Despite longstanding excitement and progress toward understanding liquid-liquid phase separation in natural and artificial membranes, fundamental questions have persisted about which molecules are required for this phenomenon. Except in extraordinary circumstances, the smallest number of components that has produced large-scale, liquid-liquid phase separation in bilayers has stubbornly remained at three: a sterol, a phospholipid with ordered chains, and a phospholipid with disordered chains. This requirement of three components is puzzling because only two components are required for liquid-liquid phase separation in lipid monolayers, which resemble half of a bilayer. Inspired by reports that sterols interact closely with lipids with ordered chains, we tested whether phase separation would occur in bilayers in which a sterol and lipid were replaced by a single, joined sterol-lipid. By evaluating a panel of sterol-lipids, some of which are present in bacteria, we found a minimal bilayer of only two components (PChemsPC and diPhyPC) that robustly demixes into micron-scale, liquid phases. It suggests an additional role for sterol-lipids in nature, and it reveals a membrane in which tie-lines (and, therefore, the lipid composition of each phase) are straightforward to determine and will be consistent across multiple laboratories.

Negatively charged lipid bilayers enhance the interaction between a chemokine and an atypical chemokine receptor.

The transport of ions through biological ion channels is regulated not only by their structural characteristics but also by the composition of the phospholipid membrane, which serves as a carrier for nanochannels. Inspired by the modulation of ion currents by lipid membrane composition, exemplified by the activation of the K+ channel of Streptomyces A by anionic lipids, we present a biomimetic nanochannel system based on combining DNA nanotechnology with two-dimensional graphene oxide (GO) nanosheets. By designing multibranched DNA nanowires, we assemble programmable DNA scaffold networks (DSNs) on the GO surface to precisely control membrane composition. Modulating the DSN layers from one to five enhances DNA composition, yielding a maximum 12-fold enhancement in ion current, primarily due to charge effects. Incorporating DNAzymes facilitates reversible modulation of membrane composition, enabling cyclic conversion of ion current. This approach offers a pathway for creating devices with highly efficient, tunable ion transport, applicable in diverse fields like mass transport, environmental protection, biomimetic channels, and biosensors.

UNASSIGNED: A better understanding of diffusion reflection (DR) behavior may allow it to be used for more noninvasive applications, including the development of in vivo non-damaging techniques, especially for medical topical diagnosis and treatments.
UNASSIGNED: For a bilayer opaque substance where the attenuation of the upper layer is larger than the attenuation of the lower layer, the DR crossover point ( C p ) is location where the photons coming from the bottom layer start affecting the DR. We aim to study the dependency of the C p on absorption changes in different layers for constant scattering and top layer thickness.
UNASSIGNED: Monolayer and bilayer optical tissue-like phantoms were prepared and measured using a DR system. The results were compared with Monte Carlo simulations.
UNASSIGNED: There is an agreement between the experiments and the simulations. C p correlates with the square root of the absorption coefficient ratio of the lower layer to the top layer.
UNASSIGNED: The experimental findings support and validate the theoretical prediction describing the dependency of the C p on the square root of the ratio of the layers\' absorption coefficients. In addition, a secondary breaking point is suggested to be observed experimentally at the entrance to the noise area.

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS\'s affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes.

Despite ongoing research on antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), their precise translocation mechanism remains elusive. This includes Buforin 2 (BF2), a well-known AMP, for which spontaneous translocation across the membrane has been proposed but a high barrier has been calculated. Here, we used computer simulations to investigate the effect of a nonequilibrium situation where the peptides are adsorbed on one side of the lipid bilayer, mimicking experimental conditions. We demonstrated that the asymmetric membrane adsorption of BF2 enhances its translocation across the lipid bilayer by lowering the energy barrier by tens of kJ mol-1. We showed that asymmetric membrane adsorption also reduced the free energy barrier of lipid flip-flop but remained unlikely even at BF2 surface saturation. These results provide insight into the driving forces behind membrane translocation of cell-penetrating peptides in nonequilibrium conditions, mimicking experiments.

Antimicrobial peptides (AMPs) are promising candidates in combating multidrug-resistant microorganisms because of their unique mode of action. Among these peptides, ultrashort AMPs (USAMPs) possess sequences containing less than 10 amino acids and have some advantages over traditional AMPs. However, one of the main limitations of designing novel and highly active USAMPs is that their mechanism of action at the molecular level is not well-known. In this article, we report the antimicrobial mechanism of the USAMP verine (R3W4V) with high antibacterial activity against Escherichia coli. Here, by using well-tempered bias-exchange metadynamics simulations and long-time conventional molecular dynamics simulations, we evaluated whether verine exhibits the same antimicrobial mode of action as that of traditional AMPs. The single verine-membrane system exhibited a relatively flat surface with multiple shallow minima separated by very small energy barriers and adopted highly dynamic structural ensembles. Although the verine sequence is very short, it can still exist briefly in the center of the cell membrane in a transmembrane state. As the concentration of verine increased, the transmembrane conformation was relatively stabilized in the membrane center or proceeded toward the membrane bottom. The lipid bilayer membrane showed relatively large deformation, including the phospholipid head groups embedded inside the lipid hydrophobic center, accompanied by a flip-flop of some lipids. Simulation results indicated that verine has a specific mechanism of action different from that of traditional AMPs. Based on this antimicrobial mechanism of verine, we can design new high-potential USAMPs by enhancing the structural stability of the transmembrane state.

Fluorescent lipid probes such as 1-palmitoyl-2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl]amino-hexanoyl)-sn-glycero-3-phosphocholine (C6 NBD-PC) have been used extensively to study the kinetics of lipid flip-flop. However, the efficacy of these probes as reliable reporters of native lipid translocation has never been tested. In this study, sum-frequency vibrational spectroscopy (SFVS) was used to measure the kinetics of C6 NBD-PC lipid flip-flop and the flip-flop of native lipids in planar supported lipid bilayers. C6 NBD-PC was investigated at concentrations of 1 and 3 mol. % in both chain-matched 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chain-mismatched 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) to assess the ability of C6 NBD-PC to mimic the behavior of the surrounding matrix lipids. It was observed that C6 NBD-PC exhibited faster flip-flop kinetics compared to the native lipids in both DPPC and DSPC matrices, with notably accelerated rates in the chain-mismatched DSPC system. SFVS was also used to measure the acyl chain orientation and gauche content of C6 NBD-PC in both DPPC and DSPC membranes. In the DSPC matrix (chain mismatched), C6 NBD-PC was more disordered in terms of both gauche content and acyl tilt, whereas it maintained an orientation similar to that of the native lipids in the DPPC matrix (chain matched). In addition, the flip-flop kinetics of C6 NBD-PC were also measured using second-harmonic generation (SHG) spectroscopy, by probing the motion of the NBD chromophore directly. The flip-flop kinetics measured by SHG were consistent with those obtained from SFVS. This study also marks the first instance of phospholipid flip-flop kinetics being measured via SHG. The results of this study clearly demonstrate that C6 NBD-PC does not adequately mimic the behavior of native lipids within a membrane. These findings also highlight the significant impact of the lipid matrix on the flip-flop behavior of the fluorescently labeled lipid, C6 NBD-PC.

Statins are among the most widely used drugs for the inhibition of cholesterol biosynthesis, prevention of cardiovascular diseases, and treatment of hypercholesterolemia. Additionally, statins also exhibit cholesterol-independent benefits in various diseases, including neuroprotective properties in Alzheimer\'s disease, anti-inflammatory effects in coronary artery disease, and antiproliferative activities in cancer, which likely result from the statins\' interaction and alteration of lipid bilayers. However, the membrane-modulatory effects of statins and the mechanisms by which statins alter lipid bilayers remain poorly understood. In this work, we explore the membrane-modulating effects of statins on model lipid bilayers and live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM) combined with viscosity-sensitive environmental probes, we demonstrate that hydrophobic, but not hydrophilic, statins are capable of changing the microviscosity and lipid order in model and live cell membranes. Furthermore, we show that hydrophobic simvastatin is capable of forming nanoscale cholesterol-rich domains and homogenizing the cholesterol concentrations in lipid bilayers. Our results provide a mechanistic framework for understanding the bimodal effects of simvastatin on the lipid order and the lateral organization of cholesterol in lipid bilayers. Finally, we demonstrate that simvastatin temporarily decreases the microviscosity of live cell plasma membranes, making them more permeable and increasing the level of intracellular chemotherapeutic drug accumulation.

Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design.