Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023900, São Paulo, SP, Brasil, and 2The Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 306027229, USA
Received on June 7, 2000; revised on September 26, 2000; accepted on September 26, 2000.
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Abstract |
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Key words: glycosphingolipid/glucosylceramide/fungus/yeast/thermal dimorphism
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Introduction |
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In addition to GIPCs, fungi also express monohexosylceramides (cerebrosides, or CMHs) having distinctive structural modifications of the ceramide moiety, some of which are also found in GSLs of plants and certain marine invertebrates, but not in those of mammals (Fujino and Ohnishi, 1976; Ballio et al., 1979
; Karlsson et al., 1979
; Fogedal et al., 1986
; Matsubara et al., 1987
; Sitrin et al., 1988
; Shibuya et al., 1990
; Jin et al., 1994
; Natori et al., 1994
; Sawabe et al., 1994
; Villas Boas et al., 1994
; Costantino et al., 1995a
,b; Duarte et al., 1998
). These modifications include addition of a characteristic
8-unsaturation and a branching 9-methyl group to the sphingoid base (see Scheme 1). In the case of fungi, such additional variations may have functional importance in growth, life cycle, morphogenesis, and hostpathogen interactions. For example, Kawai and Ikeda (Kawai and Ikeda, 1982
, 1983, 1985a; Kawai et al., 1985b
; Kawai, 1989
) reported that fungal glucocerebrosides or structurally similar analogs exhibited fruiting-inducing activity in bioassays with Schizophyllum commune. An intact 9-methyl-4,8-sphingadienine, but not the ß-glucopyranosyl residue, was essential for activity (Kawai and Ikeda, 1983
; Kawai et al., 1985b
). Glucocerebrosides extracted from the rice pathogen Magnaporthe grisea were found to be highly active elicitors of defense responses, including accumulation of phytoalexins and hypersensitive cell death, when applied to rice leaves (Koga et al., 1998
). However, although these and a number of other suggestive phenomena have been observed with fungal cerebrosides (Kawai and Ikeda, 1982
, 1983, 1985a; Kawai et al., 1985b
; Koga et al., 1998
; Mizushina et al., 1998
; Toledo et al., 1999
), very little is known about their true functions, biosynthesis, or metabolic fate in vivo.
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A key component of our efforts to understand more about the possible functional roles of GSLs has been studies directed toward defining precisely the structural similarities and variations among different species and strains of pathogenic and related non-pathogenic fungi. Such studies of secondary product structure are an essential complement to molecular genetics for defining the function of enzymes associated with their biosynthesis. One focus of these studies has been on an important group of dimorphic mycopathogens which grow in a low temperature saprophytic phase exhibiting hyphal morphology, but are found in tissues of an infected host primarily as budding yeasts. Within this group, which includes the closely related trio Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, as well as the more distantly related Sporothrix schenckii, temperature is a dominant, but not exclusive, determinant of morphology. To varying extents depending on species and strain, CO2 concentration, pH, and nutritional factors also contribute to maintenance of a particular form in vitro (Szaniszlo et al., 1983; Travassos, 1985
).
We have now characterized both GIPCs and CMHs of P. brasiliensis (Levery et al., 1998, 2000; Toledo et al., 1999
) as well as CMHs of S. schenckii (Toledo et al., 2000
). In a classic 1984 study, the structures of GIPCs of H. capsulatum were elucidated by Lester and his colleagues (Barr, 1984a
,b), but the CMHs of this fungus have never been examined. In this paper we report on the detailed characterization of cerebroside components from both mycelium and yeast forms of H. capsulatum; these studies revealed a remarkable difference in ceramide structure correlating with morphological transition in this fungus.
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Results |
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Analysis of H. capsulatum cerebrosides by electrospray ionization mass spectrometry and tandem collision-induced dissociation mass spectrometry
Major components.
In positive ion mode ESI-MS, abundant monolithiated molecular ion adducts were observed virtually exclusively at m/z 760 and m/z 762 for the mycelium and yeast form glucocerebrosides, respectively (Figure 3A,B), corresponding to nominal molecular masses of 753 and 755 Da. These molecular masses are consistent with monohexosylceramides containing (d19:2) (4E,8E)-9-methyl-4,8-sphingadienine attached to either N-2'-hydroxy-(E)-3'-octadecenoate or N-2'-hydroxyoctadecanoate, respectively. Confirmation that the observed difference of m/z 2 is due to variation in the fatty acid moiety was provided by tandem collision induced-dissociation mass spectrometry (+ESI-MS/CID-MS) experiments. In these experiments, product ion spectra were obtained from the lithiated molecular ions, either m/z 760 or m/z 762, selected in Q1 (Figure 4A,B). All spectra were characterized by highly abundant [M + Li acyl]+ (O) and [M + Li HexOH Sph-C3C19]+ (T Z0/G) fragments, as observed previously under these conditions for a variety of fungal cerebrosides (Levery et al., 2000
). In both spectra, the O ion is observed at m/z 480, while the m/z 2 difference is carried by the T fragment, containing the fatty acid moiety, observed at either m/z 330 or m/z 332 depending on the mass of the pseudomolecular ion selected. Furthermore, the masses and relative abundances of additional major and minor fragments are in each case virtually identical with those previously obtained under these conditions for fungal cerebrosides containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base in combination with N-2'-hydroxy-(E)-
3-octadecenoate and N-2'-hydroxyoctadecanoate, respectively. A number of these fragments can be considered as diagnostic for the presence and positions of characteristic functional groups such as unsaturations and fatty N-acyl 2-hydroxylation (Levery et al., 2000
). The assignments for these fragments are summarized in Table II.
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Minor components.
In addition to completing the structure elucidation of the major components, it was also possible by +ESI-MS/CID-MS to characterize minor components appearing in the molecular ion profiles of mycelium and yeast form GlcCer, which could correspond either to low abundance intermediates in the biosynthetic pathway or to structural variants differing, for example, in fatty acid chain length. Both of these possibilities are apparent in the product ion spectrum of m/z 746 (Figure 5A) selected from the molecular ion profile of the mycelium form GlcCer. The spectrum exhibits a single [M + Li hexose]+ (Y0) ion at m/z 584; as expected, this is 14 Th less than that observed for the major molecular ion (Y0 at m/z 598). However, at lower m/z, product ions arising from two isobaric components are apparent, characterized by two sets of O and T ions, one at m/z 480 and 316, the other at m/z 466 and 330. These are consistent with components in which the 14 Th decrement is carried by either the fatty N-acyl group or the sphingoid moiety, respectively, present in approximately equal amounts. In contrast with the former case, which can be clearly attributed to a variant carrying an h17:1 fatty N-acyl group, the latter case most likely corresponds to a h18:1/d18:2 component in which the sphingoid 9-methyl group is missing, which may represent a biosynthetic intermediate.
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Unlike the result with the mycelium form m/z 746 ion, products of the corresponding minor m/z 748 ion in the yeast form profile (Figure 5B) are consistent with essentially a single component in which the 14 Th decrement is carried by the sphingoid moiety, as characterized by a single set of O and T ions at m/z 466 and 332. With the exception of a very low abundance set of O and T ions at m/z 480 and 318, corresponding to the h17:0/d19:2 fatty N-acyl/sphingoid combination, the masses and relative abundances of almost all other fragments in the spectrum are consistent with a h18:0/d18:2 component lacking the sphingoid 9-methyl group.
Products of the m/z 750 component (Figure 5C) were also consistent with essentially one component lacking both the sphingoid 9-methyl group and the 8-unsaturation. In addition to the O and T fragments observed at m/z 468 and 332, respectively, which show the additional 2 Th increment to be carried by the sphingoid moiety, further evidence for the mono-unsaturated (d18:1) sphing-4-enine structure are the obvious changes in the abundance of the T and N (m/z 306) fragments relative to each other and the increase in the abundance of the Y0 (m/z 588) relative to the O fragment. A number of other characteristic changes in the spectrum are diagnostic for the 2-hydroxy fatty N-alkanoyl/sphing-4-enine combination under these conditions, and in fact the product spectrum is essentially the same as that obtained previously from a bovine brain galactocerebroside with h18:0/d18:1 ceramide (Levery et al., 2000
). Interestingly, an analogous component is not observed in significant abundance in the mycelium form profile (at m/z 748, see Figure 3A). On the other hand, a molecular ion at m/z 774 was sufficiently abundant in the mycelium form profile for a product ion spectrum to be acquired (not shown). In this case the predominant O and T ions were observed at m/z 480 and 344, respectively, consistent with an h19:1/d19:2 fatty N-acyl/sphingoid combination. Other product ions observed in the spectrum were consistent with this structure (see Table II). An analogous molecular ion m/z 776 in the yeast form profile was not sufficiently abundant to produce a useful CID spectrum.
Comparison with results from P. brasiliensis cerebrosides
The +ESI-MS profile obtained from a yeast form GlcCer of P. brasiliensis (Levery et al., 2000) was very similar to that obtained here for the corresponding H. capsulatum fraction, differing mainly in the proportions of some minor components. Aside from the appearance of an abundant m/z 762 component (yielding an identical CID product spectrum), minor ions at m/z 750 and 748 were also observed in the P. brasiliensis yeast form GlcCer profile. In the case of m/z 750, in P. brasiliensis as with H. capsulatum the only product ions observed corresponded to the h18:0/d18:1 component. On the other hand, in the case of m/z 748 from P. brasiliensis yeast form isobaric h18:0/d18:2 and h17:1/d19:2 components were comparably represented (Levery et al., 2000
), whereas the latter was barely observed in H. capsulatum (Figure 5B).
The +ESI-MS profile of the P. brasiliensis mycelium form GlcCer was also similar to that from H. capsulatum, although with P. brasiliensis the m/z 760 component was accompanied by a significant ion abundance at m/z 762 (Levery et al., 2000). This was in agreement with the NMR and fatty acid analysis, which showed incomplete conversion to the
3-unsaturated form (Toledo et al., 1999
). A minor m/z 746 ion was also observed in the P. brasiliensis mycelium form GlcCer profile. Unlike the case with H. capsulatum, in which product ions from the isobaric h18:1/d18:2 and h17:1/d19:2 components could be observed in comparable amounts, the latter was only barely detectable in the P. brasiliensis m/z 746 spectrum. A number of other minor ions were observed in the profiles of P. brasiliensis glucocerebrosides, and CID data acquired (Levery et al., 2000
), but were not sufficiently abundant in the H. capsulatum profiles for useful product spectra to be obtained; these were m/z 748 and 750 in the mycelium form profile, and m/z 734 and 790 in both profiles.
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Discussion |
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Although reports of physiological activities for fungal glucocerebrosides (Kawai and Ikeda, 1982, 1983, 1985a; Kawai et al., 1985b
; Kawai, 1989
; Koga et al., 1998
; Mizushina et al., 1998
) are not necessarily unambiguous demonstrations of true function, they are highly suggestive that significant functional roles exist for these compounds in vivo. In the present case, although the mechanism and functional implications of the observed ceramide structural dimorphism are unclear, the changes in levels of (E)-
3-unsaturation could have implications for the regulation of morphological transitions in H. capsulatum and P. brasiliensis (and possibly B. dermatitidis). We previously proposed that the (E)-
3 modification of P. brasiliensis GlcCer could serve a specific messenger function; with activation (or deactivation) of a desaturase responsible for this modification being one step in a signaling cascade directing the transition from yeast to mycelium (or the reverse) which is initiated by a change in temperature. It is possible that the observed changes in CMH composition in dimorphic fungi could simply be ascribed to temperature sensitivity of expression, stability, or activity for the enzymes involved, rather than regulation by more complex mechanisms, but this would not rule out their potential functional significance in morphogenesis. On the contrary, changes in CMH hexose and/or ceramide structure distribution, regulated directly by a simple temperature dependent parameter such as enzymatic activity, could constitute ideal chemical switches activating other processes following alterations in environmental temperature. As pointed out in the Introduction, the possibility that cerebrosides may be components of signal transduction pathways regulating morphological transitions in fungi should be investigated further.
Interestingly, (E)-3-unsaturation in the 2-hydroxy fatty N-acyl moiety so far appears to be a modification found only in CMHs of Euascomycetes, having been previously observed in varying amounts in Fusicoccum amygdali Delacroix (Ballio et al., 1979
), Pachybasium spp. (unnamed) (Sitrin et al., 1988
), Magnaporthe grisea (Koga et al., 1998
), Penicillium funiculosum (Kawai et al., 1985b
), Aspergillus spp. (Ohnishi, 1976; Villas Boas et al., 1994
; da Silva Bahia et al., 1997
; Toledo et al., 1999
; Levery et al., 2000
), Fusarium spp. (Duarte et al., 1998
), P. brasiliensis (Toledo et al., 1999
; Levery et al., 2000
), and S.schenckii (Toledo et al., 2000
), but not reported in any Basidiomycete so far investigated, including Schizophyllum commune (Kawai and Ikeda, 1982
, 1983, 1985a), Lentinus edodes (Kawai, 1989
), Hypsizigus marmoreus (Sawabe et al., 1994
), Ganoderma lucidum (Mizushina et al., 1998
), Clitocybe spp. (Fogedal et al., 1986
), and yeastlike pathogenic Cryptococcus spp. (Levery et al., 2000
); nor in the Hemiascomycete dimorphic pathogen Candida albicans (Matsubara et al., 1987
; Levery et al., 2000
). It may therefore be tentatively proposed as a chemotaxonomic marker for Euascomycetes.
Finally, the results herein constitute further evidence for the partitioning of fungal ceramide catabolism into two distinct pathways for CMH and GIPC biosynthesis, as has been pointed out independently by Lester and Dickson (Dickson and Lester, 1999) and by us (Toledo et al., 1999
; Toledo et al., 2000
). For example, unlike the CMHs from Candida albicans (Matsubara et al., 1987
), P. brasiliensis (Toledo et al., 1999
), and H. capsulatum (this work), in which the ceramides are of the type shown in Scheme 1, the GIPCs of the same fungi are found predominantly with saturated, longer chain 2-hydroxy fatty acids (h24:0 or h26:0) attached to t18:0 4-hydroxysphinganine (phytosphingosine) (Wells et al., 1996
; Levery et al., 1998
; Barr and Lester, 1984b
; Barr et al., 1984a
). It is not known how this partitioning of ceramide types into CMH and GIPC biosynthesis is accomplished, but two possibilities which have been suggested (Dickson and Lester, 1999
; Toledo et al., 1999
;) are compartmentalization of their respective biosynthetic and/or transport pathways, or selective recognition of ceramide structural elements somewhat remote from the reaction site by the putative IPC synthase (Nagiec et al., 1997
) and the as yet uncharacterized fungal cerebroside synthase(s). The latter possibility could depend, for example, on the interaction of different ceramide acceptor substrates, having either a 4-hydroxyl group or a 4-unsaturation on the sphingoid, with specific domains on the respective synthases selectively recognizing these features. On the other hand, selectivity in this step alone would not explain the difference with respect to fatty acid chain length in the two types of products. This difference implies a prior partitioning or some other discriminatory process associating a specific fatty acid chain length with its appropriate sphingoid partner.
Knowledge of the CMH biosynthetic pathway in fungi is still at a rudimentary stage, and appears to be more complex than that for GIPCs with respect to assembly of the ceramide moiety. A considerable number of associated genes and their products remain to be discovered. Some of these may be highly homologous to those found widely among eukaryotes, such as the sphingoid (E)-4-desaturase or the GlcCer and GalCer synthases. A gene encoding a plant sphingoid (E)-
8-desaturase was recently confirmed (Sperling et al., 2000
), and similar genes should eventually be found in fungi, sponges, and echinoderms; one or more 9-methyltransferases should also be found in the latter species. Still others, such as the Euascomycete-associated 2-hydroxy fatty N-acyl (E)-
3-desaturase, may have no homologues in other taxa. Recently, candidate GlcCer synthase homologs could be extracted from publicly accessible fungal genome databases via tBLASTn searching with mammalian UDP-glucose:ceramide glucosyltransferases (Ugcg) peptide sequences (Ichikawa et al., 1996
, 1998). Single-exon predicted ORFs, identified in the ongoing Candida albicans (Contig4-3104 [HSX11]; Stanford DNA Sequencing and Technology Center) and Neurospora crassa (9a3.pep1152; Münchener Informazionscentrum für ProteinsequenzenMIPS) genomic sequence databases, have stretches of significantly conserved homology encompassing the NRD2S and NRD2L motifs proposed by Kapitonov and Yu (1999)
to be characteristic for the family of glycosyltransferases which includes the mammalian Ugcg proteins; within these motifs, the homology appears closest to those in the Ugcg group (S. B. Levery and J. K. Rose, unpublished observations). Interestingly, the fungal Ugcg sequences contain numerous extra stretches of peptide interpolated between conserved motifs; some of these could comprise additional recognition domains responsible for maintaining strict specificity against Glc residue transfer to phytosphingosine-containing ceramides. The high likelihood of the involvement of cerebrosides in a variety of key fungal cellular processes should provide considerable incentive for future investigations in this area.
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Materials and methods |
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Solvents for extraction and anion exchange chromatography
Solvent A, isopropanol/hexane/water (55:20:25, v/v/v, upper phase discarded); solvent B, chloroform/methanol (2:1, v/v); solvent C, chloroform/methanol/water (30:60:8, v/v/v).
High performance thin layer chromatography
Both analytical and preparative HPTLC were performed on silica gel 60 plates (E. Merck, Darmstadt, Germany) using chloroform/methanol/water (60:40:9 v/v/v, containing 0.002% w/v CaCl2; solvent D) or chloroformmethanolaq. ammonium hydroxide (15 N)aq. ammonium chloride (0.83%) (50:36:8.6:7.2 v/v/v/v; solvent E) as mobile phases. Lipid samples were dissolved in solvent B and applied by streaking from 5 µl Micro-caps (Drummond, Broomall, PA). For analytical HPTLC, detection was made by Bials orcinol reagent (orcinol 0.55% (w/v) and H2SO4 5.5% (v/v) in ethanol/water 9:1 (v/v); the plate is sprayed and heated briefly to 200250°C). For preparative HPTLC, samples were streaked lengthwise on 10 x 20 cm plates; separated glycosphingolipid bands were visualized under UV after spraying with primulin (Aldrich; 0.01% in 80% aqueous acetone). Bands were marked by pencil and individually scraped from the plate. Glycosphingolipids were then isolated from the silica gel by repeated sonication in solvents A and B followed by centrifugation. Following concentration of the extract, primulin was removed by passage through a short column of DEAE-Sephadex A-25 in Solvent C.
Extraction and purification of glycosphingolipids
Extraction and purification of glycosphingolipids were carried out as described previously (Toledo et al., 1995, 1999). Briefly, glycosphingolipids were extracted by homogenizing yeast or mycelium forms (2535 g wet weight) in an Omni-mixer (Sorvall Inc. Wilmington, DE), three times with 200 ml of solvent A, and twice with 200 ml of solvent B. The five extracts were pooled, dried on a rotary evaporator, dialyzed against water, lyophilized, resuspended in solvent C, and applied to a column of DEAE-Sephadex A-25 (Ac form). Neutral glycosphingolipids were eluted with five volumes of solvent C. The neutral glycosphingolipid fraction was further purified from other contaminants by column chromatography on silica gel 60 using a step-wise gradient of chloroform/methanol from 9:1 to 1:1 (v/v) (Sweeley, 1969
). Fractions containing ceramide monohexosides (CMHs), as assessed by analytical HPTLC, were pooled, dried, and further purified by preparative-scale HPTLC as described above. The purity of each fraction was assessed by analytical HPTLC.
1H-nuclear magnetic resonance spectroscopy
Samples of underivatized CMH (0.51.0 mg) were deuterium exchanged by repeated evaporation from CDCl3/CD3OD (2:1 v/v) under N2 stream at 37°C, and then dissolved in 0.5 ml DMSO-d6/2% D2O (Dabrowski et al., 1980a
,b; Yamada et al., 1980
) for NMR analysis. 1-D 1H-NMR, 2-D 1H-1H-TOCSY (Braunschweiler and Ernst, 1983
; Bax and Davis, 1985
), 1H-detected, 13C-decoupled, phase sensitive, gradient (Davis et al., 1992
) 13C-1H-HSQC (Bodenhausen and Ruben, 1980
) and -HMBC (Bax and Summers, 1986
; Bax and Marion, 1988
) experiments were performed at 35°C on a Varian Unity Inova 600 MHz spectrometer using standard acquisition software available in the Varian VNMR software package. Proton-decoupled 1-D 13C-NMR spectra were acquired by direct detection on a Varian Unity Inova 500 MHz spectrometer under identical conditions. Proton chemical shifts are referenced to internal tetramethylsilane (
= 0.000 p.p.m.), carbon chemical shifts to the center line of residual DMSO (set at
= 39.82 p.p.m.).
The percentage of (E)-3 unsaturation was calculated from the integrated ratio of the vinyl proton resonances corresponding to H-4'' of (E)-
3 unsaturated fatty acid and H-5 of the sphingosine moiety (Toledo et al., 1999
, 2000). These resonances were chosen since they have similar splitting patterns and chemical shifts, but are completely resolved from each other in all spectra; although the chemical shift of H-5 is slightly affected by the presence or absence of (E)-
3 unsaturation, the total integral for this resonance was assumed to represent 1.00 mol, regardless of fatty acyl distribution.
Electrospray ionization mass-spectrometry
ESI-MS and tandem ESI-MS/CID-MS were performed in the positive ion mode on a PE-Sciex (Concord, Ontario, Canada) API-III spectrometer, with a standard IonSpray source (orifice-to-skimmer voltage (OR), 120160 V; Ionspray voltage, 5 kV; interface temperature, 45°C), using direct infusion (35 µl/min) of CMH samples dissolved (20 ng/µl) in 100% MeOH to which was added a solution of LiI (10 mM) in MeOH until the observed ratio of Li+ to Na+ molecular ion in +ESI-MS profile mode was >95:5 (the final concentration of LiI was generally 23 mM) (Levery et al., 2000
). For +ESI-MS/CID-MS experiments, precursor ions selected in Q1 were subjected to collision induced dissociation (with argon as collision gas) in Q2, while the mass range in Q3 was scanned from m/z 100800 in 0.2 u steps. OR was set to 120 V, the collision gas was argon (collision gas temperature (CGT) = 380400 [x 1012 molecules/cm2]), and collision energy was 80 eV. Other parameters were set to achieve a peak width at height of 0.60.7 Th (measured at m/z 332), deemed sufficient to assign nominal masses to all peaks in the mass range of interest (Levery et al., 2000
). Dwell time of 5 ms (2.5 ms for minor components), giving a total cycle time 19 s (or 9.5 s). In general, spectra represent summations of 510 scans for single analyzer profiles, and 1030 scans for CID experiments (50100 for minor components). Fragment nomenclature, illustrated in Scheme 2, is after Costello et al. (Domon and Costello, 1988
; Costello and Vath, 1990
; Domon et al., 1990
) as modified and expanded by Adams and Ann (1993; see also Sullards et al., 2000
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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