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Pulmonary delivery of small interfering RNA (siRNA) using nanoparticle-based delivery systems is promising for local treatment of respiratory diseases. We designed dry powder inhaler formulations of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) with aerosolization properties optimized for inhalation therapy. Interactions between LPNs and pulmonary surfactant (PS) determine the fate of inhaled LPNs, but interaction mechanisms are unknown. Here we used surface-sensitive techniques to study how physicochemical properties and pathological microenvironments influence interactions between siRNA-loaded LPNs and supported PS layers. PS was deposited on SiO2 surfaces as single bilayer or multilayers and characterized using quartz crystal microbalance with dissipation monitoring and Fourier-transform infrared spectroscopy with attenuated total reflection. Immobilization of PS as multilayers, resembling the structural PS organization in the alveolar subphase, effectively reduced the relative importance of interactions between PS and the underlying surface. However, the binding affinity between PS and LPNs was identical in the two models. The physicochemical LPN properties influenced the translocation pathways and retention time of LPNs. Membrane fluidity and electrostatic interactions were decisive for the interaction strength between LPNs and PS. Experimental conditions reflecting pathological microenvironments promoted LPN deposition. Hence, these results shed new light on design criteria for LPN transport through the air-blood barrier.

Resumen Antecedentes Se ha descrito una elevada incidencia de tromboembolismo pulmonar (TEP) durante la pandemia por coronavirus. Metodos Estudio retrospectivo unicentrico, con revision de las angiografias pulmonares por tomografia computarizada solicitadas por sospecha de tromboembolismo pulmonar durante dos periodos, del 01 de marzo del 2020 al 31de mayo del 2020 (pandemia), e igual intervalo en 2019 (control). Resultados Se diagnosticaron 22 tromboembolismos pulmonares durante el periodo control y 99 en el pandemico, 74 asociados con COVID-19. El 5,3% de los pacientes hospitalizados con COVID-19 sufrio un tromboembolismo pulmonar, con un retraso entre ambos diagnosticos de 9,1 ± 8,4 dias. Durante la pandemia, los pacientes con tromboembolismo pulmonar tenian menos condiciones predisponentes (tromboembolismo pulmonar previo 5,1 vs. 18,2%, p = 0,05, cirugia previa 2 vs. 35,4%, p = 0,0001, trombosis venosa profunda 11,1 vs. 45,5%, p = 0,0001), y los tromboembolismos pulmonares perifericos eran mas frecuentes (73,5 vs. 50%, p = 0,029). Conclusiones Existe un riesgo incrementado de sufrir un TEP durante la pandemia por SARS-CoV-2, que afecta a pacientes con perfil clinico diferente y causa mas frecuentemente TEP distales.

Background A high incidence of pulmonary embolism has been described during the coronavirus pandemic. Methods This work is a single-center retrospective study which reviewed computed tomography pulmonary angiograms ordered due to suspected pulmonary embolism during two periods: from March 1, 2020 to May 31, 2020 (pandemic) and during the same interval in 2019 (control). Results Twenty-two pulmonary embolisms were diagnosed during the control period and 99 in the pandemic, 74 of which were associated with COVID-19. Of all patients hospitalized with COVID-19, 5.3% had a pulmonary embolism, with a delay between the two diagnoses of 9.1 ± 8.4 days. During the pandemic, patients with pulmonary embolism had fewer predisposing conditions (previous pulmonary embolism 5.1 vs. 18.2%, p = .05; previous surgery 2 vs. 35.4%, p = .0001; deep vein thrombosis 11.1 vs. 45.5%, p = .0001); peripheral pulmonary embolisms were the most frequent (73.5 vs. 50%, p = . 029). Conclusions There is an increased risk of having a pulmonary embolism during the SARS-CoV-2 pandemic, which affects patients with a different clinical profile and more often causes distal pulmonary embolisms.

The purpose of this work was to determine the influence of myocardial wringing on ventricular function in patients with cardiac amyloidosis (CA).Fifteen healthy volunteers (group 1) and 34 patients with CA (17 with left ventricular ejection fractions [LVEFs] ≥ 53% [group 2] and 17 with LVEFs 53% [group 3]) were evaluated using two-dimensional speckle-tracking echocardiography. A control group of mass-matched patients (n = 20) with left ventricular (LV) hypertrophy and LVEFs ≥ 53% was also included. Longitudinal strain (LS), circumferential strain, and LV twist and torsion were calculated. Deformation index (DefI), a new parameter of wringing, calculated as twist/LS, that takes into account actions that occur simultaneously during LV systole (i.e., longitudinal shortening and twist), was evaluated. Torsional and wringing parameters were calculated according to LVEF.Lower global values of LS and circumferential strain were observed among patients with CA (LS: group 1, -20.6 ± 2.5%; group 2, -11.6 ± 4.1%; group 3, -9.0 ± 3.1%; circumferential strain: group 1, -22.7 ± 4.9%; group 2, -14.4 ± 8.0%; group 3, -13.6 ± 3.8%; P .001 for both). Torsion did not vary between group 2 and group 1 (2.5 ± 1.1°/cm vs 2.7 ± 0.8°/cm, P = NS). In contrast, DefI was greater in group 2 than in group 1 (-1.8 ± 0.8°/% vs -1.0 ± 0.3°/%, P .01). Torsion and DefI were lower in group 3 (1.2 ± 0.7°/cm and -1.1 ± 0.6°/%, respectively, P .001 for both) than in group 2. DefI was similar in patients with LV hypertrophy (-1.7 ± 0.6°/%, P = NS) and group 2.In patients with CA, preservation of LVEF depends on greater ventricular wringing. DefI, a parameter that integrates the twist and the simultaneous longitudinal shortening of the left ventricle, is a more accurate indicator of the efficacy of this mechanism.

Graphical abstract, Highlights • We describe how inhaled substances can inhibit lung surfactant function. • The inhibition of lung surfactant function leads to alveolar collapse. • We present the weight of evidence that supports the adverse outcome pathway. • We give an overview of the methods available to measure each key event of the pathway., Inhaled substances, such as consumer products, chemicals at the workplace, and nanoparticles, can affect the lung function in several ways. In this paper, we explore the adverse outcome pathway (AOP) that starts when inhaled substances that reach the alveoli inhibit the function of the lung surfactant, and leads to decreased lung function. Lung surfactant covers the inner surface of the alveoli, and regulates the surface tension at the air–liquid interface during breathing. The inhibition of the lung surfactant function leads to alveolar collapse because of the resulting high surface tension at the end of expiration. The collapsed alveoli can be re-opened by inspiration, but this re-opening causes shear stress on cells covering the alveoli. This can damage the alveolar-capillary membrane integrity, allowing blood components to enter the alveolar airspace. Blood components, such as albumin, can interact with the lung surfactant and further inhibit its function. The collapse of the alveoli is responsible for a decrease in the surface area available for blood oxygenation, and it reduces the volume of air that can be inhaled and exhaled. These different key events lead to decreased lung function, characterized by clinical signs of respiratory toxicity and reduced blood oxygenation. Here we present the weight of evidence that supports the AOP, and we give an overview of the methods available in vitro and in vivo to measure each key event of the pathway, and how this AOP can potentially be used in screening for inhalation toxicity.

Current computing platforms, including HPC, Grid and Cloud computing, offer many computational resources. These platforms can provide the scientific world with on-demand and scalable computing power. However, their use requires advanced computer skills. In our work, we summarize the main problems found by research scientists when they attempt to execute complex software on one or several of these platforms. As a solution to these problems, we propose the Science for Everyone (ScifE) framework, and an intuitive and easy-to-use web interface, to perform scientific experiments on HPC or Cloud platforms. The Community Earth System Model (CESM) has been used to test our proof-of-concept framework as it is a complex scientific software used by many climate researchers. Using our framework, a user can execute CESM with custom parameters with only a few clicks on the web interface. As ScifE is generic, other software can be imported relatively easy to our framework. Additional collaborative tools are being developed for successive versions of ScifE, enhancing the practice of science.

This study was carried out to evaluate the Life Cycle Environmental Impacts of three Pig Production Technologies (PPT), considering the activities involved in their productive systems (pig production, animal feed, avoided fertilizer production, energy and water consumption) and relevant cleaner production scenarios for the Cuban context; such as: 1) the impact of technical conditions during lagooning; and 2) the use of high efficiency anaerobic digestion technologies (e.g., Upflow Anaerobic Sludge Blanket, UASB) over small scale digester (Fixed Dome Digesters, FDD) for waste management. The province of Sancti Spiritus was taken as case study considering the existence of similar scenarios in all Cuban provinces. The Life Cycle Assessment Methodology was used to determine the environmental burdens of the three PPT (Genetic Farms, Multiplier Farms and Production Farms). Genetic Farms produced the lowest impacts due to their lower consumption of feed (soybean and corn) and energy, and the lowest global emissions of pollutants. Pig production (housing and lagooning) was the activity with the highest contributions (75–89%) to the impact categories climate change, terrestrial acidification, marine eutrophication and freshwater eutrophication, due to the emissions of greenhouse gases during lagooning. In contrast, feed production (corn and soybeans) reached the highest impacts (87%) for terrestrial ecotoxicity, which was related to the agricultural and industrial processes of corn and soybean. The main cleaner production opportunities (with an impact above 49%) were obtained from the utilization of a high efficiency anaerobic digestion technology (i.e., UASB), while low environmental benefits can be obtained when using small scale digesters (i.e., FDD). The technical conditions of the lagoons affect also the environmental burdens of the PPT, suggesting that a proper maintenance is crucial. The results obtained became a tool for decision makers and researchers about the implementation of high efficiency technologies for waste management in Pig Farms to obtain the highest environmental benefits, not only in Cuba but also in developing countries with similar situation.

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Background Sleep-disordered breathing (SDB) has been associated with a greater incidence and mortality of cancer, although such findings are inconsistent. However, no large studies are currently available to investigate this association in patients with a specific type of cancer. This study seeks to assess potential relationships between SDB severity and aggressiveness markers of cutaneous melanoma. Methods Four hundred and forty-three patients with a diagnosis of melanoma underwent a sleep study within 6 months of diagnosis. General demographics were collected, along with melanoma characteristics and polygraphic parameters consisting of the apnea-hypopnea index (AHI) and indices of both continuous and intermittent night-time oxyhemoglobin desaturation (DI4%). An exploration of independent relationships between SDB and various objective melanoma aggressiveness markers (Breslow index, presence of ulceration, presence of regression, mitotic index, stage of severity, damage to the sentinel lymph, and spreading of the melanoma) was performed. Results Patients in the upper tertiles of AHI or DI4% were 1.94 (95% CI, 1.14-3.32; P = .022) and 1.93 (95% CI, 1.14-3.26; P = .013) times more likely, respectively, to present with aggressive melanoma (Breslow index > 1 mm) than those in the lowest tertiles of these sleep attributes after adjustment for age, sex, tumor location, and BMI. This association was particularly prominent among patients 2 mm. The presence of the additional markers of aggressiveness was also associated with higher AHI and DI4% values. Conclusions The severity of SDB was independently associated with greater aggressiveness of cutaneous melanoma, particularly among younger patients.

This work explores the potential for strategizing pulmonary surfactant (PS) for drug delivery over the respiratory air-liquid interface: the interfacial delivery. The efficacy of PS- and interface-assisted drug vehiculization was determined both in vitro and in vivo using a native purified porcine PS combined with the hydrophobic anti-inflammatory drug Tacrolimus (TAC), a calcineurin inhibitor. In vitro assays were conducted in a novel double surface balance setup designed to emulate compression-expansion dynamics applied to interfacially connected drug donor and recipient compartments. In this setup, PS transported TAC efficiently over air-liquid interfaces, with compression/expansion breathing-like dynamics enhancing rapid interface-assisted diffusion and drug release. The efficacy of PS-assisted TAC vehiculization was also evaluated in vivo in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). In anesthetized mice, TAC combined with PS was intra-nasally (i.n) instilled prior administering i.n. LPS. PS/TAC pre-treatment caused greater TAC internalization into a higher number of lung cells obtained from bronchoalveolar lavages (BAL) than TAC pre-treatment alone. Additionally, the PS/TAC combination but not TAC or PS alone attenuated the LPS-induced pro-inflammatory effects reducing cells and proteins in BAL fluid. These findings indicated that PS-mediated increase in TAC uptake blunted the pro-injurious effects of LPS, suggesting a synergistic anti-inflammatory effect of PS/drug formulations. These in vitro and in vivo results establish the potential utility of PS to open novel effective delivery strategies for inhaled drugs., This work has been funded by grants from the Spanish Ministry of Science, Universities and Innovation (RTI2018-094564-B-I00, RTI2018-096410-B-C22) and the Regional Government of Madrid (P2018/NMT­4389), and by grants from the US National Institutes of Health (HL36024 and HL57556). PC acknowledges a research associate contract at the University of the Basque Country (DOCREC18/01) and a postdoctoral fellowship from the Basque Government (POS_2018_1_0066). PC also acknowledges funding from the Basque Government (IT1196-19).

Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. Statement of significance Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.

Pulmonary surfactant is a complex of lipids and proteins assembled and secreted by the alveolar epithelium into the thin layer of fluid coating the respiratory surface of lungs. There, surfactant forms interfacial films at the air-water interface, reducing dramatically surface tension and thus stabilizing the air-exposed interface to prevent alveolar collapse along respiratory mechanics. The absence or deficiency of surfactant produces severe lung pathologies. This review describes some of the most important surfactant-related pathologies, which are a cause of high morbidity and mortality in neonates and adults. The review also updates current therapeutic approaches pursuing restoration of surfactant operative films in diseased lungs, mainly through supplementation with exogenous clinical surfactant preparations. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

With the intention to move away from animal testing for the toxicological evaluation of chemicals comes the need to develop new approach methodologies which are mechanism-anchored and target relevant key events leading to an adverse outcome. To date, no validated alternative methods are available for studying the acute inhalation toxicity potential of airborne chemicals but the constrained drop surfactometer measuring the surface tension of a drop of lung surfactant presents as a promising candidate. Indeed, the correlation of the increase in minimum surface tension of lung surfactant in vitro with changes in the breathing patterns of mice after inhalation of test compounds has been shown in multiple studies. However, the causal factors leading to lung surfactant inactivation remain speculative. This paper combines molecular and biophysical methods (constrained drop and captive bubble surfactometers, Langmuir-Blodgett balance, epifluorescence microscopy, cryogenic transmission electron microscopy, and differential scanning calorimetry) applied to purified porcine lung surfactant and dipalmitoylphosphatidylcholine interfacial films to gain insights into the disruption of lung surfactant function by three chemicals known to show acute inhalation toxicity (trimethoxyoctylsilane, methyl 3-oxo-2-pentylcyclopentaneacetate, and diisopentyl ether). The results of this study suggest that the test chemicals intercalate between the phospholipids at the air-liquid interface, reduce the stability of the films, and decrease the cohesivity of interface-associated multilayered structures thereby perturbing the lung surfactant surface activity. These findings contribute to a better understanding of chemically-induced lung surfactant function disruption.

Pulmonary surfactant is a lipoprotein complex that reduces surface tension to prevent alveolar collapse and contributes to the protection of the respiratory surface from the entry of pathogens. Surfactant protein A (SP-A) is a hydrophilic glycoprotein of the collectin family, and its main function is related to host defense. However, previous studies have shown that SP-A also aids in the formation and biophysical properties of pulmonary surfactant films at the air-water interface. Humans, unlike rodents, have two genes, SFTPA1 and SFTPA2. The encoded proteins, SP-A1 and SP-A2, differ quantitatively or qualitatively in function. It has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of lung surfactant. The goal of this study was to investigate potential differences in the biophysical properties of surfactants containing human SP-A1, SP-A2, or both. For this purpose, we have studied for the first time, to our knowledge, the biophysical properties of pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1 and SP-A2, in the captive bubble surfactometer. We observed that pulmonary surfactant containing SP-A1 reaches lower surface tension after postexpansion interfacial adsorption than surfactants containing no SP-A or only SP-A2. Under interfacial compression-expansion cycling conditions, surfactant films containing SP-A1 also performed better, particularly with respect to the reorganization of the films that takes place during compression. On the other hand, addition of recombinant SP-A1 to a surfactant preparation reconstituted from the hydrophobic fraction of a porcine surfactant made it more resistant to inhibition by serum than the addition of equivalent amounts of SP-A2. We conclude that the presence of SP-A1 allows pulmonary surfactant to adopt a particularly favorable structure with optimal biophysical properties.

Many pathologies of the respiratory tract are inadequately treated with existing small molecule-based therapies. The emergence of RNA interference (RNAi) enables the post-transcriptional silencing of key molecular disease factors that cannot readily be targeted with conventional small molecule drugs. Pulmonary administration of RNAi effectors, such as small interfering RNA (siRNA), allows direct delivery into the lung tissue, hence reducing systemic exposure. Unfortunately, the clinical translation of RNAi is severely hampered by inefficient delivery of siRNA therapeutics towards the cytoplasm of the target cells. In order to have a better control of the siRNA delivery process, both extra- and intracellular, siRNAs are typically formulated in nanosized delivery vehicles (nanoparticles, NPs). In the lower airways, which are the targeted sites of action for multiple pulmonary disorders, these siRNA-loaded NPs will encounter the pulmonary surfactant (PS) layer, covering the entire alveolar surface. The interaction between the instilled siRNA-loaded NPs and the PS at this nano-bio interface results in the adsorption of PS components onto the surface of the NPs. The formation of this so-called biomolecular corona conceals the original NP surface and will therefore profoundly determine the biological efficacy of the NP. Though this interplay has initially been regarded as a barrier towards efficient siRNA delivery to the respiratory target cell, recent reports have illustrated that the interaction with PS might also be beneficial for local pulmonary siRNA delivery.

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein., This work has been supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-094564-B-I00 to J.P.-G) and the Regional Government of Madrid (P2018/NMT­4389 to J.P.-G.) F.M.-H. Acknowledges support from the Spanish Ministry of Science Innovation and Universities (project BFU2017-83794-P, (AEI/FEDER, UE)) and from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation (grant agreement No 681299). R.A. acknowledges an FPU PhD fellowship from the Spanish Ministry of Education.

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways. This work was supported by SAF2014-53209-R and SAF2017-86483-R grants from the Ministerio de Economía y Competitividadto R.B. and M.V., and to M.V., respectively, RTI2018-094564-B-I00 from the Spanish Ministry of Science and Universities andP2018/NMT4389 from the Regional Government of Madrid to J.P-G. and A.C., and RIRAAF Network RD12/0013/0015 grant and ARADyAL Network RD16/0006 grant from the Instituto de Salud Carlos III (ISCIII) co-founded by Fondo Europeo de Desarrollo Regional -FEDER-for the Thematic Networks and Co-operative Research Centres.A.J. and C.M. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. JC.L-R., P.SS-A. and C.G-M. are recipients of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. We also gratefully acknowledge the financial support from theAES-ISCIII for the PI17CIII/00045 grant to R.B. We also thank the excellent technical support of Sara Abián. Sí