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Keywords:

  • Microcystin-LR;
  • PP2A;
  • Tau;
  • VASP;
  • HL7702

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Previously, we have reported alterations to HSP27 during Microcystin-LR (MC-LR)-induced cytoskeletal reorganization in the human liver cell line HL7702. To further elucidate the detailed mechanism of MC-LR-induced cytoskeletal assembly, we focused on two cytoskeletal-related proteins, Tau and VASP. These two proteins phosphorylated status influences their ability to bind and stabilize cytoskeleton. We found that MC-LR markedly increased the level of Tau phosphorylation with the dissociation of phosphorylated Tau from the cytoskeleton. Furthermore, the phosphorylation of Tau induced by MC-LR was suppressed by an activator of PP2A and by an inhibitor of p38 MAPK. VASP was also hyperphosphorylated upon MC-LR exposure; however, its phosphorylation appeared to regulate its cellular localization rather than cytoskeletal dynamics, and its phosphorylation was unaffected by the PP2A activator. These data suggest that phosphorylated Tau is regulated by p38 MAPK, possibly as a consequence of PP2A inhibition. Tau hyperphosphorylation is likely an important factor leading to the cytoskeletal destabilization triggered by MC-LR and the role of VASP alteration upon MC-LR exposure needs to be studied further. To our knowledge, the finding that Tau is implicated in cytoskeletal destabilization in MC-LR-treated hepatocytes and MC-LR-induced VASP's alteration has not been reported previously. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 92–100, 2015.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Microcystins (MC) are a family of monocyclic heptapeptide hepatotoxins produced by cyanobacteria during water blooms. Until now, more than 80 MC congeners have been found. Microcystin-LR (MC-LR) is the most common member of this family, and it has two variable amino acids, Leu and Arg (Campos and Vasconcelos, 2010). Studies of the hepatotoxic mechanisms of MC-LR have revealed that acute exposure of human or rat hepatocytes to MC-LR induces the rearrangement or collapse of the cytoskeleton (Wickstrom et al., 1995; Khan et al., 1996; Batista et al., 2003), thereby causing cytoskeleton-related cellular effects, and that this cytoskeletal alteration may be involved in pathological hepatic changes (Hooser et al., 1991). Although the induction of cytoskeleton assembly by MC-LR has been well studied, the relevant proteins that may be involved in this stress response are not clear.

HSP27, a small heat shock protein, could be regulated by posttranslational modifications. Phosphorylated HSP27 has been shown to provide cellular stress protection and to enhance actin stability (Lavoie et al., 1995; Huot et al., 1996; Bryantsev et al., 2002). In our previous study, we found that HSP27 was hyperphosphorylated and seemed to provide cytoskeletal stability during MC-LR-induced cytoskeletal destabilization (Sun et al., 2011). Thus, we infer that some cytoskeleton-related proteins with HSP27-opposing functions, i.e., that damage rather than stabilize the cytoskeleton, may be active during MC-LR-induced cytoskeletal reassembly. Therefore, in the present study, we aimed to further investigate the proteins involved in MC-LR-induced cytoskeletal changes, which may provide helpful insights into the mechanism of MC-LR-induced cytoskeletal reorganization.

Cytoskeletal organization and dynamics depend on protein self-associations and their interactions with regulatory elements such as microtubule-associated proteins (MAPs) (Maccioni and Cambiazo 1995). Tau, is an important neural MAP that is essential to the assembly and stability of microtubules. It has been demonstrated that the ability of Tau to bind and stabilize microtubules correlates inversely with its phosphorylation status (Drewes et al., 1995; Jenkins et al., 2000). In our prior study, we demonstrated that MC-LR induced hyperphosphorylation of Tau protein which may be caused by direct PP2A inhibition and indirect p38 MAPK activation in the neuroendocrine cell line PC12 (Meng et al., 2011). Tau and/or phospho-Tau have also been found in several other cell types, such as muscle fibers (Murakami et al., 1995), in human fibroblast and in Huh-7 hepatoma cell lines (Cross et al., 2000), in addition to neurons. While no study on the effects of Tau on hepatocytes has been done yet. Therefore, in the present study, we used a human liver cell line to investigate whether Tau is phosphorylated and is involved in MC-LR-induced microtubule alterations. Besides, we further identified the roles of JNK and ERK kinases in regulation of Tau.

Vasodilator-stimulated phosphoprotein (VASP), belongs to the Enabled/VASP (Ena/VASP) protein family that links signal transduction pathways to actin cytoskeleton dynamics (Barzik et al., 2005). VASP phosphorylation affects its interaction with actin (Harbeck et al., 2000; Benz et al., 2009). The phosphorylation of VASP at Ser157 was reported to potentiate F-actin binding by Laurent et al. (1999). VASP phosphorylation at this site also appears to modulate its subcellular distribution (Benz et al., 2009). However, there is no report showing that VASP is involved in MC-LR-induced cytoskeletal reassembly. Thus, in our present study, we investigated the presence and possible role of VASP (pSer157) in MC-LR-induced microfilament reorganization.

In the present study, we examined the expression of phosphorylated Tau and VASP in the human liver cell line HL7702 during MC-LR-induced cytoskeletal rearrangement. Because we observed PP2A inhibition in this MC-LR-induced stress condition in a prior study (Sun et al., 2011), thus in the present study we further examined the role of PP2A in regulating the phosphorylation of Tau and VASP in response to MC-LR exposure. Moreover, we explored the role of MAPKs in mediating MC-LR-induced Tau phosphorylation.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Materials

MC-LR was obtained from Alexis Biochemicals (Lausen, Switzerland). Powdered RPMI-1640 was purchased from Gibco (Scotland, UK). Trypsin was obtained from Serva (Heidelberg, Germany). The antibody against phospho-Tau (Ser199/202) was purchased from Invitrogen Corporation (Camarillo, CA). The antibodies against phospho-VASP (Ser157) and VASP were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from KangChen Biotech (Shanghai, China). Horseradish peroxidase-conjugated antigoat/mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). d-erythro-sphingosine (DES) (PP2A activator) was obtained from Calbiochem (San Diego, CA). The MAPK inhibitors PD98059 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) were purchased from Calbiochem (La Jolla, CA). Alexa Fluor 488-conjugated phalloidin were purchased from Molecular Probes, Invitrogen (Eugene, OR). Mouse monoclonal alpha-tubulin antibody was purchased from Abcam (Cambridge, UK). DyLight 549-conjugated goat anti-rabbit IgG and DAPI were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and Sigma. All other chemicals and reagents were of the highest grade available from commercial sources.

Cell Culture and MC-LR Exposure

The human normal liver cell line HL-7702 (adherent cells) was obtained from the Cell Bank of the Chinese Academic of Science and cultured in RPMI-1640 media supplemented with 10% newborn bovine serum. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Logarithmic phase cells were seeded into six- or 12-well plates for 36 or 24 h at a density of 5 × 104 cells/mL prior to MC-LR treatment. Then, the cells were incubated with 5 or 10 μM MC-LR for 12 and 24 h. For signaling studies or to evaluate the effects of DES, the cells were pretreated with MAPK inhibitors (25 μM SB203580, 10 μM PD98059, or 10 μM SP600125) or DES (10 μM) for 12 h prior to MC-LR exposure. As a control, cells that were not treated with MC-LR were incubated in the same media as the treated cells.

Total Protein Extraction

The cells were collected by scraping, pelleted by centrifugation in microtubes and then lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, pH 7.6). The supernatant was removed carefully and stored at −80°C.

Protein Extraction of the Triton X-100 Soluble and Insoluble Fractions

The distribution of phosphorylated Tau into Triton-soluble and Triton-insoluble fractions was assessed according to our previous protocol (Sun et al., 2011). After MC-LR exposure, the harvested and pelleted cells were taken up in lysis buffer (5 mM Tris–Cl, 300 mM sucrose, 2 mM EDTA, 0.5% Triton X-100, 0.5 mM PMSF, 1 mM Na3VO4, pH 7.4). The supernatant was collected and designated as the Triton X-100-soluble fraction, with the pellet designated as the Triton X-100-insoluble fraction. Western blot analysis was used to measure the expression of phospho-Tau.

Western Blot Analysis

The protein concentration was determined using the Bradford assay (in the standard curve concentration range ≤25 μg/μl). The samples containing 50 μg of protein were run on a SDS–PAGE gel and were transferred to a nitrocellulose membrane for 90 min. Primary antibodies were applied at 4°C overnight: phospho-Tau 1:1000, phospho-VASP 1:500, VASP 1:500, and GAPDH 1:5000, with the corresponding HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. The details of these procedures were published previously (Sun et al., 2011). The blots were analyzed by densitometry and quantified using Quantity-One software.

Immunofluorescence

The cells were washed twice with prewarmed PBS, fixed in 3.7% formaldehyde solution in PBS for 15 min at room temperature and treated with 1% BSA for 30 min. The antibodies were all dissolved in PBS containing 0.1% saponin. Cells were then incubated with antiphospho-VASP primary antibody (1:25) at 4°C overnight. Nuclei and F-actin were visualized by staining with DAPI for 10 min and Alexa Fluor 488-conjugated phalloidin for 30 min, respectively. The cells were imaged using a Zeiss confocal microscope. Randomly chosen fields are shown.

Statistical Analysis

The data are presented as the mean ± SD from three sets of experiments. Statistical analysis was performed using SPSS software. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. Differences were considered significant at P < 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

MC-LR Upregulates Tau Phosphorylation

Because the expression of phosphorylated Tau has not been studied in human liver cell lines, thus we investigated whether Tau could be phosphorylated by MC-LR in HL7702 cells. The cells were incubated with 5 or 10 μM MC-LR for 12 and 24 h. Phosphorylation of Tau (Ser199/202) was measured by western blot analysis. A low level of Tau phosphorylation was detectable in the untreated cells. Enhanced Tau phosphorylation was observed after a 12 h treatment with 5 μM MC-LR, and the increase in phosphorylation was found to be both time and concentration dependent, with the maximal phosphorylation observed after treatment with 10 μM MC-LR for 24 h (Fig. 1).

image

Figure 1. MC-LR upregulates Tau phosphorylation. The cells were incubated with 5 or 10 μM MC-LR for 12 and 24 h or control. (A) Phospho-Tau (p-Tau), total-Tau (Tau-5) and GAPDH were detected by western blotting. GAPDH was used as a control for equal loading. (B) Values were obtained for the phosphorylated bands in comparison with total protein bands. *Significantly (P < 0.05) different from the control.

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MC-LR Upregulates the Cytoskeletal Distribution of Hyperphosphorylated Tau

Because we found that MC-LR induced microtubule reassembly in our previous study (Sun et al., 2011), and we also detected the hyperphosphorylation of Tau in the present study, therefore we were interested in the possible role of Tau in MC-LR-induced microtubule changes. Western blot analysis revealed that MC-LR induced increasing levels of Tau phosphorylation, and the phospho-Tau was distributed to the Triton-soluble fraction (Fig. 2). The ratio of the Triton-soluble fraction (cytosolic fraction) to the Triton-insoluble fraction (cytoskeletal fraction) of phosphorylated Tau rapidly increased after 10 μM MC-LR treatment for 12 h compared with the control, and the maximal cytosolic:cytoskeletal ratio was observed after treatment with 10 μM MC-LR for 24 h (Fig. 2).

image

Figure 2. Distribution of phosphorylated Tau by MC-LR exposure. (A) The cells were incubated with 10 μM MC-LR for 12 and 24 h. Western blotting was used to detect the expression of p-Tau in the Triton-soluble fraction and the Triton-insoluble fraction. (B) The ratio was calculated from the measured amounts of p-Tau in the Triton-soluble and Triton-insoluble fractions. *Significantly (P < 0.05) different from the control.

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MC-LR Upregulates VASP Phosphorylation

To determine whether VASP is altered during MC-LR-induced F-actin reorganization, we measured the expression of VASP protein by western blot. The onset of VASP phosphorylation (Ser157) was slower than that of Tau phosphorylation. A low level of VASP phosphorylation was detectable in the untreated cells. Enhanced phosphorylation of VASP was after a 12 h treatment with 10 μM MC-LR, increasing thereafter, with a maximum treatment of 10 μM MC-LR for 24 h (Fig. 3).

image

Figure 3. MC-LR upregulates VASP phosphorylation. The cells were incubated with 5 or 10 μM MC-LR for 12 and 24 h or control. (A) Phospho-VASP (p-VASP), total-VASP (t-VASP) and GAPDH were detected by western blotting. GAPDH was used as a control for equal loading. (B) Values were obtained for the phosphorylated bands in comparison with total protein bands. *Significantly (P < 0.05) different from the control.

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MC-LR Upregulates the Intranuclear Distribution of Hyperphosphorylated VASP

We used immunofluorescence to detect VASP and F-actin to simultaneously evaluate the subcellular distributions of VASP and the interaction of VASP and F-actin. Immunofluorescence showed that phosphorylated VASP was detectable and distributed ubiquitously throughout the cytoplasm and the nucleus in control group; however, in MC-LR-treated group, hyperphosphorylated VASP had an elevated intranuclear distribution compared to the control treatment. Besides, MC-LR did not influence the colocalization of VASP and F-actin (Fig. 4).

image

Figure 4. Distribution of phosphorylated VASP by MC-LR exposure. The cells were incubated with 10 μM MC-LR for 24 h and fixed in 3.7% formaldehyde solution. P-VASP (red) was stained with an anti-p-VASP antibody and a fluorescence-conjugated secondary antibody. F-actin (green) was stained with phalloidin. Nucleus (blue) was stained with DAPI. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

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Activation of PP2A by DES Suppresses MC-LR-induced Phosphorylation of Tau But has no Influence on VASP Phosphorylation

Ceramide is a membrane sphingolipid that has recently emerged as a second messenger involved in the induction of PP2A activation, cytoskeleton destabilization, and apoptosis (Hannun and Obeid, 2008; Zeidan et al., 2008). Our previous results demonstrated that ceramide may mediate MC-LR-induced PP2A activation and cytoskeleton destabilization (Li et al., 2012). Because the activity of PP2A was decreased in HL7702 cells exposed to MC-LR in our prior study (Sun et al., 2011), therefore to explore whether the activity of PP2A could be responsible for the regulation of Tau and VASP phosphorylation, we treated HL7702 cells with D-erythro-sphingosine (DES) (C2-ceramide, a nonnatural, but cell-permeable analog of the endogenous long-chain ceramides) to activate PP2A (Deng et al., 2009; Zhu et al., 2010). Preincubating the cells with DES (10 μM) before MC-LR exposure partially blocked the MC-LR-induced increase in Tau phosphorylation (Fig. 5A). However, VASP phosphorylation was unaffected in the cells that were pretreated with DES (Fig. 5B). These results suggest that MC-LR controls Tau phosphorylation through the regulation of PP2A activity.

image

Figure 5. Effects of a PP2A activator on MC-LR-induced protein phosphorylation. The cells were preincubated with 10 μM DES and then exposed to 10 μM MC-LR for 24 h; the control was not exposed to MC-LR. (A) P-Tau and GAPDH pretreated with DES were detected by western blotting. (B) P-VASP and GAPDH pretreated with DES were detected by western blotting. GAPDH was used as a control for equal loading. *Significantly (P < 0.05) different from control. #Significantly (P < 0.05) different from MC-LR exposure.

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MC-LR-induced Hyperphosphorylation of Tau is Blocked by P38 MAPK Inhibitor

We have demonstrated that MC-LR can activate members of the MAPK superfamily (Sun et al., 2011). Because MAPK kinases are known substrates for PP2A (Lee et al., 2003; Liu and Hofmann 2004; Alvarado-Kristensson and Andersson 2005; Chen et al., 2008; Zhao et al., 2008), and our findings suggest that Tau is a downstream target of PP2A, we sought to investigate the possible link between MAPKs and the regulation of Tau phosphorylation induced by MC-LR. Pre-treating the cells with 25 μM SB203580 (a specific inhibitor of p38 MAPK) before MC-LR exposure suppressed the increased phosphorylation of Tau induced by MC-LR (Fig. 6). However, Tau phosphorylation was unaffected in the cells that were pretreated with 10 μM PD98059 (a specific inhibitor of the ERK1/2 upstream kinase MEK1/2) or 10 μM SP600125 (a JNK inhibitor) (Fig. 6).

image

Figure 6. Effects of MAPK inhibitors on MC-LR-induced Tau phosphorylation. The cells were pre-incubated with 25 μM SB203580, 10 μM SP600125, or 10 μM PD98059 and then exposed to 10 μM MC-LR for 24 h; the control was not exposed to MC-LR. (A) P-Tau and GAPDH were detected by western blotting. GAPDH was used as a control for equal loading. (B) Values obtained by comparing the phosphorylated bands with total protein. *Significantly (P < 0.05) different from control. #Significantly (P < 0.05) different from MC-LR exposure.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The dynamic stability of microtubules is thought to be regulated by MAPs, such as Tau, that promote the polymerization of tubulin dimers and stabilize microtubules (Sergeant et al., 2008). Tau hyperphosphorylation was correlated with the dissociation of Tau from microtubules and a loss of Tau-induced microtubule stabilization (Sontag et al., 1996; Jenkins et al., 2000). However, whether such hyperphosphorylation is induced in MC-LR-treated hepatocytes remains unclear. In the present study, Tau phosphorylation (Ser199/202) was found to be upregulated by MC-LR in a time- and concentration-dependent manner in HL7702 cells. Moreover, an increased ratio of the Triton-soluble (cytosolic) to the Triton-insoluble (cytoskeletal) fractions of phosphorylated Tau was observed, suggesting that more phosphorylated Tau is distributed to the cytosolic fraction, which may reflect a reduction in the binding of Tau to microtubules, as previously reported (Sontag et al., 1996). Tau hyperphosphorylation is likely a key factor in the destabilization of the cytoskeleton triggered by MC-LR, which may be subsequently involved in the cytoskeleton-related cellular effects and the mechanism of MC-LR hepatotoxicity. To our knowledge, the finding that Tau is hyperphosphorylated and implicated in cytoskeletal destabilization in MC-LR-treated hepatocytes has not been reported previously.

The human VASP protein is composed of 380 amino acids and can be phosphorylated at three sites: two serine sites (Ser157 and Ser239) and one threonine site (Thr278) (Zhang et al., 2010). The phosphorylation of VASP at Ser157 was reported to potentiate its binding to F-actin (Laurent et al., 1999), which subsequently mediates cell migration(Zhang et al., 2010), or to modulate its subcellular distribution (Benz et al., 2009). In the present study, a progressive increase in VASP phosphorylation was detected (using the antiphospho-VASP (Ser157) antibody) in response to increasing MC-LR concentrations. However, phosphorylated VASP was mainly located in the cell nucleus, and increased intranuclear localization was observed in cells treated with MC-LR; the interaction of VASP and F-actin was not detected, as we predicted. Consistent with our results, previous studies have shown that the phosphorylation status of VASP appears to modulate its subcellular distribution. Comerford found that increased S157 phosphorylation of VASP promoted its accumulated at the cell periphery in human cells (Comerford et al., 2002). Benz demonstrated that phosphorylation at Ser157 does not affect Ena/VASP-driven F-actin assembly but favors VASP localization to focal adhesions and lamellipodia as cells spread (Benz et al., 2009). Additionally, VASP phosphorylation also modulates other protein–protein interactions. S157 phosphorylation abrogates the interactions between VASP and Abl (Howe et al., 2002), nSrc and aII-spectrin (Benz et al., 2008). However, the function of MC-LR-induced intranuclear translocation of VASP must be explored further. To our knowledge, the finding that MC-LR induces VASP's phosphorylation and regulates its cellular localization has not been reported previously.

PP2A activity was markedly inhibited by MC-LR in our prior study (Sun et al., 2011). To confirm the role of PP2A in regulating the phosphorylation of cytoskeleton-related proteins, we pretreated cells with DES (a PP2A activator) for 12 h before MC-LR exposure. Tau phosphorylation induced by MC-LR was partially blocked by DES, while VASP phosphorylation was unaffected. Our data demonstrated that Tau is a substrate of PP2A and that its phosphorylation status is regulated by PP2A activity, which was in agreement with previous reports (Liu et al., 2005). However, our findings show that VASP is not a downstream target of PP2A. Because MC-LR is a potent inhibitor of both PP2A and PP1, it is possible that PP1 rather than PP2A is responsible for regulating VASP phosphorylation.

In our prior study, we detected MAPK activation during MC-LR-induced cytoskeletal reassembly (Sun et al., 2011). In the current study, using inhibitors of these MAPKs (p38 MAPK, JNK, and ERK), we found that p38 MAPK regulates Tau phosphorylation. Consistent with these results, other studies have shown that Tau phosphorylation is achieved by the activation of p38 MAPK (Reynolds et al., 1997; Feijoo et al., 2005). It is well documented that MAPKs are substrates of PP2A and are either dephosphorylated by PP2A or activated by the inhibition of PP2A (Lee et al., 2003; Alvarado-Kristensson and Andersson 2005; Chen et al., 2008; Zhao et al., 2008). Therefore, the hyperphosphorylation of Tau observed in the present study is likely due to the activation of p38 MAPK as a consequence of MC-LR-induced PP2A inhibition. However, several reports have shown that the direct dephosphorylation of Tau by PP2A also exists (Liu et al., 2005; Xu et al., 2008). Taken together, these results suggest that the PP2A-driven regulation of Tau phosphorylation may not be restricted in a unique manner.

In summary, the present work demonstrates that MC-LR exposure induced Tau and VASP hyperphosphorylation during cytoskeletal reorganization in HL7702 cells. Furthermore, increasingly phosphorylated Tau was distributed in the cytosolic fraction, and disassociated from microtubules. Tau phosphorylation may be regulated by the PP2A-mediated activation of the p38 MAPK signal pathway. Although VASP phosphorylation increased markedly in response to MC-LR treatment, the phosphorylation of VASP seemed to regulate its intracellular location but not its cytoskeletal regulation activities, and it was not a target of PP2A. Our study shows that the hyperphosphorylation of Tau is likely a key factor in the destabilization of the cytoskeleton induced by MC-LR, which may be involved in the subsequent MC-LR hepatotoxicity, while MC-LR-induced VASP alteration needs to be explored further.

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  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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