Dual-monomer solvatochromic probe system (DSPS) for effectively differentiating lipid raft cholesterol and active membrane cholesterol in the inner-leaflet plasma membrane.

Monitoring active membrane cholesterol and lipid raft cholesterol in the inner leaflet of the plasma membrane is significant for understanding the membrane function and cellular physiopathological processes. Limited by existing methods, it is difficult to differentiate active membrane cholesterol and lipid raft cholesterol. A novel dual-monomer solvatochromic probe system (DSPS) that targets two types of cholesterol was developed. Acrylodan-BG/SNAP-D4 composed of SNAP-D4 cholesterol-recognizing monomers and solvatochromic acrylodan-BG-sensing monomers exhibits excellent cholesterol detecting properties in terms of selectivity, accuracy, convenience and economic benefits. Cell imaging revealed that lipid raft cholesterol emitted blue fluorescence, whereas active membrane cholesterol (which partially bobbed in aqueous cytosol) displayed green fluorescence; both the fluorescence emissions increased or decreased in a cholesterol-dependent manner. This system provides a new technology for the determination of two types of cholesterol, which is beneficial for the further study of membrane function, intracellular cholesterol trafficking, and cell signaling.

[Role and related mechanisms of LiaSR two-component system in acid tolerance and biofilm formation of Streptococcus mutans].

Objective: To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans (Sm) 593. Methods: The growth curves of various Sm strains in pH=5.5 brian heart infusion (BHI) medium were analyzed. And colony forming unit (CFU) was also performed to evaluate the acid tolerance of Sm. Laurdan probe, H+-K+adenosine triphosphate (ATP)ase activity analysis kit, proton permeability assay and real-time fluorescence quantitative PCR (RT-qPCR) were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm. Crystal violet staining, CFU, SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms. RT-qPCR was conducted to detect the expression levels of underlying regulated genes. Results: The growth of mutants in acidic BHI were inhibited (P<0.05). The acid tolerance of mutants significantly decreased compared to the wild-type strain (P<0.05). In mutants, the activity of H+-ATPase (917.06±59.53 and 469.53±47.65) were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain (127.00±50.71) (P<0.001, P<0.001) and the encoded gene atpD (3.39±0.21 and 1.94±0.17) were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain (1.00±0.15) (P<0.001, P=0.001). The Laurdan generalized polarization of mutants (0.18±0.04 and 0.18±0.05) increased significantly compared to the wild-type strain (0.08±0.05) (P=0.006, P=0.003) and the expression levels of fabM gene were decreased in mutants (0.52±0.11 and 0.57±0.05) by 1/2 (P=0.014, P=0.022). In liaR deletion mutant, the reduced terminal pH (4.76±0.01) can also be observed (P<0.001). The total amount of the biofilms of three Sm didn't show significant differences (P>0.05). But the number of viable bacteria of mutants' biofilms were decreased [Sm 593: (12.00±2.80)×107 CFU/ml; Sm ΔliaS: (2.95±1.13)×107 CFU/ml; Sm ΔliaR: (7.25±1.60)×107 CFU/ml] (P=0.001, P=0.024). The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants' biofilms (128.73±15.65 and 46.38±5.52) compared to the wild-type strain (7.16±3.62) (P<0.001, P=0.003). Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant [(138.73±10.12) µg/ml] (P=0.003) along with the expression level of gtfC gene (1.65±0.39) (P=0.014). The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants (P<0.001, P=0.010). Conclusions: The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance. The LiaR can also decrease the proton permeability and restrict the entrance of H+. The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.

Nuclear Overhauser spectroscopy in hyperpolarized water - chemical vs. magnetic exchange.

Dissolution dynamic nuclear polarization (dDNP) is a versatile hyperpolarization technique to boost signal intensities in nuclear magnetic resonance (NMR) spectroscopy. The possibility to dissolve biomolecules in a hyperpolarized aqueous buffer under mild conditions has recently widened the scope of NMR by dDNP. The water-to-target hyperpolarization transfer mechanisms remain yet unclear, not least due to an often-encountered dilemma of dDNP experiments: The strongly enhanced signal intensities are accompanied by limited structural information as data acquisition is restricted to short time series of only one-dimensional spectra or a single correlation spectrum. Tackling this challenge, we combine dDNP with molecular dynamics (MD) simulations and predictions of cross-relaxation rates to unravel the spin dynamics of magnetization flow in hyperpolarized solutions.

Signal enhancement of hyperpolarized 15 N sites in solution-increase in solid-state polarization at 3.35 T and prolongation of relaxation in deuterated water mixtures.

Hyperpolarized 15 N sites have been found to be promising for generating long-lived hyperpolarized states in solution, and present a promising approach for utilizing dissolution-dynamic nuclear polarization (dDNP)-driven hyperpolarized MRI for imaging in biology and medicine. Specifically, 15 N sites with directly bound protons were shown to be useful when dissolved in D2 O. The purpose of the current study was to further characterize and increase the visibility of such 15 N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H2 O:D2 O composition. The T1 values of hyperpolarized 15 N in [15 N2 ]urea and [15 N]NH4 Cl demonstrated similar dependences on the H2 O:D2 O composition of the solution, with a T1 of about 140 s in 100% D2 O, about twofold shortening in 90% and 80% D2 O, and about threefold shortening in 50% D2 O. [13 C]urea was found to be a useful solid-state 13 C marker for qualitative monitoring of the 15 N polarization process in a commercial pre-clinical dDNP device. Adding trace amounts of Gd3+ to the polarization formulation led to higher solid-state polarization of [13 C]urea and to higher polarization levels of [15 N2 ]urea in solution.

Effect of osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements.

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan's generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress.

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.

Solvent-Dependent Excited-State Evolution of Prodan Dyes.

Excited-state character and dynamics of two 6-(dimethylamino)-2-acylnaphthalene dyes (Prodan and Badan-SCH2CH2OH) were studied by picosecond time-resolved IR spectroscopy (TRIR) in solvents of different polarity and relaxation times: hexane, CD3OD, and glycerol-d8. In all these solvents, near-UV excitation initially produced the same S1(ππ*) excited state characterized by a broad TRIR signal. A very fast decay (3, ∼100 ps) followed in hexane, whereas conversion to a distinct IR spectrum with a ν(C═O) band downshifted by 76 cm-1 occurred in polar/H-bonding solvents, slowing down on going from CD3OD (1, 23 ps) to glycerol-d8 (5.5, 51, 330 ps). The final relaxed excited state was assigned as planar Me2N → C═O intramolecular charge transfer S1(ICT) by comparing experimental and TDDFT-calculated spectra. TRIR conversion kinetics are comparable to those of early stages of multiexponential fluorescence decay and dynamic fluorescence red-shift. This work presents a strong evidence that Prodan-type dyes undergo solvation-driven charge separation in their S1 state, which is responsible for the dynamic fluorescence Stokes shift observed in polar/H-bonding solvents. The time evolution of the optically prepared S1(ππ*) state to the S1(ICT) final state reflects environment relaxation and solvation dynamics. This finding rationalizes the widespread use of Prodan-type dyes as probes of environment dynamics and polarity.

Evaluating membrane structure by Laurdan imaging: Disruption of lipid packing by oxidized lipids.

Impact of different lipids on membrane structure/lipid order is critical for multiple biological processes. Laurdan microscopy provides a unique tool to assess this property in heterogeneous biological membranes. This review describes the general principles of the approach and its application in model membranes and cells. It also provides an in-depth discussion of the insights obtained using Laurdan microscopy to evaluate the differential effects of cholesterol, oxysterols and oxidized phospholipids on lipid packing of ordered and disordered domains in vascular endothelial cells.

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.

PAD4 inhibitor promotes DNA damage and radiosensitivity of nasopharyngeal carcinoma cells.

Peptidylarginine deiminases 4 (PAD4), a kind of enzyme capable of converting protein arginine or mono-methylarginine into citrulline, has been identified to display a key role in diverse diseases. Radiotherapy is frequently used in nasopharyngeal carcinoma (NPC) treatment and induces DNA double strand breaks. In this study, whether PAD4 inhibitor YW3-56 affects the radiosensitivity of NPC cells was explored. RT-qPCR, immunofluorescence, western blot, clonogenic survival, and flow cytometry assays were used to assess the function of PAD4 and YW3-56 in NPC. We found the upregulation of PAD4 expression in NPC cells. PAD4 overexpression suppressed NPC cell apoptosis and promoted cell cycle, while PAD4 depletion had an opposite result. Moreover, the survival of NPC cells after irradiation was increased by overexpression of PAD4. PAD4 overexpression inhibited DNA damage and sensitivity of NPC cells to irradiation. Functional assays showed that YW3-56 treatment promoted DNA damage, apoptosis, and radiosensitivity of NPC cells. Importantly, YW3-56 treatment inhibited tumor growth in vivo. Overall, this study revealed the efficacy of PAD4 inhibitor YW3-56 in promoting sensitivity of NPC cells to irradiation.

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.

Correlated flickering of erythrocytes membrane observed with dual time resolved membrane fluctuation spectroscopy under different D-glucose concentrations.

A correlated human red blood cell membrane fluctuation dependent on D-glucose concentration was found with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of red blood cells (RBCs) at distant membrane points simultaneously, enabling correlation analysis. Mechanical parameters under different D-glucose concentrations were obtained from direct membrane flickering measurements, complemented with membrane fluidity measurements using Laurdan Generalized Polarization (GP) Microscopy. Our results show an increase in the fluctuation amplitude of the lipid bilayer, and a decline in tension value, bending modulus and fluidity as D-glucose concentration increases. Metabolic mechanisms are proposed as explanations for the results.

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.

The Molecular Diversity of 1,8-diaminonaphthalene in Organic Chemistry.

BACKGROUND: 1,8-diaminonaphthalen (1,8-DAN) with special organic structure was applied in organic synthesis to provide efficient complex scaffolds, through the two or fourcomponent fashion. This review highlights its recent application in organic reactions under different conditions and heterogynous catalysts to produce various molecules, which were used as medicines, sensors, and dyes. OBJECTIVE: 1,8-diaminonaphthalene (1,8-DAN) is a bicyclic compound with two amino groups which has received much attention in organic chemistry due to their medicinal activities such as antifungal, antimicrobial, antiulcer, antitumor, oxidation dyestuff, hair color, fluorescent, and chemo-sensors. In continuation of our studies, herin, recent application of 1,8-DAN in organic synthesis was reviewed. CONCLUSION: In conclusion, the application of 1,8-DAN 1 in different reactions was reviewd through the various conditions to yield the target compounds with high medicinal activities, and potential for sensitive detection of different ions. The target compounds including 1,8-DAN 1 with various structures were provided such as perimidines, perimidinones, aminonaphthalenes which were produced through different methods as brought for further study in this review.