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Mannosylerythritol lipids (MELs) are a class of amphipathic molecules bearing a hydrophilic 4-O-β-D-mannopyranosyl-D-erythritol skeleton. Here, we designed and synthesized four kinds of MEL analogues R-MEL-A ([2R,3S]-erythritol type), S-mannosylthreitol lipid (MTL)-A ([2S,3S]-threitol type), R-MTL-A ([2R,3R]-threitol type), and α-S-MEL-A ([2S,3R]-erythritol type) using our previously reported boron-mediated aglycon delivery (BMAD) method and a neighboring group assisted glycosylation method. The selective cytotoxicity of the target compounds against cancer cells was evaluated, with R-MTL-A showing the highest selective cytotoxicity against human skin squamous carcinoma HSC-5 cells. Our findings suggest that R-MTL-A induces necrosis-like cell death against HSC-5 cells by decreasing cell membrane fluidity. R-MTL-A also exhibits an efficient recovery effect on damaged skin cells, indicating that R-MTL-A has potential as a lead compound for new cosmeceuticals with both cancer cell-selective toxicity and recovery effects on damaged skin cells., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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.

Plasma membrane damage in vitrified oocytes is closely linked to mitochondrial dysfunction. However, the mechanism underlying mitochondria-regulated membrane stability is not elucidated. A growing body of evidence indicates that mitochondrial activity plays a pivotal role in cell adaptation. Since mitochondria work at a higher temperature than the constant external temperature of the cell, we hypothesize that suppressing mitochondrial activity would protect oocytes from extreme stimuli during vitrification. Here we show that metformin suppresses mitochondrial activity by reducing mitochondrial temperature. In addition, metformin affects the developmental potential of oocytes and improves the survival rate after vitrification. Transmission electron microscopy results show that mitochondrial abnormalities are markedly reduced in vitrified oocytes pretreated with metformin. Moreover, we find that metformin transiently inhibits mitochondrial activity. Interestingly, metformin pretreatment decreases cell membrane fluidity after vitrification. Furthermore, transcriptome results demonstrate that metformin pretreatment modulates the expression levels of genes involved in fatty acid elongation process, which is further verified by the increased long-chain saturated fatty acid contents in metformin-pretreated vitrified oocytes by lipidomic profile analysis. In summary, our study indicates that metformin alleviates cryoinjuries by reducing membrane fluidity via mitochondrial activity regulation., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

TREK-1 is a mechanosensitive channel activated by polyunsaturated fatty acids (PUFAs). Its activation is supposed to be linked to changes in membrane tension following PUFAs insertion. Here, we compared the effect of 11 fatty acids and ML402 on TREK-1 channel activation using the whole cell and the inside-out configurations of the patch-clamp technique. Firstly, TREK-1 activation by PUFAs is variable and related to the variable constitutive activity of TREK-1. We observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a K D,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. Finally, we proposed a two steps mechanism: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it., (© 2024. The Author(s).)

Persister cells constitute a bacterial subpopulation able to survive to high concentrations of antibiotics. This phenotype is temporary and reversible, and thus could be involved in the recurrence of infections and emergence of antibiotic resistance. To better understand how persister cells survive to such high antibiotic concentration, we examined changes in their lipid composition. We thus compared the lipidome of Acinetobacter baumannii ATCC 19606 T persister cells formed under ciprofloxacin treatment with the lipidome of control cells grown without antibiotic. Using matrix assisted laser desorption ionisation-Fourier transform ion cyclotron resonance mass spectrometry, we observed a higher abundance of short chains and secondary chains without hydroxylation for lipid A in persister cells. Using liquid chromatography-tandem mass spectrometry, we found that persister cells produced particular phosphatidylglycerols, as LPAGPE and PAGPE, but also lipids with particular acyl chains containing additional hydroxyl group or uncommon di-unsaturation on C18 and C16 acyl chains. In order to determine the impact of these multiple lipidome modifications on membrane fluidity, fluorescence anisotropy assays were performed. They showed an increase of rigidity for the membrane of persister cells, inducing likely a decrease membrane permeability to protect cells during dormancy. Finally, we highlighted that A. baumannii persister cells also produced particular wax esters, composed of two fatty acids and a fatty diol. These uncommon storage lipids are key metabolites allowing a rapid bacterial regrow when antibiotic pressure disappears. These overall changes in persister lipidome may constitute new therapeutic targets to combat these particular dormant cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus . In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF , yycG , and pgsA . In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase ( pdhB ) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels., Importance: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to β-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus , routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus , including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA , on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance., Competing Interests: The authors declare no conflict of interest.

Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des , desK , and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions., Importance: The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro . Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity., Competing Interests: The authors declare no conflict of interest.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade., (© 2024. The Author(s).)

Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the hypothalamic‒pituitary‒adrenal (HPA) axis can cause changes in the lipid content of the cellular plasma membrane. These changes are suspected to be involved in the development of depressive disorders. St. John's wort extract (SJW) Ze 117 has long been used as an alternative to synthetic antidepressants. Part of its effect may be due to an effect on the cellular lipid composition and thus on the properties of plasma membranes and receptor systems embedded therein. In this study, we investigated the effect of Ze 117 on that of dexamethasone and simvastatin. Dexamethasone increases the fluidity of C6 cell plasma membranes. This effect is counteracted by administration of Ze 117. Here we demonstrate that this is not due to a change in C16:1/16:0 and C18:1/18:0 ratios in C6 cell fatty acids. On the other hand, Ze 117 increased the cellular cholesterol content by 42.5%, whereas dexamethasone reduced cholesterol levels similarly to simvastatin. Lowering cholesterol levels by dexamethasone or simvastatin resulted in decreased β-arrestin 2 recruitment to the 5-HT 1a  receptor. This effect was counterbalanced by Ze 117, whereas the SJW extract had little effect on β-arrestin 2 recruitment in non-stressed cells. Taken together, in C6 cells, Ze 117 induces changes in membrane fluidity through its effect on cellular cholesterol metabolism rather than by affecting fatty acid saturation. This effect is reflected in an altered signal transduction of the 5-HT 1a  receptor under Ze 117 administration. The current in vitro results support the hypothesis that Ze 117 addresses relevant parts of the cellular lipid metabolism, possibly explaining some of the antidepressant actions of Ze 117., (© 2024. The Author(s).)

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 10 7 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 μM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC 50 of 24 μM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl- sn -glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Caffeic acid (CA) has demonstrated a strong intracellular antioxidant ability by scavenging ROS, contributing to the maintenance of cell membrane structural integrity and to reduce oxidative injuries in other cell components. Nevertheless, caffeic acid has limited usage, due to its hydrophilic character. In this work, the introduction of alkyl chains in the caffeic acid molecule by esterification (methyl - C1, ethyl - C2, butyl - C4, hexyl - C6, octyl - C8 and hexadecyl - C16), significantly increased its lipophilicity. All caffeates tested showed a much higher protective activity than caffeic acid against red blood cells (RBCs) AAPH-induced oxidative stress; this protection was heavily dependent on the length of the alkyl chain of the esters, and on their concentration. At 2.5 and 5 μM, the more lipophilic compounds (C8 and C16) showed a remarkable antioxidant activity, inhibiting hemolysis; probably, their better location within the membrane leads to a better antioxidative protection; however, at 50 μM, the more hydrophilic compounds (C1-C4) showed a better activity against hemolysis than the more lipophilic ones (C8-C16). At this higher concentration, the better interaction of the more lipophilic compounds with the membrane seems to cause changes in RBC membrane fluidity, disturbing membrane integrity. Our data show that the antioxidant activity of these compounds could play an important role for the protection of different tissues and organs, by protecting cell membranes from oxidative injuries., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The specific structure and composition of the cell plasma membrane (PM) is crucial for many cellular processes and can be targeted by various substances with potential medical applications. In this context, biosurfactants (BS) constitute a promising group of natural compounds that possess several biological functions, including anticancer activity. Despite the efficiency of BS, their mode of action had never been elucidated before. Here, we demonstrate the influence of cyclic lipopeptide surfactin (SU) on the PM of CHO-K1 cells. Both FLIM and svFCS experiments show that even a low concentration of SU causes significant changes in the membrane fluidity and dynamic molecular organization. Further, we demonstrate that SU causes a relevant dose-dependent reduction of cellular cholesterol by extracting it from the PM. Finally, we show that CHO-25RA cells characterized by increased cholesterol levels are more sensitive to SU treatment than CHO-K1 cells. We propose that sterols organizing the PM raft nanodomains, constitute a potential target for SU and other biosurfactants. In our opinion, the anticancer activity of biosurfactants is directly related with the higher cholesterol content found in many cancer cells., (Copyright © 2021. Published by Elsevier B.V.)

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

Fatty acid (FA) balance is strictly related to human health. The composition of fatty acids in lipid membranes seems to be influenced by diet. Shark liver oil (SLO) supplementation has been widely used recently in the prevention and treatment of human diseases. We analyzed the impact of short-term SLO supplementation on certain biochemical parameters and erythrocyte FA composition in a group of young healthy women. Our results showed that 6 weeks of SLO supplementation led to a significant decrease in C-reactive protein levels in sera and intracellular cholesterol levels in peripheral blood mononuclear cells. SLO supplementation caused a significant increase in the content of the polyunsaturated omega-3 FAs: docosahexaenoic acid, docosapentaenoic acid and α-linolenic acid. In the group of omega-6 FAs, we observed a significant elevation of arachidonic and dihomo-gamma-linoleic acid content. Due to these alterations, the omega-3 index increased significantly from 3.6% (before) to 4.2% (after supplementation). We also observed the impact of SLO supplementation on the membrane fluidity index. The ratio between saturated and unsaturated FAs decreased significantly from 13.1 to 9.9. In conclusion, our results show that even short-term SLO supplementation can improve human erythrocyte fatty acid composition and other parameters that may have health-promoting consequences.

Clotrimazole (1-[(2-chlorophenyl)-diphenylmethyl]-imidazole) is an azole antifungal drug belonging to the imidazole subclass that is widely used in pharmacology and that can be incorporated in membranes. We studied its interaction with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicles by using differential scanning calorimetry and found that the transition temperature decreases progressively as the concentration of clotrimazole increases. However, the temperature of completion of the transition remained constant despite the increase of clotrimazole concentration, suggesting the formation of fluid immiscibility. 1 H-NMR and 1 H NOESY MAS-NMR were employed to investigate the location of clotrimazole in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes. In the presence of clotrimazole, all the resonances originating from POPC were shifted upfield, but mainly those corresponding to C2 and C3 of the fatty acyl, chains suggesting that clotrimazole aromatic rings preferentially locate near these carbons. In the same way, 2D-NOESY measurements showed that the highest cross-relaxation rates between protons of clotrimazole and POPC were with those bound to the C2 and C3 carbons of the fatty acyl chains. Molecular dynamics simulations indicated that clotrimazole is located near the top of the hydrocarbon-chain phase, with the nitrogen atoms of the imidazole ring of clotrimazole being closest to the polar group of the carbonyl moiety. These results are in close agreement with the NMR and the conclusion is that clotrimazole is located near the water-lipid interface and in the upper part of the hydrophobic bilayer.

Staphylococcus aureus is a leading pathogen responsible for mild to severe invasive infections in humans. Especially, methicillin resistant Staphylococcus aureus (MRSA) is prevalent in hospital and community associated infections. Staphyloxanthin is a golden yellow color eponymous pigment produced by S. aureus and provides resistance to reactive oxygen species (ROS) and host neutrophil-based killing. In addition, this membrane pigment contributes to membrane rigidity and helps MRSA to survive under stress conditions. Targeting virulence of pathogen without exerting selection pressure is the recent approach to fight bacterial infections without developing drug resistance. The present study for the first time evaluated the staphyloxanthin inhibitory potential of thymol against MRSA. Qualitative and quantitative analyses demonstrated 90% of staphyloxanthin inhibition at 100 µg/mL concentration of thymol without alteration in growth. Molecular docking analysis and in vitro measurement of metabolic intermediates of staphyloxanthin revealed that thymol could possibly interact with CrtM to inhibit staphyloxanthin. Absorbance and infra red spectra further validated the inhibition of staphyloxanthin by thymol. In addition, thymol treatment significantly reduced the resistance of MRSA to ROS and neutrophil-based killing as exhibited by oxidant susceptibility assays and ex vivo innate immune clearance assay using human whole blood and neutrophils. Further, reduction in staphyloxanthin by thymol treatment increased the membrane fluidity and made MRSA cells more susceptible to membrane targeting antibiotic polymyxin B. Especially, thymol was found to be non-cytotoxic to human peripheral blood mononuclear cells. Our study validated the antivirulence potential of thymol against MRSA by inhibiting staphyloxanthin and suggests the prospective therapeutic role of thymol to combat MRSA infections., (Copyright © 2021. Published by Elsevier Masson SAS.)

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N -(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Damage to the cell membrane is an effective method to prevent drug resistance in plant fungal diseases. Here, we proposed a negative remodeling model of the cell membrane structure induced by the C-coordinated O-carboxymethyl chitosan Cu (II) complex (O-CSLn-Cu). FITC-labeled O-CSLn-Cu (FITC-O-CSLn-Cu) was first synthesized via a nucleophilic substitution reaction and confirmed by FT-IR. FITC-labeled O-CSLn-Cu could pass through the fungal cell membrane, as detected by confocal laser scanning microscopy (CLSM) coupled with fluorescein isothiocyanate (FITC)-fluorescence. O-CSLn-Cu treatment led to apparent morphological changes in the membranes of P. capsici Leonian and giant unilamellar vesicles (GUVs) by transmission electron microscopy (TEM). Then, we performed component analysis of the cell membrane from the P. capsici Leonian affected by O-CSLn-Cu with a particular interest in membrane physicochemical properties. Many unsaturated fatty acids (UFAs) and key enzymes promoting UFA synthesis of the cell membrane were downregulated. Similarly, a large number of membrane proteins responsible for substance transport and biochemical reactions were downregulated. Furthermore, O-CSLn-Cu treatments increased plasma membrane permeability with significant leakage of intercellular electrolytes, soluble proteins and sugars, and lipid peroxidation with decreasing membrane fluidity. Finally, aquaporin 10 was proven to be a potential molecular target sensitive to antimicrobial agents according to composition analysis of membrane structure and immunohistochemistry., (Copyright © 2021. Published by Elsevier Ltd.)

The vitamin E derivative with side chain modification (TC6OAc) has been shown to possess anticancer activity in our earlier in vivo studies. It was hypothesized that, as Vitamin E (VE) and VE derivative are fat soluble lipophilic molecules, they exert their function by modulating the lipid metabolism and related pathways. This study aimed to evaluate the cellular impact of this VE derivative (2,5,7,8-Tetramethyl-2-(4'-Methyl-3'-Pentenyl)-6-Acetoxy Chromane-TC6OH), using α-tocopherol as a reference compound throughout the experiments. Their effects on the cellular metabolism, the biophysical properties of cellular lipids and the functional characteristics of cells were monitored in human estrogen receptor (ER) positive breast cancer cells. It has been documented that TC6OH treatment induces tumor cell apoptosis by dissipating the mitochondrial membrane potential, modulating the lipid, transportation and degradation as well as downregulating certain anti-apoptotic and growth factor related proteins. Due to resistance of ER positive cells to the established therapies, the findings of this study are of translational value.

Aging is one of the significant risk factors for Alzheimer's disease (AD). Therefore, this study aimed to propose a new hypothesis "membrane aging" as a critical pathogenesis of AD. The concept of "membrane aging" was reviewed, and the possible mechanisms of membrane aging as the primary culprit of AD were clarified. To further prove this hypothesis, a hydroxyurea-induced "membrane aging" model was established in vitro and in vivo . First, neuronal aging was validated by immunocytochemistry with age-related markers, and membrane aging phenotypes were confirmed. The alterations of membrane fluidity within APP/PS1 mice were re-proved by intracerebroventricular injection of hydroxyurea. Decreased membrane fluidity was found in vitro and in vivo , accompanied by increased total cholesterol concentration in neurons but decreased cholesterol levels within membrane fractions. The Aβ level increased considerably after hydroxyurea treatment both in vitro and in vivo . DHA co-treatment ameliorated membrane aging phenotypes and Aβ aggregation. The study revealed the AMP-activated protein kinase/acetyl CoA carboxylase/carnitine palmitoyl transferase 1 pathway involved in membrane aging processes. These results strongly supported the idea that membrane aging was a pathogenesis of AD and might serve as a new therapeutic target for AD.

The accumulation of toxic soluble oligomers of the amyloid-β peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aβ oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aβ oligomers. However, the mechanism by which structurally distinct Aβ oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aβ42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aβ42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aβ42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aβ42 without perturbing the membrane dynamics. We show that the two structurally distinct Aβ42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aβ42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aβ-membrane interactions.

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa , which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes., Competing Interests: AS, KH, AK, MB, LE, RF, DM, RM, MS, JD, GL, TC, AE No competing interests declared, (© 2021, Sabnis et al.)

Sixteen chemical constituents of Paeonia suffruticosa Andr. buds extract (PSABE) were identified by UHPLC-PDA-Q/TOF-MS, belonging to phenolic acids, flavonoids, monoterpene glycosides and gallotannins. PSABE exhibited significant antibacterial activity against six tested microorganisms. Particularly, it showed the most efficient antibacterial effect against Staphylococcus aureus and Escherichia coli O157:H7, which the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) both were 1.56 mg/mL and 6.25 mg/mL, respectively. The results showed that PSABE induced obvious alterations in membrane fatty acid composition of S. aureus and E. coli O157:H7, such as the decrease of unsaturated fatty acids, leading to the reduce of membrane fluidity. Membrane integrity was destroyed and cell morphology was obviously changed with PSABE. Furthermore, the transcription level of virulence factors was inhibited in the presence of PSABE. These results indicated that PSABE mainly exerted antibacterial effect by damaging cell membrane and inhibiting transcription level of virulence factors.[Figure: see text].

Background: Intracellular tension plays a crucial role in the destruction of the blood-brain barrier (BBB) in response to lesion stimuli. Tight junction structure could be primarily affected by tension activity. In this study, we aimed to determine the effects of extracellular BBB damage on intracellular tension activity, and elucidate the mechanism underlying the effects of intracellular protein nanoparticle-related osmotic pressure on BBB permeability., Methods: The intracellular tension for tight junction proteins occludin and ZO1 was evaluated using the fluorescence resonance energy transfer (FRET)-based tension probes and cpstFRET analysis. The changes in mobility ratios of occludin were evaluated via the fluorescence recovery after photobleaching (FRAP) test. The cytoplasmic osmotic pressure (OP) was measured using Osmometer. The count rate of cytoplasmic nanoparticles was detected by Nanosight NS300. The activation of cofilin and stathmin was examined by Western blot analysis. The BBB permeability in vivo was determined via the changes of Evans Blue (EB) injected into SD rats. The tight junction formation was assessed by the measurement of transendothelial electrical resistance (TEER). Intracellular calcium or chloride ions were measured using Fluo-4 AM or MQAE dyes., Results: BBB lesions were accompanied by changes in occludin/ZO1 tension. Increases in intracellular osmotic pressure were involved in alteration of BBB permeability, possibly through the depolymerization of microfilaments or microtubules and mass production of protein nanoparticles according to the Donnan effect. Recovery of protein nanoparticle-related osmotic pressure could effectively reverse the effects of changes in occludin/ZO1 tension under BBB lesions. Outward tension of intracellular osmotic potential also caused upregulation of membrane fluidity, which promoted nonselective drug influx., Conclusion: Our results suggest a crucial mechanical mechanism underlying BBB lesions, and protein nanoparticle-related osmotic pressure could be a novel therapeutic target for BBB lesion-related brain diseases., Competing Interests: The authors declare that they have no competing interests., (© 2021 Li et al.)

Indoxyl sulfate (IS) belongs to groups of uremic toxins binding to proteins. This compound may contribute to the generation of oxidative stress in chronic kidney disease (CKD) patients. We hypothesized that a high concentration of IS in the blood may induce structural changes of erythrocyte components and thus may contribute to CKD progression. In the present study, we evaluated the influence of IS on hemolysate and membrane proteins' conformational state, lipid membrane fluidity, and internal viscosity in erythrocytes. We examined thiols, carbonyl groups, peroxides, and TBARS levels in erythrocyte incubated with IS. The treatment of erythrocytes with IS led to increase in lipid membrane fluidity, decrease in the internal viscosity of the cells and the motion of the spin labels attached to hemolysate proteins. We did not observe conformational changes in plasma membrane proteins; however, in the plasma membranes of erythrocytes incubated with IS, a decrease in the content of thiol groups and increase in the carbonyls levels and peroxides and TBARS in comparison with the control was observed. The obtained results indicate that IS induces the oxidative damage of erythrocyte components. This may be an important factor that affects the functional properties of erythrocytes in CKD patients., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Chitosan is non-toxic, biodegradable and biocompatible. However, it is insoluble in water, which limits its applications in biomedical areas. Hydroxypropyltrimethyl ammonium chloride chitosan (HACC), a chitosan derivative, can be dissolved in physiological condition and has been widely used in the field of biomedicine and bioengineering. The biological effect of HACC has been extensively studied. However, it is rarely investigated at the subcellular level. To study the biological effect of HACC, mitochondria, energy-producing organelles in eukaryotes, were chosen as a model. The investigation mainly focused on the changes of mitochondrial membrane property in the presence of HACC. Results showed that HACC can induce the collapse of mitochondrial transmembrane potential (∆Ψ m ), the increase in mitochondrial membrane swelling and the decrease of mitochondrial membrane fluidity, demonstrating that mitochondrial membrane permeability transition pore (mPTP) opening happened. Possible mechanism of mPTP opening investigation indicated that it was occurred in a typical model. In addition, HACC can induce the release of cytochrome C (Cyt c) and affect the respiratory activity of mitochondria. The study will provide a lot of important information on biosafety evaluation of HACC., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gram-negative bacteria are becoming resistant to almost all currently available antibiotics. Systemically designed antimicrobial peptides (AMPs) are attractive agents to enhance the activities of antibiotics. We constructed a small Pro-scanning library using amphipathic model peptides. Measurements of minimum inhibitory concentration (MIC) against Escherichia coli and hemolytic activities showed that one of the Pro-hinged peptides, KL-L9P, displays the highest specificity toward E. coli . Moreover, KL-L9P sensitizes E. coli to be responsive to most antibiotics that are not active against Gram-negative bacteria. The results of biochemical experiments show that KL-L9P promotes the rearrangement of the bacterial membrane that enables hydrophobic antibiotics to permeate. Finally, the results of animal tests demonstrate that KL-L9P strongly sensitizes Gram-negative bacteria to linezolid (Lzd), rifampicin (Rif), or clarithromycin (Clr). Thus, KL-L9P operates as a sensitizer to extend the antibacterial activity of most antibiotics to Gram-negative bacteria.

Purpose: We have previously reported that Capryol 90 improves the intestinal absorption of insulin, a peptide drug, without causing serious damage to the intestinal epithelium. However, the effects of Capryol 90 and its related formulations on the intestinal absorption of other drugs, and their absorption-enhancing mechanisms are still unclear. The aim of this study is to evaluate the effects of Capryol 90 and its related formulations on the intestinal absorption of drugs and elucidate their absorption-enhancing mechanisms., Methods: The intestinal absorption of 5(6)-carboxyfluorescein, fluorescein isothiocyanate-dextrans, and alendronate was evaluated using an in situ closed loop method. Brush border membrane vesicles (BBMVs) were labeled with fluorescent probes, and the fluidity of membrane was evaluated by a fluorescence depolarization method. The expression levels of tight junction (TJ) proteins were measured using a Western blot method and immunofluorescence staining., Results: Among the tested excipients, Capryol 90 significantly improved the small and large intestinal absorption of drugs. In mechanistic studies, Capryol 90 increased the membrane fluidity of lipid bilayers in BBMVs. Additionally, Capryol 90 decreased the expression levels of TJ-associated proteins, namely claudin-4, occludin, and ZO-1., Conclusions: Capryol 90 is an effective absorption enhancer for improving the intestinal absorption of poorly absorbed drugs via both transcellular and paracellular pathways.

Atomistic molecular dynamics simulations have been carried out with a view to investigating the stability of the SARS-CoV-2 exterior membrane with respect to two common disinfectants, namely, aqueous solutions of ethanol and n -propanol. We used dipalmitoylphosphatidylcholine (DPPC) as a model membrane material and did simulations on both gel and liquid crystalline phases of membrane surrounded by aqueous solutions of varying alcohol concentrations (up to 17.5 mol %). While a moderate effect of alcohol on the gel phase of membrane is observed, its liquid crystalline phase is shown to be influenced dramatically by either alcohol. Our results show that aqueous solutions of only 5 and 10 mol % alcohol already have significant weakening effects on the membrane. The effects of n -propanol are always stronger than those of ethanol. The membrane changes its structure, when exposed to disinfectant solutions; uptake of alcohol causes it to swell laterally but to shrink vertically. At the same time, the orientational order of lipid tails decreases significantly. Metadynamics and grand-canonical ensemble simulations were done to calculate the free-energy profiles for permeation of alcohol and alcohol/water solubility in the DPPC. We found that the free-energy barrier to permeation of the DPPC liquid crystalline phase by all permeants is significantly lowered by alcohol uptake. At a disinfectant concentration of 10 mol %, it becomes insignificant enough to allow almost free passage of the disinfectant to the inside of the virus to cause damage there. It should be noted that the disinfectant also causes the barrier for water permeation to drop. Furthermore, the shrinking of the membrane thickness shortens the gap needed to be crossed by penetrants from outside the virus into its core. The lateral swelling also increases the average distance between head groups, which is a secondary barrier to membrane penetration, and hence further increases the penetration by disinfectants. At alcohol concentrations in the disinfectant solution above 15 mol %, we reliably observe disintegration of the DPPC membrane in its liquid crystalline phase.

Antimicrobial peptides (AMPs) are a class of molecules widely used in applications on eukaryotic and prokaryotic cells. Independent of the peptide target, all of them need to first pass or interact with the plasma membrane of the cells. In order to have a better image of the peptide action mechanism with respect to the particular features of the membrane it is necessary to better understand the changes induced by AMPs in the membranes. Laurdan, a lipid membrane probe sensitive to polarity changes in the environment, is used in this study for assessing changes induced by melittin, a well-known peptide, both in model and natural lipid membranes. More importantly, we showed that generalized polarization (GP) values are not always efficient or sufficient to properly characterize the changes in the membrane. We proved that a better method to investigate these changes is to use the previously described log-normal deconvolution allowing us to infer other parameters: the difference between the relative areas of elementary peak (ΔS r ), and the ratio of elementary peaks areas (R s ). Melittin induced a slight decrease in local membrane fluidity in homogeneous lipid membranes. The addition of cholesterol stabilizes the membrane more in the presence of melittin. An opposite response was observed in the case of heterogeneous lipid membranes in cells, the local order of lipids being diminished. R S proved to be the most sensitive parameter characterizing the local membrane order, allowing us to distinguish among the responses to melittin of both classes of membrane we investigated (liposomes and cellular membranes). Molecular simulation of the melittin pore in homogeneous lipid bilayer suggests that lipids are more closely packed in the proximity of the melittin pore (a smaller area per lipid), supporting the experimental observation.

The biological properties of doxyl stearate nitroxides (DSs): 5-DS, Met-12-DS, and 16-DS, commonly used as spin probes, have not been explored in much detail so far. Furthermore, the influence of DSs on the cellular changes induced by the anticancer drug doxorubicin (DOX) has not yet been investigated. Therefore, we examined the cytotoxicity of DSs and their ability to induce cell death and to influence on fluidity and lipid peroxidation (LPO) in the plasma membrane of immortalised B14 fibroblasts, used as a model neoplastic cells, susceptible to DOX-induced changes. The influence of DSs on DOX toxicity was also investigated and compared with that of a natural reference antioxidant α-Tocopherol. By employing the trypan blue exclusion test and double fluorescent staining, we found a significant level of cytotoxicity for DSs and showed that their ability to induce apoptosis and modify plasma membrane fluidity (measured fluorimetrically) is more potent than for α-Tocopherol. The most cytotoxic nitroxide was 5-DS. The electron paramagnetic resonance (EPR) measurements revealed that 5-DS was reduced in B14 cells at the fastest and Met-12-DS at the slowest rate. In the presence of DOX, DSs were reduced slower than alone. The investigated compounds, administered with DOX, enhanced DOX-induced cell death and demonstrated concentration-dependent biphasic influence on membrane fluidity. A-Tocopherol showed weaker effects than DSs, regardless the mode of its application-alone or with DOX. High concentrations of α-Tocopherol and DSs decreased DOX-induced LPO. Substantial cytotoxicity of the DSs suggests that they should be used more carefully in the investigations performed on sensitive cells. Enhancement of DOX toxicity by DSs showed their potential to act as chemosensitizers of cancer cells to anthracycline chemotherapy.

Epidemiological evidence suggests that chronic consumption of caffeine, a non-selective antagonist of adenosine A 2A R receptors (A 2A R), can be neuroprotective in a number of age-related neurodegenerative disorders including Alzheimer's disease. A growing body of work shows that this neuroprotection may act via a synergistic interaction with the glucocorticoid receptor (GR) and its associated genetic response elements. Therefore, we hypothesized that A 2A R signaling may directly stimulate glucocorticoid receptor translocation via downstream signaling elements within the cell. Surprisingly, we found no effect of A 2A R agonism on GR translocation in the absence of steroid. As expected, membrane-bound dexamethasone was capable of stimulating full GR translocation, albeit at a slower rate. This non-liganded translocation was unaffected by A 2A R ligands, providing strong evidence that GR translocation occurs independently of activation of A 2A Rs. To identify other potential mechanisms of translocation, membrane fluidity was increased significantly by benzyl alcohol, which also induced full nuclear translocation of the GR, but unlike the membrane-bound dexamethasone, benzyl alcohol did result in transcriptional upregulation of GR-dependent genes. Taken together, our data shows that the unliganded GR is sensitive to changes in membrane state and can be transcriptionally active., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

After leaving the testis, mammalian sperm undergo a sequential maturation process in the epididymis followed by capacitation during their movement through the female reproductive tract. These phenotypic changes are associated with modification of protein phosphorylation and membrane remodeling, which is requisite for sperm to acquire forward motility and induce fertilization. However, the molecular mechanisms underlying sperm maturation and capacitation are still not fully understood. Herein, we show that PPP3R2, a testis-specific regulatory subunit of protein phosphatase 3 (an isoform of calcineurin in the testis), is essential for sperm maturation and capacitation. Knockout of Ppp3r2 in mice leads to male sterility due to sperm motility impairment and morphological defects. One very noteworthy change includes increases in sperm membrane stiffness. Moreover, PPP3R2 regulates sperm maturation and capacitation via (i) modulation of membrane diffusion barrier function at the annulus and (ii) facilitation of cholesterol efflux during sperm capacitation. Taken together, PPP3R2 plays a critical role in modulating cholesterol efflux and mediating the dynamic control of membrane remodeling during sperm maturation and capacitation., (© The Author(s) (2020). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

This is a comprehensive review on the use of phospholipid nanovesicles for dermal/transdermal and nasal drug administration. Phospholipid-based vesicular carriers have been widely investigated for enhanced drug delivery via dermal/transdermal routes. Classic phospholipid vesicles, liposomes, do not penetrate the deep layers of the skin, but remain confined to the upper stratum corneum. The literature describes several approaches with the aim of altering the properties of these vesicles to improve their penetration properties. Transfersomes and ethosomes are the most investigated penetration-enhancing phospholipid nanovesicles, obtained by the incorporation of surfactant edge activators and high concentrations of ethanol, respectively. These two types of vesicles differ in terms of their structure, characteristics, mechanism of action and mode of application on the skin. Edge activators contribute to the deformability and elasticity of transfersomes, enabling them to penetrate through pores much smaller than their own size. The ethanol high concentration in ethosomes generates a soft vesicle by fluidizing the phospholipid bilayers, allowing the vesicle to penetrate deeper into the skin. Glycerosomes and transethosomes, phospholipid vesicles containing glycerol or a mixture of ethanol and edge activators, respectively, are also covered. This review discusses the effects of edge activators, ethanol and glycerol on the phospholipid vesicle, emphasizing the differences between a soft and an elastic nanovesicle, and presents their different preparation methods. To date, these differences have not been comparatively discussed. The review presents a large number of active molecules incorporated in these carriers and investigated in vitro, in vivo or in clinical human tests.

Curcumin is a yellow-orange dye widely used as a spice, food coloring and food preservative. It also exhibits a broad range of therapeutic effects against different disorders such as cancer, diabetes, or neurodegenerative diseases. As a compound insoluble in water curcumin accumulates in cell membranes and due to this location it may indirectly lead to the observed effects by structurally altering the membrane environment. To exert strong structural effects on membrane curcumin needs to adopt a transbilayer orientation. However, there is no agreement in literature as to curcumin's orientation and its structural effects on membranes. Here, we investigated the effects of curcumin on lipid order, lipid phase transition, and local polarity in a model liposome membranes made of DMPC or DSPC using electron paramagnetic resonance (EPR) spin labeling technique. Curcumin affected lipid order at different depths within the membrane: it slightly increased the phospholipid polar headgroup mobility as monitored by spectral parameters of T-PC, while along the acyl chain the ordering effect was observed in terms of order parameter S. Also, rotational correlation times τ 2B and τ 2C of 16-PC in the membrane center were increased by curcumin. Polarity measurements performed in frozen suspensions of liposomes revealed enhancement of water penetration by curcumin in the membrane center (16-PC) and in the polar headgroup region (T-PC) while the intermediate positions along the acyl chain (5-PC and 10-PC) were not significantly affected. Curcumin at a lower concentration (5 mol%) shifted the temperature of the DMPC main phase transition to lower values and increased the transition width, and at a higher concentration (10 mol%) abolished the transition completely. The observed effects suggest that curcumin adopts a transbilayer orientation within the membrane and most probably form oligomers of two molecules, each of them spanning the opposite bilayer leaflets. The effects are also discussed in terms of curcumin's protective activity and compared with those imposed on membranes by other natural dyes known for their protective role, namely polar carotenoids, lutein and zeaxanthin.

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10 6 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca 2+ , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca 2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10 6 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10 6 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10 6 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca 2+ ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca 2+ , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca 2+ , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10 6 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species., (© 2020 Blackwell Verlag GmbH.)

The antimicrobial peptide CGA-N12 (NH2-ALQGAKERAHQQ-COOH) is an active peptide derived from chromogranin A (CGA) and consists of the 65th to 76th amino acids of the N-terminus. The results of our previous studies showed that CGA-N12 exerts anti-Candida activity by inducing apoptosis without destroying the integrity of cell membranes. In this study, the effect of CGA-N12 on the cell membrane structure of Candida tropicalis was investigated. CGA-N12 resulted in the dissipation of the membrane potential, the increase in membrane fluidity, and the outflow of potassium ions in C. tropicalis without significantly changing the ergosterol level. Fluorescence quenching was applied to evaluate the membrane channel characteristics induced by CGA-N12 through detection of the following: membrane permeability of hydrated Cl- (ϕ ≈ 0.66 nm) using the membrane-impermeable halogen anion-selective fluorescent dye lucigenin, passage of the membrane-impermeable dye carboxyfluorescein (CF) (ϕ ≈ 1 nm) through the membrane, and membrane permeation of H3O+ based on the membrane non-permeable pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS). In conclusion, CGA-N12 can induce the formation of non-selective ion channels <1 nm in diameter in the membranes of C. tropicalis, resulting in the leakage of potassium ions, chloride ions, and protons, among others, leading to dissipation of the membrane potential. As a result, the fluidity of membranes is increased without destroying the synthesis of ergosterol is not affected., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM 1-3 Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM 2 Glu, whereas DDM 1 Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM 2-3 Glu, significantly affect the type and extent of interaction with biological structures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The proliferation of molds in domestic environments can lead to uncontrolled continuous exposure to mycotoxins. Even if not immediately symptomatic, this may result in chronic effects, such as, for instance, immunosuppression or allergenic promotion. Alternariol (AOH) is one of the most abundant mycotoxins produced by Alternaria alternata fungi, proliferating among others in fridges, as well as in humid walls. AOH was previously reported to have immunomodulatory potential. However, molecular mechanisms sustaining this effect remained elusive. In differentiated THP-1 macrophages, AOH hardly altered the secretion of pro-inflammatory mediators when co-incubated with lipopolysaccharide (LPS), opening up the possibility that the immunosuppressive potential of the toxin could be related to an alteration of a downstream pro-inflammatory signaling cascade. Intriguingly, the mycotoxin affected the membrane fluidity in macrophages and it synergistically reacted with the cholesterol binding agent MβCD. In silico modelling revealed the potential of the mycotoxin to intercalate in cholesterol-rich membrane domains, like caveolae, and immunofluorescence showed the modified interplay of caveolin-1 with Toll-like Receptor (TLR) 4. In conclusion, we identified the structural similarity with cholesterol as one of the key determinants of the immunomodulatory potential of AOH., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: DHA can regulate various physiological functions of cells. Our group has clarified the immunomodulatory activity and molecular mechanism of DHA on RAW264.7 cells. However, the effect of DHA on the membrane fatty acid environment and the activation of signaling pathways on the cell membrane is still not clear., Methods: In this study, we evaluated the fluidity, the potential and the fatty acid, phospholipid and protein composition of the RAW264.7 cell membrane by DHA treatment., Results: The fluidity of the RAW264.7 cell membrane was increased by DHA treatment. The results of membrane potential analysis suggested that DHA (2.4 μM) significantly reduced the surface potential of the cell membrane, which might influence the fluidity of cell membranes. In addition, the fatty acids and phospholipids were measured and the results indicated that DHA treatment (2.4 μM) altered the lipid environment and the composition of phospholipids on the RAW264.7 cell membrane. Then the LC-MS/MS-based label free quantitative proteomics approach was applied to identify a total of 86 differential proteins in the 2.4 μM DHA and control groups (>2.0-fold change or <0.5-fold change in protein expression); these proteins are most frequently related to the cell response to stimuli and the response to stress. These results suggested that DHA could alter the fluidity, the potential, the fatty acid and phospholipid composition of the RAW264.7 cell membrane, eventually affecting the proteins of the cell membrane, especially the changes in Siglec 1, iNOS, GPR120, Ras and MEK expressions (validated by western blot analysis), which are likely associated with the activation of the intracellular signaling pathway in RAW264.7 cells by DHA treatment.

The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) -3 and -5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

One of the congenital flaws of metabolism, phenylketonuria (PKU), is known to be related to the self-assembly of toxic fibrillar aggregates of phenylalanine (Phe) in blood at elevated concentrations. Our experimental findings using l-phenylalanine (l-Phe) at millimolar concentration suggest the formation of fibrillar morphologies in the dry phase, which in the solution phase interact strongly with the model membrane composed of 1,2-diacyl- sn -glycero-phosphocholine (LAPC) lipid, thereby decreasing the rigidity (or increasing the fluidity) of the membrane. The hydrophobic interaction, in addition to the electrostatic attraction of Phe with the model membrane, is found to be responsible for such phenomena. On the contrary, various microscopic observations reveal that such fibrillar morphologies of l-Phe are severely ruptured in the presence of its enantiomer d-phenylalanine (d-Phe), thereby converting the fibrillar morphologies into crushed flakes. Various biophysical studies, including the solvation dynamics experiment, suggest that this l-Phe in the presence of d-Phe, when interacting with the same model membrane, now reverts the rigidity of the membrane, i.e., increases the rigidity of the membrane, which was lost due to interaction with l-Phe exclusively. Fluorescence anisotropy measurements also support this reverse rigid character of the membrane in the presence of an enantiomeric mixture of amino acids. A comprehensive understanding of the interaction of Phe with the model membrane is further pursued at the single-molecular fluorescence detection level using fluorescence correlation spectroscopy (FCS) experiments. Therefore, our experimental conclusion interprets a linear correlation between increased permeability and enhanced fluidity of the membrane in the presence of l-Phe and certifies d-Phe as a therapeutic modulator of l-Phe fibrillar morphologies. Further, the study proposes that the rigidity of the membrane lost due to interaction with l-Phe was reinstated-in fact, increased-in the presence of the enantiomeric mixture containing both d- and l-Phe.

The polyphenolic compounds present in grape extracts have chemopreventive and anticancer properties. Here, we studied the ability of two grape skin extracts (GSEs), Autumn Royal and Egnatia, to influence the cell motility and membrane fluidity regulated by the enzyme Stearoyl-CoA desaturase-1 (SCD1) which increases with the cancer aggressiveness. Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs to evaluate cell proliferation and motility. SCD1 levels were evaluated in both treated cell lines, by membrane lipidomic analysis conducted by gas chromatography. The expression levels of SCD1 and other factors involved in the reorganization of the cytoskeleton and focal adhesions were assessed by Real-time PCR, Western Blotting, and Immunofluorescence staining. High-performance liquid chromatography (HPLC) analyses were performed to determine the phenolic composition in the GSEs, finding them more expressed in Autumn Royal than in Egnatia. Both treatments reduced the levels of SCD1, phospho-Rac1/Cdc42/Rac1/Cdc42 ratio, Cofilin, Vimentin, and phospho-Paxillin especially in Caco2 compared to SW480, showing a different behavior of the two cell lines to these natural compounds. Our findings show that GSEs block the cell migration and membrane fluidity through a new mechanism of action involving structural cellular components., Competing Interests: The authors declare no conflict of interest.