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J. Biol. Chem., Vol. 282, Issue 7, 4719-4727, February 16, 2007

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Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins^*

Tsui-Fen Chou and Carston R. Wagner¹

From the Departments of Medicinal Chemistry and Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

Received for publication, November 13, 2006 , and in revised form, December 8, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [-³²P]ATP, but not [-³²P]GTP, ³²P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg²⁺, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap₄A, believed to be synthesized by LysRS in vivo, and Zn²⁺ were shown to inhibit the formation of Hint-AMP with an IC₅₀ value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Histidine triad nucleotide binding protein (Hint)² belongs to a histidine triad (HIT) superfamily that has a characteristic C-terminal active site motif, HXHXHXX, where X is a hydrophobic residue (1). HIT enzymes are a ubiquitous superfamily consisting primarily of nucleoside phosphoramidases, dinucleotide hydrolyases, and nucleotidyltransferases (1). Five distinct branches of HIT superfamily have been recently classified by a phylogenetic study of HIT proteins (2, 3). Recently, Hint1 homologs isolated from rabbit (4), human (5), chicken (6), yeast (4), and Escherichia coli (5) have been shown to be purine nucleoside phosphoramidases. Human Hint1 has been shown to associate with, and possibly regulate several transcription factors such as TFIIH (7), MITF (8, 9), and USF2 (10). Additionally, Hint1 knock out mice have been shown to have an increased susceptibility to the induction of ovarian and mammary tumors by the carcinogen dimethylbenzanthracene and to spontaneous tumors (11). Up-regulation of Hint1 and the significantly reduced in vivo tumorigenicity of 5-aza-dC-treated non-small-cell lung cancer cell line NCI-H522 suggested that hHint1 might be a tumor suppressor (12). Recently, Weiske and Huber (13) reported that Hint1 triggers apoptosis independent of its phosphoramidase activity (13). Human Hint2, which is 61% identical to Hint1, has recently been shown to be a mitochondrial apoptotic sensitizer that is down-regulated in hepatocellular carcinomas (14). Although Hints are efficient hydrolases of purine nucleoside phosphoramidates, cellular function and biochemical relevance of the enzymatic phosphoramidase activity has not been determined.

The Fhit (fragile histidine triad) branch of the HIT superfamily is only found in eukaryotes. Like Hint, Fhit is also a homodimer with tumor suppressor activity (15). Human Fhit is a diadenosine P₁,P₃-triphosphate (Ap₃A) hydrolyase as well as phosphoramidase (16, 17). The precise mechanism of action by which it affects tumor development is not well understood, although site-directed mutagenesis studies have indicated that the Fhit tumor suppressor function is likely not dependent on its Ap₃A hydrolase activity (18). In contrast to the first two branches, the galactose-1-phosphate uridylyltransferase branch has been shown to be the second enzyme in the Leloir pathway necessary for galactose utilization (19, 20). Although, galactose-1-phosphate uridylyltransferase is a homodimer with little overall sequence homology with Hint1 or Fhit, it does share some tertiary structure similarity (21). The fourth family is aprataxin, which is mutated in ataxia-oculomotor apraxia1 (22, 23) and possesses phosphoramidase and Ap₄A hydrolase activity as well as DNA/RNA binding properties (2); recently, the physiological substrate for aprataxin has been shown to be abortive adenylated DNA ligation intermediates (3). The last family is the scavenger mRNA decapping enzyme, DcpS/DCS-1, a 7-methyl-GpppG hydrolase (24, 25).

Recently, hHint1 has been isolated from complexes with lysyl-tRNA synthetase (LysRS) and MITF or USF2 transcription factors (9, 10). To elucidate the biochemical connection between LysRS and Hint1, both E. coli LysRS (ecLysU) and human LysRS (hLysRS) were purified and shown to label echinT and hHint1 with [-³²P]ATP but not with [-³²P]ATP, [-³²P]GTP, or [-³²P]GTP. This was found to be consistent with chemical degradation and mutagenic experiments revealing that the Hint-AMP intermediate formed upon interaction with LysRS contained a phosphoramidate linkage between AMP and a strictly conserved and catalytically essential active site histidine (His-112 of hHint1, Fig. 1). Furthermore, by varying individual components in the aminoacylation reaction, the formation of the Hint-AMP intermediate was found to be dependent on the formation of the lysyl-AMP intermediate. Hence, Hints may function in part to regulate the catalytic activity of LysRS by providing a possible new mechanism of pre-transfer editing or by scavenging highly reactive inappropriately released aminoacyl-adenylates. Moreover, the interaction of Hint1 with transcription factors such as USF2 and MITF may be mediated by the adenylation of Hint1 by LysRS.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein Purification—The E. coli BS68 strain harboring the pBAS39 (derived from pET3a) plasmid for expressing ecLysU as a C-terminal His₆-tag fusion protein was a gift from Dr. Paul Schimmel (The Scripps Research Institute) (26). The plasmid pM368 encoding hLysRS expresses a fusion protein containing the N-terminal MRGSHHHHHHSSGWVD sequence appended to full-length hLysRS was a gift from Dr. Karin Musier-Forsyth (University of Minnesota) (27). Both LysRS enzymes were purified by using Ni²⁺-agarose binding according to a previously published procedure (27). Wild type and mutants of hHint1 and echinT were purified by an AMP-agarose column from echinT knock out strain (BB2) as described previously (5).

Labeling of Hints by Purified E. coli LysU—E. coli LysU (6.25 µM) was incubated with [-³²P]ATP or [-³²P]GTP (0.33 µM, 800 Ci/mmol, MP Biomedicals) in buffer A (10 µl, 25 mM Tris HCl, pH 7.8, 100 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol, protease inhibitor tablet (Roche Applied Science)) at 23 °C for 10 min followed by the addition of either buffer A (5 µl), echinT (5 µl, 1.25, 3, or 7.5 µM), hHint1 (5 µl, 1.25, 3, or 7.5 µM), or the chimera mutant (5 µl, 1.25, 3, or 7.5 µM) and incubated for 10 min. The reaction was terminated by the addition of SDS sample buffer (4x,5 µl, Invitrogen). The reaction mixture was boiled for 10 min, and the proteins were separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. Labeled proteins were visualized by subjecting dried polyvinylidene difluoride membranes to autoradiography with a storage phosphor screen for 12 h followed by scanning with a Storm 840 PhosphorImager.

Lysyl-AMP-dependent Adenylation of Hints by E. coli LysU and Human LysRS—The concentration of ecLysU, hLysRS, lysine, [-³²P]ATP, Mg ²⁺, Zn ²⁺, nucleotides, yeast inorganic pyrophosphatase (Sigma), Hint proteins, and incubation time period for each experiment is described in the legends to Figs. 3, 4, 5, 6, 7, 8, 9. The reactions were terminated by the addition of SDS sample buffer (4x), and samples were analyzed as described above. Quantitation of the intensity of the ³²P signal was carried out with ImageQuant software (GE Healthcare).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. X-ray crystallographic structure of hHint1. A, overall dimer structure with bound AMPCP (Protein Data Bank code 1AV5). Shown is the active site region in one monomer with bound AMPCP (Protein Data Bank code 1AV5) (B) and with bound AMP (C); histidine 112 of hHint1 is the nucleophilic residue that forms a covalent P-N bond with its substrate (Protein Data Bank code 1KPF) (48).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Nucleotidylation of Hint by ecLysU. A, incubation of ecLysU (6.25 µM) was carried out at 23 °C for 10 min with [-32P]ATP (0.33 µM). Labeling of echinT (0.5 µM, 1 µM, lanes 3 and 4), hHint1 (lanes 6 and 7), and the chimera (lanes 9 and 10) was observed after the addition of Hints for 10 min. Control experiments were carried out with buffer A containing 2.5 µM Hints but not ecLysU (lanes 1, 5, and 8). B, incubation of ecLysU (6.25 µM) was carried out at 23 °C for 10 min with [-32P]GTP (0.33 µM). Only weak labeling of ecLysU (59 kDa) was observed by the addition of hHint1 (2.5 µM, lane 3) and the chimera (2.5 µM lane 4) but not with buffer A (lane 1) and echinT (lane 2).

	RESULTS

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View larger version (31K): [in this window] [in a new window]   FIGURE 3. Enhanced adenylation of echinT by ecLysU by the addition of lysine. Incubation of ecLysU (0.22 µM) was carried out at 23 °C for 10 min with [-32P]ATP (0.38 µM) in buffer A followed by the addition of echinT (0.28 or 2.8 µM) for 10 min (lanes 2 and 4) and in buffer A containing lysine (2.2 µM) followed by the addition of echinT (0.28 or 2.8 µM) for 10 min (lanes 3 and 5). The addition of lysine enhanced the amount of Hint-AMP by factors of 11 or 5.

Enhanced Adenylation of Hint by ecLysU by the Addition of Lysine—To first investigate whether the lysyl-tRNA synthetase activity associated with ecLysU was responsible for the adenylation of Hints, the effect of lysine on Hint-AMP formation was evaluated. Incubation of ecLysU (0.22 µM) was carried out at 23 °C for 10 min with [-³²P]ATP (0.38 µM) in buffer A followed by the addition of echinT (0.28 or 2.8 µM) for 10 min (Fig. 3, lanes 2 and 4) and in buffer A containing lysine (2.2 µM) followed by the addition of echinT (0.28 or 2.8 µM) for 10 min (lanes 3 and 5). The addition of lysine (2.2µM) enhanced Hint-AMP formation by 11-fold, when the ecLysU (0.22 µM) and echinT (0.28 µM) were comparable in concentration. Consistent with a saturable process, when the amount of echinT was increased by 10-fold, only a 5-fold increase in labeling was observed. Because the small amount of Hint-AMP was observed in the absence of added lysine (Fig. 3, lanes 2 and 4), it is highly likely that a small amount of contaminating lysine or lysyl-AMP bound with ecLysU is present with the purified enzyme. In addition, at the higher echinT concentration, we do observe small amounts of labeled ecLysU (Fig. 3, lanes 4 and 5, 59 kDa), possibly indicating that at high enough concentrations of Hint, adenylation of ecLysU can be observed.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Time dependence of Hint adenylation by ecLysU. Hint-AMP formation was assayedat23 °Coveran8-mintimecourseafterincubationofecLysU (0.022 µM) in buffer A containing lysine (2.2 µM) with [-32P]ATP (0.35 µM) for 1 min. After the addition of echinT (1.4 µM), aliquots were withdrawn at 1-min intervals and quenched with SDS sample buffer. The steady-state level of Hint-AMP was maintained for 2 min, then the intensity was decreased from 3 to 8 min.

View larger version (64K): [in this window] [in a new window]   FIGURE 5. Adenylation of ecLysU by echinT. Incubation of ecLysU (0.022 µM) was carried out at 23 °C in buffer A containing lysine (2.2 µM) with [-32P]ATP (0.35 µM) for 1 min followed by the addition of echinT (0.056–14 µM) and incubation for 1 min. Maximal Hint-AMP formation was observed at 2.8 µM echinT. Formation of ecLysU-AMP was observed after the concentration of echinT reached 1.4 µM. The intensity of the ecLysU-AMP was increased with higher concentrations of echinT (2.8 4 µM). Moreover, the ecLysU/echinT-AMP complex or multiple adenylated ecLysU was observed at 14 µM echinT.

Adenylation of Hint Is Dependent on the Concentration of Lysine and Mg²⁺—Because lysine is one of the substrates and Mg²⁺ is absolutely required for lysyl-AMP formation (30), it is of interest to determine the effect of both molecules on adenylation of echinT in more detail. Hence, various concentrations of lysine (0.025–2500 µM) were examined to reveal a saturation-type behavior with respect to the amount of lysine; under these conditions maximal intensity was observed at 25 µM lysine (Fig. 6A). In addition, labeling was observed only when Mg²⁺ was present in the reaction mixture, with maximal intensity reached at a concentration of 1500 µM (Fig. 6B). Taken together, the observed reliance of echinT adenylation on lysine and Mg²⁺ is consistent with a labeling reaction dependent on the formation of lysyl-AMP by ecLysU.

Inhibition of Hint Adenylation by Zn²⁺,Ap₄A, and Ap₃A— Divalent Zn²⁺ has been shown to stimulate Ap₄A formation by ecLysU. The mechanism has been shown to rely on the ability of Zn²⁺ to stimulate nucleophilic attack by the -phosphate of ATP on lysyl-AMP (31). In addition, Zn²⁺, but not Ap₄A, has been shown to inhibit Hint activity with an IC₅₀ of (9.5 ± 2.1) µM.⁴ Therefore, we chose to test the effect of Zn²⁺, Ap₄A, and other nucleotides on the formation of Hint-AMP. As shown in Fig. 7, Zn²⁺ (15 µM) inhibited Hint-AMP formation by 80%, and Ap₄A inhibited Hint-AMP formation with an IC₅₀ of 15 µM. Moreover, about 70–90% inhibition was observed for incubations with 150 µM Ap₄A, Ap₃A, or ATP. Consistent with the preference of LysRS for ATP and not GTP, incubations with 150 µM GTP decreased Hint-AMP by only 50%, whereas inhibition by either AMP or GMP was not observed (supplemental Fig. 1). These results provided additional evidence that lysyl-AMP is required for adenylation of echinT.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Adenylation of echinT is dependent on the concentration of lysine and Mg2+. Incubation of ecLysU (0.025 µM) was carried out at 23 °C in buffer A containing lysine (0 2500 µM) with [-32P]ATP (0.32 µM) for 1 min followed by the addition of echinT (2.5 µM) for an additional 1 min (A) and in buffer A containing lysine (2.5 µM) and Mg2+ (0 3000 µM) with [-32P]ATP (0.32 µM) for 1 min followed by the addition of echinT (2.5 µM) for 1 min (B).

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Inhibition of Hint-AMP formation by Zn2+ and Ap4A. Incubation of ecLysU (0.025 µM) was carried out at 23 °C in buffer A containing lysine (2.5 µM) with [-32P]ATP (0.32 µM) with various concentrations of Zn2+ (0 1500 µM) for 1 min followed by the addition of echinT (2.5 µM) for 1 min (A) and various concentrations of Ap4A(0 1500 µM) for 1 min followed by the addition of echinT (2.5 µM) for 1 min (B). The IC50 of Ap4A on Hint AMP formation was about 15 µM. In addition, Ap3A and ATP exhibited similar inhibitory effects, whereas GTP displayed less effect. AMP and GMP have no-observable effect.

Effect of Hint on AMP Formation by ecLysU—To further establish the effect of Hint on LysRS-mediated reaction, a direct measurement of AMP production by ecLysU with or without echinT was compared using the described TLC assay (28, 29). The observed rate constant for AMP formation by ecLysU is 0.08 ± 0.01 min^-1 and 0.16 ± 0.02 min^-1 in the presence of echinT. Variants are represented by S.D.

Adenylation of Hint Mutants by ecLysU and hLysRS—Mutation of His-101 of echinT and His-112 of hHint1 to Ala or Gly completely abolished formation of Hint-AMP (Fig. 9), indicating that the adenylation site was the nucleophilic active site histidine. As shown in Fig. 9A, incubation of ecLysU with echinT, the chimera, and hHint1 revealed similar labeling intensities for echinT and the chimera but about 100-fold lower intensity for hHint1. Labeling by hLysRS demonstrated a 10-fold higher intensity for echinT than that for hHint1 and a 47-fold higher intensity for the chimera (Fig. 9B), suggesting that the Hint C terminus is responsible for mediating the differential labeling observed by the ecLysU and hLysRS.

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Dependence of Hint adenylation on the concentration of echinT and ecLysU. Adenylation was carried out in buffer A containing lysine (2.5 µM) with [-32P]ATP (0.32 µM) at 23 °C. A, incubations with ecLysU (0.025 µM) for 1 min followed by the addition of echinT (0.05–12.5 µM) for 1 min. Steady-state levels of Hint-AMP were observed when the concentration of echinT reached 2.5 µM. B, incubations with ecLysU (0.025 14 µM) for 1 min followed by the addition of echinT (6.25 µM) for 1 min. The intensity of Hint-AMP is proportional to the increased concentration of ecLysU.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (26K): [in this window] [in a new window]   FIGURE 9. Adenylation of Hints by ecLysU and hLysRS. Labeling was carried out in buffer A containing lysine (35 µM) and inorganic pyrophosphatase (0.02 unit/µl) with [-32P]ATP (0.9 µM) at 23 °C for 1 min followed by the addition of Hint proteins (3.6 µM) for 1 min with ecLysU (0.1 µM)(A) and hLysRS (1 µM)(B). WT, wild type.

To investigate the relationship between LysRS and Hint-AMP formation, a series of experiments was carried out under various conditions to verify the necessity for lysyl-AMP formation. Consistent with the preference of aminoacyl-tRNA synthetase for ATP over GTP, labeling of Hints was only observed for incubations with [-³²P]ATP (Fig. 2). In addition, GTP was found to be a significantly poorer inhibitor of Hint-AMP formation than ATP. In support of our findings, recent modeling studies attempting to develop a molecular rationale for the substrate specificity of ecLysU have concluded that the enzyme has a greater propensity to bind ATP in the necessary productive conformation than GTP (44).

Given the catalytic processes involving LysRS (Fig. 11), we proposed that formation of Hint-AMP is dependent on the formation of lysyl-AMP. As can be seen in Figs. 2 and 5, increasing the amount of lysine in the reaction solution resulted in enhanced and saturable Hint labeling, as expected from an enzymatic process. Hint labeling was found to be dependent on Mg²⁺, which is required for aminoacyl-adenylate formation, whereas the addition of Zn²⁺, which favors Ap₄A formation, was found to be inhibitory. In addition, our observation that Ap₄A and Ap₃A, which are neither substrates nor inhibitors of Hints, are potent inhibitors of Hint-AMP formation is consistent with the inhibitory effect of Zn²⁺ and the necessity of lysyl-AMP for labeling. That we can observe a plateau in the amount of labeling as we increase the amount of echinT is indicative of a substrate-saturable enzymatic reaction, with an apparent K_m of (0.85 ± 0.34) µM, which is about 25-fold lower than that observed for the cognate tRNA^Lys (45).

The removal of PPi, a byproduct of aminoacyl-tRNA synthetase aminoacyl-adenylate formation, by pyrophosphatase has been shown to significantly enhance tRNA aminoacylation (34). Because the binding site for both PPi and the incoming tRNA are overlapping, inorganic pyrophosphatase has been shown to aid the reaction by reducing product inhibition and the rate of the reverse reaction. Similarly, a greater amount of Hint adenylation was observed with reaction mixtures containing inorganic pyrophosphatase.

Previously, it has been proposed that Hints, like other members of the HIT family, carry out substrate hydrolysis by employing nucleophilic catalysis (1, 46). Of the four conserved active site histidines, one has been shown to be catalytically essential and presumably required for intermediate formation. Adenylated enzymes were not observed when the putative nucleophilic histidine in either echinT (His-101) or hHint1 (His-112, Fig. 1) was mutated to alanine or glycine. Chemical degradation studies and pulse-chase experiments with cold ATP have demonstrated that the Hint-adenylate is sensitive to acid, but not base, and that the intermediate has a half-life of <1 min. This result is consistent with kinetic experiments with model adenosine phosphoramidate substrate that have revealed the half-life of the echinT-AMP, hHint1-AMP, chimera-AMP to be 0.15 ± 0.05, 0.30 ± 0.02, and 7.2 ± 2.0 s, respectively⁴ (47). In addition, the rate of AMP production by ecLysU increases by 2-fold in the presence of echinT. Taken together, these results are consistent with formation of a covalent P-N bond between the active site histidine and AMP. As observed for other HIT proteins, such as Fhit and galactose-1-phosphate uridylyltransferase (19, 48, 49), which also proceed through active site histidine-AMP intermediates, the catalytic mechanism used by Hints proceeds through formation of an adenylated enzyme intermediate (Fig. 10). The extent to which Hint hydrolysis of the aminoacyl-adenylate is dependent on proteinprotein transfer or the release of lysyl-AMP from the active site of LysRS remains to be determined (Fig. 11).

View larger version (14K): [in this window] [in a new window]   FIGURE 10. Proposed catalytic mechanism for lysyl-adenylate and Hints.

View larger version (14K): [in this window] [in a new window]   FIGURE 11. Catalytic processes involving lysyl-tRNA synthetase and Hints.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: University of Minnesota, Dept. of Medicinal Chemistry, 8-174 Weaver Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455. Tel.: 612-625-2614; Fax: 612-624-0139; E-mail: wagne003{at}umn.edu.

² The abbreviations used are: Hint1, histidine triad nucleotide binding protein1; hHint1, human Hint1; AMPCP, adenosine 5'-(,-methylene)diphosphate; HIT, histidine triad; Fhit, fragile histidine triad; Ap₃A, diadenosine P₁,P₃-triphosphate; LysRS, lysyl-tRNA synthetase; hLysRS, human LysRS; ecLysU, E. coli LysRS.

³ T.-F. Chou, Y. Y. Sham, and C. R. Wagner, unpublished data.

⁴ T.-F. Chou and C. R. Wagner, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. Karin Musier-Forsyth and Dr. Robert Kennedy (University of Minnesota) for kindly providing us the expression plasmid for human LysRS and helpful discussions. We thank Dr. Paul Schimmel (The Scripps Research Institute) for providing ecLysU expression plasmid.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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