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First published online October 18, 2006
Journal of Experimental Biology 209, 4371-4378 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02524
PKC
knockout mouse lenses are more susceptible to oxidative stress damage
1 Department of Biochemistry, Kansas State University, Manhattan, KS 66506,
USA
2 Department of Diagnostic Medicine and Pathobiology, Kansas State
University, Manhattan, KS 66506, USA
3 Department of Neurobiology and Jules Stein Eye Institute, David Geffen
School of Medicine, Los Angeles, CA90095, USA
* Author for correspondence (e-mail: dtak{at}ksu.edu)
Accepted 5 September 2006
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Summary |
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(PKC) regulates gap junctions in the lens epithelium and
cortex. In the current study, we further determined whether PKC control
of gap junctions protects the lens from cataractogenesis induced by oxidative
stress in vitro, using PKC knockout and control mice as our models. The
results demonstrate that PKC knockout lenses are normal at 2 days
post-natal when compared to control. However, cell damage, but not obvious
cataract, was observed in the lenses of 6-week-old PKC knockout mice,
suggesting that the deletion of PKC causes lenses to be more
susceptible to damage. Furthermore, in vitro incubation or lens
oxidative stress treatment by H2O2 significantly induced
lens opacification (cataract) in the PKC knockout mice when compared to
controls. Biochemical and structural results also demonstrated that
H2O2 activation of endogenous PKC resulted in
phosphorylation of Cx50 and subsequent inhibition of gap junctions in the
lenses of control mice, but not in the knockout. Deletion of PKC
altered the arrangement of gap junctions on the cortical fiber cell surface,
and completely abolished the inhibitory effect of H2O2
on lens gap junctions. Data suggest that activation of PKC is an
important mechanism regulating the closure of the communicating pathway
mediated by gap junction channels in lens fiber cells. The absence of this
regulatory mechanism in the PKC knockout mice may cause those lenses to
have increased susceptibility to oxidative damage.
Key words: oxidative stress, gap junction, cataract, lens, protein kinase C
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
White et al., 1998;
Xia et al., 2006). However,
the regulatory mechanism by which gap junctions prevent lens opacification is
less understood.
Protein kinase C (PKC) is a conventional PKC, primarily
found in central nervous and peripheral nervous systems, particularly abundant
in cerebellar Purkinje cells and hippocampal pyramidal cells
(Shutoh et al., 2003). In the
lens, it is found in anterior epithelium and the fiber cells of the cortex,
but not in the deeper fibers constituting the lens nucleus
(Saleh et al., 2001). As with
most conventional PKCs, diacylglycerol, calcium and oxidative stress signals
(e.g. H2O2) activate this isoform. Upon activation, the
soluble PKC translocates to the plasma membrane, and phosphorylates
targets such as receptors, structural proteins, ion channels and gap junctions
(Lin and Takemoto, 2005;
Zampighi et al., 2005).
Numerous studies indicate that the phosphorylation of such a wide spectrum of
proteins appears to prevent brain ischemia and plays a protective role in cell
survival (Aronowski et al.,
2000).
PKC is the principal enzyme involved in the phosphorylation of gap
junctions in the lens (Lin and Takemoto,
2005; Saleh et al.,
2001; Zampighi et al.,
2005). Gap junctions regulate the cell-to-cell pathway responsible
for the movement of nutrients and waste products from the surface into the
lens interior (Zampighi et al.,
2005). This pathway consists of specialized channels, named gap
junctions, which link directly to the cytoplasm of adjacent cells. Gap
junction channels are composed of members of the connexin family arranged as
hemi-channels (hexamers) joined through their external domains in the
extra-cellular space (Kistler et al., 1988). The complete channels are
arranged in large aggregates called plaques
(Baldo et al., 2001; Kistler et
al., 1988; Konig and Zampighi,
1995; Zampighi et al.,
2005).
In lens epithelium, gap junctions consist of a dominant connexin 43 (Cx43)
and a minor Cx50. In lens fiber cells, the gap junctions are comprised of
equal amounts of Cx46 and Cx50 (Kistler et al., 1988;
Konig and Zampighi, 1995).
PKC controls gap junctions in the lens epithelium and cortex during
oxidative stress. Oxidative activation of PKC uncouples both Cx43 and
Cx50 gap junctions to the passage of fluorescent dyes, through phosphorylation
of both connexins (Lin et al.,
2004; Zampighi et al.,
2005). Cx50 gap junction channels are disassembled into
hemi-channels that remain in the plasma membrane of the fiber cells
(Zampighi et al., 2005). This
occurs within the lipid rafts, the microdomains in plasma membranes.
We took advantage of the observation that deletion of PKC produces
viable mice with a slight ataxia, less memory, reduced neuropathic pain,
decreased anxiety, impaired long-term potentiation, enhanced opioid responses,
increased alcohol consumption and less protection against brain ischemia
(Abeliovich et al., 1993;
Aronowski et al., 2000;
Bowers and Wehner, 2001;
Malmberg et al., 1997;
Narita et al., 2001;
Ohsawa et al., 2001;
Verbeek et al., 2005). Such
mice have very small litters and short breeding lifespan (D. Lin et al.
unpublished observation). We demonstrated that the loss of PKC control
of gap junctions greatly increases opacification in the lens of PKC
knockout mice. Deletion of PKC altered the arrangement of gap junctions
on the cortical fiber cell surface, caused failure of connexin 50
phosphorylation and the loss of gap junction uncoupling response after
H2O2 as measured by fluorescent dyes transfer. These
results demonstrate that PKC is essential for the protection of the
lens from environmental/oxidative damage.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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, caveolin 1 and Cx43 were obtained
from BD Biosciences (Palo Alto, CA, USA); monoclonal mouse anti-Cx50 (amino
acids 290-440) was from Zymed Laboratories (South San Francisco, CA, USA);
anti-aquaporin 0 (AQP0), polyclonal rabbit anti-phosphothreonine (pT) and
anti-phosphoserine (pS) were from Chemicon (Temecula, CA, USA); nonspecific
rabbit IgG and protein-agarose beads (A/G PLUS) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA); anti-mouse or anti-rabbit IgG conjugated
with horseradish peroxidase (HRP), and nonradioactive PKC assay system
(PepTag) were obtained from Promega (Madison, WI, USA); Dulbecco's modified
Eagle's medium (DMEM; low glucose), gentamycin, and penicillin-streptomycin
were from Invitrogen-Life Technologies (Carlsbad, CA, USA); hydrogen peroxide
(H2O2) and sodium fluoride (NaF) were purchased from
Fisher Scientific (Pittsburgh, PA, USA); phenylmethylsulfonyl fluoride (PMSF)
and protease inhibitor cocktail were from Sigma-Aldrich (St Louis, MO, USA);
optimal cutting temperature (OCT) compound was obtained from Sakura Finetec
(Torrance, CA, USA); Lucifer Yellow, rhodamine-dextran were from Molecular
Probes (Eugene, OR, USA); and 12-O-tetradecanoylphorbol 13-acetate
(TPA) was purchased from CalBiochem (San Diego, CA, USA).
Animals
Male and female mice (Mus musculus) at 2 days to 16 weeks of age
were used in this study. Both the control mice (b6129pf21j100903) and
PKC knockout mice (B6;129p-Prkcctm1St1) were initially purchased from
Jackson Laboratory (Bar Harbor, MA, USA), as breeders, then maintained as a
colony at Kansas State University Animal Resource Facility. This was necessary
as animals younger than 1 week are not available for purchase. All experiments
were performed according to an institutionally approved animal protocol.
Lens light microscopy
All lenses were removed immediately and fixed in a solution of 2%
paraformaldehyde, 2.5% glutaraldehyde, 0.1 mol l-1 cacodylate.
Lenses were post-fixed with osmium tetroxide, dehydrated, and embedded in Epon
(LX112). Sections (1 µm thick) were stained with Toluidine Blue, and viewed
and photographed under a Nikon microscope.
Freeze-fracture
The eyes from 6-week-old control or PKC knockout mice were removed
from the orbit, opened and immersed in the fixative solution composed of 3%
glutaraldehyde in 0.2 mol l-1 sodium cacodylate pH 7.3 for 2-4 h at
room temperature. The lenses were washed in 0.1 mol l-1 cacodylate
and cut in halves to improve infiltration. They were postfixed in 1%
OsO4 for 90 min at room temperature, washed in 0.1 mol
l-1 sodium acetate (pH 5.0) and immersed in 0.5% uranyl acetate in
0.1 mol l-1 sodium buffer for 12 h at 4°C. Dehydration was
performed in a graded series of ethanol, passed through propylene oxide and
infiltrated in Polybed 812 under vacuum
(Zampighi et al., 2000). Thin
sections (1 µm) were cut and were infiltrated in 20% glycerol in 0.1 mol
l-1 sodium cacodylate. The samples were frozen by immersion in
liquid propane and stored in a liquid nitrogen refrigerator. They were
transferred into a JEOL freeze-fracture-etch apparatus and fractured at
-120°C with a knife cooled at liquid nitrogen temperature. The fracture
faces were shadowed with platinum at 70° and carbon at 90°. The
replicas were cleaned with sodium hypochloride and deposited on single hole,
Formvar-coated grids (Zampighi et al.,
2000).
Lens in vitro culture
Lenses were immediately removed from eyes after mice were killed, and
incubated in serum-free DMEM medium at 37°C in a CO2 incubator
for 24 h. At that time all transparent lenses were used for further
treatments. Six lenses per group were used. The transparent lenses were
treated with H2O2 or phosphate-buffered saline (PBS) at
37°C in a CO2 incubator for an additional 24 h.
H2O2 was added into the serum-free DMEM medium every 4 h
for a total of six times. After 24 h, the lenses were washed with PBS, and
were examined for opacification as described previously
(Behndig et al., 2001).
Contrast ratios (maximum to minimum transmitted signal) were calculated from
digitized images using UN-SCAN-IT 6.1.
PKC enzyme activity assay
PKC activity was analyzed using a PepTag Assay kit. Equal amounts of
whole cell protein extracts from whole lens were immunoprecipitated with
PKC antisera. Immunoprecipitated PKC/agarose bead complexes were
incubated with a PKC reaction mixture (Lin
and Takemoto, 2005). To stop the reactions, samples were boiled at
95°C for 5 min. Then the PKC reaction products (fluorescent PepTag
peptides) were resolved by agarose gel electrophoresis and visualized under UV
light. The phosphorylated peptide bands were excised, and fluorescence
intensities were quantified by spectrophotometry, according to the
manufacturer's instructions. Results are expressed as the percentage of
nontreated specific PKC activity from an average of three experiments
± s.e.m.
Immunoprecipitation, western blot and phosphorylation of Cx50 on serines and threonines
The whole lenses were homogenized with cell lysis buffer followed by
sonication. The cell lysis buffer contained 20 mmol l-1 Tris-HCl,
pH 7.5, 0.5 mmol l-1 EDTA, 0.5 mmol l-1 EGTA, 0.5%
Triton X-100, 0.1% protease inhibitor cocktail, 5 mmol l-1 NaF, 5
µmol l-1 Na3VO4 and 2 mmol l-1
PMSF. After centrifugation at 12 000 g for 20 min, the
supernatants were collected and used as whole cell extracts. Whole cell
extracts were immunoprecipitated with anti-Cx50 at 4°C for 4 h, as
described (Lin et al., 2003).
The immunoprecipitate/agarose bead complexes were resolved by SDS-PAGE and
visualized by western blot with antisera to phosphoserine (pS),
phosphothreonine (pT), and/or connexin 50 (Cx50). In some cases, whole cell
homogenates were blotted using antisera.
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). Six-week-old PKC knockout or control mice were
killed with CO2 and the lenses were removed immediately after the
death of the animal and washed in PBS. Lenses were incubated with 50 µmol
l-1 H2O2 for 20 min in 2 ml serum-free DMEM
(low glucose) medium. Lucifer Yellow (2.5 mg ml-1 in PBS) was
microinjected as described previously
(Zampighi et al., 2005), and
the lenses were subsequently incubated in serum-free DMEM at room temperature
for 30 min, to allow dye transfer. Rhodamine-dextran (1%) was injected with
Lucifer Yellow and used as a control for nonspecific leakage. A total 126 nl
of Lucifer Yellow and rhodamine-dextran was injected into the superficial
cortical fibers (around 20 µm in depth) per injection site, with a
microinjection apparatus (Nanoliter 2000; World Precision Instruments, Inc.,
Sarasota, FL, USA). After incubation, the lenses were fixed in 2.5%
paraformaldehyde, dissected, and mounted in 3% agar. Lens gap junction dye
transfer was measured using confocal microscopy. The extent of Lucifer Yellow
dye transfer minus the distance of rhodamine-dextran (in µm) was expressed
as gap junction dye transfer activity. Each experimental group contained six
lenses and the distance of dye transfer was determined in six areas of the bow
region of each lens in coded samples. Results are expressed as mean ±
s.e.m., with P
0.05. |
Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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results in damaged lenses at 6 weeks of age knockout
mice (Fig. 1). In particular,
nuclei were lost in the typical organ free zone, indicating that loss of
PKC did not alter epithelial to fiber cell differentiation. The lenses
of 6-week-old PKC knockout mice were substantially different to those
of wild-type control mice. Large vacuoles were found in the epithelia and
outer cortex.
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knockout lens in vitro demonstrates increased tendency towards cataractogenesis causes intact lenses to be more susceptible to
environmental insults, we treated the intact lenses in culture with
H2O2, and lens opacification was determined as shown in
Fig. 2.
H2O2 induction of opacity in vitro has been
used as a model to determine lens susceptibility to opacification
(Lin et al., 2005). In each
treatment, six clear lenses were initially incubated in serum-free DMEM for 24
h. Three out of six PKC knockout lenses were opaque after this initial
incubation, whereas only two of six control lenses were opaque. For the
H2O2-triggered cataract assay only clear lenses were
incubated with H2O2 for 24 h (every 4 h, or six times,
1-10 µmol l-1). PBS was used as a control treatment. The
surprising result is that, even without H2O2, after 48 h
incubation only, one of six knockout lenses remained clear whereas four of six
controls were clear. As previously reported, control lenses showed increased
opacity with increasing H2O2 concentrations. Simple
removal of lens from knockout eyes resulted in cataract formation. This
clearly demonstrated an increased tendency to form opacities even with the
stress of surgical removal.
H2O2 activates control lens PKC enzyme activity
To address whether lack of the PKC enzyme would account for lens
opacification through improper control of gap junctions, we first determined
if activation of PKC by activators, TPA and H2O2,
could be demonstrated. In control lenses, endogenous PKC was activated
by TPA and H2O2. Very low or no PKC enzyme
activity was detected before or after activation in the PKC knockout
lenses (Fig. 3A). These results
demonstrate no detectable PKC enzyme activity in lenses from PKC
knockout mice, consistent with western blotting results
(Fig. 3B).
Phosphorylation of Cx50 on serines and threonines is stimulated by H2O2 or TPA in lenses from the control, but not the PKC knockout mice
Connexin 43 (Cx43) and Cx50 are found in lens epithelium, whereas Cx46 and
Cx50 are in lens fibers. We have found that PKC differentially
phosphorylates Cx46 and Cx50 (Lin et al.,
2004). Since the activation of PKC disassembles only Cx50
channels in whole lenses (Zampighi et al.,
2005), we measured the phosphorylation of Cx50 in the control and
PKC knockout mice. This would allow us to determine if other PKCs could
compensate. Lenses were treated with TPA or H2O2, both
of which are strong activators of PKC
and, both found in lens
(Lin et al., 2004;
Zampighi et al., 2005). The
total Cx50 from lens whole cell extracts were immunoprecipitated and resolved
by SDS-PAGE. Phosphorylation of Cx50 on serines and threonines was determined
by immunoblotting with antibodies against phosphoserine (pS) or
phosphothreonine (pT) as shown in Fig.
4. Treatment with TPA or H2O2 caused
phosphorylation of Cx50 on both serines and threonines in the lenses from
control mice (Fig. 4). However,
no detectable Cx50 phosphorylation was observed in the samples prepared in the
same manner but from knockout lenses (Fig.
4). Therefore, in lenses from control animals phosphorylation of
Cx50 is controlled primarily by activation of PKC and other PKCs do not
compensate for loss of PKC, i.e. Cx50 is not phosphorylated.
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knockout abolishes effects of H2O2 on decreases in lens gap junction dye transfer knockout lenses, the dye moved 
86 µm into the lens where
Cx50 is found (Zampighi et al., 2003). After H2O2
treatment, the dye diffused only 32 µm into the cortex in the control
lenses. In contrast, the diffusion of Lucifer Yellow into the cortex remained
unchanged (94 µm) in the PKC knockout lenses after
H2O2. The results indicate that the deletion of
PKC abolishes the effects of H2O2 on inhibition
of lens gap junctions in PKC knockout mice, however dye transfer is
observed. Therefore, the gap junctions transfer dye but are not controlled
properly and may be open.
Structural changes of gap junctions occur in PKC knockout lens
Finally, we wished to demonstrate whether the deletion of PKC alters
morphology of gap junctions in lens fiber cells. Fiber cell surface gap
junctions were revealed by freeze-fracture as shown in
Fig. 6. We found that a
significant difference between lenses of control and knockout mice was a
decrease in the frequency and arrangement of the so-called `tongue-and-groove'
junctions (Simon et al., 1982;
Zampighi et al., 1982). In
wild-type controls, these junctions appeared as hemispheres of 0.5 µm
in diameter that protrude within the cytoplasm of one of the fibers. In the
knockout mice, these junctions were smaller and were seen with a lower
frequency in the surface of the fibers. There was a similar frequency of gap
junction plaques although they appeared more fragmented in the knockout
animals. In controls, the gap junction plaques measured 0.7-0.8 µm in
diameter (red areas, Fig.
6A,C). In the knockout mice, fiber cells also contained large
plaques (Fig. 6B). In addition,
there were large numbers of small plaques that were composed of up to 50
channels and were randomly distributed in the plasma membrane (small red
areas, Fig. 6B,D). These small
plaques were not seen in fibers from controls. The density of channels in both
controls and knockout plaques was comparable (4,500 µm-2).
Taken together, our results suggest that open gap junctions in the PKC
knockout mice cause lenses to be more sensitive to stress.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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acts as an oxidative stress sensor and controls gap junctions
in the lens epithelial cells and whole lens in culture. In this work we took
advantage of PKC knockout mice, to determine effects of loss of
PKC on subsequent lens homeostasis. Our study offers conclusive
evidence that control of gap junction channels, especially Cx50 in the lens,
by activated PKC protects lenses from stress damage. Deletion of
PKC in the knockout mice eliminates phosphorylation of Cx50 and results
in an inability to uncouple Cx50 in response to H2O2
which, in turn, results in lens fiber cells being more susceptible to
oxidative stress and subsequent lens opacification. We, thus, conclude that
PKC is a critical regulatory enzyme which controls lens gap junctions,
and its loss in the knockout mice may account for the extensive fiber damage
and lens opacification through improper control of lens gap junctions by
PKC.
PKC is a neural-specific, conventional PKC isoform, which is
involved in multiple signaling pathways
(Oancea and Meyer, 1998;
Tanaka and Nishizuka, 1994;
Zeidman et al., 1999).
Numerous publications have demonstrated that PKC is also present in the
lens (Berthoud et al., 2000;
Saleh et al., 2001;
Lin et al., 2004;
Zampighi et al., 2005).
Functional studies have revealed that Cx43 gap junctions are one of the major
targets for activated PKC in the lens epithelium. PKC is
activated by oxidative stress, e.g. H2O2, through
oxidation of the C1B domain (Lin and
Takemoto, 2005). Recently, missense mutations in the PKC
C1B domain have been identified as causing a type of autosomal dominant
neurodegenerative disorder, spinocerebellar ataxia type 14 (SCA-14)
(Chen et al., 2003;
Chen et al., 2005;
Alonso et al., 2005;
Seki et al., 2005). However,
it is still unclear how these C1B mutations lead to loss of Purkinje neuron
and cerebellar dysfunction during ataxia. Our observations that PKC
targets specifically the gap junction channels might also be relevant to the
mechanisms involved in this disease.
Gap junctions are hydrophilic channels connecting adjacent cells. They
provide a free passage of both necessary metabolites and apoptotic signals
from cell to cell (Farahani et al.,
2005). Thus, gap junctions play important roles in lens
homeostasis when properly controlled (Gao
et al., 2004; Mathias and Rae,
2004; Gerido and White,
2004; Yu et al.,
2005; Zampighi et al.,
2005). However, the passage of apoptotic signals such as high
Ca2+ or ATP to an adjacent cell through open gap junctions could be
linked to cell death (Cusato et al.,
2003; Lin et al.,
1998). The process is well documented in neural cells and immune
cells (Cusato et al., 2003;
Lin et al., 1998;
Vinken et al., 2006). This
work provides an in vivo model to better understand how control of
gap junctions by PKC relates to lens growth and development. We showed
previously that PKC differentially regulates Cx43, Cx46 and Cx50 gap
junctions in the lenses. Cx50, found in the lens, is now also known to be
extensively present in neural systems, for example, in the optic nerve and
central nervous system projections
(Schutte et al., 1998). Cx50
knockout results in a small lens with less differentiation, suggesting that
Cx50 is required for lens differentiation and cell growth as well
(White et al., 1998;
Rong et al., 2002). Cx50 was
recently found to bind to calmodulin and to be regulated by Ca2+
(Zhang and Qi, 2005). We
measured PKC activation in both PKC knockout and control mice
and found that Cx50 phosphorylation and lens gap junction activity are
specifically controlled by PKC. The total lack of Cx50 phosphorylation
in the knockout mice clearly demonstrates that PKC phosphorylates and
controls Cx50. Further, this cannot be compensated by other PKCs. We have
demonstrated that the deletion of PKC causes lens fiber cells to have
altered gap junctions, which are open and uncontrolled, and this subsequently
results in fiber cell damage. A life-time accumulation of sublethal oxidative
stress damage through open gap junctions would be harmful. Oxidative stress is
one of the most common causative agents of disease
(Cutler, 2005). Prolonged
oxidative stress (such as long term H2O2 treatment for
24 h) causes protein oxidation, aggregation, degeneration and cell death. In
our current model, sublethal H2O2 induced cataract
formation in control mice after 48 h. In contrast, the lenses of knockout mice
become opaque even without H2O2. This may be due to open
gap junctions which cannot control entry of components from the medium
(Fig. 5). It is thus possible
that regulation of gap junctions via PKC is essential for
survival of cells, not just in lenses but also in other tissues where this
critical enzyme is expressed. Therefore, studies of the interplay of gap
junction channels and PKC in control and knockout animals might provide
useful information for understanding how tissues respond and deal with stress
and disease.
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Acknowledgments |
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under a light microscope. Scale is identical in each panel.
