Three dimensional mapping of neuropeptides and lipids in crustacean brain by mass spectral imaging

Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. To confirm their identities, MS/MS fragmentation was performed. Different lipid species displayed distinct localization patterns, suggesting their potential different functional roles in the nervous system.     

The determination of polychlorinated biphenyl in small samples of monkey milk and tissues. II. Extraction efficiency

The extraction efficiency of benzene, toluene, dichloromethane, acetone:hexane and chloroform:methanol with respect to lipids and polychlorinated biphenyls was investigated using small samples of monkey adipose tissue, liver, kidney, brain, skin, feces and milk. The most efficient solvents were: acetone:hexane and chloroform:methanol for brain, feces, kidney, liver and milk; acetone:hexane and dichloromethane for adipose tissue; acetone:hexane and toluene for blood and dichloromethane for skin tissue. Within these solvent pairs acetone:hexane was the most outstanding with respect to an average of 90% PCB recoveries from fortified samples in the range of 0.02-2 ppm. In addition, a comparison was made between the lipid determination before and after Florisil column chromatography. Only adipose and blood lipids were sufficiently recovered from Florisil to make a lipid determination after chromatography feasible.     

Dose response for UV-induced immune suppression in people of color: differences based on erythemal reactivity rather than skin pigmentation.

Ultraviolet radiation (UVR) is known to suppress immune responses in human subjects. The purpose of this study was to develop dose responses across a broad range of skin pigmentation in order to facilitate risk assessment. UVR was administered using FS 20 bulbs. Skin pigmentation and UVR sensitivity were evaluated using Fitzpatrick classifications, minimal erythemal dose (MED), slope of the erythemal dose response curve (sED), baseline pigmentation and tanning response. To assess immune responses dinitrochlorobenzene (DNCB) was applied to irradiated buttock skin 72 h after irradiation. Two weeks later DNCB was applied to the inside upper arm. Skin thickness was measured before and after challenge. Dose response was modeled (to obtain a regression line) for the entire group of 185 subjects. With the exception of sED none of the above-mentioned pigmentation indicators contributed significantly to variability around the regression line. Thus, differences in sensitivity for multiple skin types based on Fitzpatrick classification or MED were not observed. However, differences in immune sensitivity to UVR were detected between subjects with steep erythemal dose response curves and those with moderate or flat responses. For subjects with steep erythemal responses the dose calculated to suppress the immune response by 50% was 114 mJ/cm2. This group included individuals with Fitzpatrick skin types I-V, MED for these subjects ranged from 30 to 80 mJ/cm2. The 50% suppression dose for subjects with weak or no erythemal response could not be computed (the dose response was flat). This resistant group included subjects with skin types IV-VI and MED for these subjects ranged from 41 to > 105 mJ/cm2. This study provides a human dose response for UVR suppression of contact sensitivity that will be useful in risk assessment. It is the first study to provide this information using the FS sun lamp and is the first study to include people of color. The sED appears to be a new variable for identifying sensitive subjects at risk of UVR-induced immune suppression.     

MALDI-TOF and cluster-TOF-SIMS imaging of Fabry disease biomarkers

Fabry disease is an X-linked disorder of glycosphingolipid metabolism, in which a partial or total deficiency of α-galactosidase A, a lysosomal enzyme, results in the progressive accumulation of neutral glycosphingolipids (globotriaosylceramide and digalactosylceramide) in most fluids and tissues of the body. Few information is available about the composition and distribution in tissues of the accumulated glycosphingolipids species. Mass spectrometry imaging is an innovative technique, which can provide pieces of information about the distribution of numerous biological compounds, such as lipids, directly on the tissue sections. MALDI-TOF and cluster-TOF-SIMS imaging approaches were used to study the localization of lipids (cholesterol, cholesterol sulfate, vitamin E, glycosphingolipids …) on skin and kidney sections of patients affected by the Fabry disease. Numerous information on pathophysiology were enlightened by both techniques.     

Soapwort (Saponaria officinalis L.) Extract vs. Synthetic Surfactants—Effect on Skin-Mimetic Models

Our skin is continuously exposed to different amphiphilic substances capable of interaction with its lipids and proteins. We describe the effect of a saponin-rich soapwort extract and of four commonly employed synthetic surfactants: sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), ammonium lauryl sulfate (ALS), cocamidopropyl betaine (CAPB) on different human skin models. Two human skin cell lines were employed: normal keratinocytes (HaCaT) and human melanoma cells (A375). The liposomes consisting of a dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3, mimicking the cell membrane of keratinocytes and melanoma cells were employed as the second model. Using dynamic light scattering (DLS), the particle size distribution of liposomes was analyzed before and after contact with the tested (bio)surfactants. The results, supplemented by the protein solubilization tests (albumin denaturation test, zein test) and oil emulsification capacity (using olive oil and engine oil), showed that the soapwort extract affects the skin models to a clearly different extent than any of the tested synthetic surfactants. Its protein and lipid solubilizing potential are much smaller than for the three anionic surfactants (SLS, ALS, SLES). In terms of protein solubilization potential, the soapwort extract is comparable to CAPB, which, however, is much harsher to lipids.     

Ultrasound extraction and thin layer chromatography-flame ionization detection analysis of the lipid fraction in marine mucilage samples

This paper reports an analytical procedure based on ultrasound to extract lipids in marine mucilage samples. The experimental conditions of the ultrasound procedure (solvent and time) were identified by a FT-IR study performed on different standard samples of lipids and of a standard humic sample, before and after the sonication treatment. This study showed that diethyl ether was a more suitable solvent than methanol for the ultrasonic extraction of lipids from environmental samples because it allowed to minimize the possible oxidative modifications of lipids due to the acoustic cavitation phenomena. The optimized conditions were applied to the extraction of total lipid amount in marine mucilage samples and TLC-flame ionization detection analysis was used to identify the relevant lipid sub-fractions present in samples.     

Changes in lipid distribution in E. coli strains in response to norfloxacin

Bacterial resistance to antibiotics has become an increasing threat, requiring not only the development of new targets in drug discovery, but more importantly, a better understanding of cellular response. In the current study, three closely related Escherichia coli strains, a wild type (MG1655) and an isogenic pair derived from the wild type (DPB635 and DPB636) are studied following exposure to sub lethal concentrations of antibiotic (norfloxacin) over time. In particular, genotype similarities between the three strains were assessed based on the lipid regulation response (e.g. presence/absence and up/down regulation). Lipid identification was performed using direct surface probe analysis (matrix‐assisted laser desorption/ionization, MALDI), coupled to high‐resolution mass spectrometry (Fourier transform ion cyclotron resonance mass spectrometry, FT‐ICR MS) followed by statistical analysis of variability and reproducibility across batches using internal standards. Inspection of the lipid profile showed that for the MG1655, DPB635 and DPB636 E. coli strains, a similar distribution of the altered lipids was observed after exposure to norfloxacin antibiotic (e.g. fatty acids and glycerol phospholipids are up and down regulated, respectively). Additionally, variations in the lipid distribution resemble the extent to which each strain can combat the antibiotic exposure. That is, the topA66 topoisomerase I mutation of DPB636 translates into diminished response related to antibiotic sensitivity when compared to MG1655 and the DPB635 strains. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing the molecular-scale lipid bilayer response to shear flow using nonequilibrium molecular dynamics

A nonequilibrium molecular dynamics simulation of the response of dimyristoylphosphatidylcholine (DMPC) bilayers to a solvent shear flow is presented. Application of shear flow to planar, stationary DMPC bilayers results in a redistribution of the membrane density profile along the bilayer normal due to the alignment of the lipids in the direction of flow and an increase in average lipid chain length. An increase in the intermolecular and intramolecular order of the lipids in response to the shear flow is also observed. This study provides groundwork for understanding the mechanism of the full response of lipid bilayers to externally imposed solvent shear flows, beginning with the response in the absence of collective lipid motions such as undulations and bilayer flow.     

Interactions between Hydrolysable Tannins and Lipid Vesicles from Escherichia coli with Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) was used to study the interactions between hydrolysable tannins (HTs) and lipid vesicles prepared from a phospholipid extract of Escherichia coli (E. coli). A group of 24 structurally different HTs was selected, and structural differences affecting their affinities to interact with lipid vesicles in aqueous buffered media were identified. In general, the interactions between HTs and lipid vesicles were exothermic in nature, and ITC as a technique functioned well in the screening of HTs for their affinity for lipids. Most notably, the galloyl moiety, the structural flexibility of the entire tannin structure, the hydrophobicity of the tannin, and higher molecular weight were observed to be important for the stronger interactions with the lipids. The strongest interactions with lipids were observed for rugosins D and G. It was also observed that some HTs with moderate hydrophobicities, such as geraniin, chebulagic acid, and chebulinic acid, did not have any detectable interactions with the lipid vesicles, suggesting that a hydrophobic structure alone does not guarantee an affinity for lipids.     

MALDI-MS imaging of lipids in ex vivo human skin

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i ^TM (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS^TM system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.     

Thermal dependence of Raman descriptors of ceramides. Part II: effect of chains lengths and head group structures

The outermost layer of the mammalian skin, the stratum corneum (SC), represents the main skin barrier. The SC lipids have a very exceptional composition, as the main lipid classes are ceramides (CER), long-chain fatty acids and cholesterol. Information on the function of each CER subclass and on the relation between CER lipid organisation and composition is of great importance to unravel the mechanism controlling the skin barrier function. Raman spectroscopy has been increasingly used for the study of intra- and inter-molecular structures of long-chain lipid compounds. In this study, we employed Raman spectroscopy to evaluate the effect of (1) the chain length and (2) the polar head architecture on the conformational order and organisational behaviour of CERs. The relation between the structure and the stability of the organisation was studied by monitoring the thermotropic response of each CER in the temperature range between 25 and 95 °C. This work enabled the determination of a correlation between the gauche/trans ratio in the νCC region and the state of the lateral packing. Moreover, it was shown that –OH groups in the α position of the fatty acids reduce the stability while long alkyl chains reinforces the intra- and inter-chains order.     

Effect of UVA or UVB Irradiation on Cutaneous Lipids in Films or in Solution

The barrier function of the skin is largely due to the stratum corneum which is essentially composed of lipids. Different external factors, such as UV irradiation, affect this skin layer and are responsible for a destabilization of the supramolecular organization of its constituted lipids. In this work, mass spectrometry and infrared spectroscopy are combined to study the correlation between the formation of oxidative compounds by UV irradiation and the lipid organization. Experiments were carried out on unsaturated lipids in film or solution form, exposed to UVA or UVB irradiation. UV exposure leads to the formation of oxygenated entities in the case of lipids with an unsaturated fatty acid moiety, resulting in a decrease in their packing which is greater when the lipids are in solution. The packing decrease is even greater following UVB irradiation.     

Starch-lipid interactions studied by differential scanning calorimetry

The amylose-lipid complex shows an endothermic transition around 100 °C in excess water. Complexes were prepared by adding lipids to an amylose-solution, and the precipitated complex was studied in the DSC during a heating and cooling sequence. The thermal stability of the complex depends on the lipid part, and the reversibility during cooling depends on presence of excess lipids.The influence of lipids on the gelatinization of starch was studied by adding lipids to wheat and potato starch, respectively, before the DSC-analysis. Depending on the lipid, an earlier as well as a delayed gelatinisation could be obtained.     

Characterization of Wastes from Olive and Sugarbeet Processing Industries and Effects of Their Application upon the Organic Fraction of Agricultural Soils

Abstract

The lipidic fraction compositions of both concentrated vinasses, a by-product of the sugar industry, and a compost made basically from olive oil vegetation waters (alpechin) were studied. The alpechin lipids are composed mainly by series of n-alkanes and lineal and branched fatty acids, whereas the major lipids in vinasses were n-alkanes, n-alkanols and acetals. Concentrations and composition of lipids in both materials do not seem of concern.

Preliminary results on the effects of the application of both materials over two years on the organic status of an agricultural soil are also reported. No significant changes were observed in total organic carbon and contents in humic fractions and lipids before and after the applications. However, analysis by GC-MS of the lipid compounds present in bound forms in the subsoil layer revealed that some hydrophobic components were accumulated in the soil following the waste applications.     

Structures and rheological properties of hen egg yolk low density lipoprotein layers spread at the air-water interface at pH 3 and 7

Low density lipoproteins (LDL) from egg yolk have a classical structure of lipoprotein with a core of neutral lipids surrounded by a monolayer of apoproteins and phospholipids. This structure collapses during adsorption and all constituents spread at the interface. To understand better the nature of the interactions between apoproteins and lipids at the interface, we have deposited LDL at an air-water interface and analysed the isotherms during their compression on a Langmuir trough. Then, these LDL films were studied by atomic force microscopy (AFM) imaging. To identify the protein and lipid structures, we imaged films before and after lipid solubilisation by butanol. To study the interactions in the LDL films, we have varied the pH, ionic strength and used simplified model systems. We also studied the correlation between observed structures and interfacial rheology of the film. The isotherms of interfacial LDL films were similar for pH 3 and 7, but their structures observed in AFM were different. At surface pressures below the transition corresponding to the demixion of apoprotein-neutral lipid complexes, the LDL film structure was not governed by electrostatic interactions. However, above this surface pressure transition (45mN/m), there was an effect of charge on this structure. Around the transition zone, the rheological properties of LDL films at pH 3 were different as a function of pH (viscous at pH 3 and visco-elastic at pH 7). So, the rheological properties of LDL films could be linked to the structures formed by apoproteins and observed in AFM.     

Structure modification in hen egg yolk low density lipoproteins layers between 30 and 45 mN/m observed by AFM

We have studied the structure of films made by low density lipoproteins (LDL) from hen egg yolk, which are composed of apoproteins, neutral lipids and phospholipids. These LDL have been deposited on air–water interface to form a monolayer which has been compressed to measure an isotherm using Langmuir balance. This isotherm presented three transitions (neutral lipid (surface pressure, π = 19 mN/m), apoprotein–lipid (π = 41 mN/m) and phospholipid (π = 51 mN/m) transitions). We have studied only the apoprotein–lipid transition. In order to observe the LDL film structure before (π = 30 mN/m) and after (π = 45 mN/m) the apoprotein–lipid transition, the formed films were transferred and visualised by atomic force microscopy (AFM). Our results have shown that the structures observed in the LDL film were different depending on the surface pressure. The apoproteins and neutral lipids appeared to be miscible up to the apoprotein–lipid transition, when demixing occured. The structures observed after the apoprotein–lipid transition should be due to the demixing between apoproteins and neutral lipids. On the other hand, apoproteins and phospholipids seemed miscible whatever the surface pressure. Hence, the first transition (π = 19 mN/m) should be attributed to the free neutral lipid collapse; the second transition (π = 41 mN/m) should be attributed to the demixing of apoprotein–neutral lipid complexes; and the last transition (π = 51 mN/m) should be attributed to phospholipid collapse or to demixing of apoprotein–phospholipid complexes.     

Study of the interaction of beta-cyclodextrin with phospholipid monolayers by surface pressure measurements and fluorescence microscopy

The interaction of beta-cyclodextrin (beta-CD) with different lipids has been studied, using Langmuir monolayers kept at constant surface pressure or constant spreading surface. Results show that beta-CD, injected beneath the monolayer, is able to desorb unsaturated palmitoyloleoylphosphatidylcholine (POPC) and sphingomyelin (SM) under specific experimental conditions. In this last case, SM monolayers, labeled with the fluorescent NBD-PC probe, were also observed by fluorescence microscopy, before and after beta-CD injection. Images show that SM monolayers are more homogeneous after beta-CD injection, because of the lipid desorption. At last, it seems that lipid desorption occurs only in a restricted surface pressure range, depending on the lipid.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Imaging of human lens lipids by desorption electrospray ionization mass spectrometry

The lipid composition of the human lens is distinct from most other tissues in that it is high in dihydrosphingomyelin and the most abundant glycerophospholipids in the lens are unusual 1-O-alkyl-ether linked phosphatidylethanolamines and phosphatidylserines. In this study, desorption electrospray ionization (DESI) mass spectrometry-imaging was used to determine the distribution of these lipids in the human lens along with other lipids including, ceramides, ceramide-1-phosphates, and lyso 1-O-alkyl ethers. To achieve this, 25 μm lens slices were mounted onto glass slides and analyzed using a linear ion-trap mass spectrometer equipped with a custom-built, 2-D automated DESI source. In contrast to other tissues that have been previously analyzed by DESI, the presence of a strong acid in the spray solvent was required to desorb lipids directly from lens tissue. Distinctive distributions were observed for [M + H]⁺ ions arising from each lipid class. Of particular interest were ionized 1-O-alkyl phosphatidylethanolamines and phosphatidylserines, PE (18:1e/18:1), and PS (18:1e/18:1), which were found in a thin ring in the outermost region of the lens. This distribution was confirmed by quantitative analysis of lenses that were sectioned into four distinct regions (outer, barrier, inner, and core), extracted and analyzed by electrospray ionization tandem mass spectrometry. DESI-imaging also revealed a complementary distribution for the structurally-related lyso 1-O-alkyl phosphatidylethanolamine, LPE (18:1e), which was localized closer to the centre of the lens. The data obtained in this study indicate that DESI-imaging is a powerful tool for determining the spatial distribution of human lens lipids.     

Hydrodynamics of dielectric fluid films

We introduce a general hydrodynamic model to study the stability of lipid films against thermal fluctuations. As one novel aspect the model accounts before all for a complete intrinsic surface rheology of the film interfaces. Thus the rheological behaviour of the surface adsorbed lipids is modelled which screen the hydrophobic film interior against the aqueous exterior. For coloured films we demonstrate first the influence of electrical forces on the dynamics and film stability. For that we perform a linear stability analysis on a simplified mechanically symmetric film with i) symmetric surface charge distribution and ii) linear electric potential drop across the film. Based on the complete film model we then categorize the complete set of solutions of the linearized equations of motion and we study the growth rates of unstable film modes. Finally we discuss the stability properties of a black film after introducing a repulsive mechanism due to the steric hindrance of the interfacial lipids.