Generalized polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles.

The solvatochromic dye Laurdan is widely used in sensing the lipid packing of both model and biological membranes. The fluorescence emission maximum shifts from about 440 nm (blue channel) in condensed membranes (So) to about 490 nm (green channel) in the liquid-crystalline phase (Lα). Although the fluorescence intensity based generalized polarization (GP) is widely used to characterize lipid membranes, the fluorescence lifetime of Laurdan, in the blue and the green channel, is less used for that purpose. Here we explore the correlation between GP and fluorescence lifetimes by spectroscopic measurements on the So and Lα phases of large unilamellar vesicles of DMPC and DPPC. A positive correlation between GP and the lifetimes is observed in each of the optical channels for the two lipid phases. Microfluorimetric determinations on giant unilamellar vesicles of DPPC and DOPC at room temperature are performed under linearly polarized two-photon excitation to disentangle possible subpopulations of Laurdan at a scale below the optical resolution. Fluorescence intensities, GP and fluorescence lifetimes depend on the angle between the orientation of the linear polarization of the excitation light and the local normal to the membrane of the optical cross-section. This angular variation depends on the lipid phase and the emission channel. GP and fluorescence intensities in the blue and green channel in So and in the blue channel in Lα exhibit a minimum near 90o. Surprisingly, the intensity in the green channel in Lα reaches a maximum near 90o. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90o in So and Lα, apart from the lifetime in the blue channel in Lα where the lifetime is short with minimal angular variation. To our knowledge, these experimental observations are the first to demonstrate the existence of a bent conformation of Laurdan in lipid membranes, as previously suggested by molecular dynamics calculations.

Cell lipid droplet heterogeneity and altered biophysical properties induced by cell stress and metabolic imbalance.

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.

Phasor-based multi-harmonic unmixing forin-vivohyperspectral imaging.

Hyperspectral imaging (HSI) is a paramount technique in biomedical science, however, unmixing and quantification of each spectral component is a challenging task. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method requires only the empirical measurement of the pure species to compute the pixel-wise photon fraction of every spectral component. Using simulations, we demonstrate the feasibility of the approach for up to 5 components and explore the use of adding a 6th unknown component representing autofluorescence. The simulations show that the method can be successfully used in typical confocal imaging experiments (with pixel photon counts between 101and 103). As a proof of concept, we tested the method in living cells, using 5 common commercial dyes for organelle labeling and we easily and accurately separate them. Finally, we challenged the method by introducing a solvatochromic probe, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (LAURDAN), intended to measure membrane dynamics on specific subcellular membrane-bound organelles by taking advantage of the linear combination between the organelle probes and LAURDAN. We succeeded in monitoring the membrane order in the Golgi apparatus, Mitochondria, and plasma membrane in the samein-vivocell and quantitatively comparing them. The phasor-based multi-harmonic unmixing method can help expand the outreach of HSI and democratize its use by the community for it does not require specialized knowledge.

Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

Does fluoride disrupt hydrogen bond network in cationic lipid bilayer? Time-dependent fluorescence shift of Laurdan and molecular dynamics simulations.

Time-dependent fluorescence shift (TDFS) of Laurdan embedded in phospholipid bilayers reports on hydration and mobility of the phospholipid acylgroups. Exchange of H2O with D2O prolongs the lifetime of lipid-water and lipid-water-lipid interactions, which is reflected in a significantly slower TDFS kinetics. Combining TDFS measurements in H2O and D2O hydrated bilayers with atomistic molecular dynamics (MD) simulations provides a unique tool for characterization of the hydrogen bonding at the acylgroup level of lipid bilayers. In this work, we use this approach to study the influence of fluoride anions on the properties of cationic bilayers composed of trimethylammonium-propane (DOTAP). The results obtained for DOTAP are confronted with those for neutral phosphatidylcholine (DOPC) bilayers. Both in DOTAP and DOPC H2O/D2O exchange prolongs hydrogen-bonding lifetime and does not disturb bilayer structure. These results are confirmed by MD simulations. TDFS experiments show, however, that for DOTAP this effect is cancelled in the presence of fluoride ions. We interpret these results as evidence that strongly hydrated fluoride is able to steal water molecules that bridge lipid carbonyls. Consequently, when attracted to DOTAP bilayer, fluoride disrupts the local hydrogen-bonding network, and the differences in TDFS kinetics between H2O and D2O hydrated bilayers are no longer observed. A distinct behavior of fluoride is also evidenced by MD simulations, which show different lipid-ion binding for Cl(-) and F(-).

Chemical composition and antioxidative activity of Echinophora platyloba DC. essential oil, and its interaction with natural antimicrobials against food-borne pathogens and spoilage organisms.

This study was undertaken to determine the chemical composition and antioxidative capacity of Echinophora platyloba DC. essential oil, and its antimicrobial potency against Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis, Rhodotorula rubra, and Rhodotorula mucilaginosa. The essential oil was analyzed by GC and GC-MS; and evaluated for its antioxidative and antimicrobial (singly or in combination with chitosan, nisin, monolaurin, or amphotericin B) activity. Thirty-three components were characterized representing 95.69% of the total oil composition in which thymol, trans-ocimene, carvacrol, and (E)-sesqui-lavandulol were the major constituents. The oil exhibited high scavenging (IC(50): 49.7 ± 2.3 µg/mL) and relative antioxidative activity (RAA%: 85.21 ± 0.4) in 1,1-diphenyl-2-picrylhydrazyl radicals and ß-carotene/linoleic acid bleaching assays, respectively. The oil showed antimicrobial activity against L. monocytogenes, B. cereus, B. subtilis, S. aureus, S. typhimurium, E. coli O157:H7, P. aeruginosa, C. albicans, C. tropicalis, R. Rubra, and R. mucilaginosa. Moreover, R. mucilaginosa and P. aeruginosa were the most susceptible and most resistant organisms, respectively. Regarding the checkerboard data, 47 fractional inhibitory concentration index (FICIs) (≤ 0.5) indicated synergistic, whereas 7 FICIs (>0.5 to 1) indicated additive effect. Consequently, E. platyloba DC. essential oil could be used as a recommended natural antioxidant and antimicrobial substance for food preservation.

Detecting the quality of glycerol monolaurate: a method for using Fourier transform infrared spectroscopy with wavelet transform and modified uninformative variable elimination.

Glycerol monolaurate (GML) products contain many impurities, such as lauric acid and glucerol. The GML content is an important quality indicator for GML production. A hybrid variable selection algorithm, which is a combination of wavelet transform (WT) technology and modified uninformative variable eliminate (MUVE) method, was proposed to extract useful information from Fourier transform infrared (FT-IR) transmission spectroscopy for the determination of GML content. FT-IR spectra data were compressed by WT first; the irrelevant variables in the compressed wavelet coefficients were eliminated by MUVE. In the MUVE process, simulated annealing (SA) algorithm was employed to search the optimal cutoff threshold. After the WT-MUVE process, variables for the calibration model were reduced from 7366 to 163. Finally, the retained variables were employed as inputs of partial least squares (PLS) model to build the calibration model. For the prediction set, the correlation coefficient (r) of 0.9910 and root mean square error of prediction (RMSEP) of 4.8617 were obtained. The prediction result was better than the PLS model with full-spectra data. It was indicated that proposed WT-MUVE method could not only make the prediction more accurate, but also make the calibration model more parsimonious. Furthermore, the reconstructed spectra represented the projection of the selected wavelet coefficients into the original domain, affording the chemical interpretation of the predicted results. It is concluded that the FT-IR transmission spectroscopy technique with the proposed method is promising for the fast detection of GML content.

Studies of oxidative stress mechanisms using a morphine / ascorbate animal model and novel N-stearoyl cerebroside and laurate sensors.

The field of oxidative stress, free radicals, cellular defense and antioxidants is a burgeoning field of research. An important biomarker of oxidative stress is ascorbate and alterations in ascorbate have been shown to be a reliable measure of oxidative stress mechanisms. The purpose of this pharmacological study was to assess changes in ascorbate in a morphine/ascorbate animal model using novel sensors which selectively detect electrochemical signals for ascorbate, dopamine (DA) and serotonin (5-HT). Studies were also performed to show reversal of morphine-induced effects by the opioid antagonist, naloxone. In vivo studies were modeled after (Enrico et al. 1997, 1998) in which the oxidative biomarker, ascorbate, was reported to compensate for free radicals produced by morphine-induced increases in DA and 5-HT. In vivo studies consisted of inserting the Laurate sensor in ventrolateral nucleus accumbens (vlNAcc), in anesthetized male, Sprague-Dawley rats. In separate studies, laboratory rats were injected with (1) ascorbate, (5-35 mg/kg, ip) or (2) dehydroascorbate (DHA) (20-100 mg/kg, ip). In another study, (3) morphine sulfate (10-20 mg/kg, sc) was injected followed by a single injection of naloxone (5 mg/kg, ip) in the same animal. Results showed that in vlNAcc, (1) neither ascorbate nor DHA injections produced ascorbate release, (2) morphine significantly increased DA and 5-HT release, but did not alter ascorbate release, and (3) naloxone significantly reversed the increased DA and 5-HT release produced by morphine. Moreover, the sensors, N-stearoyl cerebroside and laurate were studied in vitro, in separate studies, in order to assess selective and separate electrochemical detection of ascorbate, DA and 5-HT, neuromolecules involved in oxidative stress mechanisms. In vitro studies consisted of pretreatment of each sensor with a solution of phosphotidylethanolamine (PEA) and bovine serum albumin (BSA) which simulates the lipid/protein composition of brain. Each new sensor was tested for stability, sensitivity and selectivity by pipetting graduated increases in concentration of ascorbate, DA and 5-HT into an electrochemical cell containing saline/phosphate buffer. Multiple and repetitive images of electrochemical signals from ascorbate, DA and 5-HT were recorded. Results showed that both sensors produced three well-defined cathodic, selective and separate electrochemical signals for ascorbate, DA and 5-HT at characteristic oxidation potentials. Dopamine and 5-HT were detected at nM concentrations while ascorbate was detected at microM concentrations. In summary, the data show that very low concentrations of ascorbate occurred in vlNAcc since novel sensors detected ascorbate at high concentrations in vitro. The data indicate that little or no change in oxidative stress mechanisms occurred in vlNAcc after morphine or naloxone administration since the oxidative biomarker, ascorbate, was not signifi cantly altered. Thus, oxidative stress mechanisms and novel N-stearoyl cerebroside and laurate sensors, which selectively detect and separate neuromolecules involved in these mechanisms, may be potentially clinically relevant.

The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and beta-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells.

The incorporation efficiencies of lutein, zeaxanthin, canthaxanthin and beta-carotene into Retinal Pigment Epithelial (RPE) cells (the human RPE cell line D 407), liver microsomes and EYPC liposomes are investigated. In RPE cells the efficiency ratio of lutein and zeaxanthin compared to canthaxanthin and beta-carotene is higher than in the other membranes. The preferential interactions of lutein and zeaxanthin with RPE cells are discussed considering special protein binding properties. Incorporation yields were obtained from the UV-Vis spectra of the carotenoids. Membrane modulating effects of the carotenoids were obtained from the fluorescence spectra of co-incorporated Laurdan (6-dodecanoyl-2-dimethylaminonaphtalene). The Laurdan fluorescence quenching efficiencies of the membrane bound carotenoids offer an access to direct determinations of membrane carotenoid concentrations. Fetal calf serum as carrier for carotenoid incorporation appears superior to tetrahydrofuran.

Minimum inhibitory concentrations of herbal essential oils and monolaurin for gram-positive and gram-negative bacteria.

New, safe antimicrobial agents are needed to prevent and overcome severe bacterial, viral, and fungal infections. Based on our previous experience and that of others, we postulated that herbal essential oils, such as those of origanum, and monolaurin offer such possibilities. We examined in vitro the cidal and/or static effects of oil of origanum, several other essential oils, and monolaurin on Staphylococcus aureus, Bacillus anthracis Sterne, Escherichia coli, Klebsiella pneumoniae, Helicobacter pylori, and Mycobacterium terrae. Origanum proved cidal to all tested organisms with the exception of B. anthracis Sterne in which it was static. Monolaurin was cidal to S. aureus and M. terrae but not to E. coli and K. pneumoniae. Unlike the other two gram-negative organisms, H. pylori were extremely sensitive to monolaurin. Similar to origanum, monolaurin was static to B. anthracis Sterne. Because of their longstanding safety record, origanum and/or monolaurin, alone or combined with antibiotics, might prove useful in the prevention and treatment of severe bacterial infections, especially those that are difficult to treat and/or are antibiotic resistant.

Effect of genistein against copper-induced lipid peroxidation of human high density lipoproteins (HDL).

Several studies have demonstrated that the isoflavone genistein exerts a protective effect against lipid peroxidation of low density lipoproteins (LDL). Aim of our study was to investigate whether genistein protects high density lipoproteins (HDL), isolated from normolipemic subjects, against Cu(++)-induced lipid peroxidation. Our results demonstrated that genistein exerts an inhibitory effect against Cu(++)-induced lipid peroxidation of HDL, as shown by the lower increase in the levels of conjugated dienes in lipoproteins oxidized after preincubation with different concentrations of genistein (0.5-2.5microM). Moreover the analysis of fluorescence emission spectra of tryptophan (Trp) and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene) demonstrated that genistein prevents the alterations of apoprotein structure and physico-chemical properties, associated with Cu(++)-triggered lipid peroxidation of lipoproteins. The protective effect exerted by genistein against oxidative damage of lipoproteins was realized at concentrations similar to those observed in plasma of human subjects consuming a traditional soy diet or receiving a soy supplement. Therefore, we suggested that antioxidant activity exerted by genistein against lipid peroxidation of HDL in vitro could be of physiological relevance.

Preparation, stability, and in vitro performance of vesicles made with diblock copolymers.

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.

Time-gated fluorescence microscopy in cellular and molecular biology.

An experimental set-up for time-gated fluorescence spectroscopy and microscopy is described, and some recent applications in cellular and molecular biology are summarized. Selective detection of intrinsic fluorophores, in particular nicotinamide adenine dinucleotide (NADH) and flavins was demonstrated in living cells. Non-radiative energy transfer from reduced NADH to the mitochondrial marker rhodamine 123 was evaluated for probing mitochondrial malfunction in living cells. An increase of "energy transfer efficacy" up to a factor 4 was detected after inhibition of enzyme complexes of the respiratory chain. Two different fluorescence lifetimes of calcium orange were evaluated, whose relative intensities depended on calcium concentration. Therefore, fluorescence measured within two different time gates appeared to be suitable for ratio fluorometry of calcium. Time-gated fluorescence spectra of the membrane marker laurdan showed more pronounced changes than steady state spectra when temperature was increased from 24 degrees C to 38 degrees C. This may improve measurements of intracellular temperature. Time-gated detection of small amounts of porphyrins and their discrimination from a large fluorescent background caused by chlorophyll in transgenic tobacco plants again proved the advantages of time-gated fluorescence spectroscopy.

[Analysis of the volatile organic acids in 5 kinds of pericarpium Trichosanthis].

The volatile organic acids in the pericarps of Trichosanthes kirilowii, T. rosthornii, T. truncata, T. hupehensis and T. cucumeroides (Cucurbitaceae) were analyzed by methylation, GC and GC-MS-DS. The results showed that they were composed of fifteen long-chain fatty acids, such as palmitic, linolenic, linoleic, lauric, myristic acid, etc.