Lipid-protein Mixture Research Articles

Vascular Endothelial Glycocalyx Damage and Potential Targeted Therapy in COVID-19.

COVID-19 is a highly infectious respiratory disease caused by a new coronavirus known as SARS-CoV-2. COVID-19 is characterized by progressive respiratory failure resulting from diffuse alveolar damage, inflammatory infiltrates, endotheliitis, and pulmonary and systemic coagulopathy forming obstructive microthrombi with multi-organ dysfunction, indicating that endothelial cells (ECs) play a central role in the pathogenesis of COVID-19. The glycocalyx is defined as a complex gel-like layer of glycosylated lipid–protein mixtures, which surrounds all living cells and acts as a buffer between the cell and the extracellular matrix. The endothelial glycocalyx layer (EGL) plays an important role in vascular homeostasis via regulating vascular permeability, cell adhesion, mechanosensing for hemodynamic shear stresses, and antithrombotic and anti-inflammatory functions. Here, we review the new findings that described EGL damage in ARDS, coagulopathy, and the multisystem inflammatory disease associated with COVID-19. Mechanistically, the inflammatory mediators, reactive oxygen species (ROS), matrix metalloproteases (MMPs), the glycocalyx fragments, and the viral proteins may contribute to endothelial glycocalyx damage in COVID-19. In addition, the potential therapeutic strategies targeting the EGL for the treatment of severe COVID-19 are summarized and discussed.

Cholesterol induced instabilities and shape transitions

Lung surfactant (LS) is a lipid-protein mixture that forms a monolayer that lines the alveolar air-liquid interface and acts to reduce the interfacial tension to a level necessary for breathing. The lack or inhibition of LS can be life threatening and can result in acute respiratory distress syndrome (ARDS) which has a 40% mortality rate. Due to the complexity of native LS, 2 or 3 component mixtures are studied to elucidate the relationship between composition, monolayer microstructure, and phase behavior and how these factors affect physiological function.

Mechanistic Insights in the Interaction of Chemicals with Surfactant Membrane Models in vitro

Every year, cases of respiratory distress are recorded in humans following exposure to chemicals. The underlying mechanisms of toxicity remain unclear. We have previously shown that pulmonary surfactant is the target of impregnation spray products, leading to lung damage in humans. Lung surfactant is a lipid-protein mixture whose main function is to reduce the surface tension in the alveoli at the end of expiration, minimizing the work of breathing. In this study, three chemicals (trimethoxyoctylsilane, 3-oxo-2-pentyl-cyclopentaneacetic acid and diisoamyl ether), classified as acute inhalation toxicants and of relevance for worker and consumer exposure, were studied with respect to their potential to perturb lung surfactant function. Two models were used: DPPC, the most abundant surface active lipid in surfactant, as well as native surfactant purified from porcine bronchoalveolar lavage. The constrained drop surfactometer and the captive bubble surfactometer were used to assess the effects of the chemicals on the biophysical activity of the two surfactant models under different exposure conditions. These two methodologies mimic the breathing dynamics of the lungs. In addition, the effect of the chemicals on the lung surfactant structure was explored by (i) Langmuir-Blodgett balance experiments coupled with epifluorescence microscopy, and (ii) cryogenic transmission electron microscopy. Lastly, the thermotropic behavior of surfactant after exposure to the chemicals was investigated using differential scanning calorimetry. The data provide evidences on the mechanism of toxicity of chemicals potentially harmful for the lungs. The identification of key event(s) for the acute inhalation toxicity pathway is crucial for the design of relevant toxicity assays in vitro, which will contribute to having safer chemicals in the workplace and on the market.

The Impact of Nonequilibrium Conditions in Lung Surfactant: Structure and Composition Gradients in Multilamellar Films.

The lipid–protein mixture that covers the lung alveoli, lung surfactant, ensures mechanical robustness and controls gas transport during breathing. Lung surfactant is located at an interface between water-rich tissue and humid, but not fully saturated, air. The resulting humidity difference places the lung surfactant film out of thermodynamic equilibrium, which triggers the buildup of a water gradient. Here, we present a millifluidic method to assemble multilamellar interfacial films from vesicular dispersions of a clinical lung surfactant extract used in replacement therapy. Using small-angle X-ray scattering, infrared, Raman, and optical microscopies, we show that the interfacial film consists of several coexisting lamellar phases displaying a substantial variation in water swelling. This complex phase behavior contrasts to observations made under equilibrium conditions. We demonstrate that this disparity stems from additional lipid and protein gradients originating from differences in their transport properties. Supplementing the extract with cholesterol, to levels similar to the endogenous lung surfactant, dispels this complexity. We observed a homogeneous multilayer structure consisting of a single lamellar phase exhibiting negligible variations in swelling in the water gradient. Our results demonstrate the necessity of considering nonequilibrium thermodynamic conditions to study the structure of lung surfactant multilayer films, which is not accessible in bulk or monolayer studies. Our reconstitution methodology also opens avenues for lung surfactant pharmaceuticals and the understanding of composition, structure, and property relationships at biological air–liquid interfaces.

Effect of Maternal Protein Restriction on the Surfactant System during Early Postnatal Life

IntroductionIntra‐uterine growth restriction (IUGR) is defined by a low birth weight and contributes to a variety of adult‐onset diseases with different severities between males and females. There is some evidence for structural and functional changes to the lung during the post‐natal period. However, the specific changes over time and the effects of IUGR on the pulmonary surfactant system are not fully elucidated. Pulmonary surfactant is critical for normal respiration, it is a lipid‐protein mixture which coats the inside of the lung and maintains the lungs' ability to expand easily during respiration. Therefore, in this study we investigated the effects of IUGR on the surfactant system and lung function during early postnatal life. It was hypothesized that IUGR leads to lung dysfunction through alterations of the pulmonary surfactant system.Materials and MethodsIUGR was induced in a pregnant rat model via a low protein diet. Briefly, pregnant rats were randomized to a control 20% or a low 8% protein diet. Rats at days 1, 7 and 21 were euthanized, and were either lavaged immediately (day 1) or were analyzed for lung compliance and elastance using a FlexiVent followed by lung lavage (day 7 and 21). Lavage was processed to obtain surfactant which was analyzed via phospholipid‐phosphorus measurements.ResultsWeights were significantly lower in IUGR offspring in all age groups compared to control offspring. Surfactant analysis demonstrated a significant decrease in the amount of phospholipid surfactant of IUGR female offspring compared to control female offspring at day 1 (p<0.01). Also, the surfactant phospholipid content at birth (day 1) for both control and IUGR males and females was significantly higher compared to animals at day 7 and 21 (p<0.01). The results showed that lung compliance in 21‐day female IUGRs (but not male IUGRs) was significantly lower compared to female controls (p<0.01), while elastance was significantly higher for this group compare to female controls (p<0.01). Day 7 male and female offspring showed no difference for compliance and elastance compare to the control groups.Discussion and ConclusionIUGR resulted in alterations in surfactant content at day 1 and lung compliance at day 21 in IUGR females compared to control females. This shows that IUGR alters the lung function, and the effect was different between males and females. No changes in the extracellular surfactant content in 21‐day female IUGRs suggests that the lower lung compliance is potentially the result of alterations happened to surfactant biophysical properties under IUGR. These potential alterations will be investigated by assessing the reducing surface tension ability of the surfactant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Intrauterine Growth Restriction (IUGR) on the Surfactant System in Response to Sepsis

IntroductionAcute respiratory distress syndrome (ARDS) is defined as severe lung dysfunction. The lung impairments in ARDS result, in part, from alterations to pulmonary surfactant; a lipid‐protein mixture coating the inside of the lung that increases the lung compliance during respiration. The most common cause of ARDS is sepsis and due to a lack of effective pharmacological therapies the mortality associated with this condition is over 30%. Our research focuses on risk factors that indicate a susceptibility to the disease, which could provide new and early therapeutic options. One such potential risk factor is intrauterine growth restriction (IUGR). IUGR is defined by a low birth weight and contributes to a variety of adult‐onset diseases with different severities between males and females. There are underlying shared mechanisms between these IUGR‐induced diseases and ARDS, such as systemic inflammation. This study investigates the IUGR effect on surfactant system in response to sepsis in adult males and females. It was hypothesized that IUGR is a risk factor for developing ARDS through surfactant alterations with different outcomes between adult males and females.MethodsIUGR was induced in pregnant rat model via a low protein diet. At postnatal day 130, control and IUGR rats were randomly assigned into sham and septic groups. Rats were anesthetized and received either an intraperitoneal injection (IP) of saline or fecal slurry solution (1m/100g at 0.5g/ml). Following injection, respiratory rate and the animal activity were monitored for 6hr. After 6hr, animals were sacrificed (sodium pentobarbital IP injection) and arterial blood and the surfactant were taken for cytokine and surfactant analysis. Some non‐lavaged lungs were fixed for histological assessments.ResultsIUGR was confirmed by significant decrease in birth weight in both male and female offspring. Septicemia was confirmed by culturing bacteria from the blood samples, while cultures from sham animals were negative. After 6hr, respiratory rates significantly decreased in septic male and female IUGRs compare to sham male and female IUGRs at time zero (before IP) (p<0.01). Surfactant analysis showed significant reduction in the total surfactant phospholipid content in septic male IUGRs compared to shams (p<0.05) with no changes in surfactant phospholipid content between female septic IUGRs and shams. Control groups demonstrated no changes in surfactant content between septics and shams in either sexes.DiscussionChanges in surfactant pools occurring between male IUGR septic animals compared to shams without changes in control animals, support our hypothesis that IUGR is a risk factor for developing surfactant impairments. Also, the results indicate that the effect is different between males and females. Reduction of surfactant content in septic IUGR males could potentially cause lower lung compliance, alter lung function and therefore, developing or exacerbating lung injury during the development of ARDS. To further study the IUGR effect on the surfactant and evaluate its role as an ARDS risk factor, future experiments will assess the surfactant biophysical properties and lung histology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Effect of Calcium Influx on PIP2 Clusters in the Inner Plasma Membrane Leaflet of Intact Cells

Phospjatidylinositol 4,5-bisphosphated(PIP2) is known to interact with many cytoskeletal proteins electrostatically. When calcium ion channels open, local calcium levels are estimated up to 500uM. Experiments in model lipid bilayer have shown that such calcium level is sufficient to affect PIP2 in the inner leaflet of plasma membrane (PM).PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between lipid acyl-chains, stabilized by cholesterol, lead to transient submicroscopic nano-domains, and head-group interactions of charged lipids with divalent ions may cluster lipids. In addition, membrane cytoskeleton may impose further structure on membrane. Here we study influence of calcium on formation of PIP2 clusters in intact cells. We study changes of domains over time and upon calcium channel activation by analyzing diffusion of GFP-tagged inner-leaflet membrane proteins. Using bimFCS, developed by us, we measure diffusion on multiple length scales simultaneously to derive information about domains. To study formation of PIP2 clusters in the PM, we use GFP-PHPLCdelta to directly mark PIP2. We observe that opening TRPV1 channels leads to a transient rise in calcium as imaged using GCaMP5G, increases interaction strength between GFP-PHPLCdelta and PIP2 domains, which decreases to base lines within 1-2 minutes. Using ionophores, we determine that the increasing interaction strength is due to calcium-PIP2 interaction. By Vinculin depletion or actin filaments (de-)polymerization, we find that both anchor proteins and actin filaments play important roles in PIP2 clusters, and the formation of larger PIP2 clusters can potentially further activate Plasma membrane Ca2+ ATPase. These results suggest that in intact cells, calcium ions are able to induce changes of PIP2 clusters that are also regulated by cytoskeleton structure, changes of PIP2 clusters can further affect other signaling events.

Transient Effect of Calcium Influx on PIP2 Clusters and Cholesterol-Stabilized Nano-Domains in the Inner Plasma Membrane Leaflet of Intact Cells

When calcium ion channels open, local calcium levels are estimated to reach up to 500uM, which may be sufficient to affect negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), in the inner leaflet of the plasma membrane (PM). The PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between the acyl-chains of the lipids, stabilized by Cholesterol, lead to transient submicroscopic nano-domains, and lipid head-group interactions of charged lipids with divalent ions may cluster lipids. Here, we study the influence of calcium on the formation of PIP2 clusters and cholesterol-stabilized nano-domains in intact cells. We study changes of these domains over time and upon calcium channel activation by analyzing diffusion of GFP-tagged inner-leaflet membrane proteins. Using bimFCS, developed by us, we measure diffusion on multiple length scales simultaneously to derive information about domains. To study the formation of PIP2 clusters we use GFP-PHPLCdelta to directly mark PIP2, and as marker for cholesterol-stabilized nano-domains, we use Lck-mGFP. We observe that opening TRPV1 channels increases the interaction both between GFP-PHPLCdelta and PIP2 domains, Lck-mGFP and cholesterol domains. The interaction between GFP-PHPLCdelta and PIP2 domains decreases to base lines within 1-2 minutes, while the interaction of Lck-mGFP and cholesterol domains takes another 2-3 minutes to decrease as the cell down-regulates the intracellular calcium level after stimulation. To control for large scale signaling, we image the membrane cytoskeleton using mCherry-alpha-actinin, and use GT46-GFP to mark transmembrane domains. These results suggest a concentration dependence of calcium-induced PIP2 clusters and cholesterol-stabilized nano-domains in the PM at calcium levels, which may be reached in intact cells locally by opening of ion channels.

Impairing effect of fibrinogen on the mono-/bi-layer form of bovine lung surfactant

Lung surfactant (LS), a lipid–protein mixture responsible for alveolar stability, is inhibited by serum proteins leaked into the lungs in disease. Interaction of bovine lipid extract surfactant (BLES), a clinical replacement lung surfactant, with serum protein fibrinogen (Fbg) was studied employing various structural and biophysical techniques in adsorbed films and bulk bilayer dispersions. Surface tension area isotherms of the adsorbed films revealed the suppression of interfacial activity of BLES by Fbg (adsorption and surface tension reduction). Fbg, predominantly associated with the fluid phase of BLES films, resulted in the aggregation of the gel lipid domains as evidenced by atomic force microscopy. BLES bilayer dispersion showed phase transition from a diffused gel to liquid–crystalline phase in the temperature range 10–35 °C as studied by differential scanning calorimetry (DSC). Fbg resulted in the shift of peak to a higher transition temperature for the maximal heat flow (Tmax) of BLES dispersions. Combined Raman and FTIR spectral studies of the BLES/Fbg dispersions revealed that Fbg altered the –CH2–, –CH3, and –PO4− vibrational modes of the phospholipids present in BLES, suggesting the condensing and dehydrating effect of the protein on surfactant. Studies suggest that Fbg, by directly interacting with the gel lipids in LS in bulk dispersions, alter the packing of the films formed at the interface, and can be used as a specific model for lung disease.

Size Influences the Effect of Hydrophobic Nanoparticles on Lung Surfactant Model Systems

The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ∼12 nm and ∼136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ∼136 nm NPs compared to minor effects in the presence of ∼12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.

Transient Effect of Calcium Influx on PIP2 Clusters and Cholesterol-Stabilized Nano-Domains in the Inner Plasma Membrane Leaflet of Intact Cells

When calcium ion channels open, local calcium levels are estimated to reach up to 500uM, which may be sufficient to affect negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2), in the inner leaflet of the plasma membrane (PM). The PM is a complex lipid protein mixture in which at least two mechanisms create lateral order: interactions between the acyl-chains of the lipids, stabilized by Cholesterol, lead to transient submicroscopic nano-domains, and lipid head-group interactions of charged lipids with divalent ions may cluster lipids.

Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza A Replication and Propagation

ABSTRACTInfluenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca2+-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis.

Deterioration of Pulmonary Surfactant by Volatile Anesthetics

Pulmonary surfactant (PS) is a lipid-protein mixture lining the alveolar air-water interface. By lowering surface tension, PS stabilizes the respiratory surface, thereby impeding alveolar collapse. Lack or alterations of PS properties are associated with acute lung injury (ALI). Sevoflurane is widely used in medical practice; however, some adverse properties were described in the lung function and PS [1,2]. The hypothesis of this study is that Sevoflurane affects native PS (NPS) system, which may contribute to ALI during anesthesia. In the present work, we have analyzed the functional, structural, and thermodynamic properties of NPS and its membranes reconstituted from the organic lipid extract (EPS) upon exposure to Sevoflurane. The compression-expansion properties of NPS films exposed to Sevoflurane were analyzed in a captive bubble surfactometer (CBS) as well as the effects of the anesthetic on the thermotropic properties of NPS and EPS membranes.Q-Static and dynamic cycles of NPS films were strongly affected upon exposure to Sevoflurane, which diminished the ability of the films to reach very low surface tension during compression. However, the equilibrium surface tension after adsorption and the maximum surface tension at the end of the expansion moieties of cycling isotherms were not affected by the presence of Sevoflurane. Spectroscopic analysis of surfactant complexes doped with LAURDAN or diphenylhexatriene indicate fluidification of EPS membranes upon exposure to Sevoflurane at room temperature. DSC experiments indicated that Sevoflurane induces a decrease of Tm and ΔH associated with the main thermotropic transition in PS membranes.In conclusion, the present results indicate that Sevoflurane impairs the biophysical properties of PS through perturbation of the lateral order of segregated phases, compromising correct compression along the respiratory cycles.[1] L. Malacrida et al. Am. J. Respir. Crit. Care Med.181;2010:A3631.[2] L. Malacrida et al. Biophysical J. 100(3);2011:505a-506a.

Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid–protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14–47μm), protein clusters exhibited a narrow size distribution (2.6–4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid–protein interactions on lateral membrane architecture.

Biophysical investigation of the interfacial properties of cationic fluorocarbon/hydrocarbon hybrid surfactant: Mimicking the lung surfactant protein C

The interfacial behavior of the newly designed Fluorocarbon Hydrocarbon Cationic Lipid (FHCL or CH3(CH2)17N+(C2H5)2(CH2)3(CF2)7CF3I−) and its mixtures with a phospholipid (DPPC, Dipalmitoylphosphatidylcholine) at different mole fractions were investigated. This new molecule was synthesized to mimic the selected properties of lung surfactant, which is a natural lipid–protein mixture which is known to play important roles in the process of respiration, by considering the structure/function relation of lung surfactant protein (SP-C). Each segment in the molecular structure was selected to affect the molecular level interaction at the interface whereas the keeping the overall structure as simple as possible. The surface pressure area isotherms obtained for the mixtures of DPPC/FHCL indicated that there was repulsive interaction between DPPC and FHCL molecules. Due to the molecular level interaction, specifically at mole fraction 0.3, the isotherm obtained from that mixture resembled the isotherm obtained from the DPPC monolayer in the presence of SP-C. High elasticity of the interface was one of the important parameters for the respiration process, therefore, shear and dilatational elasticities of two-component systems were determined and they were found to be similar to the case where SP-C protein is present. Fluorescence microscopy images were taken in order to investigate the monolayer in details. The FHCL was able to fluidize the DPPC monolayer even at high surface pressures effectively. In addition, the cyclic compression–expansion isotherms were obtained to understand the spreading and re-spreading ability of the pure FHCL and the mixed DPPC/FHCL monolayers. At a specific mole fraction, XFHCL=0.3, the mixture exhibited good hysteresis in area, compressibility, recruitment index and re-spreading ability at the interface. All these results point out that FHCL can fulfill the selected features of the lung surfactant that are attributed to the presence of SP-C protein when mixed with DPPC, even if the molecular structure of the FHCL is quite simple.

Effect of surfactant monomers on chloramphenicol association to an albumin-lecithin complex: a model for modified drug absorption.

The binding of chloramphenicol to an albumin-lecithin complex in the presence or absence of premicellar concentrations of both ionic and non-ionic surfactants has been examined. Long chain, strong ionic detergents, such as sodium dodecyl sulphate or cetyltrimethylammonium bromide, severely perturb protein structure and eventually allow full separation of the complex into lecithin and albumin-detergent complexes. The dissociation process is reversible upon the removal of the detergent by exhaustive dialysis. After the splitting of the complex, the amount of antibiotic associated with the lipid-protein mixture increases. Structural alteration of the albumin-lecithin complex and the increase in the binding of chloramphenicol have an effect on the transfer rate of this antibiotic across an artificial barrier consisting of an aqueous dispersion of the same complex, as observed in a model system. It is suggested that a reversible alteration in membrane structure, and consequently in membrane permeability, might be easily effected, at the molecular level, through a reversible dissociation of structural lipoproteins into their components, operated by premicellar concentrations of ionic surfactants. This represents a tentative picture of the possible events taking place within the membrane and modifying the absorption rate of a drug, when it is associated with surfactants in a pharmaceutical preparation.

A ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems

Pulmonary surfactant is a complex lipid–protein mixture whose main function is to reduce the surface tension at the air–liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir–Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid–lipid and lipid–protein interactions. This may further our understanding of pulmonary surfactant function.

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid–protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir–Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.

Stable isotope labelling of phosphatidylcholine synthesis in vivo in children with acute lung injury

<h3>Background</h3> Acute lung injury (ALI) is an inflammatory lung condition with significant morbidity and mortality, associated with surfactant dysfunction. Surfactant is a lipid–protein mixture composed of 80% phospholipid of which 35–55% is dipalmitoylphosphatidylcholine. The lipid profile of surfactant is significantly altered in patients with ALI,1 but it is not clear whether this is due to decreased surfactant synthesis or increased degradation. <h3>Material and Methods</h3> This study employs a novel method to quantify surfactant kinetics in children in vivo. Five children, aged 2 months to 3 years, admitted to the paediatric intensive care unit with a clinical diagnosis of ALI, were infused with methyl-D₉ choline chloride. Synthesis of phosphatidylcholine (PC) in sequential lung bronchoalveolar lavage (BAL) and serum samples were analysed by electrospray ionisation tandem mass spectrometry. <h3>Results</h3> Measurable incorporation of methyl-D₉ choline chloride into both surfactant and serum PC was demonstrated, with peak incorporations of 0.7±0.08% in BAL fluid (BALF) (figure 1) and 3.0±0.8% in serum (figure 2). The rate of surfactant synthesis varied widely and did not correlate with respective measurement of serum PC synthesis, highlighting lung and systemic effects of ALI. <h3>Conclusion</h3> This study demonstrates the feasibility of methyl-D₉ choline chloride labelling to quantify kinetics of surfactant and serum PC synthesis in children with ALI in vivo. The variation in BALF PC synthesis indicates a wide range of surfactant synthesis/secretion, while comparison with serum PC synthesis suggests that much of this variation is lung specific.

Effect of Cholesterol on Electrostatics in Lipid−Protein Films of a Pulmonary Surfactant

We report the changes in the electrical properties of the lipid-protein film of pulmonary surfactant produced by excess cholesterol. Pulmonary surfactant (PS) is a complex lipid-protein mixture that forms a molecular film at the interface of the lung's epithelia. The defined molecular arrangement of the lipids and proteins of the surfactant film gives rise to the locally highly variable electrical surface potential of the interface, which becomes considerably altered in the presence of cholesterol. With frequency modulation Kelvin probe force microscopy (FM-KPFM) and force measurements, complemented by theoretical analysis, we showed that excess cholesterol significantly changes the electric field around a PS film because of the presence of nanometer-sized electrostatic domains and affects the electrostatic interaction of an AFM probe with a PS film. These changes in the local electrical field would greatly alter the interaction of the surfactant film with charged species and would immediately impact the manner in which inhaled (often charged) airborne nanoparticles and fibers might interact with the lung interface.