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.

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.

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.

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.

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.

2-naphthylamine (NAP) was classified as a group I carcinogen associated with bladder cancer. The daily exposure is mostly from cigarette and E-cigarette smoke. NAP can lead to testicular atrophy and interstitial tissue hyperplasia; however, the outcomes of NAP treatment on spermatogenesis and the associated mechanisms have not been reported. The study aimed to investigate the effect of NAP on spermatogenesis and sperm physiologic functions after being persistently exposed to NAP at 5, 20, and 40 mg/kg for 35 days. We found that sperm motility, progressive motility, sperm average path velocity, and straight-line velocity declined remarkably in the NAP (40 mg/kg) treated group, and the sperm deformation rate rose upon NAP administration. The testis immunity- and lipid metabolism-associated processes were enriched from RNA-sequence profiling. Plvap, Ccr7, Foxn1, Trim29, Sirpb1c, Cfd, and Lpar4 involved in testis immunity and Pnliprp1 that inhibit triglyceride and cholesterol absorption were confirmed to rise dramatically in the NAP-exposed group. The increased total cholesterol and CD68 levels were observed in the testis from the NAP-exposed group. Gpx5, serving as an antioxidant in sperm plasma, and Semg1, which contributes to sperm progressive motility, were both down-regulated. We concluded that the short-term exposure to NAP caused reproductive toxicity, primarily due to the inflammatory abnormality in the testis.

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.

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.

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.
目的： 探究LiaSR双组分信号转导系统对变异链球菌（Sm）593号（Sm 593）临床株耐酸和生物膜形成的影响及相关机制。 方法： 以Sm 593及其liaS和liaR基因敲除株为研究菌株，采用全自动生长曲线测定仪检测并绘制pH=5.5环境中各菌株的生长曲线；用菌落计数法检测细菌的适应性耐酸能力；使用Laurdan探针、氢-钾腺苷三磷酸（ATP）酶试剂盒、质子通透性检测实验和实时荧光定量PCR（RT-qPCR）探究LiaSR双组分信号转导系统介导的耐酸机制。体外构建各菌株的生物膜，通过结晶紫染色法、菌落计数法、SYTOX探针检测法和蒽酮-硫酸法探究各菌株的生物膜总量、活菌数、胞外DNA（eDNA）和胞外多糖含量；采用RT-qPCR法检测胞外多糖合成相关基因的表达情况。 结果： pH=5.5环境中，liaS和liaR基因敲除株的生长受到显著抑制（P<0.05）。适应性耐酸曲线显示敲除株与野生株相比，固有耐酸和适应性耐酸能力均显著下降（P<0.05）。对三菌株无预酸化处理20和40 min以及预酸化处理20和40 min后，liaS基因敲除株生存率［（8.98±2.00）%、（0.18±0.07）%、（14.88±8.64）%、（0.82±0.91）%］和liaR基因敲除株生存率［（0.38±0.19）%、（0.34±0.18）%、（7.89±2.02）%、（1.52±0.37）%］均分别显著低于野生株［（32.49±9.75）%、（1.27±0.32）%、（62.76±29.06）%、（8.02±1.25）%］（均P<0.05）。此外，liaS和liaR敲除株膜流动性（0.18±0.04和0.18±0.05）均显著低于野生株（0.08±0.05）（均P<0.01）；不饱和脂肪酸合成基因fabM表达水平（0.52±0.11和0.57±0.05）与野生株（1.04±0.30）相比均显著降低约1/2（均P<0.001）；H+-ATP酶活性（917.06±59.53和469.53±47.65）与野生株（127.00±50.71）相比显著升高7.22和3.70倍（均P<0.001），且编码H+-ATP酶基因atpD的表达水平（3.39±0.21和1.94±0.17）也较野生株（1.00±0.15）显著升高3.39和1.94倍（均P<0.01）。liaR基因敲除株的终末pH值（4.76±0.01）显著低于野生株（4.90±0.00），liaS基因敲除株的终末pH值（5.19±0.01）显著高于野生株（均P<0.001）。liaS和liaR基因敲除株与野生株相比，Sm 593生物膜总量未发生明显变化，生物膜活菌数均显著少于野生株（均P<0.05），且eDNA含量均显著高于野生株（均P<0.01）。liaS基因敲除株生物膜水溶性多糖含量与野生株相比显著增加1.88倍（P=0.003）。liaS和liaR基因敲除株胞外多糖合成相关基因gtfD表达水平较野生株显著升高47.43和16.90倍（均P<0.05），liaS基因敲除株中gtfC基因比野生株显著高表达1.65倍（P=0.014）。 结论： LiaSR双组分信号转导系统通过调控膜不饱和脂肪酸合成和胞膜流动性，参与Sm 593的耐酸过程；LiaR反应调节蛋白还可参与调节膜质子通透性，增强Sm 593的耐酸能力；此双组分信号转导系统可负向调控生物膜胞外基质的产生。.

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.