A molecular mechanism for how pressure induces interdigitation of phospholipid bilayer membranes.

The phase transition from the ripple gel phase to the interdigitated gel phase of bilayers of phosphatidylcholines (PCs) with two saturated long-chain fatty acids under high pressure was investigated by pressure-scanning microscopy, fluorometry, and dynamic light scattering (DLS) measurements. Microscopic observation for giant vesicles (GVs) of distearoyl-PC (DSPC) under high pressure showed that spherical GVs transforms significantly into warped and distorted spherical ones instantaneously at the pressure-induced interdigitation. The fluorescence intensities of amphiphilic probe Prodan and hydrophobic probe Laurdan in the dipalmitoyl-PC (DPPC) bilayer steeply decreased and increased, respectively, at the interdigitation, suggesting that the conformational change of the polar head group of DPPC molecule in the bilayer transiently occurred at the interdigitation. Further, it was found from the high-pressure DLS measurements that the size of the vesicle particles of the DPPC and DSPC transiently increases near the interdigitation pressure, whereas the chemically induced interdigitation by adding ethanol to the DSPC bilayer membrane under atmospheric pressure produce no such change in the particle size. Taking account of the critical packing parameter of the PC molecule, the above experimental results would lead us to the conclusion that the pressure-induced interdigitation is attributable to the increase in repulsive interaction between the polar head groups of the PC molecules resulting from the orientational change of the head group from a parallel alignment to a perpendicular one with respect to the bilayer surface by applying pressure, namely the transient state: it occurs when the repulsive interaction exceeds a threshold value for the balance between the repulsive interaction and the attractive interaction among the hydrophobic acyl chains.

Reorientation of interfacial water molecules during melting of brain sphingomyelin is associated with the phase transition of its C24:1 sphingomyelin lipids.

Melting of brain sphingomyelin (bSM) manifests as a broad feature in the DSC curve that encompasses the temperature range of 25 - 45 °C, with two distinguished maxima originating from the phase transitions of two the most abundant components: C24:1 (Tm,1) and C18:0 (Tm,2). While C24:1/C18:0 sphingomyelin transforms from the gel/ripple phase to the fluid/fluid phase, the dynamics of water molecules in the interfacial layer remain completely unknown. Therefore, we carried out a calorimetric (DSC), spectroscopic (temperature-dependent UV-Vis and fluorescence) and MD simulation study of bSM in the absence/presence of Laurdan® (bSM ± L) suspended in Britton-Robinson buffer with three different pH values, 4 (BRB4), 7 (BRB7) and 9 (BRB9), and of comparable ionic strength (I = 100 mM). According to DSC, T̅m, 1 (≈ 34.5 °C/≈ 32.1 °C) and T̅m, 2 (≈ 38.0 °C/≈ 37.2 °C) of bSM suspended in BRB4, BRB7, and BRB9 in the absence/presence of Laurdan® are found to be practically pH-independent. Turbidity-based data (UV-Vis) detected both qualitative and quantitative differences in the response of bSM suspended in BRB4/BRB7/BRB9 (T̅m: ∼ 35 °C/32.0 ± 0.2 °C/36.4 ± 0.4), suggesting an intricate interplay of weakening of van der Waals forces between their hydrocarbon chains and of increased hydration in the polar headgroups region during melting. The temperature-dependent response of Laurdan® reported a discontinuous, pH-dependent change in the reorientation of interfacial water molecules that coincides with the melting of C24:1 lipids (on average, T̅m (LTC/HTC): ≈ 31.8 °C/30.6 °C/30.5 °C). MD simulations elucidated the impact of Laurdan® on a change in the physicochemical properties of bSM lipids and characterized the hydrogen bond network at the interface at 20 °C and 50 °C.

Hydration- and Temperature-Dependent Fluorescence Spectra of Laurdan Conformers in a DPPC Membrane.

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

Photophysics in Biomembranes: Computational Insight into the Interaction between Lipid Bilayers and Chromophores.

ConspectusLight is ubiquitously available to probe the structure and dynamics of biomolecules and biological tissues. Generally, this cannot be done directly with visible light, because of the absence of absorption by those biomolecules. This problem can be overcome by incorporating organic molecules (chromophores) that show an optical response in the vicinity of those biomolecules. Since those optical properties are strongly dependent on the chromophore's environment, time-resolved spectroscopic studies can provide a wealth of information on biosystems at the molecular scale in a nondestructive way. In this work, we give an overview on the multiscale computational strategy developed by us in the last eight years and prove that theoretical studies and simulations are needed to explain, guide, and predict observations in fluorescence experiments. As we challenge the accepted views on existing probes, we discover unexplored abilities that can discriminate surrounding lipid bilayers and their temperature-dependent as well as solvent-dependent properties. We focus on three archetypal chromophores: diphenylhexatriene (DPH), Laurdan, and azobenzene. Our method shows that conformational changes should not be neglected for the prototype rod-shaped molecule DPH. They determine its position and orientation in a liquid-ordered (Lo) sphingomyelin/cholesterol (SM/Chol) bilayer and are responsible for a strong differentiation of its absorption spectra and fluorescence decay times in dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) membranes, which are at room temperature in liquid-disordered (Ld) and solid-gel (So) phases, respectively. Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. Finally, the conformational versatility of azobenzene in saturated Ld lipid bilayers is simulated, along with its photoisomerization pathways. By means of nonadiabatic QM/MM surface hopping analyses (QM/MM-SH), a dual mechanism is found with a torsional mechanism and a slow conversion for trans-to-cis. For cis-to-trans, simulations show a much higher quantum yield and a so-called "pedal-like" mechanism. The differences are related to the different potential energy surfaces as well as the interactions with the surrounding alkyl chains. When tails of increased length are attached to this probe, cis is pushed toward the polar surface, while trans is pulled toward the center of the membrane.

The spectral phasor approach to resolving membrane order with environmentally sensitive dyes.

Hyperspectral imaging is a technique that captures a three-dimensional array of spectral information at each spatial location within a sample, enabling precise characterization and discrimination of biological structures, materials, and chemicals, based on their unique spectral features. Nowadays most commercially available confocal microscopes allow hyperspectral imaging measurements, providing a valuable source of spatially resolved spectroscopic data. Spectral phasor analysis quantitatively and graphically transforms the fluorescence spectra at each pixel of a hyperspectral image into points in a polar plot, offering a visual representation of the spectral characteristics of fluorophores within the sample. Combining the use of environmentally sensitive dyes with phasor analysis of hyperspectral images provides a powerful tool for measuring small changes in lateral membrane heterogeneity. Here, we focus on applications of spectral phasor analysis for the probe LAURDAN on model membranes to resolve packing and hydration. The method is broadly applicable to other dyes and to complex systems such as cell membranes.

Investigating the impact of 2-OHOA-embedded liposomes on biophysical properties of cancer cell membranes via Laurdan two-photon microscopy imaging.

2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.

Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.

Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.

Evaluation of polypropylene microporous membranes as extraction devices for determination of carcinogenic aromatic amines in smoker urine by GC-MS/MS.

Exposure to tobacco smoke is highly correlated to the incidence of different types of cancer due to various carcinogenic compounds present in such smoke. Aromatic amines, such as 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA), are produced in tobacco burning and are linked to bladder cancer. Miniaturized solid phase extraction techniques, such as microporous membrane solid phase extraction (MMSPE), have shown potential for the extraction of aromatic compounds. In this study, a bioanalytical method for the determination of 1-NA and 2-NA in human urine was developed using polypropylene microporous membranes as a sorptive phase for MMSPE. Urine samples were hydrolyzed with HCl for 1 h at 80 °C, after which pH was adjusted to 10. Ultrasound-assisted MMSPE procedure was optimized by factorial design as follows. To each sample, 750 µL of methanol was added, and ultrasound-assisted MMSPE was conducted for 1 h with four devices containing seven 2 mm polypropylene membrane segments. After extraction, the segments were transferred to 400 µL of hexane, and desorption was conducted for 30 min. Extracts were submitted to a simple and fast microwave-assisted derivatization procedure, by the addition of 10 µL of PFPA and heating at 480 W for 3 min, followed by clean-up with phosphate buffer pH 8.0 and GC-MS/MS analysis. Adequate linearity was obtained for both analytes in a range from 25 to 500 µg L-1, while the multiple reaction monitoring approach provided satisfactory selectivity and specificity. Intra-day (n = 6) and inter-day (n = 5) precision and accuracy were satisfactory, below 15 % and between 85 and 115 %, respectively. Recovery rates found were 91.9 and 58.4 % for 1-NA and 2-NA, respectively, with adequate precision. 1-NA was found in first-hand smokers' urine samples in a concentration range from 20.98 to 89.09 µg in 24 h, while it could be detected in second-hand smoker's urine samples, and 2-NA detected in all first and second-hand smokers' urine samples. The proposed method expands the applicability of low cost MMSPE devices to aromatic amines and biological fluids.

Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.

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.

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.

Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid.

By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy.

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from -40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions.

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated "PICT" (planar intramolecular charge transfer state) and solvated "TICT" (twisted intramolecular charge transfer state) excited states. While "TICT" state can be formed both as a result of the "PICT" state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained.

Impact of macromolecular crowding on the mesomorphic behavior of lipid self-assemblies.

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.

Ultrasmall Molybdenum Disulfide Quantum Dots Cage Alzheimer's Amyloid Beta to Restore Membrane Fluidity.

Alzheimer's disease (AD) is a major cause of dementia characterized by the overexpression of transmembrane amyloid precursor protein and its neurotoxic byproduct amyloid beta (Aß). A small peptide of considerable hydrophobicity, Aß is aggregation prone catalyzed by the presence of cell membranes, among other environmental factors. Accordingly, current AD mitigation strategies often aim at breaking down the Aß-membrane communication, yet no data is available concerning the cohesive interplay of the three key entities of the cell membrane, Aß, and its inhibitor. Using a lipophilic Laurdan dye and confocal fluorescence microscopy, we observed cell membrane perturbation and actin reorganization induced by Aß oligomers but not by Aß monomers or amyloid fibrils. We further revealed recovery of membrane fluidity by ultrasmall MoS2 quantum dots, also shown in this study as a potent inhibitor of Aß amyloid aggregation. Using discrete molecular dynamics simulations, we uncovered the binding of MoS2 and Aß monomers as mediated by hydrophilic interactions between the quantum dots and the peptide N-terminus. In contrast, Aß oligomers and fibrils were surface-coated by the ultrasmall quantum dots in distinct testudo-like, reverse protein-corona formations to prevent their further association with the cell membrane and adverse effects downstream. This study offers a crucial new insight and a viable strategy for regulating the amyloid aggregation and membrane-axis of AD pathology with multifunctional nanomedicine.

ICT based water-soluble fluorescent probe for discriminating mono and dicarbonyl species and analysis in foods.

A unique and highly water-soluble ICT-based fluorescent probe is developed for efficient detection and discrimination of reactive monocarbonyl formaldehyde (FA) from dicarbonyl methylglyoxal (MGO)/glyoxal (GO) by modulating the ICT process, which was confirmed by photophysical and TD-DFT analysis. The probe is applied in cellular imaging and quantifying FA in preserved food and MGO in manuka honey.

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.