1Biological Research Laboratories, Syracuse University, Syracuse, New York 13244; 2Department of Physiology, McGill University, Montreal H3G 1Y6; and 3Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2
Submitted 17 November 2003 ; accepted in final form 26 May 2004
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ABSTRACT |
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cell size; mitochondria
The existing evidence implicating changes in Fe homeostasis in HCMV infection is limited. It consists mainly of the inhibitory effects of Fe chelation on HCMV replication (11) and the blocking effect of Fe chelation on the expression of intercellular adhesion molecules (10). In addition, the HCMV US2 protein mediates the degradation of the Fe-regulatory protein HFE (52). Interestingly, changes in Fe homeostasis have been implicated in the pathogenesis of several other viruses, including hepatitis C, herpes simplex virus, human immunodeficiency virus, rhabdovirus, and vaccinia virus (11, 13, 50, 53).
The present study was undertaken to directly assess the effects of HCMV infection on the magnitude of labile Fe and its role during cytomegaly. While most cellular Fe is stored in the hollow cores of 24 ferritin subunit spherical shells or incorporated into heme and Fe-S cluster of Fe proteins, there is a small, chelatable fraction that constitutes the labile iron pool (LIP). The state of Fe inside this LIP, which is often referred to as the low-molecular-weight transit Fe pool or chelatable Fe, is still very poorly defined but can be estimated with fluorescent probes (22, 49). In the present work, we show that there is a transient increase in the size of the LIP that precedes an increase in cell size and that most of the HCMV-induced host cell enlargement is Fe dependent.
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EXPERIMENTAL PROCEDURES |
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A stock of HCMV (strain AD169; American Type Culture Collection) was generated in confluent MRC-5 cells (for details, see Ref. 15). Viral infectivity was determined by plaque reduction assay (3). Briefly, confluent MRC-5 fibroblasts, which were plated in six-well culture plates, were exposed to serial dilutions (103108) of cell lysate that was isolated from HCMV-infected cells. After the 1-h infection period, cells were washed with PBS and covered with 0.5% agarose overlay containing cell culture medium. An additional overlay was applied after 1 wk. On day 14, cells were fixed in 0.03% formalin and stained with 0.03% methylene blue, and viral plaques were counted and expressed as the number of plaque-forming units per milliliter of undiluted cell lysate.
Solutions and reagents. Standard N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution contained (in mM) 128 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 20 HEPES. The solution pH was adjusted to 7.4 (at 37°C) with N-methyl-D-glucammonium (NMDG+), and the osmolality was 285 ± 5 mosmol/kgH2O. Salicylaldehyde isonicotinoyl hydrazone (SIH) was synthesized as previously described (7) and prepared as a 50 mM stock solution in dimethyl sulfoxide. Calcein acetoxymethyl ester (calcein-AM), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and A-23187 were obtained from Molecular Probes (Eugene, OR).
Cell size.
Sets of MRC-5 cells were grown to confluence in 35-mm-diameter cell culture dishes and then either mock or HCMV infected. To assess volumetric cell size, cells were first treated for 3 min with trypsin in Ca2+-free PBS. They were then diluted in CO2-free HEPES-buffered cell medium containing FCS and 1 µM calcein-AM and separated from each other by gentle pipetting. As a result of this treatment, both the spindle-shaped, mock-infected cells and the polymorphic HCMV-infected cells took on a spherical morphology, thereby facilitating cell size determination. After a 30-min incubation period in an air 37°C incubator, the cell culture dishes containing loosely suspended mock- and HCMV-infected cells were transferred to the stage of an inverted fluorescence microscope. Images from three experiments, 20 randomly selected fields containing 70120 cells per point, were collected. Cell diameters were determined with the assistance of the semiautomated segmentation routines built into the DeltaVision software package (Applied Precision, Issaquah, WA). Cell sizes were calculated from measured cell diameters, with the cell shape assumed to be a perfect sphere. To minimize measurement errors, images were collected within 1 h of trypsinization. Beyond this time, cell morphology became more ellipsoid because of the development of filopodia and lamellipodia, thereby resulting in their attachment and flattening onto the culture dish.
Labile iron pool.
The size of the LIP in MRC-5 fibroblasts was estimated using the metallosensitive fluorescent dye calcein together with the cell-permeant iron chelator SIH. This method is widely used to assess the size of the LIP (8; for review, see Ref. 21). MRC-5 cells were grown to confluence on 25-mm-diameter coverslips placed in 35-mm cell culture dishes. Care was taken to ensure that a layer of solution remained over the cell surface when transferring coverslips to the microscope chamber. Cells were loaded with calcein by flowing (0.2 ml/min) a standard HEPES-buffered solution containing 1 µM calcein-AM until they reached
1,000 arbitrary fluorescence units (510 min). This amount of dye loading was sufficient to obtain an adequate signal-to-noise ratio without excess fluorophore, which would have resulted in calcein self-quenching. In these experiments, fluorescence was collected using a microphotometric system and a Nikon x40, 1.3 NA oil objective lens (5). The photometer's pinhole was wide open so that fluorescence was collected from multiple cells to minimize fluorescence intensity changes due to any volume perturbations. To minimize light-induced change to the fluorophore, exposure duration for each time point was limited to 100 ms and an ND2 neutral density filter was included in the excitation light pathway.
After calcein loading, the flow rate was increased to 2 ml/min. Once the raw fluorescence signal (Fraw) had stabilized, a maximal (100 µM) dose of SIH (21) was added to the superfusate (see Fig. 1A). The new elevated steady-state fluorescence level was defined as the zero metal state (F0) as SIH sequestered the labile Fe and possibly other SIH chelatable metals. To calibrate the fluorescence change at the end of the experiment, we used 5 µM Co2+ in the presence of the metal ionophore A-23187. Using this metal avoided the need to address the calibration problem associated with the autoxidization of Fe2+ to Fe3+. Background fluorescence (Fbkg) was determined by adding excess Co2+ (1 mM) to the superfusate to maximally quench calcein fluorescence. In vitro experiments showed that 1 mM Co2+ completely quenched calcein fluorescence. Experiments were performed at room temperature to improve baseline stability because at this temperature dye loss was minimal and therefore the need to correct for time-dependent fluorescence decay was avoided.
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The Stern-Volmer constant of calcein for Co2+ (), calculated from the application of 5 µM Co2+ in the presence of A-23187, was 0.116 x 106 M1. An upper estimate of the LIP can be obtained if it is assumed that Co2+ is twice as effective as Fe in quenching calcein fluorescence (8).
Mitochondrial membrane potential.
The mitochondrial voltage-sensitive dye JC-1 was used to obtain an index of mitochondrial membrane potential (M). The advantage of this dye is that a voltage-dependent shift in its emission spectra provides a means for obtaining a ratiometric emission fluorescence signal. This type of signal is less dependent on dye concentration and also permits imaging of regional differences in
M (12). Red emission fluorescence (580- to 653-nm band-pass emission filter) arises from a region of high
M. Green emission fluorescence (509- to 547-nm band-pass emission filter) comes from a region of low
M. However, because of its slow kinetics, the use of JC-1 to assess acute changes in
M is limited, and single-wavelength
M dyes would be preferable.
Mock- and HCMV-infected MRC-5 cells grown on 25-mm-diameter, 170-µm-thick coverslips (custom order; Bellco Glass, Vineland, NJ) were loaded with 0.5 µM of the dye for 20 min at 37°C in an air incubator and then mounted in a microincubator (PDMI-2; Harvard Apparatus, Holliston, MA). Image stacks were collected with an Inovision ISee imaging system (Durham, NC) that consisted of a Nikon Eclipse TE300 inverted microscope (Melville, NY) equipped with a TILL Photonics Polychrome II monochromator (Union City, CA), a Hamamatsu ORCA II cooled charge-coupled device camera (Bridgewater, NJ), and a Ludl Electronic Products emission filter wheel and stepper motor focusing control (Hawthorne, NY). Experiments were performed at room temperature to minimize mitochondrial movement during stack acquisition.
JC-1 red-to-green emission ratio fluorescence (FR/G) images were obtained by dividing red (high M) emission fluorescence by green (low
M) emission fluorescence after background values had been subtracted and mitochondria had been delineated using an appropriate threshold value for the green emission fluorescence. This procedure resulted in a small percentage of regions with predominately red emission being excluded from the analysis. Thus calculated FR/G mean values were underestimated to a minor extent. However, this methodology reduces the noise introduced from out-of-focus and nonmitochondrial fluorescence.
To improve the qualitative presentation of JC-1 fluorescence images, a deconvolution algorithm was used to reassign out-of-focus fluorescence in the image stacks back to mathematically determined points of origin using the appropriate experimentally determined point spread function for each of the emission wavelengths (DeltaVision; Applied Precision). Deconvolved images were not used for quantitative analysis, because there was considerable scatter in FR/G values. This scatter was presumably due to the varying ability of the deconvolution algorithm to appropriately reassign green and red emission fluorescence to their computed points of origin.
ATP content. Intracellular ATP content was determined using an ATP bioluminescence kit (Sigma).
Data analysis. Values are means ± SE. The unpaired t-test was used to compare two sets of data. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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Iron chelation inhibits HCMV-induced increase in cell size. An increase in the LIP is known to promote cell growth (29), and because HCMV-infected MRC-5 cells are arrested at the G1/S phase of the cell cycle (24), growth promotion of these nondividing cells may manifest as host cell enlargement. Therefore, to test whether labile Fe was required for the development of cytomegaly, the time courses of cell size changes in HCMV-infected cells were determined in both the presence and the absence of 100 µM SIH in the culture medium.
The increase in cell size that normally accompanies HCMV infection is shown in Fig. 3A. Cell size began to increase after 24 hpi and rose almost linearly to 96 hpi, achieving an 275% increase in cell size. To test whether labile Fe is required for the development of this cytomegaly, cells were incubated in a culture medium containing 100 µM SIH immediately after HCMV infection. During the cytomegalic phase (2496 hpi), cell enlargement was suppressed 6090% by SIH chelation (Fig. 3A). In separate control experiments (not shown), a stoichiometric amount of Fe was added together with SIH. Under this set of conditions, the HCMV-induced increase in cell size was similar to that observed in unchelated cells. Thus the ability of SIH to inhibit the HCMV-induced increase in cell size was specific to its chelating ability instead of nonspecific toxic effects.
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In addition to the inhibition of cytomegaly, we observed that vacuolation of the cytoplasm occurred in 13% of the HCMV-infected cells treated with SIH (data not shown). No vacuolation was observed in nonchelated HCMV-infected cells. While mock-infected cells showed an increased abundance of small granular structures, there was no evidence of vacuolation in the presence of SIH. Because vacuolation was specific to SIH-chelated HCMV-infected cells, Fe-dependent cell cycle processes may have been impaired (31).
Effect of SIH chelation on mitochondrial M after HCMV infection.
An Fe-mediated stimulation of oxidative phosphorylation (42) may underlie the development of cytomegaly. Therefore, a possible explanation for the inhibitory effect of Fe chelation on host cell enlargement could be the prevention of any Fe stimulation of mitochondrial activity.
To determine whether Fe was involved in HCMV-induced changes in mitochondria, we investigated the effects of SIH chelation on host cell M using the mitochondrial voltage-sensitive fluorophore JC-1. This dye provides a semiquantitative estimate of
M by an increase in FR/G as the
M increases (see EXPERIMENTAL PROCEDURES). The distribution of FR/G values (Fig. 4A) in mock-infected cells was broad as a result of regional differences in
M (Fig. 4B). In contrast, HCMV-infected cells at 48 hpi had a much narrower distribution of FR/G values. Furthermore, the peak FR/G values for HCMV-infected cells were much lower than they were in mock-infected cells, suggesting that these mitochondria were somewhat depolarized. However, the continuous presence of SIH in the culture medium had no effect on the histogram of FR/G values in either HCMV- or mock-infected cells (Fig. 4A). Moreover, there were numerous small, grainlike mitochondria clustered in the perinuclear regions of HCMV-infected cells (Fig. 4C), whereas the mitochondria in mock-infected cells were less numerous and more threadlike (Fig. 4B). The low contrast and tight clustering of mitochondria in infected SIH-chelated cells impeded the determination of whether Fe chelation affected the HCMV-induced appearance of grainlike mitochondria.
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Effect of HCMV infection on ATP content. A large decrease of ATP content and the appearance of mitochondrial perinuclear clustering have been observed within 1 day after herpes simplex virus and rotavirus infections (19, 39). However, these viruses have a short replication cycle and may be less dependent than long-replication-cycle viruses, such as HCMV, on maintaining ATP for efficient viral replication. Relative to mock-infected cells, the ATP content of HCMV-infected cells was 92 ± 9% at 6 hpi and 87 ± 5% at 24 hpi (n = 4). Cell loss (37) may account for the observed decreases in ATP content. Nevertheless, these values indicate that HCMV-infected cells, unlike HSV- and rotavirus-infected cells, can maintain a near-normal ATP content at these stages of HCMV infection.
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DISCUSSION |
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Under normal conditions, the level of the LIP is tightly controlled by a posttranscriptional regulatory system (49). This negative feedback system involves the binding of iron-regulatory proteins (IRP) to the iron-responsive elements (IRE) present in the untranslated region of mRNA for the key proteins responsible for functionally coordinating Fe metabolism. For example, when Fe levels are low, the IRE/IRP system decreases ferritin protein synthesis while it increases the synthesis of the transferrin receptor, thus leading to an increased LIP. The opposite occurs when Fe levels are high.
Using the metallosensitive fluorophore calcein, we estimated the LIP in mock-infected MRC-5 cells to be 1 µM (Fig. 2). Estimates of LIP in other studies using Fe-sensitive fluorophores have been in the 0.22.5 µM range (21, 44). Furthermore, LIP in K562 cells was found to be
3 µM as determined by electron paramagnetic resonance spectroscopy (14). Thus two independent methods have indicated that LIP in a variety of cells is in the low micromolar range.
As shown in Fig. 2, 24 h after HCMV infection, the amount of SIH-chelatable metals nearly doubles. This result clearly implies that viral mechanisms are able to override the normal tight cellular control of the LIP by the IRE/IRP system. Several mechanisms could be responsible for this rise in the LIP, including increased uptake from the external medium, release of stored Fe from ferritin, and release of Fe incorporated into prosthetic groups of Fe proteins. Stimulated external Fe uptake is one of the possible mechanisms for increasing the LIP because the HCMV gB virion coat protein stimulates internalization and recycling of clathrin-coated transport vesicles known to contain the transferrin receptor in human fibroblasts and retinal pigment epithelial cells (51).
Suppression of ferritin Fe storage capacity is also a possible explanation for an increase in the LIP levels. HCMV infection of primary human foreskin fibroblasts was found to decrease mRNA for both H-ferritin (4x at 8 hpi) and L-ferritin (287x at 24 hpi) (57). Our DNA microarray experiments with MRC-5 fibroblasts (36) (GEO accession nos. GSM9973GSM9975) indicated that H-ferritin and L-ferritin mRNA were likewise decreased at 72 hpi (4.2x and 2.4x, respectively). However, these changes were not significant at 30 hpi (1.8x and 1.4x, respectively).
Because various infected cells enhance free radical production, the release of Fe from Fe-S clusters could be induced (1, 34, 47, 54). Furthermore, the breakdown of heme is another potential source of increased labile Fe. A majority of these Fe proteins are located in mitochondria and, in particular, in the cytochromes and Fe-S proteins of the electron transport chain (6). Hence, the release of Fe from these mitochondrial proteins may be a component of the early postinfection LIP increase, and it could contribute to transient cellular dysfunctions that occur within the first 624 h of HCMV infection (see Labile iron pool and transient HCMV-induced host cellular dysfunctions).
Finally, HCMV infection results in degradation of the major histocompatibility complex-encoded class I-like proteins, including the hemochromatosis gene product HFE (52). The HCMV US2 gene product directs this class of proteins to the proteasomes for degradation instead of to the cell surface (26). Furthermore, mutations in the HFE gene that result in defective protein expression, as well as the knockout of this gene, are known to lead to excess Fe in multiple organs and cell types (23).
Limitations of calcein as a probe of the labile iron pool.
Although the size of the LIP is in the range measured by others in different cell types (21), it is worth discussing some of the limitations of this method. An inherent problem with using most of the ion-sensitive fluorophores, including calcein, is that their fluorescence properties are affected to some extent by multiple ions (28). Furthermore, Fe chelators bind other metals, but generally at a much lower affinity (48); therefore, other metals have the potential to contaminate the measure of the LIP. Also, it should be noted that Fe proteins, in addition to synthetic chelators, incorporate other metals into their binding site, especially when access to Fe is limited (30, 43). Thus, while in theory it would be highly desirable to have fluorophores and Fe chelators that bind only Fe, meeting this ideal is not possible at present and, in any event, may not be a major practical problem. The intracellular metals most likely to contaminate calcein measurement of the LIP are Zn2+ and Cu2+ (28). However, SIH chelation of labile Fe and the subsequent dequenching of calcein fluorescence dominate the fluorescence signal (Fig. 1) because 1) SIH has a very high affinity for Fe (pM of 50.0 compared with 27.7 for its parent compound pyridoxal isonicotinoyl hydrazone) (55), 2) the affinity of SIH for Zn2+ is similar to desferrioxamine and at pH 7.4 is multiple log units less than Fe (48), 3) Zn2+ enhances calcein fluorescence (18, 28) and would therefore attenuate measurements of the LIP rather than add to it, 4) body Cu2+ is only 2% of the Fe amount (20), and 5) only bound but not labile Cu2+ has been detected inside cells (46).
Other areas of potential concern include intracellular compartmentalization and/or binding of calcein. In this study, 25% of the total calcein fluorescence from MRC-5 cells was unquenched by 5 µM A-23187 plus 1 mM Co2+, indicating the presence of a sizable background. While this background was less than the osmotically insensitive component (
67%) that was observed in neuroblastoma cells (16), it was still rather large. Hepatocytes and hepatoma cells accumulate calcein in mitochondria, which remain fluorescent in the presence of both digitonin (10 µM) and A-23187 (2 µM) plus Co2+ (1 mM) (45). However, in MRC-5 cells, the fluorescence was diffuse in the presence of A-23187 plus Co2+. Furthermore, digitonin permeabilization reduced fluorescence to
1% of the autofluorescence level. These observations indicate that mitochondria in MRC-5 cells are much less resistant to Co2+ entry through A-23187 pores than was the case for hepatoma cells. Another possible explanation for the high background is calcein binding to intracellular proteins. The addition of bovine serum albumin in vitro slowed the rate of Co2+ quenching of calcein fluorescence but did not prevent it (unpublished observation), suggesting that proteins may impede metal binding to the fluorophore. More complex protein binding may occur in vivo and would help to explain the sizable fluorescence background.
Labile iron pool and the development of cytomegaly. An early increase in LIP could be an upstream signaling event that promotes cell growth and/or induces the later biosynthesis of mitochondrial Fe proteins (6, 33, 42). Furthermore, we suggest that the biosynthesis of mitochondrial Fe proteins might play an important role in the recovery from initial cellular dysfunctions that occurs shortly after infection. However, because some increase in cell size occurs in the presence of Fe chelation, the LIP increase is most likely not the primary signaling event responsible for promoting cell growth. Nevertheless, signaling may be disrupted because Fe chelation could impair the maturation of the heme-containing protein cyclooxygenase-2, as the progression of HCMV infection is dependent on the upregulation of this protein (58).
An increase in the rate of cellular enlargement after SIH removal from the culture medium (Fig. 3B) indicates that HCMV-induced cytomegaly depends on the availability of labile Fe. Because the inhibitory effect of SIH applied at 48 hpi required 24 h to suppress the progression of cytomegaly (Fig. 3C), a preexisting reservoir of Fe prosthetic groups and/or mature Fe-proteins is probably sufficient to support limited growth. However, the subsequent prevention of further increase in cell size may be a result of impaired maturation of the necessary additional Fe proteins. In particular, the lack of mature electron transport chain Fe proteins may limit the enhancement of energy production. This energy would be needed directly or indirectly to fuel the activities of ion transporters responsible for solute uptake, such as Na+-K+-ATPase (4, 41), Na+/H+ exchanger (15), and the Cl/HCO3 exchanger (35), that are upregulated during this period of the HCMV infection cycle. Extra energy would also be necessary to support enhanced protein synthesis and viral DNA replication during this period of productive HCMV replication.
The HCMV-induced changes in M still occurred in the presence of SIH (Fig. 4), indicating that processes other than an increase in the LIP were responsible for the HCMV-induced transformation of mitochondrial properties. However, the appearance of abundant grainlike mitochondria (Fig. 4C) suggests that mitochondrial biogenesis, in addition to HCMV-induced mitochondrial fragmentation (38), may be stimulated as a result of HCMV infection. This interpretation is supported by the report that HCMV infection stimulates mitochondrial DNA synthesis (27). Furthermore, our preliminary studies indicate that total mitochondrial mass increased approximately twofold (17), and therefore the incorporation of labile Fe into Fe proteins may explain some of the decline in the LIP at 72 hpi (Fig. 2). In this scenario, Fe chelation may impede the maturation of mitochondrial Fe proteins during mitochondrial biogenesis and thereby suppress cytomegaly and impair normal cell function.
The SIH-induced vacuolation of a subset of HCMV-infected cells, but not in mock-infected cells, indicates that other important cellular processes have also been disrupted. Fe chelators are known to have multiple effects on cellular function (31), and therefore some of these other Fe-dependent processes may also be necessary for the progression of cytomegaly.
Labile iron pool and transient HCMV-induced host cellular dysfunctions. The initial increase in the LIP may be a pathological response to viral entry into the cell rather than being part of the HCMV growth factor-like response. Support for this premise is the inability of the IRP/IRE system to prevent the initial rise in the LIP. Furthermore, excess labile Fe, and probably enhanced free radical production, may cause some of the early host cell dysfunctions. Among the early host cell perturbations are the temporary inhibition of Na+-K+-ATPase (41), cell rounding, and decrease in size (2), as well as a prolonged elevation in intracellular Ca2+ concentration (40), inhibition of Na+-K+-Cl cotransporter (37), and a 2030% reduction in cell number (37). Similar cellular dysfunctions are observed in other pathological conditions associated with excess Fe, especially when combined with acidic intracellular pH (9, 56). Although the increase in the LIP could be a consequence of these cellular dysfunctions, excess Fe is nevertheless expected to exacerbate them.
Virally induced cellular perturbations, including those induced or exacerbated by an elevated LIP, could lead to the premature loss of host cell viability. To counteract cellular dysfunctions, HCMV has evolved a number of antiapoptotic mechanisms to ensure host cell survival during its replication cycle. These antiapoptotic processes may include the upregulation of ion transporters (see above) to minimize disturbances in cellular ionic homeostasis evoked by the initial viral entry. Furthermore, we have shown that HCMV-infected cells preserve host cell ATP content and may use labile Fe for mitochondria biogenesis, whereas certain other viral infections, such as herpes simplex virus and rotavirus, result in marked decline in ATP content and mitochondrial dysfunctions (19, 39).
In conclusion, the HCMV-induced increase in the LIP and the inhibition of cytomegaly by Fe chelators are further evidence that Fe plays an important role in productive HCMV infection. Thus further elucidation of the mechanisms by which HCMV alters Fe homeostasis and mitochondrial properties may suggest new targets for combating this virus.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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