Spontaneous Formation and Fusion of Raspberry Vesicle Self-Assembled from Star Block Terpolymers in Aqueous Solution

The spontaneous formation and fusion of raspberry vesicles was studied using the dissipative particle dynamics (DPD) method. The vesicles were formed through the self-assembly of amphiphilic E12O6F2 star terpolymers in selective solvent. E and F blocks are solvophobic and the O block is solvophilic. The shortest F block plays a major role in the formation of raspberry vesicles. Distinct vesicle formation mechanisms were observed at different polymer concentrations. At higher concentrations, vesicles form via the bending and closure of an oblate F-bump-E bilayer. At lower concentrations, the formation pathway contains: the initial formation of a vesicle with a core, the combination of such vesicles into cylindrical micelles, and the bending of the cylindrical micelles to form a hollow vesicle. In addition, raspberry vesicle fusion is regulated by F bumps through the continuous coalescence of them from apposed vesicle membranes. The contact area bends, followed by the formation of a fusion pore and a tilted inner layer. As the pore sealed, the hemifusion structure appears, which further restructures to form a vesicle. Our results provide guidance on understanding the dynamic processes of complex vesicles and biological membrane fusion.

Comparison of photodynamic efficiency of cholesterol, selected cholesterol esters, metabolites and oxidation products on lipid peroxidation processes

Cholesterol (Ch) is one of the most important components of biological membranes, which has a significant impact on their biophysical properties. As a key component of lipid membranes, Ch along with other unsaturated lipids present in a biological membrane undergoes oxidation reaction during oxidative stress. Cholesterol oxidation products, cholesteryl esters and metabolites are also localise in lipid membranes, where they may modify membrane properties. In this work the impact of cholesterol, selected cholesteryl esters, cholesterol oxidation products and metabolites on lipid peroxidation induced by photodynamic action has been studied using EPR oximetry and direct detection of singlet oxygen phosphorescence at 1270 nm. The obtained rate constants values of interaction of selected lipids and sterols with singlet oxygen indicate that the tested compounds are not efficient singlet oxygen quenchers. Nevertheless, the presence of sterols modifies to different extend the oxygen photoconsumption rate in peroxidisable liposomes.

Flipping the switch: reverse-demand voltage-sensitive fluorophores

Fluorescence microscopy with fluorescent reporters that respond to environmental cues are a powerful method for interrogating biochemistry and biophysics in living systems. Photoinduced electron transfer (PeT) is commonly used as a trigger to modulate fluorescence in response to changes in the biological environment. PeT based indicators rely either on PeT into the excited state (acceptor PeT) or out of the excited state (donor PeT). Our group has been developing voltage-sensitive fluorophores (VF dyes) that respond to changes in biological membrane potential. We hypothesize that the mechanism of voltage sensitivity arises from acceptor PeT (a-PeT) from an electron-rich aniline-containing molecular wire into the excited state fluorophore, resulting in decreased fluorescence at negative membrane potentials. Here, we can reverse the direction of electron flow to access donor-excited PeT (d-PeT) VF dyes by introducing electron-withdrawing (EWG), rather than electron-rich molecular wires. Similar to first-generation aniline containing VF dyes, EWG-containing VF dyes show voltage-sensitive fluorescence, but with the opposite polarity: hyperpolarizing membrane potentials now give fluorescence increases. We use a combination of computation and experiment to estimate a ΔE of ~0.6 eV for voltage sensitivity in d-PeT indicators, show that two of the new reverse VF dyes are voltage sensitive, and provide the first example, to our knowledge, of a molecular sensor that can be tuned across energy regimes to access bi-directional electron flow for fluorescence sensing in living systems.

Enthalpic and Entropic Contributions to Interleaflet Coupling Drive Domain Registration and Anti-registration in Biological Membrane

Biological membrane is a complex self-assembly of lipids, sterols and proteins organized as a fluid bilayer of two closely stacked lipid leaflets. Differential molecular interactions among its diverse constituents give rise to heterogeneities in the membrane lateral organization. Under certain conditions, heterogeneities in the two leaflets can be spatially synchronised and exist as registered domains across the bilayer. Several contrasting theories behind mechanisms that induce registration of nanoscale domains have been suggested[1–3]. Following a recent study[4] showing the effect of position of lipid tail unsaturation on domain registration behavior, we decided to develop an analytical theory to elucidate the driving forces that create and maintain domain registry across leaflets. Towards this, we formulated a Hamiltonian for a stacked lattice system where site variables encapsulate the lipid molecular properties including the position of unsaturation and various other interactions that could drive phase separation and interleaflet coupling. We solve the Hamiltonian using Monte Carlo simulations and create a complete phase diagram that reports the presence or absence of registered domains as a function of various Hamiltonian parameters. We find that the interleaflet coupling should be described as a competing enthalpic contribution due to interaction of lipid tail termini, primarily due to saturated-saturated interactions, and an interleaflet entropic contribution from overlap of unsaturated tail termini. We find that higher position of unsaturation provides weaker interleaflet coupling. We also find points in our parameter space that allow thermodynamically stable nanodomains in our bilayer model, which we have verified by carrying out extended Monte Carlo simulations. These persistent non-coalescing registered nanodomains close to the lower end of the accepted nanodomain size range also point towards a possible “nanoscale” emulsion description of lateral heterogeneities in biological membrane leaflets.

Molecular interpretation of the carbon nitride performance as a template for the transport of anti-cancer drug into the biological membrane

Evaluation of interaction mechanism between 2-dimensional (2D) nanomaterials and cell membranes is a critical issue in providing guidelines for biomedical applications. Recent progress in computer-aided molecular design tools, especially molecular dynamics (MD) simulation, afford a cost-effective approach to achieving this goal. In this work, based on this hypothesis, by utilizing theoretical methods including MD simulation and free energy calculations, a process is evaluated in which the Doxorubicin (DOX)-loaded onto carbon nitride (CN) nanosheet faced with bilayer membrane. It should be mentioned that to achieve an efficient CN-based drug delivery system (DDS), in the first place, the intermolecular interaction between the carrier and DOX is investigated. The obtained results show that the DOX prefers a parallel orientation with respect to the CN surface via the formation of π–π stacking and H-bond interactions. Furthermore, the adsorption energy value between the drug and the carrier is evaluated at about − 312 kJ/mol. Moreover, the investigation of the interaction between the CN-DOX complex and the membrane reveals that due to the presence of polar heads in the lipid bilayer, the contribution of electrostatic energy is higher than the van der Waals energy. The global minimum in free energy surface of the DDS is located between the head groups of the cell membrane. Overall, it can be concluded that the CN nanosheet is a suitable candidate for transfer and stabilize DOX on the membrane.

Quinolone Resistance of Actinobacillus pleuropneumoniae Revealed through Genome and Transcriptome Analyses

Actinobacillus pleuropneumoniae is a pathogen that infects pigs and poses a serious threat to the pig industry. The emergence of quinolone-resistant strains of A.pleuropneumoniae further limits the choice of treatment. However, the mechanisms behind quinolone resistance in A.pleuropneumoniae remain unclear. The genomes of a ciprofloxacin-resistant strain, A. pleuropneumoniae SC1810 and its isogenic drug-sensitive counterpart were sequenced and analyzed using various bioinformatics tools, revealing 559 differentially expressed genes. The biological membrane, plasmid-mediated quinolone resistance genes and quinolone resistance-determining region were detected. Upregulated expression of efflux pump genes led to ciprofloxacin resistance. The expression of two porins, OmpP2B and LamB, was significantly downregulated in the mutant. Three nonsynonymous mutations in the mutant strain disrupted the water–metal ion bridge, subsequently reducing the affinity of the quinolone–enzyme complex for metal ions and leading to cross-resistance to multiple quinolones. The mechanism of quinolone resistance in A. pleuropneumoniae may involve inhibition of expression of the outer membrane protein genes ompP2B and lamB to decrease drug influx, overexpression of AcrB in the efflux pump to enhance its drug-pumping ability, and mutation in the quinolone resistance-determining region to weaken the binding of the remaining drugs. These findings will provide new potential targets for treatment.

Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed ‘split borosins’), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.