Lung (1999) 177:127–138

© Springer-Verlag New York Inc. 1999

Influence of Phospholipid Composition on the Properties of Reconstituted Surfactants R. Dhand,1 J. Young,1 S. Krishnasamy,1,2 F. Possmayer,3 and N. J. Gross1,2 1

Division of Pulmonary and Critical Care Medicine and 2Cellular and Molecular Biochemistry, Edward Hines Jr. Veterans Affairs Hospital and Loyola University of Chicago Stritch School of Medicine, Hines, IL 60141, USA, and the 3Department of Obstetrics/Gynecology, University of Western Ontario, London, ON, NGA 5A5 Canada

Abstract. The influence of phospholipids on the ultrastructure and metabolism of reconstituted surfactants has not been well defined. The aim of this study was to determine if changes in the phospholipid composition of reconstituted surfactants altered their biophysical properties, ultrastructure, and conversion to light subtype by cycling. We prepared various surfactants containing radiolabeled dipalmitoylphosphatidylcholine ([14C]DPPC). The addition of phosphatidylglycerol (PG) or dipalmitoylphosphatidic acid (PA) to DPPC increased conversion to light subtype. In contrast, the addition of dipalmitoylphosphatidylglycerol (DPPG) to DPPC markedly reduced conversion to light subtype on cycling. DPPC and DPPC+PG produced large liposomes (∼1,000 nm), whereas DPPC+PA or DPPC+DPPG formed multilamellar membranes. Mixtures of DPPC and PA were highly surface active in vitro, whereas the surface activity of DPPC+DPPG was similar to that of DPPC. In conclusion, the ultrastructure, metabolism, and surface active properties of DPPC+PG mixtures were influenced markedly by alterations in the fatty acid composition or polar head group of PG. Key words: Surfactant artificial—Phosphatidylcholine—Phosphatidylglycerol— Phosphatidic acid—Cycling—Electron microscopy—Surface tension. Introduction The metabolism of exogenous surfactants within alveoli could be a major factor influencing their efficacy. Exogenous surfactants used for replacement therapy have varying composition [4]. The influence of surfactant apoproteins A, B, and C (SP-A, SP-B, and SP-C) on the surface active properties, ultrastructure, and metabolism of Offprint requests to: Rajiv Dhand

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surfactant [5, 25, 27, 29, 32–34] have been well described. Changes in the phospholipid composition influence the biophysical properties of surfactant [3, 30], but the effects of phospholipid composition on the ultrastructure and metabolism of surfactant have not been elucidated. Cyclic expansion and contraction of the surface of a suspension of nascent surfactant (cycling) recreates the events in the life cycle of surfactant [8, 9]. The phospholipid composition, protein/phospholipid ratio, ultrastructure, and surface tension of the material in heavy and light subtypes of natural surfactant generated by cycling correspond to the similar fractions of surfactant in alveolar lavage [8, 9, 27]. Moreover, alterations in surfactant metabolism in acute lung injury from a variety of causes correlate with the conversion of heavy to light subtype on cycling [16, 26, 31]. Artificial surfactants also convert to light subtype on cycling [5, 32]. Similarly, conversion to light subtype after intratracheal administration of artificial surfactant was greater in rabbits receiving mechanical ventilation with higher tidal volumes vs those receiving low tidal volumes [18]. Thus, a greater cyclic expansion and contraction of the alveolar surface with higher tidal volumes enhanced conversion of artificial surfactant to light subtype in vivo. These data suggest that light subtype generation on cycling artificial surfactants in vitro may reflect their metabolism in vivo. Phosphatidylglycerol (PG), an anionic phospholipid, constitutes about 10% of the phospholipids in surfactant. This unusually high percentage of PG is typical of mature surfactant [12]. Various investigators have shown that the properties of a reconstituted surfactant containing synthetic dipalmitoylphosphatidylcholine (DPPC) in combination with PG, SP-A, and SP-B mimic those of natural surfactant [5, 25, 30, 34]. Previously, we showed that conversion to light subtype on cycling DPPC+PG [5] was reduced significantly by the addition of surfactant apoproteins (SP-A 5% of phospholipid, w/w, and SP-B 0.02% of phospholipid, w/w). In the present study, we investigated the influence of changes in PG on the properties of DPPC. We were intrigued to find that replacing PG with dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (PA) altered the surface properties, ultrastructure, and cycling behavior of DPPC. Materials and Methods Isotopes were obtained from NEN Life Science Products (Boston, MA), reagents from Sigma (St. Louis, MO) or Fisher Chemicals (Itasca, IL), and phospholipids from Avanti Polar Lipids (Alabaster, AL). PG (phosphatidyl-DL-glycerol, sodium salt) was derived from egg lecithin. DPPC (1,2-dipalmitoyl-sn-glycero3-phosphocholine), DPPG (1,2 dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol), sodium salt], and PA (1,2-dipalmitoyl-sn-glycero-3-phosphate, monosodium salt) were synthetic products. All lipids were obtained in chloroform. SP-A and SP-B for these experiments were purified from the alveolar lavages of patients with alveolar proteinosis [2, 5, 11, 15].

Preparation of Surfactant The synthetic phospholipids were mixed in chloroform and incubated with 0.1 ␮Ci of [14C]DPPC/mg of the phospholipid mixture at 37°C for 60 min ([14C]DPPC, 80–120 mCi/mmol, Life Science Products). The organic solvents were evaporated at 43°C for 10 min, the residue was suspended in 3 ml of buffer (2%

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octylglucopyranoside [OGP] in 5 mM Tris, 75 mM NaCl, and 1 mM EDTA) at 57°C, and OGP was removed by dialysis against 5 mM Tris, pH 7.4, for 48 h at 4°C. Six different surfactants were prepared: DPPC, DPPC+ egg PG (7:3, w/w), DPPC+DPPG (7:3, w/w), DPPC+DPPG+PA (7:2:1, w/w), DPPC+DPPG+PA (7:1:2, w/w), and DPPC+PA (7:3, w/w). We also prepared surfactants containing DPPC+PG (7:3, w/w) or DPPC+DPPG (7:3, w/w) in combination with SP-B (0.02% of phospholipid, w/w) and SP-A (5% of phospholipid, w/w) as described earlier [5].

Cycling For cycling, approximately 100 ␮g of phospholipid was used in each tube and the volume made up to 2 ml with AF buffer (0.15 M NaCl, 5 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, pH 7.4). The purpose of these experiments was to determine the proportion of heavy surfactant which converted to light subtype under various cycling conditions. A typical cycling experiment consisted of an uncycled tube, to determine the buoyant density of the starting material, and five other tubes cycled for 4 h at 37°C [8, 9]. After cycling, the cycling mix was centrifuged to equilibrium in continuous sucrose gradients (0.1–0.9 M) at 190,000 × g in an SW 55 rotor at 8°C for 20 h. The bottom of the cycling tube was pierced and the gradient dripped out in 0.4-ml fractions. The density of alternate fractions was estimated from the refractive index. Scintillation fluid was added to each fraction, and radioactivity determined in a Beckman LS 5801 liquid scintillation counter [10]. In a few confirmatory experiments, we measured inorganic phosphorus in the phospholipids extracted from each fraction [1, 6] to verify that the distribution of radioactivity corresponded with the phospholipid concentration in various fractions.

Cycling with Convertase Partially purified convertase was obtained by subjecting mouse alveolar lavage to affinity chromatography with concanavalin A-Sepharose [5]. The cycling experiment was performed with approximately 100 ␮g of phospholipid in each tube. An uncycled tube and two tubes that were cycled without enzyme were used as controls. The remaining three tubes were cycled with approximately 30 ␮g of the partially purified protein/ cycling tube. The volume of the mixture in each tube was made up to 2 ml by adding AF buffer.

Surface Activity The surface activity of the surfactants was measured at 37°C and 100% humidity in a modified all-Teflon Wilhelmy balance with a movable Teflon barrier (Kimray Inc., Oklahoma City, OK). The balance was calibrated on each occasion, and aliquots containing approximately 250 ␮g of each surfactant were applied below the surface of 50 ml of cleaned subphase buffer (AL buffer). The subphase was stirred continuously at maximal surface area for 10 min. Compression of the surface from 100 to 15% of the maximum area was carried out six times, with a cycle time of 3 min. Surface tension was recorded continuously by using a platinum flag connected through a simple force transducer to a recording pen. Maximum and minimum surface tensions from the first and sixth cycle were analyzed. Three experiments were performed with each surfactant.

Electron Microscopy Phospholipid mixtures were processed for electron microscopy according to Williams et al. [34]. Each surfactant (1–2 ml) was fixed in suspension in 2.5% glutaraldehyde, 1% tannic acid in 0.1 M sodium cacodylate buffer, pH 7.4, for 1 h at room temperature. The surfactants were pelleted (10,000 × g), stored overnight in fixative at 4°C, washed twice in 0.1 M sodium cacodylate buffer, pH 7.4, at 4°C, postfixed in 1% osmium tetroxide in the same buffer for 1 h at 4°C, dehydrated in acetone, and embedded in 812 resin

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(Electron Microscopy Sciences). Thin sections (70–80 nm) stained with uranyl acetate and lead citrate were examined and photographed with a Hitachi H600 transmission electron microscope.

Statistical Analysis Phospholipid peaks generated by cycling were analyzed with computerized software (PeakFit, Jandel Scientific, San Rafael, CA). The buoyant densities and percentage area of each radiolabeled peak were determined and the mean ± S.D. calculated [8, 9]. Because the light subtype has a buoyant density below 1.04 g/ml, we compared the proportion of radioactivity in the gradient below this value. Differences in light subtype generation after cycling with different types of reconstituted surfactants were determined by Student’s t-test. The hysteresis in the surface area–surface tension loop was determined by cutting out the loops and weighing the paper in a chemical balance. The weight of the loop during the sixth compression was expressed as a percentage of the weight of the first loop. Statistical significance was accepted with p < 0.05.

Results Cycling with Phospholipid Mixtures The phospholipid composition of surfactant had a marked influence on light subtype generation. Representative profiles of the uncycled and cycled surfactants are shown in Figure 1. The uncycled phospholipid mixtures had a heavy buoyant density (∼1.06; range of peak buoyant density 1.059–1.065 g/ml). About one third of DPPC converted to light subtype on cycling for 4 h (艋1.04 g/ml) (Table 1). The addition of PG to DPPC increased conversion (p < 0.05 compared with DPPC), whereas the addition of DPPG to DPPC markedly reduced conversion to light subtype (p < 0.001 compared with DPPC or DPPC+PG) (Table 1). Light subtype generation on cycling mixtures of DPPC+PA was greater than that observed with DPPC alone (p < 0.05) but comparable to that observed with DPPC+PG (p > 0.05) (Table 1). Similarly, the addition of PA to mixtures of DPPC+DPPG increase light subtype generation (Fig. 1 and Table 1). Cycling with Convertase We investigated the effect of adding SP-A and SP-B to the cycling properties of DPPC+PG and DPPC+DPPG. Light subtype generation on cycling DPPC+PG+SPA+SP-B was lower than that with DPPC+PG (Tables 1 and 2) but was increased in the presence of convertase in the cycling mixture (p < 0.001 compared with cycling without convertase). Minimal light subtype was formed after cycling DPPC+DPPG+SP-A+SP-B. Although light subtype generation increased on cycling DPPC+DPPG+SP-A+SP-B with convertase (p < 0.01 compared with cycling without convertase) (Table 2), the amount of light subtype formed was significantly lower than that observed on cycling DPPC+PG+SP-A+SP-B with convertase (p < 0.001). Surface Activity The plots of surface area–surface tension loops for the first and sixth compression are shown in Figure 2. By the sixth compression the shapes of the surface area–surface

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Fig. 1. Profiles of surfactants radiolabeled with [14C]DPPC. Each point represents radioactivity in a fraction as a percentage of total gradient activity plotted against buoyant density. Best-fit computer estimates of subtype distributions are shown. Profiles of uncycled DPPC and various cycled surfactants are shown. All uncycled surfactants were of heavy buoyant density (peak buoyant density 1.059–1.065 g/ml). DPPC+DPPG showed negligible conversion, whereas the addition of PA to DPPC increased conversion to light subtype. The generation of light subtype observed after cycling DPPC+PG, DPPC+DPPG+PA (7:1:2 w/w), and DPPC+PA was significantly greater (p < 0.001 for each) than that observed on cycling DPPC, DPPC+DPPG, or DPPC+DPPG+PA (7:2:1 w/w).

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Table 1. Light subtype generation on cycling various surfactants. Results are the mean ± S.D. of six cycling experiments. Phospholipids

% Light subtype generateda

DPPC DPPC+PG DPPC+DPPG DPPC+DPPG+PA (7:2:1) DPPC+DPPG+PA (7:1:2) DPPC+PA

33.0 ± 2.6* 54.1 ± 2.9 5.8 ± 1.2* 13.1 ± 2.6* 56.7 ± 4.8 59.3 ± 3.5

a

Percent of total radioactivity in the gradient below a buoyant density of 1.040 g/ml *p < 0.001 compared with DPPC+PG, DPPC+PA, or DPPC+DPPG+PA (7:1:2).

Table 2. Light subtype generation on cycling reconstituted surfactants containing phospholipids, SP-A, and SP-B. Results are the mean ± S.E. of 8–12 cycling experiments. Surfactant and cycling DPPC+PG+SP-A+SP-B Cycled Cycled with convertase DPPC+DPPG+SP-A+SP-B Cycled Cycled with convertase a

% Light subtype generateda

11.6 ± 3.4 49.5 ± 6.3 9.5 ± 2.4 16.2 ± 4.7

Percent of total radioactivity in the gradient below a buoyant density of 1.040 g/ml.

tension loops were reproducible. Hysteresis in various surfactants ranged between 39 and 112 arbitrary units on the first compression. Hysteresis during the first compression was greater compared with the sixth compression, and this difference was particularly notable with DPPC or DPPC+PA, whereas it was minimal with DPPC+PG+SP-A+SPB. Reproducibility of the hystereses (ratio of sixth vs first compression) ranged from 28% with DPPC to 103.2% with DPPC+PG+SP-A+SP-B, with values for the other phospholipids ranging from 79.2 to 95.7%. Marked hysteresis in the surface area– surface tension loop was observed with DPPC+PA compared with the other mixtures (Figs. 2 and 3). DPPC did not reduce surface tension before compression; DPPC+PG, DPPC+DPPG, and DPPC+PG+SP-A+SP-B reduced it minimally; and DPPC+PA reduced surface tension to ∼35 mN/m before the first compression (Fig. 2). At minimum surface area (15% of the maximum) DPPC, DPPC+PG, and DPPC+DPPG achieved surface tensions between 10 and 20 mN/m, whereas DPPC+PA and DPPC+PG+SPA+SP-B reduced surface tension to <10 mN/m (Fig. 2). Electron Microscopy The phospholipid composition altered the ultrastructure of the various surfactants. Thus, DPPC or DPPC+PG consisted of large liposomes varying from 500 to 2,000 nm

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Fig. 2. The surface activities of DPPC, DPPC+PG, DPPC+DPPG, DPPC+PA, and DPPC+PG+SP-A+SP-B were determined in a modified Wilhelmy balance (250 ␮g of phospholipid (PL) of each; 5 ␮g/ml). The surface area–surface tension loop is plotted for the first (solid line) and sixth compression (dashed line). Marked hysteresis in the surface area–surface tension loop was observed with DPPC+PA compared with the other mixtures. Hysteresis during the first compression was greater compared with the sixth compression, and this difference was particularly notable with DPPC or DPPC+PA, whereas it was minimal with DPPC+PG+SP-A+SP-B. The surfactants showed differences in their ability to lower surface tension. DPPC did not reduce surface tension before compression; DPPC+PG, DPPC+DPPG, and DPPC+PG+SP-A+SP-B reduced it minimally; and DPPC+PA reduced surface tension to ∼35 mN/m before the first compression. At minimum surface area (15% of the maximum) DPPC, DPPC+PG, and DPPC+DPPG achieved surface tensions between 10 and 20 mN/m, whereas DPPC+PA and DPPC+PG+SP-A+SP-B reduced surface tension to 艋6 mN/m.

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Fig. 3. The surface activities of DPPC+DPPG+PA (7:1:2) and DPPC+DPPG+PA (7:2:1) were determined in a modified Wilhelmy balance (250 ␮g of phospholipid of each; 5 ␮g/ml). The surface area–surface tension loop is plotted for the first (solid line) and sixth compression (dashed line). Marked hysteresis in the surface area–surface tension loop was observed with both mixtures. Hysteresis during the sixth compression was lower (86.7% and 79.2% for DPPC+DPPG+PA (7:1:2) and DPPC+DPPG+PA (7:2:1), respectively) compared with the first compression. The surfactants showed differences in their ability to lower surface tension. DPPC+DPPG+PA (7:1:2) did not reduce surface tension before compression, whereas DPPC+DPPG+PA (7:1:2) reduced it to 59 mN/m. At minimum surface area (15% of the maximum) DPPC+DPPG+PA (7:1:2) and DPPC+DPPG+PA (7:2:1) achieved surface tensions of 11 and 7 mN/m, respectively.

in size (average 1,000 nm), whereas DPPC+DPPG formed sheets of membranes (Fig. 4). The formation of multilamellar vesicles was notable in preparations of DPPC+PA (Fig. 4). DPPC+PG+SP-A+SP-B consisted of large liposomes whose membranes showed multiple lamellae (Fig. 4), with few areas showing tubular structures in a lattice formation. Discussion The phospholipid composition influences the calorimetric behavior, phase structure, molecular shape, and function of lipid mixtures [3, 30]. To our knowledge, the present report is the first to show that changes in the fatty acid composition or the polar head group of PG influence the ultrastructure and metabolism of surfactant. Thus, the addition of DPPG or PA to DPPC altered the ultrastructure and cycling properties of DPPC, whereas the addition of PG did not produce a significant change (Figs. 1–4 and Table 1). DPPG did not influence the surface activity of DPPC, but it markedly reduced conversion of DPPC to the light subtype on cycling (Figs. 1–3 and Table 1). In contrast, PA enhanced the surface activity of DPPC and increased its conversion to the light subtype on cycling (Figs. 1–3 and Table 1). Previously, we showed that a mixture of DPPC+PG underwent a time-dependent conversion to light subtype on cycling at 37°C [5]. In the present study, we found that conversion of DPPC+PG (54.1 ± 2.9%) was greater than that of DPPC alone (33.0 ± 2.6%), whereas DPPC+DPPG showed minimal conversion (5.8 ± 1.2%) (Table 1). DPPC and DPPG contain saturated long chain fatty acids (palmitate) at both C-1 and

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Fig. 4. Ultrastructure of various surfactants before cycling. Liposomes of the various surfactants were prepared by the dialysis-detergent method. DPPC formed bilayered vesicles approximately 1,000 nm in size (A). Similar vesicles were observed after the addition of PG to DPPC, except that some areas showed formation of multilayered vesicles (B). Mixtures of DPPC and DPPG formed compactly arranged sheets of multilayered membranes (C). DPPC+PA produced sheets of multilayered membranes as well as large multilayered vesicles (D). The ultrastructure of DPPC+PG+SP-A (5% of phospholipids, w/w)+SP-B (0.02% of phospholipids, w/w) is shown for comparison (E). The surfactant reconstituted with SP-A and SP-B formed large multilayered vesicles with few areas showing the formation of a tubular myelin like lattice (magnification × 35,000; bar represents 1 ␮m).

C-2 positions, whereas PG contains other acyl side chains besides palmitic acid at these positions. Moreover, unsaturated fatty acids like oleic and linoleic acid constitute 40–50% of the fatty acids in egg PG. We do not fully understand the reasons for the difference in the behavior of DPPC+DPPG from that DPPC+PG, but the differences are so striking that they must have physiological relevance. Conversion of heavy to light subtype during cycling requires the intermediary formation of a surface monolayer, and material that is squeezed out during compression of the surface monolayer during cycling is believed to form the light subtype [8–10]. An increase in the fluidity of the surface monolayer by the addition of PG to DPPC may facilitate the exclusion of phospholipids during compression of the surface [14, 22]. We speculate that DPPC+DPPG mixtures contain tightly packed phospholipids [3, 4, 17] which resist squeeze-out during compression. Therefore, the tight packing of the rigid saturated fatty acid side chains of DPPC+DPPG may be responsible for the markedly decreased light subtype generation observed on cycling this mixture. Convertase is a carboxylesterase [19] that promotes light subtype generation on cycling natural surfactant [9, 10] or reconstituted surfactant containing DPPC+PG+SPA+SP-B [5]. The heavy subtype of surfactant is surface active, whereas the light subtype does not lower surface tension [8–10, 27]. Therefore, the reduction in light

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subtype generation on cycling DPPC+DPPG+SP-A+SP-B with convertase suggests that reconstituted surfactants containing DPPC+DPPG may be more resistant to inactivation than DPPC+PG mixtures. However, further studies in mechanically ventilated animals are needed to verify these findings. The PA employed in these experiments was similar to DPPG except that it lacked a glycerol head group. The addition of PA to DPPC almost doubled light subtype generation on cycling vs DPPC alone (Table 1). The addition of PA to DPPC+DPPG mixtures also increased light subtype formation (Fig. 1) in proportion to the content of PA in the mixture (Fig. 1 and Table 1). PA is an anionic phospholipid that facilitates rearrangement of lipid bilayers to a hexagonal II (HII) phase [3]. Such a rearrangement could facilitate lipid transfer to the air-water interface through unstable lipid intermediates [23]. Moreover, PA facilitates fusion of membranes and vesicles in the presence of calcium ions [21] and may increase light subtype generation by promoting the formation of stable small vesicles from phospholipids that are squeezed out during compression of the surface monolayer. The effect of PA to reduce surface tension before the first compression was probably caused by enhanced adsorption of DPPC to the surface monolayer (Fig. 2). The low amounts of surfactant used in these experiments may have influenced the ability of phospholipids to adsorb quickly (within 10 min) to the monolayer and to achieve very low surface tension during compression. Nevertheless, DPPC+PA lowered surface tension to <10 mN/m. The effect of adding PA to DPPC was comparable to that observed with the addition of SP-A and SP-B to mixtures of DPPC+PG (Fig. 2). Interestingly, the results with mixtures of DPPC+DPPG+PA were intermediate between those with DPPC+DPPG and DPPC+PA (Fig. 3). In general, the effects of changing the phospholipid composition on the surface properties of various surfactants were not as striking as the effects on their cycling properties and ultrastructure. Examination of the heavy subtype of natural surfactant under the electron microscope reveals multilamellar vesicles with some areas showing tubular myelin formation [8, 9, 27, 32], whereas after cycling the heavy subtype is composed mainly of sheets of membranes [27]. Reconstituted surfactants of heavy buoyant density are composed of large vesicles [5, 32]. We found that various phospholipid and phospholipid + protein mixtures differed in their ultrastructure (Fig. 4). Changes in the properties of the various lipid mixtures may be related to differences in their morphology, but the correlation between structure and function is unclear. The properties of artificial surfactants were influenced profoundly by subtle changes in the fatty acid and polar head groups of PG. The addition of PA to DPPC enhanced conversion, whereas a combination of DPPC with DPPG decreased conversion of heavy to light subtype compared with DPPC or DPPC+PG. Thus alterations in the phospholipid composition may help in modulating the metabolism of artificial surfactants used for replacement therapy. In addition, changes in the phospholipid composition of natural surfactant which occur in various physiological and pathological states [20] have been considered as markers of surfactant immaturity [28] or disease [7, 13, 24]. Whether the changes in surfactant phospholipids alter the metabolism of surfactant and are responsible for changes in the pool size, rate of turnover, and function of surfactant needs further investigation.

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Acknowledgments. We thank Paresh Savani, Syed Irfan, and Linda Fox for assistance with the experiments. This work was supported by an RAG and merit review grant from the Department of Veteran’s Affairs (to R.D. and N.J.G., respectively) and by a grant from the Chicago Association for Research and Education in Science (to R.D.).

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