adhesin to the fimbria
breaks away, as shown in panel C.
Determining Spatial Organization
in Living Bacteria with Nanoscopy
To address the interactions between single
molecules or molecular complexes, it is also
important to probe their spatial organization
in live bacteria. Optical microscopy has been
the major workhorse harnessed by biologists
for imaging and probing dynamic events in
eukaryotic cells. However, its resolution of
about 0.5 ␮m—which is set by the diffraction
limit of light and is almost the size of an
individual bacterium—is not suitable for
probing the spatiotemporal organization of
bacterial cells. Visualizing their functional
modules thus requires alternative methods
with nanometer resolution. Although electron
microscopy provides such resolution, it is lim-
Volume 70, Number 3, 2004 / ASM News Y 117
Some of these AFM images help to
show, for example, how drying alters
the appearance of live bacteria, acA Confocal nanoscope
B Optical fluorescence
cording to Mitchel Doktycz and his
micrograph of
Bacillus megatorium
colleagues at Oak Ridge National
Laboratory in Oak Ridge, Tenn.
Other images enable investigators to
visualize photosynthetic core complexes in their native membranes.
Red-shifted waveEven in such ex-vivo systems, AFM
length mask
provides a fantastic advantage over
electron microscopy because samples
are not static, allowing dynamic
movements of supramolecular protein
700
nm
complexes to be observed. Thus, because single bacterial ATP synthase
Membrane,
rotors can be observed diffusing within
30 nm optical
Fluorescence
a membrane at nanometer resolution,
resolution
observing dynamic processes of other
systems cannot be far behind.
Sample
Other imaging schemes similarly
enable scientists to circumvent the dif30
nm
fraction limit of light, thus resolving features of living cells at submicron resolution. For example, in near-field
microscopy, light passes through an aperture whose diameter is much smaller
Confocal microscope
than the wavelength of light. By scanobjective
ning samples with the evanescent tail of
light passing the aperture, a lateral resOptical nanoscope. A new method called STED-4Pi microscopy increases the resolution of
fluorescence confocal microscopy over 10-fold, to give 3-D images at 30- to 40-nm
olution of 50 nm is currently possible.
resolution. Breaking the diffraction limit of light microscopy is accomplished by using a
While near-field optical microscopy
red-shifted wavelength that masks out the fluorescence in all but a small area, as shown
in the figure. This mask is created by positioning two objectives opposite to each other so
is confined to the imaging of specimen
that they create an interference pattern in the image plane where the two beams cancel
surfaces by definition, Stefan Hell and
each other in a “null node.” The fluorescence (shown here in green) can only be observed
colleagues at the Max Planck Institute
in the center of this hole where the red-shifted intensity is too low to mask it. The effective
size of the doughnut hole can be made arbitrarily small by increasing the laser intensity.
of Biophysical Chemistry in GöttinThis figure shows an optical micrograph of the fluorescent membrane of a Bacillus
gen, Germany, have introduced physmegaterium. (This image is courtesy of Marcus Dyba and Stefan Hell and is taken from
ical methods to overcome the diffracNature Biotechnol. 21:1347, 2003.)
tion barrier, with focused light and
regular lenses. The first far-field
method with conceptually unlimited
spatial resolution is stimulated emission depletion (STED) microscopy. STED miited to using dried or frozen samples, and so
croscopy relies on the saturated depletion of the
cannot probe dynamic events.
In contrast, atomic force microscopy (AFM)
excited state of the fluorescent marker with a
enables biologists to produce spatial images of
red-shifted “depletion” wavelength. To boost
living bacteria at the nanoscale (Fig. 2). While
the axial resolution, they have developed 4Pi
the flexibility and movements of live bacteria
microscopy, which improved the axial resolucurrently appear to limit resolution with AFM
tion by three- to sevenfold in live cells through
to hundreds of nanometers, subnanoscale resothe coherent use of two opposing lenses. The
lution is possible with isolated microbial memcombination of the two methods has displayed a
branes or reconstituted components.
resolution of 30 nm (Fig. 4). Yet another develFIGURE 4
118 Y ASM News / Volume 70, Number 3, 2004
opment in imaging, called fluorescence resonance energy transfer (FRET), is being widely
used in cell biology to probe distances and distance changes between energy donors and acceptors in the 1–10 nm range, including those
induced by conformational changes and in
building molecular assemblies.
These new high-resolution methods in optical
microscopy often involve fluorescence, and the
intense laser excitation needed to excite small
numbers of molecules accentuates the limitations
posed by photobleaching. However, NT offers
solutions for this, such as the development of lightemitting quantum dots (QDs), consisting of semiconductor nanocrystals 1 to 10 nm in diameter.
When coated properly, QDs resist photobleaching and have higher absorption coefficients than fluorophores. Equally important, as
QD particle size increases wavelength emission
increases, while excitement wavelength remains
constant, which makes multifunctional imaging
feasible even in dynamic experiments. Thus, investigators may introduce as many as 10 colors
to visualize and distinguish among different
classes of target molecules simultaneously,
while using a single excitation wavelength.
For use in vivo, the surfaces of QDs are modified to carry biomolecules that can bind specifically to target molecules. For example, QDs
conjugated to specific lectins provide strain-specific labeling, while other properly functionalized QDs can enter bacterial cells, according to
Jay Nadeau at the Jet Propulsion Laboratory in
Pasadena, Calif., and his collaborators at several
institutions. In their experiments, QDs were degraded after they entered bacterial cells, destroying their utility as intracellular labels and pointing to the need to develop coatings that extend
the lifetime of such QDs.
One disadvantage of QDs is the difficulty of
engineering them with single binding sites that
can be specifically conjugated to only one molecule of interest. Instead, during the photolabeling step, QDs tend to bind to several molecules
simultaneously. One approach that might circumvent this problem would involve trapping
QDs within the cylindrical cavity of the chaperonin protein GroEL from E. coli. Daisuke Ishii
at the University of Tokyo in Tokyo, Japan, and
his collaborators showed that GroEL (or other)
proteins, which typically encapsulate denatured
proteins to facilitate their proper folding, can
entrap QDs. Entrapment of QDs by proteins
might be one approach to site-specifically link
QDs to other molecules of interest.
Test and Technology: Functional
Reconstitutions
One solid proof of whether we truly understand
any particular biomolecular chess game, with all
its rules and exceptions, would come from our
ability to reconstitute specific supramolecular
entities and their functions ex vivo. Doing so
would provide additional benefits beyond learning more about how biological systems work.
We might also learn from these schemes how to
develop new nonbiological technologies.
Starting at a simple level, an early goal is to
learn how to assemble individual molecules in
synthetic environments, and later to assemble
multicomponent systems with increasing complexity. Bacteria are inspiring nanoscale engineers, and many of their designs can inspire new
technology.
For example, archaebacteria stabilize their
membranes against thermal agitation by integrating lipids that span across the two lipid
leaflets, acting as molecular “staples.” This molecular scheme pointed drug-delivery engineers
toward new ways for stabilizing synthetic membranes that encapsulate therapeutic agents.
Another example is provided by the proteins
of the crystalline layer, also called the S-layer,
with which most walled bacteria and archaebacteria surround themselves. These proteins have
been assembled ex vivo into two-dimensional
protein arrays by Uwe Sleytr and colleagues at
the Center for Nanobiotechnology at the University of Vienna, Austria. The isoporous protein lattices assembled from S-layer proteins allow for selected nutrient transport across
bacterial membranes in vivo, and are now being
used in synthetic devices as ultrafiltration membranes with defined sieving properties.
Other engineers, who are tackling more complex nanosystems, also use insights gained from
studying bacterial molecules. For example, the
ATP synthase complex reversibly couples proton fluxes across cell membranes with synthesis
of ATP molecules or, the reverse, couples hydrolysis of ATP molecules to pumping protons
against concentration gradients. Using singlemolecule spectroscopy, Hiroyuki Noji and colleagues from the Tokyo Institute for Technology
Volume 70, Number 3, 2004 / ASM News Y 119
in Tokyo, Japan, demonstrated that the central
shaft of F1-ATPase rotates with respect to the
surrounding barrel when ATP is present. Subsequently these F1-ATPase were assembled on
microfabricated posts where each one turns an
inorganic rod of a nanoscale propeller bound to
the central shaft.
Reconstituting more complex assemblies that
contain multiple cooperating biomolecules ex
vivo looms as a far more challenging task. NT
researchers are only beginning to predict how
relatively simple molecules, including amphiphilic thiols, lipids, or certain polymeric systems,
assemble. In general, questions about how molecules with more structural complexity assemble continue to be addressed empirically because
the interactions among larger molecules are too
complex for humans to intuit. Recent advances
involving computer simulations are making it
possible to simulate systems containing several
hundred thousand atoms—a degree of complexity that finally opens the door for simulating
larger biomolecular assemblies and the water
molecules in which they are immersed.
Considering the multitude of molecular interactions, will it ever be possible to reassemble
complex functional modules or entire networks
ex vivo? Recent findings which indicate that the
number of interactions between molecules within a functional module is high compared to
those between molecules from different modules
are thus promising indicators that the goal of
ex-vivo assembly of functional modules is
within reach.
The active propulsion system of bacteria,
which has been studied extensively by Howard
Berg at Harvard University in Cambridge, Mass.,
and several other research groups, is one that is
particularly worth trying to reconstitute in a
synthetic environment. It contains a flagellar
filament that is driven at its base by a rotary
motor that is anchored in the cytoplasmic membrane and is powered by a proton gradient. Such
flagella convert chemical fuel into mechanical
motion with an efficiency that far exceeds that
found in manmade motors.
Consequently, it would be a major technological breakthrough if the mechanisms at work in
such flagellar motors could somehow be harnessed. This reconstitutive task will not be easy,
knowing that each flagellum contains more than
20 different proteins and that another 30 proteins are required for their assembly and regulation. However, once we learn precisely how this
and other supramolecular assembly processes
work, it may reveal new design principles that
could be exploited in boosting the efficiency of
human-designed nanoscale engines.
The tools developed by the nanotechnology
communities will be major players in deciphering how bacteria and other living systems work.
The knowledge obtained is poised to innovate
medicine and to serve as inspiration for developing new technologies.
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Soong, R. K., G. D. Bachand, H. P. Neves, A. G. Olkhovets, H. G. Craighead, and C. D. Montemagno. 2000. Powering an
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