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Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 acyl ester linkage in phospholipid, producing lysophospholipid and fatty acid in the presence of Ca2+. The hydrolysis mediated by PLA2 has attracted much interest in various fields, such as pharmacy and biotechnology. It is recognized that PLA2 cannot hydrolyze phospholipid monolayers at high surface coverage. However, the origin of different PLA2 activities is not fully understood yet. The present study investigated the interaction between DPPC (16:0 PC) monolayer and PLA2 using heterodyne-detected sum frequency generation spectroscopy, which is interface-specific spectroscopy and highly sensitive to molecular symmetry based on a second-order nonlinear optical process. It was revealed that PLA2 adsorbs to the DPPC monolayer on the aqueous solution surface only when the surface coverage is low. The adsorption at the low surface coverage significantly changes the interfacial structures of PLA2 and the hydration, which are stabilized by the presence of Ca2+. Therefore, the restriction of the hydrolysis of phospholipid monolayers at high surface coverage can be rationalized by the inhabitation of the PLA2 adsorption. The present study deepens our molecular-level understanding of the hydrolysis of phospholipids by PLA2., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Lung surfactant is inactivated in acute respiratory distress syndrome (ARDS) by a mechanism that remains unclear. Phospholipase (PLA 2 ) plays an essential role in the normal lipid recycling processes, but is present in elevated levels in ARDS, suggesting it plays a role in ARDS pathophysiology. PLA 2 hydrolyzes lipids such as DPPC-the primary component of lung surfactant-into palmitic acid (PA) and lyso-PC (LPC). Because PA co-crystallizes with DPPC to form rigid, elastic domains, we hypothesize that PLA 2 -catalyzed degradation establishes a stiff, heterogeneous rheology in the monolayer, and suggests a potential mechanical role in disrupting lung surfactant function during ARDS. Here we study the morphological and rheological changes of DPPC monolayers as they are degraded by PLA 2 using interfacial microbutton microrheometry coupled with fluorescence microscopy. While degrading, domain morphology passes through qualitatively distinct transitions: compactification, coarsening, solidification, aggregation, network percolation, network erosion, and nucleation of PLA 2 -rich domains. Initially, condensed domains relax to more compact shapes, and coarsen via Ostwald ripening and coalescence up until the domains solidify, marked by a distinct roughening of domain boundaries that does not relax. Domains aggregate and eventually form a percolated network, whose elements then erode and whose connections are broken as degradation continues. The relative enzymatic activity of PLA 2 , set by the age of the sample, impacts the order and the duration of morphology transitions. The fresher the PLA 2 , the faster the overall degradation, and the earlier the onset of domain solidification: domains solidify before aggregating with fresh PLA 2 samples, but aggregate and percolate before solidification with aged PLA 2 . Irrespective of the activity of the PLA 2 , all measured linear viscoelastic surface shear moduli obey the same dependence on condensed phase area fraction (log| G *| ∝ ϕ ) throughout monolayer degradation. Moreover, the onset of domain solidification coincides with the time when the relative surface elasticity begins to increase.

Adaptation to hypoxia has attracted much public interest because of its clinical significance. However, hypoxic adaptation in the body is complicated and difficult to fully explore. To explore previously unknown conserved mechanisms and key proteins involved in hypoxic adaptation in different species, we first used a yeast model for mechanistic screening. Further multi-omics analyses in multiple species including yeast, zebrafish and mice revealed that glycerophospholipid metabolism was significantly involved in hypoxic adaptation with up-regulation of lysophospholipid acyltransferase (ALE1) in yeast, a key protein for the formation of dipalmitoyl phosphatidylcholine [DPPC (16:0/16:0)], which is a saturated phosphatidylcholine. Importantly, a mammalian homolog of ALE1, lysophosphatidylcholine acyltransferase 1 (LPCAT1), enhanced DPPC levels at the cell membrane and exhibited the same protective effect in mammalian cells under hypoxic conditions. DPPC supplementation effectively attenuated growth restriction, maintained cell membrane integrity and increased the expression of epidermal growth factor receptor under hypoxic conditions, but unsaturated phosphatidylcholine did not. In agreement with these findings, DPPC treatment could also repair hypoxic injury of intestinal mucosa in mice. Taken together, ALE1/LPCAT1-mediated DPPC formation, a key pathway of glycerophospholipid metabolism, is crucial for cell viability under hypoxic conditions. Moreover, we found that ALE1 was also involved in glycolysis to maintain sufficient survival conditions for yeast. The present study offers a novel approach to understanding lipid metabolism under hypoxia and provides new insights into treating hypoxia-related diseases., (© 2024 Federation of European Biochemical Societies.)

The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC., Competing Interests: Declaration of competing interest Authors declare no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Understanding the transport mechanism of the peptide Asn-Cys-Trp (NCW) is crucial to improving its intestinal absorption and bioavailability. This study investigated the absorption of NCW through Caco-2 cell monolayers and its interaction with the DPPC bilayers. Results revealed that after a 3 h incubation, the Papp (AP-BL) and Papp (BL-AP) values of NCW at a concentration of 5 mmol/L were (22.24 ± 4.52) × 10 -7 and (6.63 ± 2.31) × 10 -7 cm/s, respectively, with the transport rates of 1.59 ± 0.32 and 0.62 ± 0.20%, indicating its moderate absorption. NCW was found to be transported via PepT1 and paracellular transport pathways, as evidenced by the significant impact of Gly-Pro and cytochalasin D on the Papp values. Moreover, NCW upregulated ZO-1 mRNA expression. Further investigation of the ZO-1-mediated interaction between NCW and tight junction proteins will contribute to a better understanding of the paracellular transport mechanism of NCW. The interaction between NCW and the DPPC bilayers was predominantly driven by entropy. NCW permeated the bilayers through electrostatic, hydrogen bonding, and hydrophobic interactions, resulting in increased fluidity, flexibility, and disorder as well as phase transition and phase separation of the bilayers.

The size, dispersibility, and fluidity of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoy-2-oleoyl-sn-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes doped with β-sitosteryl sulfate (PSO 4 ) were comparatively studied. In all three types of liposomes, PSO 4 reduced sizes and enhanced the negative values of the ζ-potential. However, the effect on sizes quantitatively differed in the three cases in a manner dependent on their phase behaviors. PSO 4 rigidified each type of membrane in its liquid crystalline phase and fluidized the gel phase. It enhanced the glucose trapping efficiency (TE) of both DPPC and DOPC liposomes. The TE of DPPC first increased with the increasing concentration of PSO 4 , then decreased gradually. On the other hand, in the case of DOPC, the TE increased significantly upon addition of PSO 4 , then remained nearly constant. Though the exact dependence of TE on the PSO 4 concentration differed in the two cases, its effect, in each case, was more than the effect of β-sitosterol (POH). The ability of PSO 4 for reducing the size and enhancing dispersibility and TE of liposomes can be useful for preparing cosmetics and pharmaceutical formulations.

Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1'-O-octacosamethylene-2,2'-di-O-tetradecyl-bis-(sn-glycero)-3,3'-diphosphocholine (AC-(di-O-C14PC) 2 ), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC) 2 monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC) 2 vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC) 2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules., (Copyright © 2021. Published by Elsevier B.V.)

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their "real" biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Amyloid-beta (Aβ) protein is related to Alzheimer disease (AD), and various experiments have shown that oligomers as small as dimers are cytotoxic. Recent studies have concluded that interactions of Aβ with neuronal cell membranes lead to disruption of membrane integrity and toxicity and they play a key role in the development of AD. Molecular dynamics (MD) simulations have been used to investigate Aβ in aqueous solution and membranes. We have previously studied monomeric Aβ40 embedded in dipalmitoylphosphatidylcholine (DPPC) membrane using MD simulations. Here, we explore interactions of two Aβ40 peptides in DPPC bilayer and its consequences on dimer distribution in a lipid bilayer and on the secondary structure of the peptides. We explored that N-terminals played an important role in dimeric Aβ peptide aggregations and Aβ-bilayer interactions, while C-terminals bound peptides to bilayer like anchors. We did not observe exiting of peptides in our simulations although we observed insertion of peptides into the core of bilayer in some of our simulations. So it seems that the presence of Aβ on membrane surface increases its aggregation rate, and as diffusion occurs in two dimensions, it can increase the probability of interpeptide interactions. We found that dimeric Aβ, like monomeric one, had the ability to cause structural destabilization of DPPC membrane, which in turn might ultimately lead to cell death in an in vivo system. This information could have important implications for understanding the affinity of Aβ oligomers (here dimer) for membranes and the mechanism of Aβ oligomer toxicity in AD., (© 2020 Wiley Periodicals LLC.)

In this work, we studied the mechanisms of classical activation and inactivation of signal transduction by the histamine H3 receptor, a 7-helix transmembrane bundle G-Protein Coupled Receptor through long-time-scale atomistic molecular dynamics simulations of the receptor embedded in a hydrated double layer of dipalmitoyl phosphatidyl choline, a zwitterionic polysaturated ordered lipid. Three systems were prepared: the apo receptor, representing the constitutively active receptor; and two holo-receptors-the receptor coupled to the antagonist/inverse agonist ciproxifan, representing the inactive state of the receptor, and the receptor coupled to the endogenous agonist histamine and representing the active state of the receptor. An extensive analysis of the simulation showed that the three states of H3R present significant structural and dynamical differences as well as a complex behavior given that the measured properties interact in multiple and interdependent ways. In addition, the simulations described an unexpected escape of histamine from the orthosteric binding site, in agreement with the experimental modest affinities and rapid off-rates of agonists.

Melatonin is a neurohormone that has been shown to be protective in Alzheimer's diseases against amyloid-β (Aβ) toxicity, which involves interaction of Aβ with neuronal membrane. Non-specific interactions of melatonin with cell membrane may play a physiological role in this process by preserving membrane fluidity. In the brain, melatonin is derived from the amino acid tryptophan through a pathway that includes serotonin and N-acetylserotonin (NAS). How these molecules affect the membrane properties is not understood. In this work, we studied interactions of melatonin and its metabolic precursors tryptophan, serotonin and NAS with dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface using Langmuir monolayer technique. Analysis of compression isotherms, phase transitions and compressibility moduli indicate that all four molecules alter the DPPC monolayer properties in a structure and concentration dependent manner. This effect was most pronounced for melatonin followed by NAS. Melatonin and NAS both decreased the compressibility modulus and shifted the LE/LC phase transition suggesting an increase in the membrane fluidity. Tryptophan and serotonin caused less pronounced effects on the DPPC isotherm. These differences suggest different interaction mechanisms and may be attributed to the interplay between electrostatic and hydrophobic interactions of these molecules with the zwitterionic DPPC headgroups which correlate with water solubility and oil partition coefficients (LogS and LogP) of each the four molecules. The results here demonstrate how the physiochemical properties of indoles can affect lipid membranes which may shed light on the functional significance of these important neurochemicals and the neuroprotective mechanisms of melatonin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lung lipid metabolism participates both in infant and adult pulmonary disease. The lung is composed by multiple cell types with specialized functions and coordinately acting to meet specific physiologic requirements. The alveoli are the niche of the most active lipid metabolic cell in the lung, the type 2 cell (T2C). T2C synthesize surfactant lipids that are an absolute requirement for respiration, including dipalmitoylphosphatidylcholine. After its synthesis and secretion into the alveoli, surfactant is recycled by the T2C or degraded by the alveolar macrophages (AM). Surfactant biosynthesis and recycling is tightly regulated, and dysregulation of this pathway occurs in many pulmonary disease processes. Alveolar lipids can participate in the development of pulmonary disease from their extracellular location in the lumen of the alveoli, and from their intracellular location in T2C or AM. External insults like smoke and pollution can disturb surfactant homeostasis and result in either surfactant insufficiency or accumulation. But disruption of surfactant homeostasis is also observed in many chronic adult diseases, including chronic obstructive pulmonary disease (COPD), and others. Sustained damage to the T2C is one of the postulated causes of idiopathic pulmonary fibrosis (IPF), and surfactant homeostasis is disrupted during fibrotic conditions. Similarly, surfactant homeostasis is impacted during acute respiratory distress syndrome (ARDS) and infections. Bioactive lipids like eicosanoids and sphingolipids also participate in chronic lung disease and in respiratory infections. We review the most recent knowledge on alveolar lipids and their essential metabolic and signaling functions during homeostasis and during some of the most commonly observed pulmonary diseases.

The bidirectional force transmission process of integrin through the cell membrane is still not well understood. Several possible mechanisms have been discussed in literature on the basis of experimental data, and in this study, we investigate these mechanisms by free and steered molecular dynamics simulations. For the first time, constant velocity pulling on the complete integrin molecule inside a dipalmitoyl-phosphatidylcholine membrane is conducted. From the results, the most likely mechanism for inside-out and outside-in signaling is the switchblade model with further separation of the transmembrane helices., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Phenothiazines are very effective antipsychotic drugs, which also have anticancer and antimicrobial activities. Despite being used in human treatment, the molecular mechanism of the biological actions of these molecules is not yet understood in detail. The role of the interactions between phenothiazines and proteins or lipid membranes has been much discussed. Herein, fourier-transform infrared (FTIR) spectroscopic studies were used to investigate the effect of three phenothiazines: fluphenazine (FPh); chlorpromazine (ChP); and propionylpromazine (PP) on the structures of a positively charged poly-l-lysine (PLL) peptide, a negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membrane, and on the mutual interactions between electrostatically associated PLL molecules and DPPC/DPPG membranes. Phenothiazine-induced alterations in the secondary structure of PLL, the conformational state (trans/gauche) of the hydrocarbon lipid chains, and the hydration of the DPPC/DPPG membrane interface were studied on the basis of amide I' vibrations, antisymmetric and symmetric stretching vibrations of the CH 2 groups of the lipid hydrocarbon chains (ν s CH 2 ), and stretching vibrations of the lipid C=O groups (νC = O), respectively. It was shown that in the presence of negatively charged DPPC/DPPG membranes, the phenothiazines were able to modify the secondary structure of charged PLL molecules. Additionally, the effect of PLL on the structure of DPPC/DPPG membranes was also altered by the presence of the phenothiazine molecules., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Controlled shock wave has its application in drug delivery via induced membrane permeability. The magnitude of the impulse force to influence the membrane permeability can be abridged via communion effect of shock wave and external applied electric field of reduced threshold. Controlled shock wave have application at targeting membrane site and are used in drug delivery. Electric field influences the phospholipid bilayer structure by creating transient nanometer-sized pores and has application in targeted chemotherapeutic drug delivery. The synergistic input compensates for increased membrane permeability, reduced threshold magnitude and time for transient poration. The hypothesis is analyzed via Molecular Dynamic (MD) simulation. MARTINI coarse grain force field is used to evaluate the changes in the permeability region of the Dipalmitoyl phosphatidylcholine (DPPC) bilayers during the effect. DPPC has been used in the previous literature to model biological membranes. The hydrophobic DPPC region showed an increased permeability during the synergistic effect via transient nanopores formed due to the perturbation. The study of the time-variant synergistic effect will allow molecular-level understanding of the dynamics of the cell membrane permeability for future drug delivery procedure.

Cultured human lung epithelial cells, particularly A549 cells, are commonly used as the in vitro model to evaluate the inhalational toxicity of nanoparticles (NPs). However, A549 cells are cancer cells that might not reflect the response of normal tissues to NP exposure. In addition, the possible influence of pulmonary surfactant also should be considered. This study used silica NPs as model NPs, and evaluated the toxicity of silica NPs to both 16HBE human bronchial epithelial cells and A549 adenocarcinomic cells, with or without the presence of pulmonary surfactant component dipalmitoyl phosphatidylcholine (DPPC). We found that silica NPs induced cytotoxicity at the concentration of 128 μg/mL in 16HBE cells but not A5490 cells, and the cytotoxicity of silica NPs to 16HBE cells was inhibited by DPPC. Intracellular reactive oxygen species (ROS) was only induced in 16HBE cells, accompanying with decreased thiol levels. Moreover, 16HBE cells internalized more silica NPs compared with A549 cells, and the internalization was reduced with the presence of DPPC in both types of cells. The retention of ABC transporter substrate Calcein was only significantly induced by silica NPs at high concentrations in 16HBE cells, and was partially reduced due to the presence of DPPC. In addition, ABC transporter inhibitor MK571 increased the toxicity of silica NPs to both types of cells, with 16HBE cells being more sensitive. Our data revealed that the cell types and pulmonary surfactant components could influence the toxicological consequences of silica NPs to human lung cells. Therefore, it is recommended that in vitro studies should carefully select suitable models to evaluate the inhalational toxicity of NPs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: After the discovery of membrane-reversible electroporation decades ago, the procedure has been used extensively in biology, biotechnology and medicine. The research on the basic mechanism has increasingly attracted attention. Although most research has focused on models that consider all atomic and molecular interactions and much atomic-level information can be obtained, the huge computational demand limits the models to simulations of only a few nanometers on the spatial scale and a few nanoseconds on the time scale. In order to more comprehensively study the reversible electroporation mechanism of phospholipid membrane on the nanoscale and at longer time intervals of up to 100 ns, we developed a dipalmitoylphosphatidylcholine (DPPC) phospholipid membrane model with the coarse-grained Martini force field. The model was tested by separately examining the morphology of the phospholipid membrane, the hydrophilic channel size, the distribution of the voltage potential on both sides of the membrane, and the movement of water molecules and ions during electroporation., Results: The results showed that the process went through several stages: (1) the formation of the pore with defects originating on the surface. (2) The maintenance of the pore. The defects expanded to large pores and the size remains unchanged for several nanoseconds. (3) Pore healing stage due to self-assembly. Phospholipid membrane shrunk and the pore size decreased until completely closed. The pores were not circular in cross-section for most of the time and the potential difference across the membrane decreased dramatically after the pores formed, with almost no restoration of membrane integrity even when the pores started to close., Conclusions: The mechanism of the reversible electroporation process on the nanoscale level, including defects, expansion, stability, and pore closing stages on a longer time scale of up to 100 ns was demonstrated more comprehensively with the coarse-grained Martini force field, which took both the necessary molecular information and the calculation efficiency into account.

Matrix vesicles (MVs) are a special class of extracellular vesicles that drive bone and dentin mineralization by providing the essential enzymes and ions for the nucleation and propagation of mineral crystals. Tissue-nonspecific alkaline phosphatase (TNAP) is an integral protein of MV membrane and participates in biomineralization by hydrolyzing extracellular pyrophosphate (PP i ), a strong mineralization inhibitor, and forming inorganic phosphate (P i ), necessary for the growth of mineral crystals inside MVs and their propagation once released in the extracellular matrix. MV membrane is enriched in cholesterol (CHOL), which influences the incorporation and activity of integral proteins in biologic membranes; however, how CHOL controls the incorporation and activity of TNAP in MV membrane has not yet been elucidated. In the present study, Langmuir monolayers were used as a MV membrane biomimetic model to assess how CHOL affects TNAP incorporation and activity. Surface pressure-area (π- A ) isotherms of binary dipalmitoilphosphatidylcholine (DPPC)/CHOL monolayers showed that TNAP incorporation increases with CHOL concentration. Infrared spectroscopy showed that CHOL influences the conformation and orientation of the enzyme. Optical-fluorescence micrographs of the monolayers revealed the tendency of TNAP to incorporate into CHOL-rich microdomains. These data suggest that TNAP penetrates more efficiently and occupies a higher surface area into monolayers with a lower CHOL concentration due to the higher membrane fluidity. However, the quantity of enzyme transferred to solid supports as well as the enzymatic activity were higher using monolayers with a higher CHOL concentration due to increased rigidity that changes the enzyme orientation at the air-solid interface. These data provide new insights regarding the interfacial behavior of TNAP and CHOL in MVs and shed light on the biochemical and biophysical processes occurring in the MV membrane during biomineralization at the molecular level.

Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, 'a respiratory quinolone', and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Two 15 μs all-atom simulations of the A 2A adenosine receptor were obtained in a ternary mixture of cholesterol, saturated phosphatidylcholine lipids, and unsaturated phosphatidylcholine lipids. An analysis of local lipid solvation is reported on the basis of a Voronoi tessellation of the upper and lower leaflets, identifying first and second solvation shells. The local environments of both the inactive state and the partially active state of the receptor are significantly enriched with unsaturated chains but depleted of cholesterol and saturated chains, relative to the bulk membrane composition. In spite of the local depletion of cholesterol, the partially active receptor binds cholesterol at three locations during the entire simulation trajectory. These long-lived interactions represent the extreme of a very broad distribution of first-solvation shell lipid lifetimes, confounding sharp distinctions between lipid interactions. The broad distributions of lifetimes also make equilibrating the local lipid environment difficult, necessitating long simulation times.

Cu(i) binds to the N-terminal metal binding domain (MBD) of hCTR1 and induces its conformational change, which promotes the interaction of the MBD with cell membranes. The membrane interaction was confirmed in living cells. This process could be the first step to initiate the cellular uptake of copper ions by hCTR1.

Toxicity is an essential parameter for drug development process and drug design. In this context, the effects of concentration of two Benzodiazepine drugs (diazepam, clonazepam) on fully hydrated dipalmitoylphosphatidylcholine (DPPC) has been studied at 323 K using molecular dynamics simulations. Various properties of bilayer such as membrane area per lipid, mass density distributions, order parameters, radial distribution functions, lateral diffusion, and electrostatic potential have been examined at three different concentrations of each drug. The location of drugs in the membrane are estimated by free energy profiles and to evaluate the results at a more detailed theoretical level, density functional theory (DFT) calculations have been carried out. It was revealed that penetration into the bilayer for diazepam is more favorable than clonazepam. On the other hand, within the fatty acid tail area both drugs are located in the middle of membrane at ∼0.75-1.5 nm from center of bilayer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Agents capable of scavenging ROS have attracted attention recently because of their potential use as antioxidative agents. Amifostine, a ROS scavenger, has the potential to be used as an antioxidant in therapeutic applications. In this study, the effect of amifostine on neutral zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic dipalmitoylphosphatidylglycerol (DPPG) model membranes' structure and dynamics is aimed to be examined by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Our results revealed that amifostine at concentrations used (1-24 mol%) does not induce any important alteration in the shape of phase transition curve and phase transition temperature in the DPPC and DPPG membranes. High concentrations of amifostine slightly increased the acyl chain flexibility of DPPC membranes in the liquid crystalline phase and DPPG membranes in the gel phase. A lessening in the dynamics of DPPC liposomes was observed for all concentrations of amifostine in both phases but slight dual effect was observed only in the gel phase as a decrease in dynamics at low concentrations and an increase at higher concentrations of amifostine in DPPG liposomes. Additionally, strong hydrogen bonding was observed for both CO and PO 2 - groups in case of DPPC and for PO 2 - groups in case of DPPG. Dehydration around the CO regions occurred in case of DPPG. Accordingly, amifostine is proposed to be interacting strongly with zwitterionic and negatively charged membrane head groups and glycerol backbone in all concentrations and because of this interaction it causes some changes in lipid order and dynamics especially at high concentrations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heroin, or diamorphine (C 21 H 23 NO 5 ), is an opium product used for various pharmaceutical and euphoric purposes. In this work, the molecular dynamics simulation study of the heroin inside the two lipid bilayers, dipalmitoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) are presented. The whole study was conducted at three different temperatures. The location of the heroin drug, the nature of the diffusion, rotational correlation function and structural variation inside both lipid bilayers is studied. Moreover, the free energy of the solvation of the drug inside both lipid bilayers is calculated. It is found that during the whole molecular dynamics study, the drug locates at the center of both lipid membranes. The effect of the temperature is not seen at the drug location. The nature of the diffusion of the heroin drug is anomalous. The radius of gyration is calculated to study the structural variations of the heroin molecule inside both lipid bilayers. It is found that the heroin molecule does not change its structure at three temperatures. From the rotational correlation function, it is seen that the drug is more hindered for rotation inside the DPPC lipid bilayer as compared to the DMPC lipid bilayer. It is applicable for all three temperatures. The rotational correlation time of the drug is decreased while the temperature of the system is increased. In the case of DMPC, there is an abrupt change in rotational correlation time while the phase is changed.

Bacillus thuringiensis is a bacterium that produces many insecticidal proteins including cytolytic proteins or Cyt toxins. Although the Cyt toxin shows specific toxicity against Dipteran insect species, the toxin binds directly to the lipid membrane without a specific protein receptor requirement. In this work, we have investigated the interaction of Cyt2Aa2 toxin with lipid bilayers composed of different lipid phases. By means of atomic force microscopy (AFM), lipid phase separation was observed for 1:1 and 4:1 M mixtures of DPPC/POPC bilayers. The exposure of Cyt2Aa2 to these lipid bilayers revealed that the toxin selectively bound to L d lipid bilayer (corresponding to POPC). In turn, it did not bind to the L o and S o phases (corresponding to DPPC). Interestingly, for the bilayer of 4:1 DPPC/POPC mixture, the binding of Cyt2Aa2 was localized at the lipid phase boundary instead of L d domain as occurred for the 1:1 DPPC/POPC bilayer. In addition, quartz crystal microbalance with dissipation experiments confirmed AFM results. In particular, the measurements showed that amount of protein bound to 1:1 DPPC/POPC (with phase separation) was half of the binding quantified for the L d phase lipid bilayer (pure POPC and 1:4 DPPC/POPC mixture). These results indicate that the lipid phase (lipid acyl chain) influences the Cyt2Aa2-lipid interaction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Computer simulations of four lipid membranes of different compositions, namely neat DPPC and PSM, and equimolar DPPC-cholesterol and PSM-cholesterol mixtures, are performed in the presence and absence of the general anesthetics diethylether and sevoflurane both at 1 and 600 bar. The results are analyzed in order to identify membrane properties that are potentially related to the molecular mechanism of anesthesia, namely that change in the same way in any membrane with any anesthetics, and change oppositely with increasing pressure. We find that the lateral lipid density satisfies both criteria: it is decreased by anesthetics and increased by pressure. This anesthetic-induced swelling is attributed to only those anesthetic molecules that are located close to the boundary of the apolar phase. This lateral expansion is found to lead to increased lateral mobility of the lipids, an effect often thought to be related to general anesthesia; to an increased fraction of the free volume around the outer preferred position of anesthetics; and to the decrease of the lateral pressure in the nearby range of the ester and amide groups, a region into which anesthetic molecules already cannot penetrate. All these changes are reverted by the increase of pressure. Another important finding of this study is that cholesterol has an opposite effect on the membrane properties than anesthetics, and, correspondingly, these changes are less marked in the presence of cholesterol. Therefore, changes in the membrane that can lead to general anesthesia are expected to occur in the membrane domains of low cholesterol content., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Post-translational lipid modification of integral membrane proteins is recognized as a key mechanism to modulate protein-protein and membrane-protein associations. Despite numerous reports of lipid-modified proteins, molecular-level understanding of the influence of lipid-modification of key membrane proteins remains elusive. This study focuses on the lipid modification of one such protein-claudin-5, a critical component of the blood-brain barrier tight junctions. Claudin-5 proteins are responsible for regulating the size and charge-selective permeability at the blood-brain interface. Palmitoylation of the claudin family of proteins is implicated in influencing the tight junction permeability in prior experimental studies. Here, we investigate the impact of palmitoylation on claudin-5 self-assembly using multiscale molecular simulations. To elucidate protein-membrane interactions, we used three model membrane compositions (endoplasmic reticulum, cholesterol-enriched endoplasmic reticulum, and plasma membrane) that mimic the complexity of cell organelles encountered by a typical membrane protein in its secretion pathway. The results show that palmitoylation enhances protein's affinity for cholesterol-rich domains in a membrane, and it can elicit a site-specific response based on the location of the palmitoyl chain on the protein. Also, in claudin-5 self-assembly, palmitoylation restricts specific protein-protein conformations. Overall, this study demonstrates the significance of post-translational lipid modification of proteins in cellular and subcellular membranes, and the impact palmitoylation can have on critical cellular functions of the protein.

The human mitochondrial outer membrane protein voltage-dependent anion channel isoform 2 (hVDAC2) is a β-barrel metabolite flux channel that is indispensable for cell survival. It is well established that physical forces imposed on a transmembrane protein by its surrounding lipid environment decide protein structure and stability. Yet, how the mitochondrial membrane and protein-lipid interplay together regulate hVDAC2 stability is unknown. Here, we combine experimental biophysical investigations of protein stability with all-atom molecular dynamics simulations to study the effect of the most abundant mitochondrial phosphocholine (PC) lipids on hVDAC2. We demonstrate experimentally that increasing the PC lipid acyl chain length from diC14:0 to diC18:0-PC has a nonlinear effect on the β-barrel. We show that protein stability is highest in diC16:0-PC, which exhibits a negative mismatch with the hVDAC2 barrel. Our simulations also reveal that structural rigidity of hVDAC2 is highest under optimal negative mismatch provided by diC16:0-PC bilayers. Further, we validate our observations by altering the physical properties of PC membranes indirectly using cholesterol. We propose that VDAC plasticity and stability in the mitochondrial outer membrane are modulated by physical properties of the bilayer., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

This study focuses on the interaction of human amyloid β-peptide (Aβ) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aβ is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aβ-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aβ. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aβ under noncrowding conditions. However, the addition of Aβ under crowding conditions induced shape deformation and aggregation to SUV resulting in multilamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aβ peptide into the interbilayer region. This phenomenon was only observed at the gel (L β ) phase. These results suggest that an intracellular crowding environment promotes Aβ-membrane interaction and a selective accumulation of Aβ peptides into the interbilayer regions.

Purpose: The inhibitors of the human isoform 5 of lactate dehydrogenase (hLDH5) have attracted growing interest as efficient anti-cancer agents. In the present paper, the interactions between an efficient hLDH5 inhibitor (N-hydroxyindole-2-carboxylic derivative) and lipid bilayers based on dipalmitoylphosphatidylcholine (DPPC) were investigated. Additionally, since interstitial acidification plays a key role in tumor pathogenesis and tumor drug therapy, the effect of acidic pH was assessed and correlated to DPPC/drug interaction., Methods: Four different techniques were used: differential scanning calorimetry, dynamic light scattering, UV-VIS second derivative spectrometry and attenuated total reflection Fourier transformed infrared spectroscopy., Results: All techniques concur in highlighting a structural change of lipid assembly, susceptible both to pH change and to the presence of the antitumor compound. Lipid vesicles appeared more compact at the lower pH, since the thermal pre-transition from the lamellar gel phase to the ripple gel phase was absent at pH 7.4 and the infrared analysis revealed a stronger acyl chain packing as well as a different hydration degree. Drug interaction was mainly detected in the lipid region including the ester linkages and the first portion of the acyl chains. Furthermore, a lower drug partitioning was recorded at pH 6.6., Conclusions: The investigated antitumor agent possesses a stable negative charge at the investigated pH values, thus the lower interaction at the acidic pH is mainly ascribable to an environmental effect on lipid assembly. Therefore, drug efficacy under tumor acid conditions may be hampered by the observed lipid membrane constraints, and suggest for the development of suitable prodrugs.

Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.

Lipid molecules and trace heavy metals are enriched in sea surface microlayer and can be transferred into the sea spray aerosol. To better understand their impact on marine aerosol generation and evolution, we investigated the interaction of trace heavy metals including Fe 3+ , Pb 2+ , Zn 2+ , Cu 2+ , Ni 2+ , Cr 3+ , Cd 2+ , and Co 2+ , with dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface. Phase behavior of the DPPC monolayer on heavy metal solutions was probed with surface pressure-area (π-A) isotherms. The conformation order and orientation of DPPC alkyl chains were characterized by infrared reflection-absorption spectroscopy (IRRAS). The π-A isotherms show that Zn 2+ and Fe 3+ strongly interact with DPPC molecules, and induce condensation of the monolayers in a concentration-dependent manner. IRRAS spectra show that the formation of cation-DPPC complex gives rise to conformational changes and immobilization of the headgroups. The current results suggest that the enrichment of Zn 2+ in sea spray aerosols is due to strong binding to the DPPC film. The interaction of Fe 3+ with DPPC monolayers can significantly influence their surface organizations through the formation of lipid-coated particles. These results suggest that the sea surface microlayer is capable of accumulating much higher amounts of these metals than the subsurface water. The organic and metal pollutants may transfer into the atmosphere by this interaction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The capability of coarse-grained models based on the MARTINI mapping to reproduce the gel-liquid phase transition in saturated and unsaturated model lipids was investigated. We found that the model is able to reproduce a lower critical temperature for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with respect to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Nonetheless, the appearance of a gel phase for DOPC is strictly dependent on the intramolecular parameters chosen to model its molecular structure. In particular, we show that the bending angle at the coarse-grained bead corresponding to the unsaturated carbon-carbon bond acts as an order parameter determining the temperature of the phase transition. Structural analysis of the molecular dynamics simulations runs evidences that in the gel phase, the packing of the lipophilic tails of DOPC assume a different conformation than in the liquid phase. In the latter phase, the DOPC geometry resembles that of the relaxed free molecule. DPPC:DOPC mixtures show a single phase transition temperature, indicating that the observation of a phase separation between the two lipids requires the simulation of systems with sizes much larger than the ones used here., (Copyright © 2017. Published by Elsevier Inc.)

Thermodynamics of the permeation of amino acids from water to lipid bilayers is an important first step for understanding the mechanism of cell-permeating peptides and the thermodynamics of membrane protein structure and stability. In this work, we employed bias-exchange metadynamics simulations to simulate the membrane permeation of all 20 amino acids from water to the center of a dipalmitoylphosphatidylcholine (DPPC) membrane (consists of 256 lipids) by using both directional and torsion angles for conformational sampling. The overall accuracy for the free energy profiles obtained is supported by significant correlation coefficients (correlation coefficient at 0.5-0.6) between our results and previous experimental or computational studies. The free energy profiles indicated that (1) polar amino acids have larger free energy barriers than nonpolar amino acids; (2) negatively charged amino acids are the most difficult to enter into the membrane; and (3) conformational transitions for many amino acids during membrane crossing is the key for reduced free energy barriers. These results represent the first set of simulated free energy profiles of membrane crossing for all 20 amino acids., Competing Interests: The authors declare no conflict of interest.

The interaction of pancreatic lipase-related protein 2 (PLRP2) with various micelles containing phospholipids was investigated using pHstat enzyme activity measurements, differential light scattering, size exclusion chromatography (SEC) and transmission IR spectroscopy. Various micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and lysophosphatidylcholine were prepared with either bile salts (sodium taurodeoxycholate or glycodeoxycholate) or Triton X-100, which are substrate-dispersing agents commonly used for measuring phospholipase activities. PLRP2 displayed a high activity on all phospholipid-bile salt micelles, but was totally inactive on phospholipid-Triton X-100 micelles. These findings clearly differentiate PLRP2 from secreted pancreatic phospholipase A2 which is highly active on both types of micelles. Using an inactive variant of PLRP2, SEC experiments allowed identifying two populations of PLRP2-DPPC-bile salt complexes corresponding to a high molecular weight 1:1 PLRP2-micelle association and to a low molecular weight association of PLRP2 with few monomers of DPPC/bile salts. IR spectroscopy analysis showed how DPPC-bile salt micelles differ from DPPC-Triton X-100 micelles by a higher fluidity of acyl chains and higher hydration/H-bonding of the interfacial carbonyl region. The presence of bile salts allowed observing changes in the IR spectrum of DPPC upon addition of PLRP2 (higher rigidity of acyl chains, dehydration of the interfacial carbonyl region), while no change was observed with Triton X-100. The differences between these surfactants and their impact on substrate recognition by PLRP2 are discussed, as well as the mechanism by which high and low molecular weight PLRP2-DPPC-bile salt complexes may be involved in the overall process of DPPC hydrolysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Usual methods for the continuous assay of lipolytic enzyme activities are mainly based on the titration of free fatty acids, surface pressure monitoring or spectrophotometry using substrates labeled with specific probes. These approaches only give a partial information on the chemistry of the lipolysis reaction and additional end-point analyses are often required to quantify both residual substrate and lipolysis products. We used transmission infrared (IR) spectroscopy to monitor simultaneously the hydrolysis of phospholipids by guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the release of lipolysis products. The substrate (DPPC, 1,2-Dipalmitoyl phosphatidylcholine) was mixed with sodium taurodeoxycholate (NaTDC) to form mixed micelles in D 2 O buffer at pD 6 and 8. After hydrogen/deuterium exchange, DPPC hydrolysis by GPLRP2 (100nM) was monitored at 35°C in a liquid cell by recording IR spectra and time-course variations in the CO stretching region. These changes were correlated to variations in the concentrations of DPPC, lysophospholipids (lysoPC) and palmitic acid (Pam) using calibration curves established with these compounds individually mixed with NaTDC. We were thus able to quantify each compound and its time-course variations during the phospholipolysis reaction and to estimate the enzyme activity. To validate the IR analysis, variations in residual DPPC, lysoPC and Pam were also quantified by thin-layer chromatography coupled to densitometry and similar hydrolysis profiles were obtained using both methods. IR spectroscopy can therefore be used to monitor the enzymatic hydrolysis of phospholipids and obtain simultaneously chemical and physicochemical information on substrate and all reaction products (H-bonding, hydration, acyl chain mobility)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effect of cholesterol (CHOL) content on the permeability and fluidity of dipalmitoylphosphatidylcholine (DPPC) liposome membrane was investigated. Liposomes encapsulating sulforhodamine B (SRB), a fluorescent dye, were prepared by reverse phase evaporation technique (REV) at various DPPC:CHOL molar ratios (from 100:0 to 100:100). The release kinetics of SRB was studied during 48 h in buffer (pH 7.4) containing NaCl at 37 °C. The DPPC:CHOL formulations were also characterized for their size, polydispersity index and morphology. Increasing CHOL concentration induced an increase in the mean liposomes size accompanying with a shape transition from irregular to nanosized, regular and spherical vesicles. The release kinetics of SRB showed a biphasic pattern; the release data was then analyzed using different mathematical models. On the overall, the SRB release was governed by a non-Fickian diffusion during the first period (0-10 h) while it followed a Fickian diffusion between 10 and 48 h. Changes in DPPC liposome membrane fluidity of various batches (CHOL% 0, 10, 20, 30 and 100) were monitored by using 5- and 16 doxyl stearic acids (DSA) as spin labels. CHOL induced a decrease in the bilayer fluidity. Concisely, CHOL represents a critical component in modulating the release of hydrophilic molecules from lipid vesicles., (Copyright © 2018. Published by Elsevier Ltd.)

Lensoside Aβ, representing the flavonol glycosides, is a compound isolated from the aerial parts of edible lentil (Lens culinaris) cultivar Tina. This substance arouses interest because so far there is very little data about secondary metabolites isolated from the leaves and stems of this plant. Additionally, bioactive potential of flavonoids is directly coupled with the membranes as a primary target of their physiological and pharmacological activity. The aim of this study was to investigate the effect of lensoside Aβ on lipid membranes. Interaction of examined compound with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated with application of FTIR spectroscopy and 1 H NMR technique. Molecular localization and orientation of lensoside Aβ in a single lipid bilayer system represented by giant unilamellar vesicles, was also investigated with application of confocal fluorescence lifetime imaging microscopy (FLIM). FTIR analysis revealed that the tested compound incorporates into DPPC membranes via hydrogen bonding to lipid polar head groups in the PO 2 group region and the COPOC segment. Furthermore 1 H NMR analysis showed ordering effect in both the hydrophobic alkyl chains region and the polar heads of phospholipids. FLIM investigation has revealed roughly parallel orientation of its molecules in the membranes. This suggests that one of the possible physiological functions of this flavonol could be screening a cell against short-wavelength radiation., (Copyright © 2017. Published by Elsevier B.V.)

The purpose of the present work was to assess the ability of five new oxicam analogues to interact with the lipid bilayers. To characterize the interaction of newly synthesized NSAIDs (non-steroidal anti-inflammatory drugs) analogues with DPPC lipid bilayers the two following techniques were applied - differential scanning calorimetry (DSC) and fluorescence spectroscopy. The results obtained by these experimental approaches show that new oxicams analogues interact with the lipid model membranes under consideration. As demonstrated both in calorimetric and spectroscopic studies, the greatest influence on the thermotropic properties of the lipid membrane and on the quenching of fluorescence of Laurdan and Prodan was exerted by a derivative named PR47 containing in its structure a two-carbon aliphatic linker with a carbonyl group, as well as bromine and trifluoromethyl substituents.

The present work describes the photophysical behavior of a saturated fatty acid (palmitic acid) containing N-acetylated dansylamide derivative (DAN-PA) into biologically important organized assembly such as β-cyclodextrin (β-CD), Tween-20 (T-20) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayer membrane. The results were compared by using another N-acetylated dansylamide conjugate having a short hydrophobic tail, DAN-ACYL. Long hydrophobic tail (saturated fatty acid) containing dansylamide conjugate (DAN-PA) shows more efficient binding interactions with the β-CD as compared to the short tail containing dansylamide derivative (DAN-ACYL). The calculated binding constants values of DAN-PA and DAN-ACYL probes are 1.35 × 10 2 M -1 and 0.31 × 10 2 M -1 respectively. The DAP-PA is a sensitive fluorophore for understanding the micellization process in T-20, as compared to the DAN-ACYL because it shows a significant change in fluorescent properties (steady-state and time-resolved both) with changing in T-20 concentrations. The calculated CMC value for T-20 surfactant is 0.07 mM. While the DAN-ACYL does not show any change in the fluorescent properties while changing the T-20 concentrations. Fluorescent parameters like steady-state and time-resolved of DAN-PA are quite sensitive towards the thermo-tropic phase transitional changes into lipid bilayer membrane properties. And the calculated thermo-tropic phase transition temperature by using DAN-PA fluorophore is 42 °C.

Biological membranes are a crucial aspect of living systems, controlling the organization and distribution of different chemical components. Control of membrane permeability is especially important for processes such as electron transport in metabolism and signal propagation in nerve cells. In this work, we show that the amino acid phenylalanine produces increased membrane permeability, which is likely responsible for some of the deleterious symptoms associated with high biological phenylalanine concentrations that occur with the genetic disorder phenylketonuria.

Membrane tension plays various critical roles in the cell. We here asked how fast and how far localized pulses of mechanical stress dynamically propagate through biological lipid bilayers. In both coarse-grained and all-atom molecular dynamics simulations of a dipalmitoylphosphatidylcholine lipid bilayer, we observed nanometer-wide stress pulses, propagating very efficiently longitudinally at a velocity of approximately 1.4 ± 0.5 nm/ps (km/s), in close agreement with the expected speed of sound from experiments. Remarkably, the predicted characteristic attenuation time of the pulses was in the order of tens of picoseconds, implying longitudinal stress propagation over length scales up to several tens of nanometers before damping. Furthermore, the computed dispersion relation leading to such damping was consistent with proposed continuum viscoelastic models of propagation. We suggest this mode of stress propagation as a potential ultrafast mechanism of signaling that may quickly couple mechanosensitive elements in crowded biological membranes.

An alternative formulation for the treatment of diabetic foot wounds that heal slowly is a requirement in pharmaceutical field. The aim of this study was to develop a dermal matrix consisting of skin proteins and lipids with an antioxidant that will enhance healing and balance the oxidative stress in the diabetic wound area due to the high levels of glucose. Thus a novel three dimensional collagen-laminin porous dermal matrix was developed by lyophilization. Resveratrol-loaded hyaluronic acid and dipalmitoylphosphatidylcholine microparticles were combined with this dermal matrix. Characterization, in vitro release, microbiological and in vivo studies were performed. Spherical microparticles were obtained with a high RSV encapsulation efficacy. The microparticles were well dispersed in the dermal matrix from the surface to deeper layers. Collagenase degraded dermal matrix, however the addition of RSV loaded microparticles delayed the degradation time. The release of RSV was sustained and reached 70% after 6h. Histological changes and antioxidant parameters in different treatment groups were investigated in full-thickness excision diabetic rat model. Collagen fibers were intense and improved by the presence of formulation without any signs of inflammation. The highest healing score was obtained with the dermal matrix impregnated with RSV-microparticles with an increased antioxidant activity. Collagen-laminin dermal matrix with RSV microparticles was synergistically effective due to presence of skin components in the formulation and controlled release achieved. This combination is a safe and promising option for the treatment of diabetic wounds requiring long recovery., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Familial Alzheimer's disease (FAD) is passed down in family, which account for 2-3% of about 40 million AD cases worldwide. The Flemish (A21G) mutant of amyloid β (Aβ) exhibits unique properties among all hereditary mutants of FAD, including the lowest aggregation rate. Recent studies showed that Aβ oligomers play a key role in Alzheimer's disease (AD) pathogenesis. They could insert themselves in brain cell membrane, disrupting the membrane integrity and ion homeostasis. However, experimental studies of transmembrane Aβ oligomers have been limited due to their intrinsic heterogeneity. In this work, we extensively studied the A21G mutant of the transmembrane 3Aβ 11-40 (A21G 3Aβ 11-40 ) using temperature replica exchange molecular dynamics (REMD) simulations. Results provide detailed information on the conformational distribution and dynamics of transmembrane A21G 3Aβ 11-40 . Minimal local change from A to G leads to significant conformational changes and wider free energy holes on the free energy surface as well as altered surface charges that lead to weaker affinity to the dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. These results are consistent with experimental data that showed that A21G mutants of Aβ peptides have lower aggregation rates and membrane binding rates.

Lung surfactant proteins (SPs) play critical roles in surfactant function and innate immunity. SP-A and SP-D, members of the collectin family of C-type lectins, exhibit distinct ligand specificities, effects on surfactant structure, and host defense functions despite extensive structural homology. SP-A binds to dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SP-D recognize widely divergent pathogen-associated molecular patterns. Previous studies suggested that a ligand-induced surface loop conformational change unique to SP-A contributes to lipid binding affinity. To test this hypothesis and define the structural features of SP-A and SP-D that determine their ligand binding specificities, a structure-guided approach was used to introduce key features of SP-D into SP-A. A quadruple mutant (E171D/P175E/R197N/K203D) that introduced an SP-D-like loop-stabilizing calcium binding site into the carbohydrate recognition domain was found to interconvert SP-A ligand binding preferences to an SP-D phenotype, exchanging DPPC for PI specificity, and resulting in the loss of lipid A binding and the acquisition of more avid mannan binding properties. Mutants with constituent single or triple mutations showed alterations in their lipid and sugar binding properties that were intermediate between those of SP-A and SP-D. Structures of mutant complexes with inositol or methyl-mannose revealed an attenuation of the ligand-induced conformational change relative to wild-type SP-A. These studies suggest that flexibility in a key surface loop supports the distinctive lipid binding functions of SP-A, thus contributing to its multiple functions in surfactant structure and regulation, and host defense.

Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)