3Department of Biology, Temple University, Philadelphia, PA 19122, USA; 4Department of Chemical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; and 5Laboratory of Bacterial Toxins and Laboratory of Bacterial Polysaccharides, Food and Drug Administration, Bethesda, MD 20892, USA
Received on August 17, 2001; revised on September 20, 2001; accepted on September 21, 2001.
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Abstract |
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Key words: insects/KDN/N-acetylneuraminic acid/sialic acid synthase
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Introduction |
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Expression of sialic acids was originally believed to be restricted to the deuterostome lineage, having been observed in echinoderms, hemichordates, cephalochordates, and vertebrates (Warren, 1963; Corfield and Schauer, 1982
). Certain pathogenic and commensal bacteria, viruses, and fungi have been found to contain sialic acids as well (Corfield and Schauer, 1982
; Schauer and Kammerling, 1997
). Whether the ability of bacteria to synthesize sialic acids is ancestral or reflects the later acquisition of this capability by horizontal transfer via phages (Roggentin et al., 1993
) is not resolved. Recently, sialic acids have also been detected in organisms of the protostome lineage (including nematodes, arthropods, and mollusks) (Roth et al., 1992
; D'Amico and Jacob, 1995
; Malykh et al., 1999
; Park et al., 1999
).
Staining of Drosophila early embryonic stages with sialic acidbinding lectins from Limulus polyhemus (LPA) and Limas flavus (LFA) has been reported and is neuraminidase-sensitive (Roth et al., 1992). In later embryonic stages, LFA stains both neural and general ectodermal cells and western blot analysis with a poly
2,8 sialic acid (PSA)specific monoclonal antibody detects a high molecular weight band between 14 and 18 h of embyogenesis. Because this developmental period corresponds to active nervous system development, it was suggested (Roth et al., 1992
) that PSA expression modifies axonal and cell adhesion molecule function during Drosophila development as it does during formation of the mammalian nervous system (Acheson et al., 1991
). Sialic acid was detected in the vacuoles of the Malpighian tubules of larvae of the cicada Philaenus spumarius (Malykh et al., 1999
) by cytochemical staining using lectins from Sambucus nigra and LFA, as well as by using a monoclonal antibody specific for PSA. The presence of sialic acid was confirmed by gas-liquid chromatographymass spectroscopy (GLC-MS) in both of these studies (Roth et al., 1992
; Malykh et al., 1999
). However, the direct detection of sialic acids is not definitive proof of cellular activity due to potential contamination by pathogens, dietary sources, or serum from cell culture media (Warren, 1963
; Corfield and Schauer, 1982
; Park et al., 1999
; Angata and Varki, 2000
), reviewed in Marchal et al. (2001)
. In fact, the vast majority of glycoproteins from insects contain truncated (paucimannosidic) or hybrid structures terminating in mannose or N-acetylglucosamine (Williams et al., 1991
; Altmann et al., 1999
; Marchal et al., 2001
), and the majority of secreted and membrane-bound mammalian glycopoteins contain complex oligosaccharides terminating in sialic acid. So far, glycosphingolipids with sialic acid have not been detected in Drosophila (Seppo et al., 2000
; Seppo and Tiemeyer, 2000
). Furthermore, Hooker et al. (1999)
and Tomiya et al. (2001)
were unable to detect any CMP-Neu5Ac donor substrates nor sialyltransferase activity in a variety of insect cell lines.
Consequently there is still considerable ambiguity as to whether insects possess the intrinsic biosynthetic capacity to produce sialic acids; if they do, then what is the nature of the glycoconjugates and their biological roles? We have taken advantage of the nearly completed genome of Drosophila melanogaster (Adams et al., 2000) to perform homology searches for orthologues of key enzymes in the sialic acid pathway. Using the sequence information from the Escherichia coli (neuB) (Annunziato et al., 1995
) and human (human SAS) (Lawrence et al., 2000
) sialic acid synthase genes, we identified a homologous genomic sequence in Drosophila and subsequently cloned and characterized the corresponding cDNA encoding the first N-acetylneuraminic acid phosphate synthase gene for any protostome species. Futhermore, we show that the Drosophila enzyme (DmSAS) uses phosphorylated substrates to generate phosphorylated sialic acids as observed in human and mouse (Lawrence et al., 2000
; Nakata et al., 2000
), rather than the unphosphorylated substrates utilized by the E. coli enzyme (Vann et al., 1997
). Similar to the human homologue, this enzyme exhibits multifunctional substrate activity and uses both N-acetylmannosamine-6-phosphate (ManNAc-6-P) and mannose-6-phosphate (Man-6-P) for the generation of phosphorylated forms of both N-acetylneuraminic acid (Neu5Ac) and deaminated Neu5Ac (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid; KDN), sialic acids, respectively. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that the DmSAS mRNA is ubiquitously expressed across different stages of development. The identification of a functional sialic acid synthase gene in Drosophila offers compelling evidence that insects can utilize an endogenous biosynthetic pathway to produce sialic acids. We also discuss the identification of other putative genes encoding key enzymes in the sialic acid pathway of Drosophila. With this knowledge, it should now be possible to elucidate the biological roles of these newly identified genes using the sophisticated genetic approaches available in Drosophila.
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Results |
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To prove that the sialic acid synthase activity observed in the presence of ManAc-6-P was due to the cloned gene, DmSAS carrying a His tag was purified from TOP10:pTrcHis2DmSAS cells using Ni-NTA resin affinity chromatography. The production of TBA-reactive sialic acids using the affinity purified DmSAS was measured at various time intervals for 1 h using ManNAc-6-P as the substrate (Table I). Sialic acid production increased linearly (R2 = 0.996) throughout the 1-h analysis time with an overall specific activity of 420 nmol Neu5Ac synthesis per h per mg protein. The fact that the specific activity significantly increased after affinity purification confirmed that the observed sialic acid synthase activity was due to DmSAS.
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Drosophila genome encodes other key enzymes in the sialic acid pathway
In mammals, sialic acid is typically transferred to a galactosyl, N-acetylgalactosaminyl, or sialyl residue depending on the glycoconjugate acceptor. The early steps in the N-linked glycosylation pathway are similar in mammals and insects (Williams et al., 1991; Seppo and Tiemeyer, 2000
; Marchal et al., 2001
). However, in insects the terminal N-acetylglucosamine (GlcNAc) residue of the core structure is typically removed by a Golgi-associated N-acetylglucosaminidase, which produces a paucimannose type N-glycan that lacks galactose (Altmann et al., 1995
, 1999; Wagner et al., 1996
; Marchal et al., 2001
). In mammals, the substrate for the sialic acid phosphate synthase is ManNAc-6-P, which is produced by a bifunctional enzyme, UDP-GlcNAc 2-epimerase/ManNAc kinase, that converts UDP-N-acetylglucosamine to ManNAc-6-P (Stasche et al., 1997
). For sialylation to occur, Neu5Ac is converted to the activated nucleotide sugar, cytidine monophosphate Neu5Ac (CMP-Neu5Ac) by a nuclear enzyme, CMP-Neu5Ac synthase (Kean, 1991
; Munster et al., 1998
). Translocation of the donor CMP-Neu5Ac to the trans-Golgi requires a CMP-Neu5Ac/CMP antiporter (Eckhardt et al., 1996
; Hirschberg et al., 1998
), and finally sialyltransferases located in the trans-Golgi transfer the CMP-Neu5Ac to glycoconjugates.
For Drosophila to sialylate glycoconjugates it should possess orthologues of the above-mentioned enzymes in the sialic acid pathway. A recent report (Angata and Varki, 2000) based on homology searches of the Drosophila genome was unable to find evidence that flies possess complete genes encoding enzymes involved in sialic acid metabolism, although several fragments of genes were identified. We carried out similar homology searches of the Drosophila database using the amino acid sequences of the bacterial and mammalian enzymes as query sequences and identified the following putative fruit fly orthologues: galactosyl transferase, AF132158 (CG8536); CMP-Neu5Ac synthase, AE003515; CMP-Neu5Ac/CMP, UDP-Gal/UMP, or UDP-GlcNAc/UMP antiporter, AL023874, AF397530 (CG2675), and AC020227 (CG14040); and sialyltransferase, AE003695, AF397532 (CG5232). In each case, the homology was deemed significant if it extended over a substantial portion of the entire coding region. The annotators of the Berkeley Drosophila Genome Project similarly identified these sequences as encoding orthologues of the mammalian enzymes (Gadfly Acc indicated in parentheses), except for the CMP-Neu5Ac synthase. However, we have found that the annotation program often does not accurately predict the protein coding region as determined by sequencing of cDNA clones. Therefore, for enzymes that had available corresponding cDNA clones in the EST database, we submitted their DNA sequences to GenBank (the accession numbers for cDNAs follow those of the genomic clones). The only sequence we could find no corresponding orthologue for was the UDP-GlcNAc 2-epimerase/ManNAc kinase. Similar to the previous report (Angata and Varki, 2000
), we obtained a hit to AE003811, but the homology was restricted only to the kinase domain, which is shared by other kinases, including hexokinase. Therefore, we do not believe this homology to be meaningful. One possible reason for the difference between our results and that of Angata and Varki (2000)
may be that we typically used only short conserved domains of the query sequences rather than the entire coding region. Although it remains to be shown that the orthologues we identified are functional, we think the results suggest that the sialic acid phosphate synthase gene identified in this study is part of a complete pathway that may result in the sialylation of glycoconjugates in Drosophila.
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Discussion |
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Expression of DmSAS was observed at all developmental stages and in S2 cells when examined by PCR amplification of staged total RNA or cDNA (Figure 3A, B). Expression of the human SAS has also been shown to be ubiquitous in all tissues (Lawrence et al., 2000). The DmSAS expression pattern is consistent with the reported presence of sialic acid from blastoderm to third-instar larvae stage detected by lectin-gold histochemistry (Roth et al., 1992
). The same report, however, showed that detection of
2,8-linked PSA by western blot was restricted to 1418-h embryos. Because DmSAS mRNA is present at all stages in Drosophila, the regulated expression of PSA is most likely determined by another enzyme in the pathway, such as the sialyltransferase. Control of sialylation in vertebrates is controlled by the expression of the sialyltransferase rather than the sialic acid synthase (Paulson and Colley, 1989
; van den Eijnden and Joziasse, 1993
). Our preliminary evidence indicates that the Drosophila sialyltransferase, which we named DmST, is also developmentally regulated (Park and Palter, unpublished data). In vertebrates the major target for PSA addition is the NCAM, a regulator of cellcell and cellsubstratum interactions that affects neurite growth, cell migration, and synaptic plasticity (reviewed in Rutishauser, 1998
; Rutishauser and Landmesser, 1996
). In mice, NCAM does not carry PSA during early fetal development, but the PSA-NCAM isoforms predominate at later stages, reaching a maximum in the perinatal phase. After birth, the amount of PSA declines and later appears only in restricted populations of the adult brain that are still undergoing neurogenesis, cell migration, axonal outgrowth, and synaptic plasticity (Rutishauser and Landmesser, 1996
; Eckhardt et al., 2000
). Two related enzymes are responsible for sialic acid polymerization in vertebrates, STX (now ST8SiaII), (Livingston and Paulson, 1993
) and PST (now ST8SiaIV) (Eckhardt et al., 1995
; Nakayama et al., 1995
; Muhlenhoff et al., 1996
; Nakayama and Fukuda, 1996
). ST8SiaII is the predominant form in the embryo, whereas ST8SiaIV is present at high levels in the postnatal brain (Hildebrandt et al., 1998
; Ong et al., 1998
). Drosophila has an NCAM orthologue, fasciculin II, which plays a similar role in development of the fly nervous system (Mendoza and Faye, 1999
) but it has not been reported to be modified by PSA. In vertebrates, constitutive expression of 2,3 and 2,6 sialyltransferases are responsible for synthesis of the complex oligosaccharide structures at the termini of glycoproteins, and distinct 2,3, 2,6, and 2,8 sialyltransferases modify glycolipids (Nagai and Iwamori, 1995
; Schauer et al., 1995
). It remains to be determined whether the role of sialic acid in Drosophila is a PSA addition to specific receptors or other types of modifications of glycoproteins or glycolipids.
When DmSAS was expressed using either a bacterial or a baculoviral expression system in the presence of various sugar substrates, we found that highest levels of Neu5Ac synthesis were obtained when ManNAc-6-P was the substrate. In addition, we showed that DmSAS could also use Man-6-P to produce the deaminated sialic acid KDN. These results are similar to those obtained for both the human and mouse SAS, except that the mouse enzyme was not reported to produce KDN (Lawrence et al., 2000; Nakata et al., 2000
). This is in contrast to the E. coli sialic acid synthase (the product of neuB), which uses ManNAc and PEP directly to produce Neu5Ac but cannot use ManNAc-6-P or Man as substrates (Vann et al., 1997
).
We additionally confirm that the primary product of DmSAS activity is a phosphorylated intermediate, and therefore Neu5Ac is produced from ManNAc-6-P and PEP through a three-step pathway, as it is in mammals (Lawrence et al., 2000; Nakata et al., 2000
). The first and last steps involve the production of ManNAc-6-P by a specific kinase and ATP and the removal of the phosphate from Neu5Ac-9-phosphate by a specific phosphatase, respectively. Therefore, DmSAS should be considered a sialic acid phosphate synthase enzyme, with greater functional similarity to the human enzyme than the form observed in E. coli. We noted that in the bacterial expression system a minor sialic acid synthetic ability was observed when ManNAc and PEP were used as substrates. ManNAc may be a low-level substrate for the DmSAS enzyme, or the presence of high concentrations of PEP may result in ATP production by a reversible pyruvate kinase followed by ManNAc phosphorylation in the partially purified protein fraction.
The Drosophila sialic acid synthase shares 48.7% identity with the human synthase but only 35.3% identity with the bacterial synthase, consistent with the bacterial enzyme being the ancestor to both insects and mammals. PSA in bacteria is generally found in the capsular polysaccharide of pathogenic organisms, especially those that are neuroinvasive (Troy, 1995). PSA is therefore not present in all bacterial strains and is not essential for viability. It has been proposed that bacteria acquired this pathway by horizontal transfer from host organisms to expand the bacterias host range (Roggentin et al., 1993
). Using homology searches, we did not detect genes involved in sialic acid metabolism in the completed yeast (Saccharomyces cerevisiae) or nematode (Caenorhabditis elegans) databases. Does the absence of the sialic acid biosynthetic pathway in certain bacteria and organisms believed to be ancestral to insects necessarily mean that the pathway could not have originated in bacteria? We can imagine a scenerio in which PSA was first used as a component of bacterial capsules, where it was nonessential, and later evolved uses in general cellcell signaling or recognition when it became integral to the viability of organisms. Therefore, certain evolutionary branches could have lost the sialic acid pathway before it assumed uses that were essential for viability.
Our finding that the Drosophila genome encodes a functional sialic acid phosphate synthase enzyme suggested that other genes encoding sialic acid biosynthetic enzymes would also be present. We think it improbable that in the absence of selective pressure Drosophila would have maintained a functional synthase gene. Typically, if enzyme pathways are no longer utilized by an organism, the affected genes rapidly accumulate mutations in their coding regions or are deleted (Lynch and Conery, 2000). The results of our homology searches of the Drosophila genome revealed putative orthologues of galactosyl transferase, CMP-Neu5Ac synthase, CMP-Neu5Ac/CMP, UDP-Gal/UMP, or UDP-GlcNAc/UMP antiporter and sialyltransferase. The nucleotide-sugar antiporters comprise a family of related proteins. Without direct genetic evidence or reconstitution experiments using proteoliposomes, it is difficult to establish the nucleotide-sugar specificity of each member (Abeijon et al., 1997
; Hirschberg et al., 1998
). We note that both genomic sequences related to the CMP-Neu5Ac transporter (AL023874 and AC020227) share homology with the mammalian UDP-galactose and UDP-N-acetylglucosamine transporters as well, with the first accession number corresponding to the sequence most similar to the mouse CMP-Neu5Ac transporter. In mammals, the individual sugars each have designated transporters, however, we do not know if this is the case in Drosophila. Interestingly, investigators doing a structural analysis of transporters created a chimeric UDP-galactose and CMP-Neu5Ac transporter that had dual sugar specificity (Aoki et al., 2001
). The Drosophila genomic sequence related to CMP-Neu5Ac synthase (AE003515) shares homology with the mammalian enzyme only in the N-terminal half, as has been previously reported (Angata and Varki, 2000
). However, when we compared the sequence of the E. coli CMP-Neu5Ac synthase with the sequences of the mammalian enzyme or with enzymes from other bacterial species, such as Neissera meningitidis, Campylobacter jejuni, Legionella pneumophila, and Haemophilus dycreyi, we noted that only the N-terminal half was conserved, similar to the results with Drosophila. We could find no corresponding orthologue for the UDP-GlcNAc2-epimerase/ManNAc kinase, although low-level endogenous epimerase activity has been reported in an Sf-9 cell line (Effertz et al., 1999
). A negative result of a homology search can be a consequence of the genomic sequence having numerous introns, creating gaps that depress the significance of the alignments found using the BLAST algorithms. The fly sialyltransferase, DmST (AE003695), shares highest amino acid identity with the 2,6 sialyltransferases of N-glycans. However, there were numerous residues conserved between DmST and
2,8 sialyltransferases that were not present in the 2,6 sialyltransferases. A determination of the glycoconjugate specificity of DmST must therefore await further enzymatic studies currently in progress in our laboratories.
The expression of sialic acidcontaining glycoconjugates in Drosophila may be regulated by the tissue-type and/or developmental stage (Marchal et al., 2001). Staining of Drosophila embryos using sialic acidbinding lectins showed staining of germ cell precursors, general ectodermal staining, and intense staining of the nervous system at a time when the nervous system was forming (Roth et al., 1992
). The failure of many groups to obtain biochemical or enzymatic evidence of sialic acid metabolism in insects may be due to a temporally restricted pattern of glycoconjugate expression in a low percentage of cells. However, using the sophisticated molecular genetic approaches available in Drosophila one can now readily create loss-of-function mutations in specific genes in the sialic acid pathway using P-element insertion (Spradling and Rubin, 1982
) or RNA interference (Kennerdell and Carthew, 1998
) coupled with targeted gene expression (Brand and Perrimon, 1993
). These techniques should allow an assessment of loss of function in specific tissues and times in development and enable us to elucidate the biological role of sialic acid glycoconjugates in Drosophila development.
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Material and methods |
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Amplification of staged Drosophila RNA and cDNA
Total RNA was isolated from staged Drosophila or S2 cells as described. RT-PCR was performed using the conditions described above, except that 0.6 µg of RNA was DNase Itreated and directly added to the RT reaction without prior ethanol precipitation. PCR of the Drosophila Rapid-Scan panels (Origene, Gaithersburg, MD) was performed in 25-µl reactions using the same PCR conditions already described except that 35 cycles were used. PCR in 48-well microtiter plates (panels) was performed using an Applied Biosystems GeneAmp 9600 thermal cycler.
Cloning, expression, and activity of DmSAS in bacteria
The DmSAS coding region was amplified by PCR using pBlueBac-DmSAS as the template with the following primers: The forward primer (JP1), 5'-CACTGAATTCATGCTGTTAAACGATATCATAAGC, had an EcoRI site (italics) upstream of the initiator ATG, and the reverse strand primer (JP2), 5'-AGTGAAGCTTTCAATTGATTATACT, contained a HindIII restriction site (italics). PCR reactions were performed as described except that reactions included 2.5 mM MgCl2 and only 25 cycles were used. All final constructs were sequenced to verify that no base changes had been introduced during PCR.
After appropriate enzyme digestion, the PCR product was ligated into the E. coli expression vector pTrc99a (Amersham Pharmacia, Piscataway, NJ) and the construct was introduced into E. coli TOP-10 cells. An overnight culture of TOP10:pTrc99A-DmSAS was used to inoculate 1.5 L Luria-Bertani broth, which was grown until A600 = 0.6. Expression was induced with 0.5 mM IPTG for 2 h at 37°C, after which the culture was harvested by centrifugation at 5500 x g for 10 min, resuspended in 20 ml 0.05 M Bicine, 1 mM dithiothreitol (DTT), pH 8.0, and lysed in a French pressure cell. The lysate was centrifuged at 27,000 x g for 15 min and the supernatant was brought to 60% (by weight) ammonium sulfate and centrifuged at 27,000 x g for 15 min to pellet the enzyme. The precipitate was dissolved in 0.05 M Bicine, 1 mM DTT, pH 8.0, and assayed for sialic acid synthase activity using the TBA assay (Vann et al., 1997). The substrates ManNAc-6-P, ManNAc, and PEP were used at a final concentration of 12.5 mM.
The DmSAS gene was also inserted into the E. coli His-tag expression vector pTrcHis2TOPO (Invitrogen) to facilitate enzyme purification. The gene was amplified by PCR with the forward primer 5'-CTGTTAAACGATATCATAAGCGGAA, the reverse strand primer 5'-ATTGATTATACTATTCCCAAGTATGGG, and the template pTrc99a-DmSAS using the following settings: 94°C 45 s; 30 cycles at 92°C 45 s, 42°C 1 min, 72°C 2 min; 72°C 10 min; hold at 4°C. The PCR product was ligated directly into pTrcHis2TOPO and the resulting plasmid was introduced into TOP-10 cells. A 1.5 L Luria-Bertani culture of TOP10:pTrcHis2TOPO-DmSAS was grown and induced with IPTG, and the cell lysate was prepared as described. The clarified cell lysate was applied to a 6 mL column packed with Ni-NTA resin (Qiagen, Valencia, CA), washed with 0.05 M Bicine, 1 mM DTT, pH 8.0, buffer and eluted with 250 mM imidazole in wash buffer. The eluent was assayed for sialic acid production over time by measuring the sialic acid content with the TBA assay (Vann et al., 1997).
Cloning, expression, and activity of DmSAS in insect cells
Using the Invitrogen Bac-N-BlueTM kit instructions, pBlueBac4.5-DmSAS plasmid DNA was transfected into Sf-9 cells (ATCC, Manassas, VA) and the resulting virus (AcDmSAS) was amplified. Baculovirus DNA was isolated using the Invitrogen procedure and probed for inclusion of the synthase gene by PCR with primers SA4 and SA5. Infected cell lysates were analyzed by SDSPAGE followed by Coomassie blue staining. For N-terminal sequencing, the Pierce NE-PERTM kit was used to isolate cytoplasmic extracts that were separated by SDSPAGE. Bands were transferred to a polyvinylidene difluoride membrane, stained with Ponceau S, and sequenced from the N-terminus with an Applied Biosystems Procise sequencer. Cells were prepared, infected, and harvested at 72 h postinfection as previously described (Lawrence et al., 2000). Sugar feeding supplementation was done with sterile-filtered 10 mM Man, mannosamine, and ManNAc from 1 M stocks. All cells were grown in serum-free HyClone (Logan, UT) SFX medium. The Neu5Ac and KDN content of Sf-9 cell lysates was detected using DMB labeling and normalized on a protein basis using a previously published procedure (Lawrence et al., 2000
). For in vitro assays, cells were prepared, infected, harvested, and assayed using previously described procedures (Lawrence et al., 2000
). ManNAc-6-phosphate was synthesized using previously published methods (Liu and Lee, 2001
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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