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Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis whether calcium preconditioning (CPC) reduces reoxygenation injury by inhibiting mitochondrial permeability transition (MPT). Cultured myocytes were preconditioned by a brief exposure to 1.5 mM calcium (CPC) and subjected to 3 h of anoxia followed by 2 h of reoxygenation (A-R). Myocytes were also treated with 0.2 µM/l cyclosporin A (CsA), an inhibitor of MPT, before A-R. A significant increase of viable cells and reduced lactate dehydrogenase release was observed both in CPC- and CsA-treated myocytes compared with the A-R group. Cytochrome c release was predominantly observed in the cytoplasm of myocytes in the A-R group in contrast with CPC- or CsA-treated groups, where it was restricted only to mitochondria. Similarly, the cell death by apoptosis was also markedly attenuated in these groups. Electron-dense Ca2+ deposits in mitochondria were also less frequent. Atractyloside (20 µM/l), an adenine nucleotide translocase inhibitor, caused changes similar to those in the A-R group, suggesting a role of MPT in A-R injury. Protection by inhibition of MPT by CsA and CPC suggests that MPT plays an important role in reoxygenation/reperfusion injury. The data further suggest that preconditioning inhibits MPT by inhibiting Ca2+ accumulation by mitochondria.
anoxia-reoxygenation; cytochrome c; neonatal rat myocytes
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS WELL KNOWN that a brief exposure to ischemia protects the myocardium against infarction from a subsequent longer ischemic insult (25). This phenomenon was defined as ischemic preconditioning (IPC). IPC may have significant clinical impact, because it may occur in certain surgical procedures or by multiple periods of brief ischemia. Because IPC is a strong endogenous protective mechanism, triggering molecules and effectors of IPC are under intense investigation. The mechanism by which IPC induces protection is not yet clear. It has been reported that the effects of preconditioning could be mediated by attenuation of apoptosis (20, 28), reduction of Ca2+ accumulation by mitochondria (35), and increased ATP synthesis due to activation of mitochondrial ATP-sensitive K+ (KATP) channels (36, 37).
Necrotic and apoptotic cell death is thought to be related to altered mitochondrial function because mitochondria possess a latent nonspecific pore in the inner membrane, known as the mitochondrial permeability transition (MPT) pore (12, 16,17). Halestrap et al. (12) proposed that the MPT is formed by mitochondrial cyclophilin-D binding to the adenine nucleotide translocase (ANT), causing a conformational change in the ANT due to matrix Ca2+. It appears that intracellular ionic alterations may contribute to the IPC, leading to improved postischemic function. IPC has been shown to attenuate the detrimental rise in intracellular free Ca2+ concentration ([Ca2+]i) caused by subsequent sustained ischemia (7, 33). However, a brief period of ischemia-reperfusion induces an increase in [Ca2+]i (24, 34). This transient increase in [Ca2+]i could be important trigger for the preconditioning-induced protection against lethal ischemia (22, 29) and Ca2+ overload injury (1). A transient ischemic stimulus leads to a rapid adaptation of [Ca2+]i homeostasis during subsequent longer ischemic conditions. Such a rapid adaptation might be an important mechanism of the IPC phenomenon in protecting against ischemic injury (32). Therefore, it appears that the transient and reversible increase in cytosolic calcium concentration could be beneficial, whereas a massive increase in total calcium content is a hallmark for irreversible injury in ischemia-reperfusion (27). Therefore, the regulation of ion homeostasis, particularly calcium, may be manipulated for endogenous protection against lethal ischemia. Cain et al. (4) demonstrated that Ca2+ preconditioning protected the human myocardium against ischemia-reperfusion injury, and concurrent protein kinase C inhibition abolished the salutary effect of Ca2+ preconditioning in the human myocardium.
Because cell death in the myocyte is closely related to mitochondrial function and calcium preconditioning (CPC) conferred cardioprotection against ischemia-reperfusion injury, we, therefore, in the present study proposed that MPT is an important factor in the ischemia-induced cell death and CPC could ameliorate cell injury by prevention of MPT in cultured neonatal rat myocytes.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, Revised 1985). Cytochrome c (from the bovine heart), atractyloside (ATR), bromodeoxyuridine (BrdU), and goat anti-mouse IgG (Fab fragment) peroxidase conjugate were purchased from Sigma Chemical (St. Louis, MO); cyclosporin A (CsA) was purchased from Biomol Research Laboratories (Plymouth Meeting, PA).
Preparation of Myocyte-Rich Culture
Primary cultures of the neonatal rat myocytes were prepared as described previously (38). To selectively enrich myocytes, dissociated cells were preplated for an hour to allow nonmyocytes to attach to the bottom of the culture dish. The resultant suspension of myocytes was transferred onto collagen-coated 60-mm or 100-mm culture dishes. BrdU (100 µM) was added during the first 24 to 36 h to prevent proliferation of nonmyocytes. Cultured cells were further confirmed using immunofluorescence staining with a monoclonal antibody against sarcomeric
-actinin (14).
Experimental Protocols
The experiments were performed on myocyte-rich cultures on the third day. Cultured myocytes were divided into five groups (Fig. 1), and the medium was replaced by Tyrode solution (pH 7.4 at 37°C), which contained (in mM) 125 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 25 HEPES with or without glucose.
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Group 1: control. Myocytes were incubated in aerobic Tyrode solution with glucose (25 mM) during the entire experimental period.
Group 2: anoxia-reoxygenation. Myocytes were incubated with anaerobic glucose-free Tyrode solution for 3 h of anoxia followed by 2 h of reoxygenation (A-R). Myocytes in Tyrode solution were transferred into the anoxic chamber (Forma 1025 anaerobic system). To induce complete anoxia, Tyrode solution was deoxygenated by bubbling with purified nitrogen for 1 h before the experiments. Reoxygenation was carried out by returning the myocytes to the incubator.
Group 3: CsA + A-R. Myocytes were preincubated with CsA (0.2 µM/l) for 20 min before anaerobic incubation and reoxygenation. CsA is a strong inhibitor of MPT (12) and thus reduces cell necrosis.
Group 4: CPC. group 4a. Myocytes were exposed two times each for 4 min with Tyrode solution containing 1.5 mM/l Ca2+ followed by 6 min of normal Ca2+-containing Tyrode solution (1.0 mM/l) before the A-R protocol.
GROUP 4B: CPC + ATR. To determine whether inhibition of MPT is the mechanism of CPC, ATR (20 µM/l), an inhibitor of ANT, which opens the pore, was added during the preconditioning protocol.Cell Viability and ATP Assay
Determination of myocyte injury was carried out at the end of the A-R. The extent of A-R-induced injury was quantitated based on the number of dead cells, ATP content, and lactate dehydrogenase (LDH) release as well as morphological examination.Cell viability was calculated by dividing the number of trypan blue-negative cells from the total number of cells examined and then multiplied by 100%. Ultrastructural assessment of myocytes was carried out by transmission electron microscopy. Myocytes cultured on the coverslips were immersed in 2.5% buffered glutaraldehyde for 4 h, rinsed in 0.1 mol/l sodium cacodylate buffer (pH 7.3), and postfixed in 1% buffered osmium tetroxide for an hour. The cells were embedded in epon resin and cut into 600-nm-thick sections with a Sorvall MTB2 ultramicrotome. The sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-600 electron microscope at 75 kV.
ATP was determined as previously described (23). Isolated myocytes were homogenized with a sonic dismembrator in 6% trichloroacetic acid. The homogenates were centrifuged at 25,000 g for 10 min. The extracts were neutralized with potassium carbohydrate. ATP was analyzed at 340 nm in a Beckman spectrophotometer by using an ATP detection kit (Sigma). The results were expressed in nanomoles per milligram protein. LDH release from myocytes was measured by using a LDH detection kit (Sigma) and expressed as milliunits per milligram protein.
Detection of Apoptotic Cells
To visualize apoptotic nuclei in cardiac myocytes in situ, the ApoTag in situ apoptosis detection kit (Oncor) was used. The cultured myocytes were fixed in 4% paraformaldehyde (pH 7.4) and subjected to terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. In brief, myocytes were incubated for 1 h at 37°C in TdT buffer (pH 7.2) containing (in mM) 140 sodium cacodylate, 1 cobalt chloride, 30 Tris · HCl, 50 units of terminal deoxynucleotide transferase, and 1 nM of FITC-conjugated dUTP. After the TdT reaction, myocytes were washed three times in phosphate-buffered saline (PBS) and mounted on glass slides. As negative control, sections were incubated in the absence of TdT enzyme. Individual nuclei were visualized at a magnification of ×400 for quantitative analysis. An average of 400 nuclei from random fields was analyzed in each slide as the apoptotic index (percentage of apoptotic nuclei) × 100%. Sample identities were concealed during scoring, and samples from at least three independent experiments were scored per group.Quantitation of Cytochrome c
Isolation of mitochondrial and cytosolic fractions. Cells were harvested by centrifugation at 600 g for 10 min at 4°C. The pellet was washed with PBS, and cells were scraped into HEPES buffer (pH 7.5) [containing (in mM/l) 10 HEPES, 200 mannitol, and 70 sucrose], which contained protease and phosphatase inhibitors. After chilling on ice for 3 min, the cells were disrupted by 40 strokes of a sonic dismembrator (Fisher model 60). The samples were centrifuged (500 g) to pellet nuclei, unbroken cells, and plasma membrane debris (nuclear fraction). The supernatants were recentrifuged (10,000 g) to separate the mitochondrial fraction from the cytosolic fraction. The mitochondrial fraction was resuspended in HEPES buffer containing 1% (vol/vol) Triton X-100. The supernatants were recentrifuged once more at 200,000 g for 30 min, resulting a supernatant (cytosolic fraction).
Western blot analysis. Cytochrome c release from the mitochondria into cytosol was determined at the end of the experiment by using a modified method of Cook et al. (5). Aliquots of mitochondria and cytosol were boiled, and 10 µg of protein from both fractions was added on 13% SDS-polyacrylamide gels. After electrophoresis, the samples were transferred to Trans-Blot transfer medium-supported nitrocellulose membranes (Bio-Rad). The blots were then blocked with 5% nonfat milk and incubated with the first antibody and second antibody at room temperature. The first antibody was mouse monoclonal antibody to denatured cytochrome c (1:500; Pharmingen). The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse antibody (1:2000; Sigma). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech), and blots were exposed to Hyperfilm MP for 30 s-2 min. Laser scanning densitometry was used for the semiquantitative determination of the proteins.
Immunocytochemistry. The distribution of cytochrome c in intact myocyte was assayed as described by Roberg et al. (30). Cultured myocytes on coverslips were fixed in 2% formaldehyde and blocked with 10% normal goat serum. Mouse monoclonal anti-cytochrome c antibody was used at a dilution of 1:100. After washing coverslips with PBS-0.1% Tween 20, cells were incubated with secondary antibody consisting of FITC-conjugated anti-mouse IgG antibody. For mitochondrial staining, unfixed cells were incubated with 500 µM Mito Tracker Orange CMTMRos (Molecular Probes) for 30 min at 37°C. After washing and fixation in 2% formaldehyde, cells were stained further with anti-cytochrome c antibody as described above.
Statistical Analysis
All the experiments were carried out from at least three independent myocyte cultures with replicates of two for each condition. The data were expressed as means ± SE. Group comparisons were analyzed by a two-way analysis of variance. Statistical significance between groups was determined by Student's t-test. Spearman's analysis was used for the correlation between the parameters. A value of P < 0.05 was considered significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CPC Prevents A-R Injury
The purification of cardiac myocytes was assessed with the mouse anti--actinin and FITC-labeled mouse anti-IgG antibody. With the use
of the present method, we obtained 90% myocytes in the cultured cells.
The effect of A-R on the cell viability was examined by using a trypan
blue exclusion assay (Fig. 2). In the
control neonatal myocytes, 86.5 ± 2.7% cells excluded trypan
blue and were considered normal, whereas in the A-R group only
38.5 ± 3.1% excluded trypan blue. CPC significantly increased
the number of viable cells (54.3 ± 3.3 vs. 38.5 ± 3.1%, P < 0.05) compared with A-R alone. In the transmission electron microscope, the cell membrane was intact, and
well-defined rows of mitochondria were observed between the compact
myofibrils or scattered loosely throughout the cytoplasm. Nuclear
chromatin material was uniformly dispersed (Fig.
3A). The
myocytes subjected to A-R were characterized by calcified mitochondria,
clumped chromatin material, wavy myofibers, and granularity of
cytoplasm with distorted subcellular organelles (Fig. 3B).
The cellular structures were extremely well preserved in CPC cells
subjected to the A-R and were similar to structures in the control
cells. Mitochondria were usually elongated. Nuclear chromatin material
was uniformly dispersed (Fig. 3C).
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LDH release was significantly increased (130.3 ± 11.0 mU/mg
protein) in the A-R group compared with that of control group (45.5 ± 6.5 mU/mg protein) (Fig.
4A). In the CPC group, the LDH release was similar to that of control group (66.9 ± 11.9 mU/mg protein). ATP in the myocytes subjected to A-R was significantly decreased when compared with the cells in the control group (7.6 ± 2.5 vs. 27.5 ± 1.8 nM/mg protein in control, P < 0.05) (Fig. 4B). The ATP content was well be preserved in
the CPC group (17.3 ± 1.4 nM/mg protein).
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Cell shrinkage and nuclear fragmentation, which are the typical
morphological features of apoptosis, were commonly observed. In our
experimental conditions, the number of TUNEL-positive nuclei in the
control was <10% (Fig. 5A).
However, in the A-R group, the number of TUNEL-positive cells was
markedly increased (39.2 ± 3.0%) (Fig. 5B). In the
CPC group (Fig. 5C), the number of TUNEL-positive cells was
significantly decreased (20.2 ± 2.1%) compared with that in the
A-R group.
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CPC Inhibits MPT
A-R has adverse effects on mitochondrial structure and function. We used a specific inhibitor of MPT to observe whether the induction of MPT is critical for A-R-induced injury. We then tested whether a specific agonist for the mitochondrial pore induces cell injury similar to A-R. Finally, the distribution of cytochrome c was determined.The treatment with CsA (0.2 µM/l) inhibited the killing of cells and increased the cell survival (56.3 ± 2.8 vs. 38.5 ± 3.1%, P < 0.05, CsA group vs. A-R group) similar to that of the CPC group (Fig. 2). The release of LDH from myocytes was significantly reduced (56.9 ± 9.0 mU/mg protein) compared with the A-R group (130.3 ± 11.0 mU/mg protein) (Fig. 4A). In addition, CsA also significantly inhibited the A-R-stimulated apoptosis (19.5 ± 2.0 vs. 39.2 ± 3.0%) (Fig. 5) and reduced the morphological changes (Fig. 3).
However, the effect of CPC was inhibited by administration of ATR (20 µmol/l) during CPC. The cell survival rate was significantly decreased in the treated group compared with the CPC group (39.3 ± 3.2 vs. 54.3 ± 3.3%) (Fig. 2). The LDH release from myocytes was increased (100.9 ± 9.4 vs. 66.9 ± 11.9 mU/mg protein, ATR group vs. CPC group), and ATP was exhausted (Fig. 4B). Furthermore, the percentage of apoptotic cardiac myocytes was increased (27.5 ± 2.2 vs. 20.2 ± 2.1%) (Fig. 5), and myocytes underwent severe structural changes (Fig. 3D).
The mitochondrial impairments may lead to the induction of apoptosis
through the release of cytochrome c. As expected, cytochrome c release was increased in the cytosol when myocytes were
subjected to A-R compared with the control myocytes or CPC group (Fig.
6). Pretreatment of myocytes with CsA
significantly blocked the release of cytochrome c from cells
subjected to A-R (P < 0.05) and was similar to the CPC
group. In contrast, ATR aggravated the release of cytochrome
c from mitochondria (Fig. 6). The concentration of
cytochrome c in mitochondria displayed a negative linear
correlation with the percentage of apoptosis in the myocytes (Fig.
7). To confirm translocation of
cytochrome c to the cytosol in individual apoptotic cells,
immunocytochemistry was performed (Fig.
8). In control cells with normal nuclear
morphology, cytochrome c immunostaining was observed in oval
to elongated or punctate bodies, which coincided the distribution of
mitochondria that were stained with Mito Tracker Orange CMTMRos. In
contrast, the myocytes that underwent A-R exhibited a diffuse
cytochrome c immunostaining (Fig. 8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Molecules released after a brief ischemic or Ca2+ stress (so-called preconditioning phemenon) are known to induce endogenous protection against prolonged ischemia. Among these molecules, Ca2+ is an important molecule for both cell death and life. A slight increase in [Ca2+]i, as observed in preconditioning (21, 22), is beneficial and an important trigger for the CPC. Intracellular Ca2+ can activate several important enzymes, which can initiate different signaling pathways for the cardiac protection. Some of these enzymes include protein kinase C (36, 37, 41), inducible nitric oxide synthase (2), and mitogen-activated protein kinases (19, 20). On the other hand, the excessive Ca2+ accumulation can lead to cell death under different pathological conditions (10, 18).
This study utilized Ca2+ as a stress molecule to activate intracellular machinery for the attenuation of anoxic injury. Calcium-mediated pathways have provided protection against ischemia using neonatal myocytes (this study) and intact hearts (22, 29) similar to that provided by IPC. It has been suggested that free radicals are important intracellular signaling components in producing IPC in cardiocytes (9, 31, 40). Although increased free radicals production during hypoxic preconditioning appears to originate from the mitochondrial electron transport system (9), more experiments are needed to confirm the mechanism by which CPC affects intracellular free radical production.
One of the mechanisms involved in the protection is the preservation of mitochondrial structure and function. Once the mitochondria accumulate Ca2+, the ATP synthesis ceases, and the myocytes undergo irreversible cell injury after lethal ischemia (15). This is preceded by the opening of a pore in the inner mitochondrial membrane, and it occurs soon after reoxygenation, which is accompanied by Ca2+ overload (8). This phenomenon of opening of mega channels in mitochondrial inner membrane is called MPT.
MPT induces cytochrome c release and, consequently, apoptosis (26), and this is in agreement with the data of this study. Apoptosis was significantly reduced in the preconditioned myocytes. MPT was inhibited by CsA, an inhibitor of MPT, and was promoted by ATR, an ANT inhibitor (11). Thus it is clear that CPC maintains the mitochondrial Ca2+ homeostasis and prevents MPT. MPT was accompanied by cytochrome c release from mitochondria, which is a typical feature of apoptosis. Cytochrome c was seen distributed throughout the cytoplasm in the myocytes after A-R and in myocytes treated with ATR. Cytochrome c release can also result from changes in mitochondrial membrane permeability after loss of membrane potential during apoptosis (42). This may result in the swelling of the mitochondria, rupturing the outer membrane and releasing cytochrome c into cytosol (3). It has been demonstrated that the induction of MPT causes release of cytochrome c from mitochondria, which is required for the genesis of apoptosis, caspase 3 activation, and nuclear laddering (39). Our study further confirmed this observation that release of cytochrome c from mitochondria preceded the apoptosis in cultured myocytes during A-R.
The mechanism by which preconditioning inhibits MPT is not yet totally clear. It appears that initial excessive Ca2+ accumulation by mitochondria after reoxygenation promotes the opening of pores in the inner membranes of mitochondria, which subsequently allows unlimited entry of Ca2+ during reoxygenation. Crompton and Costi (6) have shown that Ca2+ accelerated the opening of pores in isolated mitochondria and that it was dependent on the level of cellular ATP. We have previously shown that preconditioning preserves ATP contents in the ischemic myocardium (36) and maintains the structural integrity of mitochondria (35). Therefore, it is likely that preconditioning prevents mitochondrial pore opening. Similarly, activation of mitochondrial KATP channels during preconditioning leads to reduction in Ca2+ accumulation by mitochondria, as observed by electron microscopy (35). The study by Holmuhamedov et al. (13), in which isolated preloaded mitochondria released their Ca2+ contents on opening of the mitochondrial KATP channel, is also in agreement with our conclusions that the degree of Ca2+ accumulation by mitochondria may be a determinant of MPT. Further direct measurements of mitochondrial Ca2+ and its role in the opening of mitochondrial pores during reoxygenation are needed to determine its role in cell death and apoptosis.
In summary, MPT is a leading determinant of myocyte cell death, and preconditioning suppresses the pore opening and apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Nancy Koster for fluorescence microscopy.
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FOOTNOTES |
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This work was supported by the National Heart, Lung, and Blood Institute Grants HL-23597 and HL-55678.
Address for reprint requests and other correspondence: M. Ashraf, Dept. of Pathology and Laboratory Medicine, Univ. of Cincinnati, 231 Bethesda Ave. Cincinnati, OH 45267-0529 (E-mail: Muhammad.Ashraf{at}UC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 August 2000; accepted in final form 28 September 2000.
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TOP
ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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