1Department of Physiology II, Nara Medical University, Kashihara, Nara 634-8521; Departments of 2Physiology I and 3Surgery II, Nagoya University Graduate School of Medicine, Tsurumai, Nagoya 466-8550; and 4Department of Basic Human Sciences, School of Human Sciences, Waseda University, Tokorozawa, Saitama 359-1192, Japan
Submitted 15 September 2003 ; accepted in final form 10 February 2004
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ABSTRACT |
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intracellular calcium concentration oscillation; interstitial cells of Cajal; peristalsis
One of the characteristics of GI smooth muscle is the generation of rhythmic spontaneous contractions. Recent investigations have demonstrated that the ICC network in the musculature of the GI tract is responsible for the generation of electrical pacemaker activity and also controls the frequency and propagation characteristics of GI motility (6, 19, 25, 29). ICC comprise a cell population that is unique to the GI tract, and its pacemaker activity results in rhythmic oscillations of smooth muscle membrane potential, called slow waves (2, 23). Enteric neurons also innervate smooth muscle and are essential for peristalsis in GI motility (1). Thus ICC and/or enteric neurons could coordinate GI motility (8, 9, 14, 21, 22). ES cells have a pluripotent ability to differentiate into a wide range of cell types, and thus the types of ES clusters are heterogeneous. Characterization of comprehensive physiological and morphological properties of spontaneously differentiated ES clusters in the absence of exogenously added factors that could influence differentiation has not fully been performed.
The aim of the present study was to characterize physiological and morphological properties of the contracting ES gut on approximately day 21 of outgrowth culture. We performed morphological characterization by immunohistochemistry and electron microscopy and performed physiological characterization by analyses of spontaneous rhythmic contractions, intracellular Ca2+ movements, and electrical activities. Our results should provide the basis for developing appropriate models to study the origin of the rhythmicity in the mammalian GI tract (20, 25, 27).
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MATERIALS AND METHODS |
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Undifferentiated ES cells (EB3) were maintained on gelatin-coated dishes without feeder cells in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL, Grand Island, NY), 0.1 mM 2-mercaptoethanol (Sigma), 0.1 mM nonessential amino acids (GIBCO-BRL), 1 mM sodium pyruvate (Sigma), and 1,000 U/ml leukemia inhibitory factor (LIF; GIBCO-BRL). The EB3 cells (a kind gift from Dr. Hitoshi Niwa, Center for Developmental Biology, RIKEN, Kobe, Japan) carried the blasticidin S-resistant selection marker gene driven by the Oct-3/4 promoter (active under undifferentiated status) and were maintained in medium containing 10 µg/ml blasticidin S to eliminate differentiated cells (17). To induce embryoid body (EB) formation, we cultured dissociated ES cells in hanging drops (10, 18) with minor modifications. The cell density of one drop was 500 cells per 15 µl of ES cell medium in the absence of LIF. After 6 days in a hanging drop culture, the resulting EBs were plated onto plastic 100-mm gelatin-coated dishes and allowed to attach for the outgrowth culture (4, 17, 28). The culturing in hanging drops was the most important process for differentiating ES gut. Each contracting cluster underwent a dramatic transformation into a cystlike structure, with a cavity containing fluid and solids. On approximately day 14, these clusters proliferated to form more prominent three-dimensional structures with lumens and began rhythmic contractions that were not necessarily frequent or regular. On approximately day 21, the clusters (ES gut) showed coordinated contraction patterns with relatively regular rhythms, although there were some incomplete cystlike structures even on day 21.
Motility Analysis of Video Images
We monitored and recorded video images of ES guts with a microscope video recording system (Olympus IX-70 and Victor cassette recorder BR-S605B; Tokyo, Japan). We counted the number of spontaneous contractions during a 5-min period at least three times from the reproduced videotape. The temperature of the dish was kept at 35°C using a micro-warm plate system (U HP-100; Kitazato, Tokyo, Japan).
Immunohistochemistry
For Kit and connexin43 immunohistochemistry, the whole mount preparations of ES guts were fixed in acetone (4°C, 5 min). After fixation, preparations were washed for 30 min in PBS (0.1 M, pH 7.4). Nonspecific antibody binding was reduced by incubation for 2 h in 10% normal goat serum in PBS containing 0.3% (vol/vol) Triton-X 100 at room temperature. Tissues were incubated overnight at 4°C with a rat monoclonal antibody raised against c-Kit protein (ACK2, 5 µg/ml in PBS; eBioscience, San Diego, CA) and with a rabbit polyclonal antibody raised against mouse connexin43 (Cx43, gap junction 1 protein, 5 µg/ml in PBS; Chemicon International, Temecula, CA). Immunoreactivity for Kit was detected using Alexa Flour 488-conjugated secondary antibody (Alexa Flour 488 goat anti-rat; Molecular Probes, Eugene, OR) diluted 1:200 in PBS for 2 h in the dark at room temperature. Immunoreactivity for Cx43 was detected using Alexa Flour 546-conjugated secondary antibody (Alexa Flour 546 goat anti-rabbit; Molecular Probes) diluted 1:200 in PBS for 2 h in the dark at room temperature. Tissues were examined with a Bio-Rad MRC 600 (Hercules, CA) confocal microscope. Confocal micrographs are digital composites of Z-series scans of 1015 optical sections through a depth of 1020 µm. Final images were constructed with Comos software (Bio-Rad).
Electron Microscopy
Tissues were fixed with 4% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at room temperature. After being rinsed in the same buffer, tissues were postfixed in 1% osmium tetroxide for 0.5 h at 4°C. Tissues were rinsed in distilled water, block-stained with 3% uranyl acetate solution for 3 h, dehydrated in a graded series of ethyl alcohols, and embedded in Epon epoxy resin. Ultrathin sections were cut with a Reichert ultramicrotome, double-stained with 3% uranyl acetate and lead citrate, and observed with a JEM1200 EXII electron microscope (JEOL, Tokyo, Japan).
Ca2+ Imaging
The ES guts were incubated for 4 h at room temperature in a modified Krebs solution containing 10 µM fluo 3-acetoxymethyl ester (Dojindo, Kumamoto, Japan) and detergents [0.02% Pluronic F-127 (Dojindo) and 0.02% Cremophor EL (Sigma)]. A digital imaging system (Argus HiSCA; Hamamatsu Photonics, Shizuoka, Japan) combined with an inverted microscope was used to monitor oscillation of the intracellular Ca2+ concentration ([Ca2+]i). The ES guts were illuminated at 488 nm, and fluorescent emissions of 515565 nm were recorded at an intensity of fluo 3. Digital Ca2+ images (328 x 247 pixels) were normally collected at 300-ms intervals. The temporal fluorescence intensity of the dye (Ft) was normalized by the fluorescence intensity at the start (F0). These relative values represent integrated [Ca2+]i. In some ES guts, after the fluorescence intensity was recorded, the distribution of ICC was examined by staining with an anti-c-Kit antibody. During Ca2+ imaging, the temperature was kept at 30°C. Although [Ca2+]i oscillation is temperature dependent, [Ca2+]i oscillation could be observed at this temperature, but the motility in all ES guts was moderately depressed. This is advantageous to Ca2+ imaging, because high motility would disturb the detection of real [Ca2+]i signal changes.
Electrophysiological Studies
The culture dish was perfused with warmed (35°C) Tyrode solution bubbled with 100% oxygen gas at a constant flow rate of 2.0 ml/min. The temperature of the dish was kept at 35°C using the U HP-100 micro-warm-plate system. Conventional microelectrode techniques were used to record electrical responses of single cells from each ES cluster under an inverted microscope (Olympus IX-70). Glass capillary microelectrodes (1.0- to 1.2-mm outer diameter) filled with 3 M KCl had tip resistances ranging between 50 and 80 M
. The intracellular potentials thus recorded were displayed on a cathode ray oscilloscope (SS-7602; Iwatsu, Tokyo, Japan). The data were also acquired by a personal computer (Fujitsu, Tokyo, Japan) through an analog-to-digital converter (Axon Instruments, Foster City, CA) at 500 Hz, filtered at 100 Hz, and analyzed with AxoScope 7 (Axon Instruments, Foster City, CA). Maximal amplitude and rate of rise of electrical activities were calculated on the basis of digital data with Clampfit 8.1 in AxoScope 7.
Drugs
Nifedipine, tetrodotoxin (TTX), nickel ions (Ni2+), and ryanodine were purchased from Sigma (St. Louis, MO). 2-Aminoethoxydiphenyl borate (2-APB) was generously donated by Dr. K. Mikoshiba (University of Tokyo, Tokyo, Japan). Nifedipine, ryanodine, and 2-APB were dissolved in DMSO at concentrations of 510 mM. Other chemicals were dissolved in distilled water as a stock solution and diluted further with Tyrode or Krebs solution to the desired concentrations (ratios of dilution were >1:1,000).
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RESULTS |
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On day 21 of EB outgrowth culture, at 35°C, the mean frequency of spontaneous motility in ES guts was 13.5 ± 8.8 cycles per minute (cpm) (n = 45 ES guts). Motility was also evaluated at a lower temperature because pacemaker activity in ICC is temperature dependent (19, 24). At 24°C, the mean frequency was reduced to 1.0 ± 0.9 cpm (15.2 ± 13.9% of control; n = 14). The voltage-gated L-type Ca2+ channel blocker nifedipine (0.110 µM) decreased the mean frequency of motility to 0.8 ± 1.3 cpm (n = 20) (Fig. 1D). At a concentration of 10 µM, nifedipine abolished spontaneous motility in 8 of 11 ES guts, but motility persisted in the remaining 3 guts.
Immunohistochemistry
When immunohistochemistry was used as a means to identify and localize ICC in ES guts, c-Kit-immunoreactive cells were found to be abundant in these preparations. Figure 2A shows a hemispherical domelike cyst (ES gut on day 21 of EB culture) that exhibited a large number of c-Kit-positive (c-Kit+) cells on the wall of the domelike structure surrounding the lumen. c-Kit+ cells did not form a single layer but were scattered throughout the muscle layer (Fig. 2A). In general, most of the c-Kit+ cells were multipolar, and they formed a distinct and dense network (Fig. 2, A, b and B). The network structure of c-Kit+ cells was similar to that of ICC at the level of the myenteric plexus in the pylorus, small intestine, and colon of a murine embryo or neonate, as described previously (25). Immunoreactivity for Cx43, a useful marker for gap junction (15), was also identified on the wall of the domelike structure in another ES gut that had potent, rhythmic spontaneous contractions (Fig. 2C).
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As previously reported, the walls of the ES guts evaluated in this investigation were composed of three layers: epithelium, submucosa, and musculature (28). The musculature consisted primarily of smooth muscle cells. Although the musculature was not well organized, several smooth muscle cells were oriented in the same direction and the musculature was divided into two or more layers (Fig. 2D, a). Cells showing some ultrastructural features of ICC were observed within the musculature, as previously reported (28). These cells were characterized by electron-dense cytoplasm and the presence of many mitochondria (Fig. 2D, a and b), and the Golgi apparatus as well as smooth and rough endoplasmic reticulum (ER) were well detected in the perinuclear cytoplasm (Fig. 2D, b and c). Caveolae were also observed along the cell membrane (Fig. 2D, d). ICC frequently formed close contacts with the neighboring smooth muscle cells (Fig. 2D, c). Gap junctions between the very thin cytoplasmic processes of unidentified cells, probably ICC, were occasionally observed (Fig. 2D, c, inset). Enteric neurons were not detected in this moving ES gut.
Ca2+ Oscillation in ES Guts
In Ca2+ imaging studies, spontaneous waves of elevated [Ca2+]i were observed originating at the upper pole in the ES gut. Figure 3 shows an example of one such spontaneous [Ca2+]i oscillation, referred to as a global wave, originating at the upper area in the ES gut and propagating widely to the middle region of the ES gut (Fig. 3A, b and c). Many c-Kit+ cells aggregated in the upper pole of the body of this same preparation (Fig. 3A, a). The aggregates of c-Kit+ cells in the upper pole are thought to be ICC and may be responsible for initiation of the global wave. Figure 3A, dg, shows a series of pseudocolor ratio images, demonstrating the global wave from the upper pole to the middle area of this ES gut.
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This local wave was not synchronous with the global wave over the body in this ES gut. The frequency of the global wave recorded at point 1 was 1.5 cpm and was quite different from that (13 cpm) of the local wave recorded at point 2 (Fig. 3C, d). These results suggest that the global and local [Ca2+]i oscillations originate independently.
In another ES gut, the widely propagated [Ca2+]i oscillation (global wave) originated at the same site (Fig. 4B, b and i) and propagated over the body even after the treatment with 10 µM nifedipine (Fig. 4B, c and j), indicating that nifedipine does not affect the generation or propagation of this global wave. Additional treatment with 40 µM Ni2+ abolished this global wave, although 40 µM Ni2+ alone did not abolish it (data not shown).
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In three other ES guts, similar global waves were observed, although the frequency was variable. The mean frequency of the global waves of four ES guts was 11.8 ± 4.3 (718.5) cpm. In comparison, no significant difference was observed in the mean frequency of the global waves in the presence of nifedipine [11.3 ± 4.0 (518.5) cpm]. Taken together, these results suggest that nifedipine does not affect the generation or propagation of the global waves in domelike ES guts, although 210 µM nifedipine abolishes global waves in sheetlike ES clusters (n = 2) (data not shown).
Electrophysiological Studies
We investigated the electrical activity of the contracting ES clusters at various differentiation stages by using a conventional intracellular recording technique at 35°C, and we focused on ES guts with rhythmic contractions present on day 21 of EB outgrowth culture.
Various types of electrical activities. Electrical slow waves were frequently recorded in these preparations (Fig. 5A). The mean frequency and amplitude of these events were 8.0 ± 4.7 (118) cpm and 12.3 ± 6.0 (5.721.2) mV, respectively (n = 17). The shape of the electrical slow waves was very similar to that recorded in cultured ICC of mouse jejunum (27).
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In general, the regularity and noise level of the electrical waves in individual ICC and smooth muscle cells of the ES gut were not homogeneous (Fig. 5, AC). This might be the result of a pluripotent ability to develop into a wide range of cell types and a different degree of differentiation without exogenously added growth factor even on the same day 21 of EB outgrowth culture.
Effects of the L-type Ca2+ channel blocker nifedipine. Electrical slow wave is considered to be generated by pacemaker current from ICC and to be insensitive to L-type Ca2+ channel blockers (19, 20, 27). Pacemaking in ICC is thought to involve oscillations in [Ca2+]i involving ER and mitochondria (27). To test the sensitivity of ES gut slow waves to L-type Ca2+ channel blockers, we applied nifedipine (5 µM) to ES guts. The slow waves persisted in the presence of nifedipine (Fig. 5B). Similar effects were obtained in two different ES guts.
After treatment with nifedipine (15 µM), plateau potentials and transient potentials did not change significantly, though the amplitude of the plateau potentials slightly decreased (Fig. 5C). In another two ES guts, plateau potentials similar to this plateau potential in shape were recorded. Mean frequency and rate of rise were 4.8 cpm and 24.1 ± 7.1 mV/s (n = 11). These potentials were also unaffected by nifedipine (5 µM).
Resting membrane potentials including spontaneously active and inactive cells were 26.2 ± 6.5 (n = 12 ES guts in 12- to 17-day outgrowth culture), 35.1 ± 6.7 (n = 24 ES guts in 18- to 24-day outgrowth culture), and 37.2 ± 6.7 mV (n = 13 ES guts in 25-day outgrowth culture), indicating that the resting membrane potential becomes progressively more hyperpolarized with time in culture.
Additional Observations in Video Images and [Ca2+]i Oscillations
To assess whether intracellular Ca2+ release channels are involved in spontaneous contractions, we examined the effect of ryanodine. Ryanodine (0.110 µM) caused a concentration-dependent decrease in the mean frequency of periodical contractions in 18 ES guts, although the effects of ryanodine in individual ES guts were variable. Ryanodine (1 µM) significantly decreased mean frequency to 3.4 ± 1.7 cpm (70.2 ± 37.1% of control) in 10 ES guts (in 6 ES guts: 45.8 ± 16.9% of control, P < 0.05; in 4 ES guts: 106.8 ± 28.1% of control). Ryanodine (10 µM) further significantly decreased contraction frequency to 2.9 ± 1.7 cpm (64.3 ± 32.1% of control) in 5 ES guts (in 2 ES guts: 15.0 ± 1.7% of control, P < 0.05; in 3 ES guts: 84.5 ± 25.9% of control) (Fig. 5D).
In contrast, the inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-APB (110 µM) only slightly decreased the frequency from 6.0 ± 4.5 to 5.4 ± 3.6 cpm (89.8 ± 23.2% of control) in all 6 ES guts (Fig. 5E).
To assess whether intracellular Ca2+ release channels are involved in [Ca2+]i oscillation, we examined the effect of ryanodine. Final application of 10 µM ryanodine significantly decreased the frequency of [Ca2+]i oscillation in 13 ES guts, indicating the contribution of intracellular Ca2+ release channels to [Ca2+]i oscillation. TTX (0.11 µM), which inhibits neural activity via Na+ channel blockade, did not affect the frequency of spontaneous periodic contractions in 12 ES guts. Characteristics of each phenomenon in domelike ES guts are summarized in Table 1.
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DISCUSSION |
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ES guts have similar spontaneous rhythmic contractions, although they have a variety of structures. In the present study, we demonstrate for the first time that the voltage-dependent L-type Ca2+ channel contribution to these spontaneous contractions is similar to that previously described in the mature mouse gut. This finding indicates that functional voltage-dependent L-type Ca2+ channels are probably expressed in smooth muscle cells of the ES gut and that the muscle contraction is dependent on activation of L-type Ca2+ channels.
The most striking finding of the present study is that widely propagated Ca2+ oscillations (global waves) generated at an aggregate of many c-Kit+ cells are frequently observed in the ES gut even in the presence of nifedipine. This finding indicates that the network between ICC and smooth muscle cells is well differentiated in the ES gut. Electron microscopic analysis confirmed that ICC frequently formed close contacts with the neighboring smooth muscle cells and occasionally formed gap junctions between what were probably ICC. Furthermore, the presence of immunoreactivity for Cx43 (gap junction 1 protein) scattered on the wall in the body of the ES gut supports the possibility that the network between ICC and smooth muscles is well differentiated in the ES gut. Ca2+ oscillations that were generated from small clusters of c-Kit+ cells varied in frequency and did not propagate widely.
L-type Ca2+ channels are known to scarcely affect electrical slow waves recorded in the mouse small intestinal ICC (11) and smooth muscle cells (26). Furthermore, it has been confirmed (23) that a single cell in the mouse small intestine, identified as an ICC by light microscopy, electron microscopy, and expression of Kit mRNA, generates a rhythmic, inward current that is insensitive to L-type Ca2+ channel blockers. Plateau potentials and electrical slow waves evaluated in the present study were hardly affected by nifedipine, indicating that these events are generated by the pacemaker current in ICC and propagated electrotonically to smooth muscles, respectively.
Periodic activation of plasmalemmal ion channels of ICC to generate pacemaker current triggers intracellular Ca2+ release from the ER, although Ca2+ release is mediated through IP3 type 1 receptor in the ER and subsequent Ca2+ entry into mitochondria (20, 27). These processes are very similar to those in cultured cell clusters isolated from mouse intestine (24). Several candidates such as nonselective cation channels, including transient receptor potential channels (16, 24), Cl channels (7, 12), and/or Ca2+-activated K+ channels (3), have been reported (5, 6).
In the present study, 2-APB did not affect the frequency of spontaneous motility in ES guts, but ryanodine significantly decreased the frequency of both Ca2+ oscillations and spontaneous motility in ES guts. However, ryanodine did not affect the frequency of spontaneous motility in a subset of ES guts. These findings suggest that ryanodine receptors differentiate and contribute to intracellular Ca2+ movements in the majority of ES guts on 21-day outgrowth, which is different from previous findings in mouse stomach (20) and in cultured mouse myenteric ICC (27). These previous studies indicate that Ca2+ release from IP3 receptor-operated stores (but not ryanodine receptor-operated stores) is linked to initiation of pacemaker current (20, 27). Although intracellular Ca2+ movements certainly contribute to generate pacemaker current among sarcolemma, ER, and mitochondria in ICC of ES guts, what regulates the periodicity of the firing of pacemaker activity in ICC and what initiates and terminates the firing remain to be elucidated.
The present results reveal physiological and morphological characterization of ES guts spontaneously formed from mouse ES cells in the absence of exogenously added growth factors: pacemaker activity generated by intracellular Ca2+ movements in ICC propagates to the smooth muscle layer through gap junctions over the body and drives spontaneous rhythmic contractions of domelike ES guts (Table 1). Because ES cells have a pluripotent ability to develop into a wide range of cell types in the present experimental conditions (without exogenously added growth factors), various ES guts could have been differentiated showing heterogeneous physiological and morphological properties such as various structures (dome, twin dome, tubular cyst, or sheet), variable sensitivities to ryanodine, and various regularity and noise levels of electrical activities. For example, in domelike ES guts, nifedipine did not abolish, whereas additional Ni2+ did abolish, [Ca2+]i oscillations (Table 1); however, in sheetlike ES clusters, nifedipine did abolish [Ca2+]i oscillations.
We have revealed that Ca2+ influx via L-type Ca2+ channels contributes to smooth muscle contraction in the domelike ES guts. On the other hand, it is possible that either different types of Ca2+ channels or intracellular Ca2+ movements contribute to [Ca2+]i oscillations in ICC. [Ca2+]i oscillations in smooth muscle cells are driven indirectly by electrical slow waves of ICC. Electrical slow waves in ICC are not dependent on L-type Ca2+ channels but are dependent on intracellular Ca2+ movements, and they electrotonically propagate over the smooth muscle layers. Such ICC were well differentiated and were distributed densely throughout the small body of the domelike ES guts presently studied, suggesting that electrical slow waves in ICC could propagate over the body with less decline. Therefore, we believe that this ICC network generates the [Ca2+]i oscillations that persist in the presence of nifedipine. Furthermore, application of Ni2+ abolished [Ca2+]i oscillations in some domelike ES guts, indicating that T-type Ca2+ channels might have a role on the origination and/or propagation of [Ca2+]i oscillations of a subpopulation of the ES guts.
Finally, at 30°C for Ca2+ imaging, the motility in the ES guts (of smaller size) was moderately depressed, as mentioned in MATERIALS AND METHODS. At 35°C, the motility of the ES guts (of larger size) was normal. This difference of 5°C in temperature would seem unlikely to affect the contribution of L-type Ca2+ channels to [Ca2+]i oscillations if the temperature sensitivity in these ES guts is not higher than in the well-developed mouse gut.
In conclusion, the information obtained from the present study should provide the basis for future studies of the development of the elements of the gut that underlie GI motility. Improved technology (EB culture), combined with various exogenously added factors, can now be developed to make appropriate models from ES cells. This approach could facilitate significant advances in studies on gut organ physiological functions such as spontaneous rhythmicity.
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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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