Transgenic salmon overexpressing growth hormone exhibit decreased myostatin transcript and protein expression
1 Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543,
USA
2 Fisheries and Oceans Canada, 4160 Marine Drive, West Vancouver, BC V7V
1N6, Canada
* Author for correspondence (e-mail: sroberts{at}mbl.edu)
Accepted 26 July 2004
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Summary |
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In transgenic and control coho embryos, MSTN1 and MSTN2 RNA expression were initially observed at about the time of eying, and a 42 kDa MIP was just detected prior to hatching. Expression of the MSTN1 transcript in transgenic salmon was not different from that in wild-type adult coho salmon muscle and brain tissue. However, expression of the MSTN2 transcript was less in white muscle, and greater in red muscle, from transgenic fish compared to wild-type salmon of the same size. Northern analysis revealed that expression of the MSTN2 transcript was less in white muscle from wild-type, age-matched salmon than in transgenic fish. In addition, there was less presumed bioactive MIP in muscle taken from adult transgenic fish compared to controls and evidence of differential protein processing. Decreased MSTN expression in faster growing fish suggests that MSTN does act as a negative regulator of muscle growth in fish, as it does in mammals. The results of this study also suggest that the anabolic effects of GH could be mediated through MSTN.
Key words: myostatin, growth hormone, muscle, salmon, transgenic
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Introduction |
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While GH has been extensively investigated for decades, myostatin (MSTN), a
transforming growth factor ß (TGF-ß) family member and negative
regulator of muscle growth, has only recently been identified
(McPherron et al., 1997). Just
as for other proteins in the TGF-ß family, the MSTN precursor protein is
synthesized and proteolytically cleaved, resulting in an N-terminal propeptide
and a C-terminal, mature, biologically active peptide
(Lee and McPherron, 2001
;
McPherron et al., 1997
;
Zimmers et al., 2002
).
Skeletal muscle mass in MSTN gene knockout mice is 23 times
larger than in wild-type counterparts, and this increase is a result of
hyperplasia and hypertrophy (McPherron et
al., 1997
). Naturally occurring mutations in the MSTN
gene have been attributed to a `double muscle' phenotype, observed in cattle
(Grobet et al., 1997
;
Kambadur et al., 1997
;
McPherron and Lee, 1997
).
Without the ability to knock-out genes in fish, a clear biological action of
MSTN has not been demonstrated in these vertebrates. However, recent research
using transgenic zebrafish overexpressing the MSTN prodomain suggests that
MSTN does inhibit muscle growth in fish
(Xu et al., 2003
).
Even though GH and MSTN have been shown to have significant roles in the
control of muscle growth, there has been limited research on the interaction
of these hormones. Most research has focused on isolated human clinical
studies, with varying results. GH administration to human subjects did not
affect MSTN expression in male subjects suffering from hypogonadism
(Hayes et al., 2001) or healthy
older men (Brill et al., 2002
).
This is similar to the results observed in pigs
(Ji et al., 1998
) and
GH-deficient rats (Kirk et al.,
2000
), where GH administration did not alter MSTN expression. In
contrast, Liu et al. (2003
)
demonstrated that GH treatment suppresses MSTN expression in GH-deficient
hypopituitary adult subjects and in skeletal muscle cell culture.
Additionally, in a study of healthy elderly men, a significant negative
relationship between skeletal muscle myostatin and GH receptor gene expression
was observed (Marcell et al.,
2001
).
Given that the relationship between MSTN and GH has not been clearly
defined, the present study used transgenic coho salmon overexpressing GH to
better characterize the role of MSTN in muscle growth. The strain of salmon
used in this study was produced using the all-salmon GH/metallothionen (MT)
gene construct (OnMTGH1; Devlin et al.,
1994). These fish grow at a significantly higher rate relative to
controls, and attain a size more than 11-fold larger than the controls by 15
months (Devlin et al., 1994
).
Compared to the controls, the transgenic coho salmon have significantly higher
numbers of small-diameter muscle fibers, thus suggesting that their larger
size is a result of increased muscle hyperplasia
(Hill et al., 2000
). In the
present study, levels of MSTN in muscle, brain and whole-body embryos from
transgenic coho salmon were compared to those in wild-type controls to
determine if they were significantly altered in fish overexpressing GH. In
addition, this model provided a system in which the potential interaction of
GH and MSTN in the regulation of muscle development could be examined.
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Materials and methods |
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Total RNA was extracted from red muscle, white muscle and brain tissue from
eight transgenic and eight size-matched fish using Tri Reagent (Molecular
Research Center, Inc., Cincinnati, OH, USA) as previously described
(Chomczynski, 1993;
Chomczynski and Sacchi, 1987
).
Transgenic (0.9 years) and wild-type (2 years) fish had mean masses of 59.1
and 57.0 g, respectively. In addition, total RNA was extracted from white
muscle tissue from five age-matched fish (0.9 years old, mean mass 5.8 g) for
northern analysis. Unfertilized eggs and three embryos were also taken from
transgenic and control fish at nine different sampling ages throughout
development and processed for total RNA. Protein was extracted from 20 red and
20 white muscle samples (10 fish/transgenic fish and size-control),
unfertilized eggs and three embryos from transgenic and control fish,
corresponding to each of the nine sampling ages above. Adult fish harvested
for protein samples were not the same as those used for RNA analysis, but were
the same age and size. Protein was extracted using a tissue lysis/extraction
reagent (CelLytic, Sigma, St Louis, MO, USA) containing protease inhibitors
(Protease Inhibitor Cocktail, Sigma) and was used at a ratio of 1:20 (1 g
tissue/20 ml reagent). The protein quantity of each sample was determined by a
Coomassie dye-based assay (Coomassie Plus, Pierce, Rockford, IL, USA).
Cloning MSTNs
Total RNA from a coho muscle pool was reverse transcribed using AMV reverse
transcriptase (Promega, Madison, WI, USA). The resulting cDNA was used to
amplify two MSTN genes using PCR [95°C 1 min, (95°C 1 min,
60°C 2 min, 72°C 1 min 30 s) x 40 cycles, 72°C 10 min].
Primers were designed to amplify the MSTN ortholog of rainbow trout
MSTN1 (F1, 1R1) and the MSTN ortholog of rainbow trout
MSTN2 (F1, 2R1) (Table
1). It should be noted that the MSTN genes, isolated from
three different salmonid species, have been named using different
nomenclature, as a result of three research groups working independently. In
the present paper, we have used the same nomenclature as for rainbow trout
MSTN (1 and 2; Rescan et al.,
2001). Therefore, the two MSTN genes in coho salmon will
be referred to as MSTN1 and MSTN2. Polymerase chain
reactions were separated on agarose gels, visualized under UV light, and the
appropriate size band cut, gel-purified and cloned in pCR 2.1-Topo
(Invitrogen, Carlsbad, CA, USA). Positive clones were grown for plasmid
preparation and DNA template was prepared in a Rev Prep Orbit (GeneMachines,
Ann Arbor, MI, USA). The resulting cDNAs were sequenced using a modified
dideoxy chain termination method using Big Dye Terminator (Applied Biosystems,
Foster City, CA, USA). Sequencing reactions were precipitated and pellets
resuspended in Hi-Di Formamide with EDTA (Applied Biosystems) and analyzed
using the 3730 Sequencer (Applied Biosystems).
|
Quantitative real time reverse transcriptionpolymerase chain reaction
To examine the two forms of MSTN in coho salmon tissues and embryos, total
RNA was analyzed quantitatively using real time RT-PCR (Brilliant SYBR Green
QRT-PCR Master Mix Kit, 1-Step, Stratagene, La Jolla, CA, USA) in the Opticon
Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). Only
transgenic and size-matched wild-type coho salmon were used for the
quantitative RT-PCR analysis because the small size of age-matched wild-type
salmon made it difficult to accurately obtain sufficient quantities of the red
muscle and brain tissue required for precise quantitative analysis. However,
white muscle RNA from age-matched fish was analyzed by northern blotting.
Reverse transcription (RT) and polymerase chain reaction (PCR) were performed
consecutively in the same reaction wells as follows: 30 min RT at 50°C, 10
min initial denaturation at 95°C, 40 cycles of 30 s denaturation at
95°C, 1 min annealing at 6365°C, and 30 s extension at
72°C, with fluorescence measured at the end of every annealing and
extension step. Primers were designed to amplify MSTN1 (1F2 and 1R2),
MSTN2 (2F2 and 2R2), or 18s RNA (18s F and 18s R)
(Table 1). Both MSTN1
and MSTN2 primer pairs were designed across an intron to ensure that
contaminating DNA (if present) was not amplified. MSTN1 and
MSTN2 primers were also designed to amplify regions of different
lengths to ensure that melting curve temperatures were easily differentiated.
Each reaction was performed in a separate well of a 96-well plate with a final
volume of 25 µl. Immediately after each PCR, a melting curve analysis was
performed to determine if the desired product was amplified by increasing the
temperature from 55°C to 95°C at a rate of 0.2°C
s1, and measuring fluorescence at every 0.5°C step.
Complementary DNA plasmid preparations of MSTN1 and MSTN2 were run to confirm that primers were specific and there was no cross reactivity between MSTN1 and MSTN2 primers. Each total RNA sample was run in duplicate with all three primer pairs. Serially diluted standard curves of MSTN1 and MSTN2 cDNA plasmid preparations were assayed so that data could be quantified. Additionally, a coho muscle RNA pool was assayed using 18S primers. Reactions with each primer pair were run on an agarose gel to verify size.
For all real-time assays, melting curves were analyzed to verify that no primer dimers were formed and that CT values represented the desired amplicon. CT values were then converted to relative MSTN abundance levels based on their respective standard curves and were normalized to the corresponding 18S RNA values.
Northern analysis
Northern blot analysis was used to examine RNA expression of MSTN2
in muscle tissue. Total RNA (10 µg/lane) from red and white muscle tissue
was separated on formaldehydeagarose gels [1.6% agarose, 2.2 mol
l1 formaldehyde, 1x MOPS
(3-(nmorpholino)propanesulfonic acid)] and transferred to nylon
membranes (Magna Charge Nylon Transfer Membranes, Micron Separations Inc.,
Westboro, MA, USA) by downward capillary elution using 20x SSC (3 mol
l1 NaCl, 0.3 mol l1 sodium citrate, pH
7.2). Nylon membranes were pre-hybridized for at least 2 h in roller tubes at
42°C in a buffer containing 5 x SSPE (0.75 mol l1
NaCl, 0.05 mol l1 sodium phosphate monobasic, 5 mmol
l1 EDTA, pH 7.4), 0.1% SDS, 5x Denhardt's solution,
50% formamide and 150 mg ml1 calf thymus DNA. Northern blots
were probed with radiolabeled ([-32P]dATP; 3000 Ci
mmol1; ICN Biomedicals, Irvine, CA, USA) double-stranded
partial MSTN2 (714 bp). The cDNA was labeled using Klenow (Prime-It II,
Stratagene) and the denatured probe was added directly to the
pre-hybridization buffer and incubated with the northern mixture at 42°C
overnight. Following hybridization, the blots were washed twice (15 min each)
under medium stringency (1x SSPE, 0.1% SDS, 45°C) and twice (15 min
each) under high stringency (0.1% SSPE, 0.5% SDS, 65°C). Northern blots
were dried, exposed to phosphorimaging screens, and visualized using a Storm
840 phosphorimager (Molecular Dynamics, Amersham, Piscataway, NJ, USA).
Protein quantification
The levels of MSTN in coho salmon muscle and eggs/embryos were determined
by western analysis using a primary antibody produced against brook trout
recombinant MSTN. This antibody has been characterized previously with brook
trout muscle and embryos and recognizes the precursor and C-terminal mature
peptide (Roberts and Goetz,
2003). For western analysis, 6 µg of muscle or whole egg/embryo
protein extract were electrophoresed on NuPage 412% Bis-Tris gels
(Invitrogen). Gels were transferred to nitrocellulose membranes, blocked and
incubated with the diluted (1:1000) primary MSTN antibody. After rinsing in
1x TBS-T, the membranes were incubated with a horseradish
peroxidase-labeled secondary antibody. For detection, the ChemiGlow detection
kit (Alpha Innotech, San Leandro, CA, USA) was used in conjunction with a
SuperChemiNova 12-bit CCD camera (ChemiImager, Alpha Innotech). A 16 bit per
pixel image was captured with a 1 min exposure time and detection level
linearity was verified by image saturation analysis software (ChemiImager 5500
v3.1, Alpha Innotech). Integrated density values were calculated, the
background subtracted, and all data normalized with a pooled muscle sample run
in duplicate on each gel.
Statistical analysis
All data are given in terms of relative abundance levels and expressed as
means ± standard errors (S.E.M.).
One-way analyses of variance (ANOVAs) were performed for adult tissue and
two-way ANOVAs for developing coho embryo samples. For statistical analysis,
RNA samples from developing coho embryos in which MSTN1 or
MSTN2 was undetected were given a value of 5% less than the lowest
relative abundance level within each respective MSTN form, rather
than a zero value. When there were significant differences within factors
(i.e. tissue analysis: fish group, tissue type; developing embryo analysis:
treatment, time) a post hoc comparison test (HolmSidak) was
performed. All significance levels were set at P0.05.
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Results |
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RNA analysis
Total RNA levels of both MSTN1 and MSTN2 were measured
using quantitative real-time RTPCR in white muscle, red muscle and
brain from transgenic and size-matched control coho. Among samples tested for
MSTN1 expression, there was no significant treatment or tissue effect
(Fig. 1). There were however,
differences observed in the expression of MSTN2
(Fig. 1). Specifically,
expression of MSTN2 in white muscle was significantly higher in
size-control fish than in transgenic fish. The opposite was seen in red
muscle, where MSTN2 expression was significantly higher in transgenic
fish than in size-control fish. The same results were observed with northern
analysis (Fig. 2) and with
conventional RT-PCR analysis (data not shown). Northern analysis also showed
that MSTN2 expression was lower in age-control fish than in
size-control and transgenic fish (Fig.
2). In the brain, expression levels between control and transgenic
fish were not significantly different. In transgenic fish, expression of
MSTN2 was significantly higher in red muscle than brain and white
muscle, and expression in both red muscle and brain was significantly higher
than in white muscle.
|
|
In transgenic and control coho salmon embryos, expression of both MSTN forms (1 and 2) was measured quantitatively from total RNA throughout development (Fig. 3). In general, there was no significant difference in MSTN expression between embryos from control and transgenic fish for either MSTN1 or MSTN2. Therefore, only RNA levels within fish groups were further compared. MSTN1 was first detected in embryos from control and transgenic fish on day 23 and day 34, respectively. The highest mean values (not significant) of MSTN1 RNA in transgenic samples were observed at day 52 whereas the highest mean levels of MSTN1 in control samples were observed at day 67. Just as with MSTN1, expression of MSTN2 was not detected in embryos from control and transgenic fish until eying at day 23 and day 34, respectively. The highest levels of MSTN2 were not observed until day 67 for both transgenic and control samples.
|
Protein quantification
Protein samples from red and white muscle obtained from transgenic and
size-matched control coho salmon, were analyzed by western analysis using an
antibody generated against recombinant brook trout MSTN (tMSTNAb)
(Roberts and Goetz, 2003). In
both muscle types, 42 kDa and 14 kDa protein bands were detected. Expression
of the 42 kDa MSTN immunoreactive protein (MIP) was significantly higher than
the 14 kDa MIP in all tissues (Fig.
4). Levels of the 42 kDa MIP were significantly higher in white
muscle tissue in transgenic compared to control samples. Expression of the 14
kDa MIP was significantly higher in red muscle tissue than in white muscle
tissue for both transgenic and control samples
(Fig. 4). Levels of the 14 kDa
MIP were significantly lower in transgenic fish than in size-controls for both
red and white muscle tissue.
|
In developing coho salmon embryos, a 42 kDa MIP was first evident 45 days after fertilization, just after eying (Fig. 3). Expression levels increased through hatching. In contrast, there was no expression of the 14 kDa MIP throughout the entire embryo sampling period.
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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MSTN is synthesized as a precursor protein that is proteolytically cleaved
to produce an N-terminal propeptide and a smaller C-terminal peptide, which is
the mature biologically active form. In the present study, the two
immunoreactive bands of 42 and 14 kDa recognized in coho muscle by western
analysis presumably represent the precursor MSTN protein and the C-terminal,
bioactive domain, respectively (Roberts
and Goetz, 2003). This conclusion is based on the predicted
molecular masses and results reported for other species using different
antibodies (Rodgers et al.,
2001
; Vianello et al.,
2003
; Zimmers et al.,
2002
), and is consistent with our previous studies on brook trout
(Roberts and Goetz, 2003
).
There was less mature MSTN protein (14 kDa MIP) in muscle taken from
transgenic fish compared to controls. In addition, expression of
MSTN2 RNA was significantly less in white muscle from transgenic fish
compared to size controls, and in northern blots, expression of MSTN2
was lowest in age control fish. These data support the hypothesis that MSTN
acts as an inhibitor of muscle growth in teleosts, since fish with the slowest
growth rates (size controls) had the highest expression levels of
MSTN2 RNA and bioactive protein (14 kDa MIP). The faster growth rates
in the transgenic fish used in this study have been attributed to hyperplasia
(Hill et al., 2000
). Recently,
Xu et al. (2003
) reported that
zebrafish overexpressing the MSTN prodomain exhibited an increased number of
muscle fibers. The MSTN prodomain is able to bind to the mature MSTN domain,
presumably inhibiting MSTN from binding to a receptor and blocking its
function (Hill et al., 2002
).
Therefore, the decreased expression of MSTN in coho salmon with
increased muscle hyperplasia suggests that MSTN2 is an inhibitor of muscle
hyperplasia in teleosts.
The results of the present study also suggest that MSTN could be a
mechanism by which the growth-regulating effects of GH are realized, at least
in part. That is, transgenic salmon that have elevated GH levels also had
decreased levels of MSTN. This is consistent with the finding of Liu et al.
(2003), who found that in
vitro and in vivo treatment with GH decreased MSTN mRNA
expression. Growth hormone response elements and GH cell-specific elements
have been identified in the promoter region of the MSTN gene
(Taylor et al., 2001
;
Roberts and Goetz, 2003
). GH
signal transduction pathways could therefore directly inhibit MSTN
transcription, increasing growth. In addition to transcriptional regulation,
GH could also regulate MSTN at the translational level. In the present study,
western analysis of the unprocessed and processed MIPs did indicate that
differential protein processing of MSTN occurred. At the time of sampling,
there were higher levels of precursor MSTN (42 kDa) in transgenic white muscle
tissue and lower levels of processed MSTN (14 kDa) compared to controls. One
explanation for this is that in the faster growing fish, less MSTN is being
proteolytically cleaved to the mature 14 kDa protein. Regulation of MSTN
proteolysis is not well characterized. Researchers working with the C2C12 cell
line of mouse myoblasts have shown that MSTN processing could be influenced by
hydroxamate-based inhibitors of metalloproteinases (HIMPs;
Huet et al., 2001
). Wolfman et
al. (2003
) have shown that
members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of
metalloproteinases may be involved in activating myostatin in vivo.
Ultimately, myostatin activity is likely to be controlled by several processes
including transcription, translation, binding proteins and receptor
binding.
In transgenic and control coho embryos, MSTN1 and MSTN2
RNA expression was initially observed at about the time of eying, and the 42
kDa MIP was just detected prior to hatching. These results are similar to what
has been observed previously in wild-type brook trout embryos
(Roberts and Goetz, 2003). In
teleost species with only one form of MSTN, RNA expression has been observed
from stages just after fertilization throughout development
(Kocabas et al., 2002
;
Rodgers et al., 2001
;
Vianello et al., 2003
;
Xu et al., 2003
).
Interestingly, in the present study MSTN1 RNA expression increased
above baseline values in transgenic coho salmon embryos earlier than in
controls (Fig. 3). While the
biological role of MSTN1 is unknown, this suggests that MSTN1 might be
involved in distinct developmental processes, as certain events might be
initiated earlier in fast-growing transgenic fish. The absence of the mature
14 kDa MIP in newly hatched coho embryos is identical to the result obtained
in brook trout (Roberts and Goetz,
2003
), and suggests either a rapid turnover of the active peptide
or the build-up of the precursor without proteolytic processing. In general,
the expression of MIP just after hatching could be related to changes that
occur in the embryo as it starts to use somatic musculature for conventional
locomotion
While the present study suggests roles for MSTN and GH in muscle
development and growth in fish, there are still some aspects of MSTN
expression that are not fully understood. One of the most complex aspects of
MSTN expression is its differential expression across muscle tissue
type. MSTN2 ortholog RNA levels are higher in red muscle than white
muscle in brook trout and rainbow trout
(Rescan et al., 2001;
Roberts and Goetz, 2001
). In
the present study, this was also the case in transgenic coho salmon. However,
in size-control coho salmon, levels of MSTN2 were not different
between muscle types. MSTN2 ortholog expression levels were also the
same in white and red muscle for Atlantic salmon
(Ostbye et al., 2001
). One
explanation for the variation in MSTN2 ortholog expression in fish
could be that expression is associated with changes that occur in developing
muscle tissue. Salmonids undergo a distinct transformation in behavior and
physical characteristics between the first juvenile stage (parr) and older
juveniles (known as smolts in anadromous salmonids;
Hoar, 1988
). During this
transformation, significant changes occur in red muscle kinetics, including
activation time, relaxation time and maximum shortening velocity
(Coughlin et al., 2001
). In
addition, there are biochemical changes occurring in red muscle that are
apparent from shifts in myosin heavy chain expression
(Weaver et al., 2001
). In
nontransgenic coho and Atlantic salmon, in which the levels of MSTN2
ortholog were similar between muscle fiber types, the fish were generally
smaller and younger than the brook and rainbow trout studied. Thus, it is
possible that the differential expression is related to the age of the fish
from which samples were obtained. Wild-type coho and Atlantic salmon might not
have completed this transformation, whereas the other salmonids that were
studied had. With coho salmon, Hill et al.
(2000
) observed that the
effects of GH transgene expression on muscle anatomy were more pronounced in
red muscle. Thus, it is possible that the changes associated with development
(mentioned above) had already occurred in transgenic fish and that is why the
MSTN2 expression pattern in transgenic coho salmon muscle type is
more like that observed in the older rainbow and brook trout. In the present
study, western analysis indicated higher expression of the bioactive 14 kDa
MIP in red muscle compared to white muscle, regardless of GH transgene
expression. Higher levels of MSTN immunoreactivity in red muscle have also
been observed in other fish (Radaelli et
al., 2003
). However, the difference in muscle expression may not
be related to the same factors affecting RNA expression. Instead, decreased
levels of the bioactive MSTN protein in white muscle compared to red muscle
could be associated with the continual fiber recruitment and hypertrophy that
occur in adult salmonids. Therefore, lower levels of the active protein would
be expected to occur if MSTN is a negative regulator of muscle growth.
The present study used GH transgenic salmon as an experimental system to characterize the role of MSTN in fast growing animals and to examine the relationship of MSTN and GH expression. Decreased MSTN expression in faster growing fish suggests that MSTN does act as a negative regulator of muscle growth in fish, as it does in mammals. Specifically, MSTN2 RNA expression appears to be involved in muscle hyperplasia, as this has been shown to be a primary mechanism of increased growth in GH transgenic coho salmon. The results of this study also provide evidence that the anabolic effects of GH could be mediated through MSTN. Further research is needed to characterize the significance of the GHMSTN signaling pathway in controlling vertebrate muscle growth.
![]() |
Acknowledgments |
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![]() |
Footnotes |
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Present address: Great Lakes WATER Institute, University of
Wisconsin-Milwaukee, 600 East Greenfield Avenue, Milwaukee, WI 53204, USA
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