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.

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.

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.

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.

Analysis of aromatic amines in human urine using comprehensive multi-dimensional gas chromatography-mass spectrometry (GCxGC-MS).

Several aromatic amines (AA) are classified as human carcinogens, and tobacco smoke is one of the main sources of exposure. Once in the human body, they undergo different metabolic pathways which lead to either their excretion or ultimately to the formation of DNA and protein adducts. The aim of this study was to investigate AA in 68 urine samples (aged 29-79, 47% female), including 10 smokers (S), 28 past-smokers (PS) and 30 never-smokers (NS), and to study if there was a relation between the smoking status and the amount of the AA present. GCxGC-MS was used to analyze AA in complex urine samples due to its high peak capacity and the fact that it provides two sets of retention times and structural information, which facilitates the separation and identification of the target analytes. First, a qualitative comparison of an example set of a NS, PS and S sample was carried out, in which 38, 45 and 46 AA, respectively, could be tentatively identified. Afterwards, seven AA were successfully quantified in the samples. Of these, 4-ethylaniline (4EA, p = 0.015), 2,4,6-trimethylaniline (2,4,6TMA, p = 0.030), 2-naphthylamine (2NA, p = 0.014) and the sum of 2,4- and 2,6-dimethylaniline (DMA, p = 0.017) were found in significantly different (α = 0.05) concentrations for the S, 29 ± 14, 87 ± 49, 41 ± 26, and 105 ± 57 ng/L respectively, compared to the NS, 15 ± 6, 42 ± 30, 16 ± 6, and 48 ± 28 ng/L. And 2,4,6TMA (39 ± 26, p = 0.022), 2NA (18 ± 9, p = 0.025) and DMA (53 ± 46, p = 0.030), were also found at significantly higher concentrations in samples from S when compared to PS. However, some samples had AA concentrations outside the calibration curve and could not be taken into account, especially for 2-methylaniline (2MA). Therefore, all the samples were evaluated using a quantitative screening approach, by which the intensities of 4EA (p = 0.019), 2,4,6TMA (p = 0.048), 2NA (p = 0.016), DMA (p = 0.019) and 2MA (p = 0.006) in S were found to be significantly (α = 0.05) higher than in the NS, and 2MA (p = 0.019) and 4EA (p = 0.023) in S were found to be significantly higher than in the PS. An association between the smoking status and the amount of certain AA present could therefore be found. This information could be used to study the relation between the smoking status, the amount of AA present, and smoking related diseases like bladder cancer.

[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.

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.

Multiplicity of solvent environments in lipid bilayer revealed by DAS deconvolution of twin probes: Comparative method of Laurdan and Prodan.

Laurdan and Prodan were designed for the evaluation of the surrounding hydration state. When inserted into lipid bilayer systems, both probes are located at different positions and their fluorescence properties are drastically varied, depending on their surrounding environment. In this study, a novel method using the above fluorescence probes was proposed on the basis of fluorescence lifetime (τ) and emission peak (λ), called as τ vs. λ plot, determined by global analysis of their multiple fluorescence decays and deconvolution of these decay-associated spectra. According to the evaluation of τ vs. λ plot, the existence of multiple fluorescence components in the membrane was revealed. In addition, their fluorescence distribution properties, described on τ vs. λ plot, of each probe tended to correspond to the phase state and vertical direction of the lipid membrane. To assess the contribution of environmental effect to each distribution, we defined the region in the τ vs. λ plot, which was modeled from a series of solvent mixtures (hexane, acetone, ethanol and water) to emulate the complex environment in the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer system. The distributions of fluorescence components of Laurdan and Prodan in lipid membranes were classified into each solvent species, and Prodan partition into bulk water was distinguished. The sensitivity of Prodan to the phase pretransition of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer system was also observed in increasing the temperature. Noticeably, most of the fluorescence components was assigned to the solvent model, except for a single component that has longer lifetime and shorter emission wavelength. This component was dominant in solid-ordered phase; hence, it is assumed to be a specific component in lipid membranes that cannot be represented by solvents. Although these are still qualitative analytical methods, the unique approach proposed in this study provides novel insights into the multi-focal property of the membrane.

Influence of DNP Polarizing Agents on Biochemical Processes: TEMPOL in Transient Ischemic Stroke.

Hyperpolarization of 13C by dissolution dynamic nuclear polarization (dDNP) boosts the sensitivity of magnetic resonance spectroscopy (MRS), making possible the monitoring in vivo and in real time of the biochemical reactions of exogenously infused 13C-labeled metabolic tracers. The preparation of a hyperpolarized substrate requires the use of free radicals as polarizing agents. Although added at very low doses, these radicals are not biologically inert. Here, we demonstrate that the presence of the nitroxyl radical TEMPOL influences significantly the cerebral metabolic readouts of a hyperpolarized [1-13C] lactate bolus injection in a mouse model of ischemic stroke with reperfusion. Thus, the choice of the polarizing agent in the design of dDNP hyperpolarized MRS experiments is of great importance and should be taken into account to prevent or to consider significant effects that could act as confounding factors.

Nitrogen-15 dynamic nuclear polarization of nicotinamide derivatives in biocompatible solutions.

Dissolution dynamic nuclear polarization (dDNP) increases the sensitivity of magnetic resonance imaging by more than 10,000 times, enabling in vivo metabolic imaging to be performed noninvasively in real time. Here, we are developing a group of dDNP polarized tracers based on nicotinamide (NAM). We synthesized 1-15N-NAM and 1-15N nicotinic acid and hyperpolarized them with dDNP, reaching (13.0 ± 1.9)% 15N polarization. We found that the lifetime of hyperpolarized 1-15N-NAM is strongly field- and pH-dependent, with T1 being as long as 41 s at a pH of 12 and 1 T while as short as a few seconds at neutral pH and fields below 1 T. The remarkably short 1-15N lifetime at low magnetic fields and neutral pH drove us to establish a unique pH neutralization procedure. Using 15N dDNP and an inexpensive rodent imaging probe designed in-house, we acquired a 15N MRI of 1-15N-NAM (previously hyperpolarized for more than an hour) in less than 1 s.

Synthesis and pharmacodynamic evaluation of naphthalene derivatives against influenza A virus in vitro and in vivo.

Influenza A virus is a highly mutable pathogenic pathogen that could cause a global pandemic. It is necessary to find new anti-influenza drugs to resist influenza epidemics due to the seasonal popularity of a certain area every year. Naphthalene derivatives had potential antiviral activity. A series of naphthalene derivatives were synthesized via the metal-free intramolecular hydroarylation reactions of alkynes. Evaluation of their biological efficacy showed that compound 2-aminonaphthalene 4d had better antiviral activity in vitro than ribavirin. By studying the mechanism of action of 2-aminonaphthalene 4din vivo and in vitro, we found that 4d had antiviral activity to three different subtype influenza viruses of A/Weiss/43 (H1N1), A/Virginia/ATCC2/2009 (H1N1) and A/California/2/2014 (H3N2). Compound 4d had the best effect after viral adsorption, and mainly played in the early stage of virus replication. 2-Aminonaphthalene 4d could reduce the replication of virus by inhibiting the NP and M proteins of virus. Compound 4d cut down ROS accumulation, autophagy and apoptosis induced by influenza virus. Inflammatory response mediated by RIG-1 pathway were suppressed in the cell and mice. In addition, the pathological changes of lung tissue and virus titer in mice were reduced by the administration of 4d. Therefore, naphthalene derivative 4d is a potential drug for the treatment of influenza A virus infection.

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.

Laurdan Discerns Lipid Membrane Hydration and Cholesterol Content.

Studies of biological membrane heterogeneity particularly benefit from the use of the environment-sensitive fluorescent probe Laurdan, for which shifts in the emission, produced by any stimulus (e.g., fluidity variations), are ascribed to alterations in hydration near the fluorophore. Ironically, no direct measure of the influence of the membrane hydration level on Laurdan spectra has been available. To address this, we investigated the fluorescence spectrum of Laurdan embedded in solid-supported lipid bilayers as a function of hydration and compared it with the effect of cholesterol─a major membrane fluidity regulator. The effects are illusively similar, and hence the results obtained with this probe should be interpreted with caution. The dominant phenomenon governing the changes in the spectrum is the hindrance of the lipid internal dynamics. Furthermore, we unveiled the intriguing mechanism of dehydration-induced redistribution of cholesterol between domains in the phase-separated membrane, which reflects yet another regulatory function of cholesterol.

Short- and Long-Term Stability of Aromatic Amines in Human Urine.

Several aromatic amines (AAs) are established by the International Agency for Research on Cancer as carcinogenic (group 1) or probable/possible carcinogens to humans (group 2A/2B). AAs can be found in mainstream and sidestream smoke from combustible tobacco products, as well as in certain environmental pollution and occupational exposure from several chemical industry sectors. Exposure to AAs can be estimated by measuring their concentrations in urine; however, information about the short-term and long-term stabilities of AAs in urine need to be characterized before conducting large-scale population studies on AA exposure and the potentially harmful effects of AA exposure. In this report, the storage stability of o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, and 4-aminobiphenyl fortified in pooled, filtered, non-smokers' urine is analyzed by isotope dilution gas chromatography-triple quadrupole mass spectrometry (ID GC-MS/MS). The six AAs were measured in urine samples stored at ~20 °C (collection temperature), 4 °C and 10 °C (short-term transit temperatures), and -20 °C and -70 °C (long-term storage temperatures) over a 10-day period. All six analytes were stable for 10 days at transit and long-term storage temperatures but showed reduced recovery at 20 °C. The instability of the target AAs at 20 °C suggests that immediate storage of freshly voided urine at low temperatures is needed to attenuate degradation. A subset of the urine samples was analyzed following a longer storage duration at -70 °C: all AAs were stable for up to 14 months at this temperature. The stability of the six AAs in urine samples can be maintained at the various temperature levels and storage times expected in a typical study set.

Experimental, Spectroscopic, and Computational Insights into the Reactivity of "Methanal" with 2-Naphthylamines.

The reactions of 2-naphthylamine and methyl 6-amino-2-naphthoate with formalin and paraformaldehyde were studied experimentally, spectrally, and by quantum chemical calculations. It was found that neither the corresponding aminals nor imines were formed under the described conditions but could be prepared and spectrally characterized at least in situ under modified conditions. Several of the previously undescribed intermediates and by-products were isolated or at least spectrally characterized. First principle density functional theory (DFT) calculations were performed to shed light on the key aspects of the thermochemistry of decomposition and further condensation of the corresponding aminals and imines. The calculations also revealed that the electrophilicity of methanal was significantly greater than that of ordinary oxo-compounds, except for perfluorinated ones. In summary, methanal was not behaving as the simplest aldehyde but as a very electron-deficient oxo-compound.

Dasabuvir suppresses esophageal squamous cell carcinoma growth in vitro and in vivo through targeting ROCK1.

Esophageal squamous cell carcinoma (ESCC) is an upper gastrointestinal cancer with high morbidity and mortality. New strategies are urgently needed to prolong patients' survival. Through screening FDA-approved drugs, we found dasabuvir, a drug approved for hepatitis C virus (HCV) treatment, suppressed ESCC proliferation. Dasabuvir could inhibit the growth of ESCC cells in a time and dose-dependent manner and arrested cell cycle at the G0/G1 phase. The antitumor activity was further validated in vivo using patient-derived xenograft tumor models. In terms of mechanism, we unveil that dasabuvir is a Rho-associated protein kinase 1 (ROCK1) inhibitor. Dasabuvir can bind to ROCK1 and suppress its kinase activity, thus downregulating the phosphorylation of ERK1/2 by ROCK1 and the expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1. These results provide evidence that dasabuvir suppresses ESCC growth in vivo and in vitro through blocking ROCK1/ERK signaling pathway.

C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy.

Membrane order is a biophysical characteristic dependent on cellular lipid makeup. Cells regulate the membrane structure as it affects membrane-bound protein activity levels and membrane stability. Spatial organization of membrane lipids, such as lipid rafts, is a proposed theory that has been indirectly measured through polarity-sensitive fluorescent dyes. C-Laurdan is one such dye that penetrates plasma and internal membranes. C-Laurdan is excited by a single 405 nm photon and emits in two distinct ranges depending on membrane order. Herein, we present a protocol for staining HEK293t cells with C-Laurdan and acquiring ratiometric images using a revised ImageJ macro and confocal microscopy. An example figure is provided depicting the effects of methyl-ß-cyclodextrin, known to remove lipid rafts through cholesterol sequestration, on HEK293t cells. Further image analysis can be performed through region of interest (ROI) selection tools.

Evidence for a transition in the cortical membranes of Paramecium.

Ever since the pioneering studies in the 1960s and 70s, the importance of order transitions for cell membrane functions has remained a matter of debate. Recently, it has been proposed that the nonlinear stimulus-response curve of excitable cells, which manifests in all-or-none pulses (action potentials (AP)), is due to a transition in the cell membrane. Indeed, evidence for transitions has accumulated in plant cells and neurons, but studies with other excitable cells are expedient in order to show if this finding is of a general nature. Herein, we investigated intact, motile specimens of the "swimming neuron" Paramecium. The cellular membranes were labelled with the solvatochromic fluorophores LAURDAN or Di-4-ANEPPDHQ. Subsequently, a cell was trapped in a microfluidic channel and investigated by fluorescence spectroscopy. The generalized polarization (GP) of the fluorescence emission from cell cortical membranes (probably plasma and alveolar membranes) was extracted by an edge-finding algorithm. The thermo-optical state diagram, i.e. the dependence of GP on temperature, exhibited clear indications for a reversible transition. This transition had a width of ~10-15 °C and a midpoint that was located ~4 °C below the growth temperature. The state diagrams with LAURDAN and Di-4-ANEPPDHQ had widely identical characteristics. These results suggested that the cortical membranes of Paramecium reside in an order transition regime under physiological growth conditions. Based on these findings, membrane potential fluctuations, spontaneous depolarizing spikes, and thermal excitation of Paramecium was interpreted.

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.