The biophysical function of pulmonary surfactant.

The type II pneumocytes of the lungs secrete a mixture of lipids and proteins that together acts as a surfactant. The material forms a thin film on the surface of the liquid layer that lines the alveolar air sacks. When compressed by the decreasing alveolar surface area during exhalation, the films reduce surface tension to exceptionally low levels. Pulmonary surfactant is essential for preserving the integrity of the barrier between alveolar air and capillary blood during normal breathing. This review focuses on the major biophysical processes by which endogenous pulmonary surfactant achieves its function and the mechanisms involved in those processes. Vesicles of pulmonary surfactant adsorb rapidly from the alveolar liquid to form the interfacial film. Interfacial insertion, which requires the hydrophobic surfactant protein SP-B, proceeds by a process analogous to the fusion of two vesicles. When compressed, the adsorbed film desorbs slowly. Constituents remain at the surface at high interfacial concentrations that reduce surface tensions well below equilibrium levels. We review the models proposed to explain how pulmonary surfactant achieves both the rapid adsorption and slow desorption characteristic of a functional film.

Effects of cholesterol on the structure and collapse of DPPC monolayers.

Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, Xchol, exceeded 0.20. Structural measurements of grazing incidence X-ray diffraction at ambient laboratory temperatures and a surface pressure of 44 mN/m, just below the onset of collapse, showed that the major structural change in an ordered phase occurred at lower Xchol. A centered rectangular unit cell with tilted chains converted to an untilted hexagonal structure over the range of Xchol = 0.0-0.1. For Xchol = 0.1-0.4, the ordered structure was nearly invariant; the hexagonal unit cell persisted, and the spacing of the chains was essentially unchanged. That invariance strongly suggests that above Xchol = 0.1, cholesterol partitions into a disordered phase, which coexists with the ordered domains. The phase rule requires that for a binary film with coexisting phases, the stoichiometries of the ordered and disordered regions must remain constant. Added cholesterol must increase the area of the disordered phase at the expense of the ordered regions. X-ray scattering from dipalmitoyl phosphatidylcholine/cholesterol fit with that prediction. The data also show a progressive decrease in the size of crystalline domains. Our results suggest that cholesterol promotes adsorption not by altering the unit cell of the ordered phase but by decreasing both its total area and the size of individual crystallites.

Changes in membrane elasticity caused by the hydrophobic surfactant proteins correlate poorly with adsorption of lipid vesicles.

To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used X-ray diffuse scattering (XDS) to determine an order parameter of the lipid chains (Sxray) and the bending modulus of the lipid bilayers (KC). Samples contained different amounts of the proteins with two sets of lipids. Dioleoylphosphatidylcholine (DOPC) provided a simple, well characterized model system. The nonpolar and phospholipids (N&PL) from extracted calf surfactant provided the biological mix of lipids. For both systems, the proteins produced changes in Sxray that correlated well with KC. The dose-response to the proteins, however, differed. Small amounts of protein generated large decreases in Sxray and KC for DOPC that progressed monotonically. The changes for the surfactant lipids were erratic. Our studies then tested whether the proteins produced correlated effects on adsorption. Experiments measured the initial fall in surface tension during adsorption to a constant surface area, and then expansion of the interface during adsorption at a constant surface tension of 40 mN m-1. The proteins produced a sigmoidal increase in the rate of adsorption at 40 mN m-1 for both lipids. The results correlated poorly with the changes in Sxray and KC in both cases. Disordering of the lipid chains produced by the proteins, and the softening of the bilayers, fail to explain how the proteins promote adsorption of lipid vesicles.

The Anionic Phospholipids of Bovine Pulmonary Surfactant.

The only known compositional change in the phospholipids (PL) of pulmonary surfactant in response to a physiologic stimulus occurs around the time of birth. In most species, the predominant anionic PL changes from phosphatidylinositol (PtdIns) to phosphatidylglycerol (PtdGro). Because prior studies have shown that the change in the headgroup itself is functionally insignificant, we tested the hypothesis that the PtdIns and PtdGro contain different diacyl pairs. Experiments used electrospray-ionization mass spectrometry to determine the molecular species in PtdIns, PtdGro, and phosphatidylcholine (PtdCho) in surfactant from newborn calves and cows. The profiles for the two anionic PL were distinct. The PtdIns contained long, unsaturated fatty acid chains and no disaturated species. The PtdGro more closely resembled the profile from PtdCho. For each headgroup, the molecular species for calf and cow were similar. The differences between the two anionic PL indicate that the switch from PtdIns to PtdGro during maturation involves more than simple substitution of the headgroup, and suggest that the functional significance of the shift may reflect the different pool of diacyl pairs.

Suppression of Lα/Lß Phase Coexistence in the Lipids of Pulmonary Surfactant.

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting Lα and Lß phases. In the Lß structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the Lß composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The Lß chains, however, lack the tilt of the Lß' phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the Lß phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.

Structural Changes in Films of Pulmonary Surfactant Induced by Surfactant Vesicles.

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactant in situ reduce surface tension to levels at which surfactant monolayers collapse from the surface in vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes induced by the added vesicles. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and X-ray reflection showed that the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from enlargement by the additional material of the ordered phase.

Location of the Hydrophobic Surfactant Proteins, SP-B and SP-C, in Fluid-Phase Bilayers.

The hydrophobic surfactant proteins, SP-B and SP-C, promote rapid adsorption by the surfactant lipids to the surface of the liquid that lines the alveolar air sacks of the lungs. To gain insights into the mechanisms of their function, we used X-ray diffuse scattering (XDS) and molecular dynamics (MD) simulations to determine the location of SP-B and SP-C within phospholipid bilayers. Initial samples contained the surfactant lipids from extracted calf surfactant with increasing doses of the proteins. XDS located protein density near the phospholipid headgroup and in the hydrocarbon core, presumed to be SP-B and SP-C, respectively. Measurements on dioleoylphosphatidylcholine (DOPC) with the proteins produced similar results. MD simulations of the proteins with DOPC provided molecular detail and allowed direct comparison of the experimental and simulated results. Simulations used conformations of SP-B based on other members of the saposin-like family, which form either open or closed V-shaped structures. For SP-C, the amino acid sequence suggests a partial α-helix. Simulations fit best with measurements of XDS for closed SP-B, which occurred at the membrane surface, and SP-C oriented along the hydrophobic interior. Our results provide the most definitive evidence yet concerning the location and orientation of the hydrophobic surfactant proteins.

The Lγ Phase of Pulmonary Surfactant.

To determine how different components affect the structure of pulmonary surfactant, we measured X-ray scattering by samples derived from calf surfactant. The surfactant phospholipids demonstrated the essential characteristics of the Lγ phase: a unit cell with a lattice constant appropriate for two bilayers, and crystalline chains detected by wide-angle X-ray scattering (WAXS). The electron density profile, obtained from scattering by oriented films at different relative humidities (70-97%), showed that the two bilayers, arranged as mirror images, each contain two distinct leaflets with different thicknesses and profiles. The detailed structures suggest one ordered leaflet that would contain crystalline chains and one disordered monolayer likely to contain the anionic compounds, which constitute ∼10% of the surfactant phospholipids. The spacing and temperature dependence detected by WAXS fit with an ordered leaflet composed of dipalmitoyl phosphatidylcholine. Physiological levels of cholesterol had no effect on this structure. Removing the anionic phospholipids prevented formation of the Lγ phase. The cationic surfactant proteins inhibited Lγ structures, but at levels unlikely related to charge. Because the Lγ phase, if arranged properly, could produce a self-assembled ordered interfacial monolayer, the structure could have important functional consequences. Physiological levels of the proteins, however, inhibit formation of the Lγ structures at high relative humidities, making their physiological significance uncertain.

The Equilibrium Spreading Tension of Pulmonary Surfactant.

Monomolecular films at an air/water interface coexist at the equilibrium spreading tension (γ(e)) with the bulk phase from which they form. For individual phospholipids, γ(e) is single-valued, and separates conditions at which hydrated vesicles adsorb from tensions at which overcompressed monolayers collapse. With pulmonary surfactant, isotherms show that monolayers compressed on the surface of bubbles coexist with the three-dimensional collapsed phase over a range of surface tensions. γ(e) therefore represents a range rather than a single value of surface tension. Between the upper and lower ends of this range, rates of collapse for spread and adsorbed films decrease substantially. Changes during adsorption across this narrow region of coexistence between the two- and three-dimensional structures at least partially explain how alveolar films of pulmonary surfactant become resistant to collapse.

Hydrophobic surfactant proteins strongly induce negative curvature.

The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote adsorption by inducing lipids to adopt a more negative curvature, closer to the configuration of the hypothetical intermediate. Our experiments used x-ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), the proteins produced a dose-related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With an increasing mol fraction of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w/w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Analysis of the results in terms of a Langmuir model for binding to a surface suggests that the effect of the lipids is consistent with a change in the maximum binding capacity. Our findings show that surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism.

An anionic phospholipid enables the hydrophobic surfactant proteins to alter spontaneous curvature.

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.

Aligning pitch for measurements of the shape of captive bubbles.

The first step required for the determination of surface tension from the shape of a captive bubble is the correct alignment of both the solid support against which the bubble floats and the camera used to record its profile. The solid support should be perpendicular to the gravitationally vertical axis. The camera used to visualize the bubble must be aligned to its axis of symmetry. Alignment of roll for both the camera and solid support is straightforward. For well-collimated light, yaw is unimportant. We show here how to align pitch, first adjusting the camera relative to the gravitational vertical, and then adjusting an agarose dome used as a ceiling above captive bubbles within the visual frame of reference.

Differential effects of the hydrophobic surfactant proteins on the formation of inverse bicontinuous cubic phases.

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. Previous studies have shown that SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce faster adsorption and formation of the bicontinuous structures by similar mechanisms, then they should also have different abilities to form the cubic phases. To test this hypothesis, we measured small-angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the dose-related ability of the combined proteins to induce the cubic phases at temperatures more than 25 °C below the point at which POPE alone forms the curved inverse-hexagonal phase. With SP-C, diffraction from cubic structures was either absent or present at very low intensities only with larger amounts of protein. The correlation between the structural effects of inducing curved structures and the functional effects on the rate of adsorption fits with the model in which SP-B promotes adsorption by facilitating formation of an inversely curved, rate-limiting structure.

Optical factors in the rapid analysis of captive bubbles.

Bubbles and droplets offer multiple advantages over Langmuir troughs for compressing interfacial films. Experiments, however, that manipulate films to maintain constant surface tension (γ) present problems because they require feedback. Measurements of bubbles and droplets calculate γ from the shape of the interface, and calculations in real time based on finding the Laplacian shape that best fits the interface can be difficult. Faster methods obtain γ from only the height and diameter, but the bubbles and droplets rest against a solid support, which obscures one section of the interface and complicates measurements of the height. The experiments here investigated a series of optical variables that affect the visualized location of the different surfaces for captive bubbles. The pitch of the support and camera as well as the collimation of illuminating light affected the accuracy of the measured dimensions. The wavelength of illumination altered the opacity of turbid subphases and hydrated gel used to form the solid support. The width of all visualized edges depended on the spectral width and collimation of the illuminating light. The intensity of illumination had little effect on the images as long as the grayscale remained within the dynamic range of the camera. With optimization of these optical factors, the width of all edges narrowed significantly. The surfaces away from the solid support approached the infinite sharpness of the physical interface. With these changes, the grayscale at the upper interface provided the basis for locating all surfaces, which improved real-time measurements based on the height and diameter.

The accelerated late adsorption of pulmonary surfactant.

Adsorption of pulmonary surfactant to an air-water interface lowers surface tension (γ) at rates that initially decrease progressively, but which then accelerate close to the equilibrium γ. The studies here tested a series of hypotheses concerning mechanisms that might cause the late accelerated drop in γ. Experiments used captive bubbles and a Wilhelmy plate to measure γ during adsorption of vesicles containing constituents from extracted calf surfactant. The faster fall in γ reflects faster adsorption rather than any feature of the equation of state that relates γ to surface concentration (Γ). Adsorption accelerates when γ reaches a critical value rather than after an interval required to reach that γ. The hydrophobic surfactant proteins (SPs) represent key constituents, both for reaching the γ at which the acceleration occurs and for producing the acceleration itself. The γ at which rates of adsorption increase, however, is unaffected by the Γ of protein in the films. In the absence of the proteins, a phosphatidylethanolamine, which, like the SPs, induces fusion of the vesicles with the interfacial film, also causes adsorption to accelerate. Our results suggest that the late acceleration is characteristic of adsorption by fusion of vesicles with the nascent film, which proceeds more favorably when the Γ of the lipids exceeds a critical value.

Hydrophobic surfactant proteins induce a phosphatidylethanolamine to form cubic phases.

The hydrophobic surfactant proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface. Previous evidence suggests that they achieve this effect by facilitating the formation of a rate-limiting negatively curved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipid leaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by 1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transition at 71 degrees C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57 degrees C, the proteins induce formation of bicontinuous inverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidence of other structures above 0.1% and 62 degrees C, but no change in the lattice-constants of the lamellar or cubic phases. The induction of the bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypothetical stalk-intermediate, supports the proposed model of how the surfactant proteins promote adsorption.

Recent advances in alveolar biology: some new looks at the alveolar interface.

This article examines the manner in which some new methodologies and novel concepts have contributed to our understanding of how pulmonary surfactant reduces alveolar surface tension. Investigations utilizing small angle X-ray diffraction, inverted interface fluorescence microscopy, time of flight-secondary ion mass spectroscopy, atomic force microscopy, two-photon fluorescence microscopy and electrospray mass spectroscopy are highlighted and a new model of ventilation-induced acute lung injury described. This contribution attempts to emphasize how these new approaches have resulted in a fuller appreciation of events presumably occurring at the alveolar interface.

The biophysical function of pulmonary surfactant.

Pulmonary surfactant lowers surface tension in the lungs. Physiological studies indicate two key aspects of this function: that the surfactant film forms rapidly; and that when compressed by the shrinking alveolar area during exhalation, the film reduces surface tension to very low values. These observations suggest that surfactant vesicles adsorb quickly, and that during compression, the adsorbed film resists the tendency to collapse from the interface to form a 3D bulk phase. Available evidence suggests that adsorption occurs by way of a rate-limiting structure that bridges the gap between the vesicle and the interface, and that the adsorbed film avoids collapse by undergoing a process of solidification. Current models, although incomplete, suggest mechanisms that would partially explain both rapid adsorption and resistance to collapse as well as how different constituents of pulmonary surfactant might affect its behavior.

Effects of hydrophobic surfactant proteins on collapse of pulmonary surfactant monolayers.

To determine if hydrophobic surfactant proteins affect the stability of pulmonary surfactant monolayers at an air/water interface, the studies reported here compared the kinetics of collapse for the complete set of lipids in calf surfactant with and without the proteins. Monomolecular films spread at the surface of captive bubbles were compressed at 37 degrees C to surface pressures above 46 mN/m, at which collapse first occurred. The rate of area-compression required to maintain a constant surface pressure was measured to directly determine the rate of collapse. For films with and without the proteins, higher surface pressures initially produced faster collapse, but the rates then reached a maximum and decreased to values <0.04 min(-1) above 53 mN/m. The maximum rate for the lipids with the proteins (1.22 +/- 0.28 min(-1)) was almost twice the value for the lipids alone (0.71 +/- 0.15 min(-1)). Because small increments in surface pressure produced large shifts in the rate close to the fastest collapse, compressions at a series of constant speeds also established the threshold rate required to achieve high surface pressure as an indirect indication of the fastest collapse. Both samples produced a sharply defined threshold that occurred at slightly faster compression with the proteins present, supporting the conclusion of the direct measurements that the proteins produce a faster maximum rate of collapse. Our results indicate that at 47-53 mN/m, the hydrophobic surfactant proteins destabilize the compressed monolayers and tend to limit access to the higher surface pressures at which the lipid films become metastable.

The anatomy, physics, and physiology of gas exchange surfaces: is there a universal function for pulmonary surfactant in animal respiratory structures?

(Orgeig and Daniels) This surfactant symposium reflects the integrative and multidisciplinary aims of the 1st ICRB, by encompassing in vitro and in vivo research, studies of vertebrates and invertebrates, and research across multiple disciplines. We explore the physical and structural challenges that face gas exchange surfaces in vertebrates and insects, by focusing on the role of the surfactant system. Pulmonary surfactant is a complex mixture of lipids and proteins that lines the air-liquid interface of the lungs of all air-breathing vertebrates, where it functions to vary surface tension with changing lung volume. We begin with a discussion of the extraordinary conservation of the blood-gas barrier among vertebrate respiratory organs, which has evolved to be extremely thin, thereby maximizing gas exchange, but simultaneously strong enough to withstand significant distension forces. The principal components of pulmonary surfactant are highly conserved, with a mixed phospholipid and neutral lipid interfacial film that is established, maintained and dynamically regulated by surfactant proteins (SP). A wide variation in the concentrations of individual components exists, however, and highlights lipidomic as well as proteomic adaptations to different physiological needs. As SP-B deficiency in mammals is lethal, oxidative stress to SP-B is detrimental to the biophysical function of pulmonary surfactant and SP-B is evolutionarily conserved across the vertebrates. It is likely that SP-B was essential for the evolutionary origin of pulmonary surfactant. We discuss three specific issues of the surfactant system to illustrate the diversity of function in animal respiratory structures. (1) Temperature: In vitro analyses of the behavior of different model surfactant films under dynamic conditions of surface tension and temperature suggest that, contrary to previous beliefs, the alveolar film may not have to be substantially enriched in the disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), but that similar properties of rate of film formation can be achieved with more fluid films. Using an in vivo model of temperature change, a mammal that enters torpor, we show that film structure and function varies between surfactants isolated from torpid and active animals. (2) Spheres versus tubes: Surfactant is essential for lung stabilization in vertebrates, but its function is not restricted to the spherical alveolus. Instead, surfactant is also important in narrow tubular respiratory structures such as the terminal airways of mammals and the air capillaries of birds. (3). Insect tracheoles: We investigate the structure and function of the insect tracheal system and ask whether pulmonary surfactant also has a role in stabilizing these minute tubules. Our theoretical analysis suggests that a surfactant system may be required, in order to cope with surface tension during processes, such as molting, when the tracheae collapse and fill with water. Hence, despite observations by Wigglesworth in the 1930s of fluid-filled tracheoles, the challenge persists into the 21st century to determine whether this fluid is associated with a pulmonary-type surfactant system. Finally, we summarize the current status of the field and provide ideas for future research.