Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

Bound Phospholipids Assist Cholesteryl Ester Transfer in the Cholesteryl Ester Transfer Protein.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that assists the transfer of cholesteryl esters (CEs) from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs), initiating cholesterol plaques in the arteries. Consequently, inhibiting the activity of CETP is therefore being pursued as a novel strategy to reduce the risk of cardiovascular diseases (CVDs). The crystal structure of CETP has revealed the presence of two CEs running in the hydrophobic tunnel and two plugged-in phospholipids (PLs) near the concave surface. Other than previous animal models that rule out the PL transfer by CETP and PLs in providing the structural stability, the functional importance of bound phospholipids in CETP is not fully explored. Here, we employ a series of molecular dynamics (MD) simulations, steered molecular dynamics (SMD) simulations, and free energy calculations to unravel the effect of PLs on the functionality of the protein. Our results suggest that PLs play an important role in the transfer of neutral lipids by transforming the unfavorable bent conformation of CEs into a favorable linear conformation to facilitate the smooth transfer. The results also suggest that the making and breaking interactions of the hydrophobic tunnel residues with CEs with a combined effort from PLs are responsible for the transfer of CEs. Further, the findings demonstrate that the N-PL has a more pronounced effort on CE transfer than C-PL but efforts from both PLs are essential in the transfer. Thus, we propose that the functionally important PLs can be considered with potential research interest in targeting cardiovascular diseases.

Universal phase behaviors of intracellular lipid droplets.

Lipid droplets are cytoplasmic microscale organelles involved in energy homeostasis and handling of cellular lipids and proteins. The core structure is mainly composed of two kinds of neutral lipids, triglycerides and cholesteryl esters, which are coated by a phospholipid monolayer and proteins. Despite the liquid crystalline nature of cholesteryl esters, the connection between the lipid composition and physical states is poorly understood. Here, we present a universal intracellular phase diagram of lipid droplets, semiquantitatively consistent with the in vitro phase diagram, and reveal that cholesterol esters cause the liquid-liquid crystal phase transition under near-physiological conditions. We moreover combine in vivo and in vitro studies, together with the theory of confined liquid crystals, to suggest that the radial molecular alignments in the liquid crystallized lipid droplets are caused by an anchoring force at the droplet surface. Our findings on the phase transition of lipid droplets and resulting molecular organization contribute to a better understanding of their biological functions and diseases.

Mechanistic Insight into How PEGylation Reduces the Efficacy of pH-Sensitive Liposomes from Molecular Dynamics Simulations.

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE-CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.

Cervidins A-D: Novel Glycine Conjugated Fatty Acids from the Tarsal Gland of Male Whitetail Deer, Odocoileus virginianus.

Sexually mature male deer are known to rub-urinate, a process where urine is deposited on the tarsal gland. The resulting mixture of compounds from urine and secretions from the tarsal gland are used to signal sex, age, maturation status, and other information at close distance. We examined the difference in metabolites of tarsal gland extracts from male and female whitetail deer, Odocoileus virginianus, harvested during the mating season. Using NMR spectroscopy and high-pressure liquid chromatography linked to high resolution mass spectrometry (HPLC/HR-MS) we identified a homologous series of four male-specific compounds. The compounds are novel glycine conjugates of 10-hydroxy-6,9-oxido fatty acids, which we term cervidins A-D. Cervidins were deemed to possess the absolute configuration 6S,9R,10R through comparison of their spectroscopic data with those of known compounds. In addition, cholesterol 3-sulfate and 3-(3-hydroxyphenyl)-propanoic acid were found to be present in the extracts. Our results clearly demonstrate the diversity of potential semiochemicals contained in the mammalian integument.

Mutations in SULT2B1 Cause Autosomal-Recessive Congenital Ichthyosis in Humans.

Ichthyoses are a clinically and genetically heterogeneous group of genodermatoses associated with abnormal scaling of the skin over the whole body. Mutations in nine genes are known to cause non-syndromic forms of autosomal-recessive congenital ichthyosis (ARCI). However, not all genetic causes for ARCI have been discovered to date. Using whole-exome sequencing (WES) and multigene panel screening, we identified 6 ARCI-affected individuals from three unrelated families with mutations in Sulfotransferase family 2B member 1 (SULT2B1), showing their causative association with ARCI. Cytosolic sulfotransferases form a large family of enzymes that are involved in the synthesis and metabolism of several steroids in humans. We identified four distinct mutations including missense, nonsense, and splice site mutations. We demonstrated the loss of SULT2B1 expression at RNA and protein levels in keratinocytes from individuals with ARCI by functional analyses. Furthermore, we succeeded in reconstructing the morphologic skin alterations in a 3D organotypic tissue culture model with SULT2B1-deficient keratinocytes and fibroblasts. By thin layer chromatography (TLC) of extracts from these organotypic cultures, we could show the absence of cholesterol sulfate, the metabolite of SULT2B1, and an increased level of cholesterol, indicating a disturbed cholesterol metabolism of the skin upon loss-of-function mutation in SULT2B1. In conclusion, our study reveals an essential role for SULT2B1 in the proper development of healthy human skin. Mutation in SULT2B1 leads to an ARCI phenotype via increased proliferation of human keratinocytes, thickening of epithelial layers, and altered epidermal cholesterol metabolism.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation.

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3ß-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Steering the Lipid Transfer To Unravel the Mechanism of Cholesteryl Ester Transfer Protein Inhibition.

Human plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron microscopy studies have suggested that CETP penetrates its N- and C-terminal domains in HDL and LDL to form a ternary complex, which facilitates the lipid transfer between different lipoproteins. Inhibition of CETP lipid transfer activity has been shown to increase the plasma HDL-C levels and, therefore, became an effective strategy for combating cardiovascular diseases. Thus, understanding the molecular mechanism of inhibition of lipid transfer through CETP is of paramount importance. Recently reported inhibitors, torcetrapib and anacetrapib, exhibited low potency in addition to severe side effects, which essentially demanded a thorough knowledge of the inhibition mechanism. Here, we employ steered molecular dynamics simulations to understand how inhibitors interfere with the neutral lipid transfer mechanism of CETP. Our study revealed that inhibitors physically occlude the tunnel posing a high energy barrier for lipid transfer. In addition, inhibitors bring about the conformational changes in CETP that hamper CE passage and expose protein residues that disrupt the optimal hydrophobicity of the CE transfer path. The atomic level details presented here could accelerate the designing of safe and efficacious CETP inhibitors.

Circulating fatty acids as biomarkers of dairy fat intake: data from the lifelines biobank and cohort study.

Background: C14:0, C15:0, C17:0 and trans-C16:1(n-7) are often used as biomarkers for dairy fat intake. Trans-C18:1(n-7) and CLA, two fatty acids which are also present in dairy, have hardly been explored. We investigated whether trans-C18:1(n-7) and CLA can enrich the existing biomarker portfolio. Methods: Data were obtained from Lifelines (n = 769). Dairy fat intake was determined by FFQ. Fatty acids were measured in fasting plasma triglycerides (TG), phospholipids (PL) and cholesterol esters (CE). Results: Median (25th-75th percentile) intakes of dairy and dairy fat were 322(209-447) and 12.3(8.4-17.4) g/d respectively. A pilot study showed that trans-C18:1(n-7) and CLA were only detectable in TG and PL. Of the established markers, TG C15:0 was most strongly associated with dairy fat intake (standardized ß (std.ß) = 0.286, R2 = 0.111). Of the less established markers, TG trans-C18:1(n-7) was most strongly associated with dairy fat intake (Std.ß = 0.292, R2 = 0.115), followed by PL CLA (Std.ß = 0.272, R2 = 0.103) and PL trans-C18:1(n-7) (Std.ß = 0.269, R2 = 0.099). In TG, a combination of C15:0 and trans-C18:1(n-7) performed best (R2 = 0.128). In PL, a combination of C14:0, C15:0, trans-C18:1(n-7) and CLA performed best (R2 = 0.143). Conclusion: Trans-C18:1(n-7) and CLA can be used as biomarkers of dairy fat intake. Additionally, combining established with less established markers allowed even stronger predictions for dairy fat intake.

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2.

Export of LDL-derived cholesterol from lysosomes requires the cooperation of the integral membrane protein Niemann-Pick C1 (NPC1) and a soluble protein, Niemann-Pick C2 (NPC2). Mutations in the genes encoding these proteins lead to Niemann-Pick disease type C (NPC). NPC2 binds to NPC1's second (middle), lumenally oriented domain (MLD) and transfers cholesterol to NPC1's N-terminal domain (NTD). Here, we report the 2.4-Å resolution crystal structure of a complex of human NPC1-MLD and NPC2 bearing bound cholesterol-3-O-sulfate. NPC1-MLD uses two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. Docking of the NPC1-NPC2 complex onto the full-length NPC1 structure reveals a direct cholesterol transfer tunnel between NPC2 and NTD cholesterol binding pockets, supporting the "hydrophobic hand-off" cholesterol transfer model.

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1.

Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface-bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell-cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141-249, which does not include the serine protease inhibitor domain, has the cell aggregation-inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1-mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7-catalyzed generation of sHAI-1. Considering that MMP-7-induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7-induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies.

PEG-Benzaldehyde-Hydrazone-Lipid Based PEG-Sheddable pH-Sensitive Liposomes: Abilities for Endosomal Escape and Long Circulation.

PURPOSE: To fabricate an acid-cleavable PEG polymer for the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), and to investigate their ability for endosomal escape and long circulation. METHODS: PEG-benzaldehyde-hydrazone-cholesteryl hemisuccinate (PEGB-Hz-CHEMS) containing hydrazone and ester bonds was synthesised and used to fabricate a dual pH-sensitive CL-pPSL. Non-cleavable PEGylated pH-sensitive liposome (pPSL) was used as a reference and gemcitabine as a model drug. The cell uptake and endosomal escape were investigated in pancreatic cancer Mia PaCa-2 cells and pharmacokinetics were studied in rats. RESULTS: The CL-pPSL showed accelerated drug release at endosomal pH 5.0 compared to pPSL. Compared to pPSL, CL-pPSL released their fluorescent payload to cytosol more efficiently and showed a 1.4-fold increase in intracellular gemcitabine concentration and higher cytotoxicity. In rats, injection of gemcitabine loaded CL-pPSL resulted in a slightly smaller Vd (149 ± 27 ml/kg; 170 ± 30 ml/kg) and shorter terminal T1/2 (5.4 ± 0.3 h; 5.8 ± 0.6 h) (both p > 0.05) but a significantly lower AUC (p < 0.01), than pPSL, due to the lower PEGylation degree (1.7 mol%) which means a 'mushroom' configuration of PEG. A five-time increase in the dose with CL-pPSL resulted in a 11-fold increase in AUC and a longer T1/2 (8.2 ± 0.5 h). CONCLUSION: The PEG-detachment from the CL-pPSL enhanced endosome escape efficiency compared with pPSL, without significantly compromising their stealth abilities.

Structural Plasticity of Cholesteryl Ester Transfer Protein Assists the Lipid Transfer Activity.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CEs) and triglycerides between different lipoproteins. Recent studies have shown that blocking the function of CETP can increase the level of HDL cholesterol in blood plasma and suppress the risk of cardiovascular disease. Hence, understanding the structure, dynamics, and mechanism by which CETP transfers the neutral lipids has received tremendous attention in last decade. Although the recent crystal structure has provided direct evidence of the existence of strongly bound CEs in the CETP core, very little is known about the mechanism of CE/triglyceride transfer by CETP. In this study, we explore the large scale dynamics of CETP by means of multimicrosecond molecular dynamics simulations and normal mode analysis, which provided a wealth of detailed information about the lipid transfer mechanism of CETP. Results show that the bound CEs intraconvert between bent and linear conformations in the CETP core tunnel as a consequence of the high degree of conformational flexibility of the protein. During the conformational switching, there occurred a significant reduction in hydrophobic contacts between the CEs and CETP, and a continuous tunnel traversing across the CETP long axis appeared spontaneously. Thus, our results support the recently proposed "tunnel mechanism" of CETP from cryo-EM studies for the transfer of neutral lipids between different lipoproteins. The detailed understanding obtained here could help in devising methods to prevent CETP function as a cardiovascular disease therapeutic.

A calorimetric and spectroscopic comparison of the effects of cholesterol and its sulfur-containing analogs thiocholesterol and cholesterol sulfate on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes.

We performed differential scanning calorimetric (DSC) and Fourier transform infrared (FTIR) spectroscopic studies of the effects of cholesterol (Chol), thiocholesterol (tChol) and cholesterol sulfate (CholS) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. Our DSC results indicate that Chol and tChol incorporation produce small temperature increases in the main phase transition broad component while CholS markedly decreases it, but Chol decreases cooperativity and enthalpy more strongly than CholS and especially tChol. Hence, Chol and tChol thermally stabilize fluid DPPC bilayer sterol-rich domains while CholS markedly destabilizes them, and CholS and particularly tChol are less miscible in such domains. Our FTIR spectroscopic results indicate that Chol incorporation increases the rotational conformational order of fluid DPPC bilayers to a slightly and somewhat greater degree than tChol and CholS, respectively, consistent with our DSC findings. Also, Chol and CholS produce comparable degrees of H-bonding (hydration) of the DPPC ester carbonyls in fluid bilayers, whereas tChol increases H-bonding. At low temperatures, Chol is fully soluble in gel-state DPPC bilayers, whereas tChol and CholS are not. Thus tChol and CholS incorporation can produce considerably different effects on DPPC bilayers. In particular, the tChol thiol group markedly reduces its lateral miscibility and increases DPPC carbonyl H-bonding without significantly affecting the other characteristic effects of Chol itself, while the CholS sulfate group significantly reduces its ability to thermally stabilize and order fluid DPPC membranes. This latter result suggests that the molecular basis for the purported ability of CholS to "stabilize" various biological membranes should be re-examined.

Tear film lipid layer: A molecular level view.

Human cornea is covered by an aqueous tear film, and the outermost layer of the tear film is coated by lipids. This so-called tear film lipid layer (TFLL) reduces surface tension of the tear film and helps with the film re-spreading after blinks. Alterations of tear lipids composition and properties are related to dry eye syndrome. Therefore, unveiling structural and functional properties of TFLL is necessary for understanding tear film function under both normal and pathological conditions. Key properties of TFLL, such as resistance against high lateral pressures and ability to spread at the tear film surface, are directly related to the chemical identity of TFLL lipids. Hence, a molecular-level description is required to get better insight into TFLL properties. Molecular dynamics simulations are particularly well suited for this task and they were recently used for investigating TFLL. The present review discusses molecular level organization and properties of TFLL as seen by these simulation studies. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

High density lipoprotein efficiently accepts surface but not internal oxidised lipids from oxidised low density lipoprotein.

OBJECTIVE: Oxidised low density lipoprotein (oxLDL) contributes to atherosclerosis, whereas high density lipoprotein (HDL) is known to be atheroprotective due, at least in part, to its ability to remove oxidised lipids from oxLDL. The molecular details of the lipid transfer process are not fully understood. We aimed to identify major oxidised lipid species of oxLDL and investigate their transfer upon co-incubation with HDL with varying levels of oxidation. APPROACH AND RESULTS: A total of 14 major species of oxidised phosphatidylcholine and oxidised cholesteryl ester from oxLDL were identified using an untargeted mass spectrometry approach. HDL obtained from pooled plasma of normolipidemic subjects (N=5) was oxidised under mild and heavy oxidative conditions. Non-oxidised (native) HDL and oxidised HDL were co-incubated with oxLDL, re-isolated and lipidomic analysis was performed. Lipoprotein surface lipids, oxidised phosphatidylcholines and oxidised cholesterols (7-ketocholesterol and 7ß-hydroxycholesterol), but not internal oxidised cholesteryl esters, were effectively transferred to native HDL. Saturated and monounsaturated lyso-phosphatidylcholines were also transferred from the oxLDL to native HDL. These processes were attenuated when HDL was oxidised under mild and heavy oxidative conditions. The impaired capacities were accompanied by an increase in a ratio of sphingomyelin to phosphatidylcholine and a reduction in phosphatidylserine content in oxidised HDL, both of which are potentially important regulators of the oxidised lipid transfer capacity of HDL. CONCLUSIONS: Our study has revealed the differential transfer efficiency of surface and internal oxidised lipids from oxLDL and their acceptance onto HDL. These capacities were modulated when HDL was itself oxidised.