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Department di Biologia e Patologia Cellulare e Molecolare, Istituto di Endocrinologia e Oncologia Sperimentale ‘G. Salvatore’-CNR, Universita' di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy1 Proteomics and Mass Spectrometry Laboratory, ISPAAM-CNR, 80147, Napoli, Italy
(Correspondence should be addressed to M Zannini; Email: s.zannini{at}ieos.cnr.it)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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Pax8 is one of the transcription factors involved in thyroid-specific gene expression (Damante & Di Lauro 1994, De Felice & Di Lauro 2004). It is a member of the Pax family of genes that are characterized by the presence of a 128-amino acid DNA binding domain, the paired domain (PD; Plachov et al. 1990). These genes encode for DNA-binding proteins that are involved in the regulation of the development of a variety of tissues in different species. In particular, Pax8 has been shown to be required for both the morphogenesis of the thyroid gland (Mansouri et al. 1998) and for the maintenance of the thyroid-differentiated phenotype (Pasca di Magliano et al. 2000). Interestingly, in Pax8 knockout mice the thyroid gland is barely visible and lacks the follicular cells (Mansouri et al. 1998). Recently, we have demonstrated that Pax8 interacts biochemically and functionally with TIF1/NKX2.1 (NK2 homeobox 1), another transcription factor involved together with Pax8 in the regulation of thyroid-specific genes (Di Palma et al. 2003).
Given the critical role played by Pax8 in thyrocyte differentiation, we focused our attention on the molecular mechanisms by which Pax8 modulates thyroid gene expression. Our hypothesis was that Pax8, similarly to other members of the Pax gene family (Fitzsimmons et al. 1996, Wheat et al. 1999), could play a role in the recruitment of other factors and/or co-factors to the gene target promoters by specific protein–protein interactions. In this study, we report on a nuclear protein, namely poly(ADP-ribose) polymerase 1 (PARP1), which was isolated during GST-pull down experiments with thyroid cell lysates using a bait consisting of Pax8 fused to GST. Further experiments confirmed that PARP1 directly interacts with Pax8 in vitro and in vivo, although it does not poly(ADP-ribosyl)ate it. Transactivation assays finally demonstrated that PARP1 behaves as a Pax8 repressor.
PARP1 is a nuclear enzyme activated by DNA strand breakage that catalyzes the transfer of ADP-ribose units from NAD+ to numerous nuclear proteins (de Murcia & Menissier de Murcia 1994, D'Amours et al. 1999). It participates in several cellular catalytic activities, including anti-recombination and genomic stability (Ding & Smulson 1994), DNA replication (Simbulan-Rosenthal et al. 1998), apoptosis regulation and DNA repair (Jeggo 1998, Pieper et al. 1999). Recent studies have also underlined its role in the regulation of gene transcription (D'Amours et al. 1999, Chiarugi 2002, Kraus & Lis 2003) and as a partner of several transcription factors and (co)-factors, including NF-kB (Hassa et al. 2003, Nakajima et al. 2004), AP-2 (Kannan et al. 1999), B-MYB (Cervellera & Sala 2000), E2F-1 (Simbulan-Rosenthal et al. 2003), RAR/RXR (Miyamoto et al. 1999, Pavri et al. 2005) and p53 (Kumari et al. 1998). Recently, another member of the PARP family, namely PARP2, and PARP1 were identified as interacting proteins of the transcription factor TIF1/NKX2-1 (Maeda et al. 2006).
Accordingly, the results here reported suggest that the interaction between PARP1 and Pax8 may significantly affect a series of molecular events related to Pax8-dependent gene expression, thus disclosing important scenarios on mechanisms playing a central role in the regulation of thyroid-specific transcription.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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The plasmids used have been previously described and are as follows: Tg-CAT (Sinclair et al. 1990), CP5-CAT (Missero et al. 1998), GST-Pax8 (Di Palma et al. 2003), GST-PD (pGEX-Pax8PB; Zannini et al. 1992), pCMV5-Pax8 (Zannini et al. 1992), and pPARP31 (Van Gool et al. 1997). The latter was kindly provided by Dr Kannan (MetroHealth Medical Center, Cleveland, OH, USA).
Protein identification by mass spectrometry
GST and GST–Pax8 proteins were purified from BL21 (DE) LysS bacterial cells as previously described (Zannini et al. 1992, Di Palma et al. 2003). The purified proteins were loaded onto glutathione–Sepharose beads (GE Healthcare, Waukesha, WI, USA) at a concentration of 1 µg/µl packed beads for 20 min at 4 °C.
A total of 5x108 PC Cl3 cells were lysed; after a pre-clearing step on of GST-coated beads, the total extract was adsorbed with GST and GST–Pax8 beads. After washing, the bound proteins were resolved by SDS-PAGE, and visualized by ammoniacal silver staining according to Shevchenko's protocol (Shevchenko et al. 1996). For identification, protein bands were excised from the gel, triturated, in-gel S-alkylated and digested with trypsin as previously reported (Caratu et al. 2007, Vascotto et al. 2007). Gel particles were extracted with 25 mM NH4HCO3/acetonitrile (1:1 v/v) by sonication. Samples were desalted using microZipTipC18 pipette tips (Millipore, Bredford, MA, USA) before MALDI-TOF-MS analysis. Peptide mixtures were loaded on the MALDI target together with a-cyano-4-hydroxycinnamic acid as matrix, using the dried droplet technique. Samples were analyzed with a Voyager-DE PRO spectrometer (Applera, Foster City, CA, USA; Caratu et al. 2007, Vascotto et al. 2007). Mass spectra were acquired in reflectron mode; internal mass calibration was performed with peptides from trypsin autoproteolysis. Data were elaborated using the DataExplorer 5.1 software (Applera, USA). ProFound software (Zhang & Chait 2000) was used to identify protein bands from an NCBI non-redundant database using peptide mass fingerprint data. Candidates with program Est'd Z scores more than 2 were further evaluated by comparison with Mr experimental values obtained from SDS-PAGE. Eventual occurrence of protein mixtures was ascertained by sequential searches for additional protein components using unmatched peptide masses.
Protein extracts and immunoblotting
For reporter assays, cells were washed twice with ice-cold PBS and lysed in a buffer containing 10 mM HEPES pH 7.9, 400 mM NaCl, 0.1 mM EGTA pH 7.8, 5% v/v glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF). For GST-pull down assays, PC Cl3 cells were washed twice with PBS and lysed in whole cell extract (WCE) buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 0.1% Triton X100, 10% v/v glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors (Sigma). For immunoprecipitation experiments, cells were washed twice with PBS and lysed in EBC buffer containing 50 mM Tris pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors (Sigma).
Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories Inc). For western blot analysis, proteins were separated by SDS-PAGE and the gels were blotted onto Immobilon P (Millipore) for 2 h; membranes were blocked in 5% nonfat dry milk in Tris-bufferd saline for 2 h or overnight before the addition of antibody for 1 h. The primary antibodies used were anti-PARP1 (46D11; Cell Signaling, Boston, MA, USA), anti-Pax8 (kindly provided by Di Lauro), anti-PAR (96-10-04; Alexis ,San Diego, CA, USA) and anti-FLAG-M2 (Sigma).
Pull down assays and co-immunoprecipitation
GST–PD and GST–Pax8 proteins were purified from BL21 (DE) LysS bacterial cells as previously described (Zannini et al. 1992, Di Palma et al. 2003).
GST, GST–Pax8 or GST-PD bound to glutathione beads were incubated with total protein extract prepared from PC Cl3 cells, or with 25 ng recombinant human PARP1 (ALX-201-063; Alexis). The binding reactions were carried out for 90 min at 4 °C on a rotating wheel. After extensive washing with WCE buffer, bound proteins were detected by western blot.
Co-immunoprecipitation experiments were performed by incubating 3 mg total protein extract with 40 µl anti-FLAG agarose affinity gel (Sigma), overnight, at 4 °C, on a rotating wheel. Bound proteins were washed several times with EBC buffer, resuspended in SDS-PAGE sample buffer and heated at 95 °C for 5 min, before loading on the gel. Alternatively, 3 mg total protein extracts were pre-cleared with 100 µl Protein A-Sepharose (GE Healthcare) and immunoprecipitated with PARP1 polyclonal antibody (46D11, Cell Signaling) overnight at 4 °C on a rotating wheel in EBC buffer. Hundred microliters protein A-Sepharose were added, and the samples were rocked for 1 h at 4 °C. After several washes with EBC buffer the bound protein were resuspended in 2X SDS-PAGE sample buffer and heated at 95 °C for 5 min before loading on the gel.
Poly(ADP-ribosyl)ation in vitro
6His-Pax8 purified recombinant protein (50, 100, and 150 ng) and recombinant human PARP1 (Alexis; 25 ng) were incubated for 10 min at 37 °C in binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM DTT) plus 10 µg/ml of sonicated DNA. In some reaction mixtures, 10 mM 3-aminobenzamide (3-AB; Sigma) was included. The reactions were started by adding 5 µCi (0.25 µM) 32P-labeled NAD+ (GE Healthcare), and incubated at 37 °C for 10 additional minutes. After terminating the reactions with SDS sample buffer, the proteins were fractionated by SDS-PAGE through a 4–15% gradient gel. Poly(ADP-ribosyl)ated proteins were visualized by autoradiography.
For pull down experiments with purified proteins, recombinant human PARP1 (Alexis; 25 ng) was incubated for 10 min at 37 °C in a mixture (20 µl) containing 10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM DTT, plus 10 µg/ml of sonicated DNA. In this case, the reaction was started by adding 200 µM βNAD+ (Sigma) and further incubated for another 15 min. The SDS sample buffer was added to stop the reaction that was diluted 10-fold in WCE buffer and then used in the pull down assay.
Electrophoretic mobility shift assay (EMSA)
Double-stranded oligodeoxynucleotide C (5'-CACTGCCCAGTCAAGTGTTCTTGA-3') derived from the Tg promoter and containing one Pax8 binding site was labeled with 
-32P ATP (GE Healthcare) and T4 polynucleotide kinase. Commercially available human PARP1, GST–Pax8, and GST–PD purified proteins were incubated for 20 min, at room temperature, in a binding buffer containing 10 mM HEPES (pH 7.9), 10% v/v glycerol, 0.1 mM EDTA, 8 mM MgCl, 1 mM DTT, 0.15 µg/ml of poly (dI-dC). In some reaction mixtures, βNAD+ (Sigma), 3-AB (Sigma), or both were included. Thus, 32P-labeled oligodeoxynucleotide C (0.8 ng) was added, and the mixtures (20 µl) were incubated for additional 20 min. DNA–protein complexes were resolved on a 5% non-denaturing polyacrylamide gel and visualized by autoradiography.
Cell culture and transient transfection assays
PC Cl3 cells (Fusco et al. 1987, Berlingieri et al. 1988) were grown in Coon's modified F-12 medium (Euroclone) supplemented with 5% v/v calf serum and a six-hormone mixture (6H), as described by Ambesi-Impiombato & Coon (1979).
HeLa cells were grown in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% v/v fetal calf serum (Hyclone). For transient transfection experiments, cells were plated at 3x105 cells/60-mm tissue culture dish 5–8 h prior to transfection, whereas PC Cl3 cells were plated at a density of 5x105 cells/60-mm tissue culture dish 18 h prior to transfection. All transfections were carried out with the FuGENE6 reagent (Roche Diagnostics) according to the manufacturer's directions. The DNA/FuGENE ratio was 1:2 in all the experiments. The plasmid CMV-LUC was used as internal control in the transfection assays. Cells extracts were prepared 48 h after transfection to determine either the levels of the CAT protein with a CAT ELISA kit (Roche Diagnostics) or the LUC activity by a Luciferase assay, as previously described (de Wet et al. 1987). Transfection experiments were done in duplicate and repeated at least three times. CAT activity values on the graph are the means of all experiments (±) S.D. Statistical analysis uses t-test (P<0.002).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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To identify proteins that are able to interact with the transcription factor Pax8, we used the GST–Pax8 fusion protein in pull down assays with a total protein extract from 5x108 PC Cl3 thyroid cells. The extract was pre-cleared onto GST-loaded beads and then was divided and incubated with GST and GST–Pax8 loaded beads. Bound proteins were eluted, analyzed by SDS-PAGE and visualized by ammoniacal silver staining. Several proteins bound specifically to the bait (Fig. 1). Peptide mass fingerprint analysis identified the protein migrating at 113 kDa, which was specifically purified via GST-Pax8, as the PARP1 nuclear protein (SwissProt code P27008 [GenBank] ; Fig. 1). The experiment was performed twice, verifying in the mass spectra the occurrence of 11 and 10 peptides, corresponding to sequence coverage of 19 and 14%, respectively. Other bands of lower molecular weight were identified as GST–Pax8 degradation products (data not shown).
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To confirm PARP1 as a Pax8-interacting protein, we performed small-scale GST-pull down assays incubating the recombinant GST-Pax8 protein with total protein extract from 1x106 thyroid cells. The eluted material was loaded onto an SDS-PAGE mini-gel and subjected to western blot with a PARP1 specific polyclonal antibody. As shown in Fig. 2A, only the eluate from GST–Pax8 contained PARP1. To further investigate the hypothesis that Pax8 and PARP1 are able to interact directly, we performed pull down experiments using the fusion protein GST–Pax8 and purified human PARP1 protein. The beads loaded with GST–Pax8 were indeed able to co-precipitate the purified PARP1 protein, while the beads loaded with the GST protein did not co-precipitate any protein (Fig. 2B). These results demonstrated that the interaction between Pax8 and PARP1, already observed in pull down assays with total protein extracts, is indeed a direct protein–protein interaction.
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Poly(ADP-ribosyl)ation of purified Pax8 protein
PARP1 is a nuclear protein that binds to DNA strand breaks and catalyzes ADP-ribosylation of itself and other nuclear protein, using NAD+ as a co-factor. In order to examine whether Pax8 is directly modified by PARP1, we incubated purified human PARP1 and 6His-Pax8 purified protein in a reconstituted poly(ADP-ribosyl)ating enzyme system containing 32PNAD+ to label the modified proteins. The reaction products were then analyzed by SDS-PAGE. As shown in Fig. 3, PARP1 was significantly auto-poly(ADP-ribosyl)ated, as indicated by the radioactive smear visible at a molecular weight higher than 113 kDa. The addition of an inhibitor of poly(ADP-ribosyl)ation, namely 3-aminobenzamide, abolished the automodification reaction of PARP1 (Fig. 3, lane 3), as already described. The presence of increasing amount of Pax8 did not exert a positive effect on the catalytic activity of PARP1 (Fig. 3, lanes 4–6). No poly(ADP-ribosyl)ation signals were instead detected at Pax8 molecular weight. Re-probing of the filter with antibodies against PARP1 and Pax8 confirmed the presence of the proteins in all the reactions and allowed us to conclude that while PARP1 is modified, Pax8 remains unmodified by poly(ADP-ribosyl)ation (Fig. 3).
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To investigate the effect of the interaction with PARP1 on the Pax8 DNA-binding activity, EMSA assays were performed with bacterially expressed and purified proteins. The results reported in Fig. 4A show that the addition of increasing amounts of PARP1 to Pax8 caused a strong reduction of Pax8 binding to the double-stranded oligonucleotide containing its consensus sequence. At the same time, no retarded bands were observed in the reaction with PARP1 protein alone, thus suggesting that PARP1 cannot bind to the same oligonucleotide. We considered the possibility of explaining the results reported above on the basis of a putative interaction of PARP1 with Pax8 DNA-binding domain, the PD. To test this hypothesis, we performed GST-pull down assays using the GST–PD (GST-PD) fusion protein and purified PARP1 protein. Results of the binding reactions demonstrated that GST–PD was indeed able to co-precipitate PARP1 protein with the same efficiency as GST–Pax8 (Fig. 4B). In order to elucidate whether poly(ADP-ribosyl)ated PARP1 retained an equal ability to interact with Pax8, PARP1 extensively modified in vitro was incubated with matrix-bound GST or GST–Pax8. Results of the binding reactions showed that poly(ADP-ribosyl)ated PARP1 interacts with Pax8 to the same extent as unmodified PARP1 (Figs 2A and 4C). The same result was obtained when GST-PD was used in place of GST–Pax8 (data not shown). Altogether, these data demonstrated that the interaction between Pax8 and PARP1 occurs via Pax8 PD and it is not affected by PARP1 automodification.
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In conclusion, these data consistently suggested that, independent of the poly(ADP-ribosyl)ation reaction, the PARP1 interaction with Pax8 negatively influences the DNA-binding activity of the transcription factor.
PARP1 behaves as a transcriptional repressor for Pax8
Since we demonstrated that PARP1 binds to Pax8 DNA-binding domain, we hypothesized that the interaction between the two proteins could affect Pax8 transactivating properties. To elucidate whether this physical interaction between Pax8 and PARP1 could affect Pax8 transcriptional activity, we performed transactivation assays in HeLa cells co-transfecting the reporter construct CP5-CAT, an artificial promoter responsive only to Pax8, together with the expression vectors encoding for Pax8 and PARP1, separately or in combination. As expected, the transfection of Pax8 alone led to a significant activation of the CP5-CAT promoter, while the transfection of PARP1 alone decreased by the basal activity of the promoter only a little, due to its suppressive activity on RNA polymerase (Oei et al. 1998; Fig. 5A). Interestingly, the co-expression of Pax8 and PARP1 led to an inhibition of Pax8-dependent transcription (Fig. 5A). The same effect was observed in the thyroid differentiated cell line PC Cl3. Also in this case, over-expression of PARP1 produced a significant reduction of the transcriptional activity of endogenously expressed Pax8 on the transfected CP5-CAT promoter (Fig. 5B). To further investigate whether PARP1 could inhibit Pax8 transcriptional activity not only on the synthetic promoter CP5-CAT, but also in a more physiological context represented by the Tg promoter, we repeated the same transactivation assays described before, this time using the reporter Tg-CAT.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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In this study, we provide evidence of a physical and functional interaction of PARP1 with the transcription factor Pax8. GST-pull down and co-immunoprecipitation assays indicated that PARP1 binds to Pax8 in vitro and in vivo, and in vitro binding studies with purified proteins showed that the interaction between Pax8 and PARP1 is direct. The identification of PARP1 as a partner of Pax8 is consistent with many reports that suggest the role of PARP1 in the control of gene expression (Oei et al. 1998, D'Amours et al. 1999, Kraus & Lis 2003). Data reported in the literature propose that PARP1 influences gene transcription modifying directly histones and structural proteins of the chromatin (Poirier et al. 1982, D'Amours et al. 1999, Huang et al. 2006) and/or regulating transcription by directly interacting with other DNA binding factors and co-activators (D'Amours et al. 1999, Hassa & Hottiger 2002). It has been suggested that PARP1 can behave both as an inhibitor or an activator of the bound transcription factors. In fact, PARP1 binds to and inactivates the DNA binding ability of p53 by covalent poly(ADP-ribosyl)ation (Kumari et al. 1998, Mendoza-Alvarez & Alvarez-Gonzalez 2001), and it functions as a suppressor of the RAR/RXR signaling (Miyamoto et al. 1999) and of the RNA polymerase II (Oei et al. 1998). On the other hand PARP1 has been reported to act as a transcriptional co-activator for NF-kB (Hassa & Hottiger 1999, 2002, Hassa et al. 2001, Nakajima et al. 2004), AP-2 (Kannan et al. 1999) and B-MYB (Cervellera & Sala 2000). Depending on the different situation, its enzymatic activity has been shown to be necessary as well as unnecessary to regulate gene expression (Poirier et al. 1982, Meisterernst et al. 1997, Oei et al. 1998, Miyamoto et al. 1999, Cervellera & Sala 2000, Hassa et al. 2003). Our findings indicate that PARP1 acts as a potent inhibitor of Pax8 transcriptional activity by interfering with the formation of the Pax8–DNA complex. In particular, our results suggest that PARP1 elicits its repressor activity on Pax8 independently of its automodification.
DNA repair and transcription are apparently closely related; in fact, association of DNA repair factors with the RNA polymerase II complex has been reported (Bardwell et al. 1994, Roy et al. 1994). Data obtained from mice lacking the PARP1 gene demonstrated its role in DNA repair (Wang et al. 1995, de Murcia et al. 1997) and in vitro assays with the purified enzyme and cellular extracts indicated the effect of poly(ADP-ribosyl)ation on transcriptional repression (Oei et al. 1998). Furthermore, it has been reported that not only PARP1 activation but also its over-expression correlates with various diseases such as malignant melanomas, hepatocellular carcinoma, colorectal carcinoma, and patho-physiological cardiac hypertrophy (Shimizu et al. 2004, Pillai et al. 2005, Staibano et al. 2005, Nosho et al. 2006). Taken together, all these observations indicate that cell stressor, not necessarily associated with DNA damage, could modify the expression level of PARP1 and, in this way, regulate transcription by both a specific and/or a general mechanism.
In this work, we propose that PARP1 acts a co-factor of Pax8, which is essential to preserving the physiological thyroid context and to allow the fine-tuning of the differentiated thyroid phenotype. Nevertheless, the observed interaction between PARP1 and Pax8 calls for additional studies to define the exact role of PARP1 in the transcriptional regulation of thyroid-expressed genes. It will be of interest to investigate the effect of PARP1 over-expression with respect to proliferation and differentiation of thyroid cells, and to analyze the effect of PARP1 absence, both in vitro by RNAi in differentiated thyroid cells and in vivo by studying the thyroid phenotype of PARP1 knockout mice.
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Declaration of interest |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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Funding |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
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Received in final form 30 July 2008
Accepted 3 September 2008
Made available online as an Accepted Preprint 3 September 2008
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